def motion_correct_file(args, n_iter=0, max_iter=10):
try:
in_fn, file_index, template, mc_args, \
subidx, apply_subidx, apply_ofn = args
max_shifts, strides, overlaps, \
max_deviation_rigid, shifts_opencv, border_nan = mc_args
mc = MotionCorrect(in_fn,
dview=None,
max_shifts=max_shifts,
strides=strides,
overlaps=overlaps,
max_deviation_rigid=max_deviation_rigid,
shifts_opencv=shifts_opencv,
nonneg_movie=True,
splits_els=1,
splits_rig=1,
border_nan=border_nan,
subidx=subidx)
mc.motion_correct(template=template, save_movie=False)
if apply_subidx is not None:
applied_mov = mc.apply_shifts_movie(in_fn, apply_subidx)
applied_mov.save(apply_ofn)
return (file_index, np.mean(mc.templates_rig, axis=0))
except Exception as e:
if n_iter < max_iter:
print("Retrying %s due to %s" % (in_fn, e))
return motion_correct_file(args, n_iter=n_iter + 1)
else:
return Exception("Failed max_iter: %s times" % max_iter)
def caiman_motion_correct(fnames, opts):
'''do the motion correction of the Caiman pipeline and save results.
Parameters
----------
fnames : string
name of tiff movie
opts : caiman object with the options for motion correction'''
#%% start a cluster for parallel processing (if a cluster already exists it will be closed and a new session will be opened)
if 'dview' in locals():
cm.stop_server(dview=dview)
c, dview, n_processes = cm.cluster.setup_cluster(backend='local',
n_processes=None,
single_thread=False)
# first we create a motion correction object with the parameters specified
mc = MotionCorrect(fnames, dview=dview, **opts.get_group('motion'))
mc.motion_correct(save_movie=True)
border_to_0 = 0 if mc.border_nan is 'copy' else mc.border_to_0
# memory map the file in order 'C'
fname_new = cm.save_memmap(mc.mmap_file,
base_name='memmap_',
order='C',
border_to_0=border_to_0,
dview=dview) # exclude borders
cm.stop_server(dview=dview)
def motion_correct_file(folder, dview):
import caiman as cm
from caiman.motion_correction import MotionCorrect
from caiman.source_extraction.cnmf import params as params
print(folder)
mc_dict = {
'fnames': [os.path.join(folder, 'movie_total.hdf5')],
'fr': 7,
'decay_time': 1.5,
'dxy': [1.15, 1.15],
'pw_rigid': False,
'max_shifts': [5,5],
'strides': None,
'overlaps': None,
'max_deviation_rigid': None,
'border_nan': 'copy'
}
opts = params.CNMFParams(params_dict=mc_dict)
mc = MotionCorrect(mc_dict['fnames'], dview=dview, **opts.get_group('motion'))
mc.motion_correct(save_movie=True)
def motion_correct(self):
"""
Do motion correction.
"""
# Set up motion correction object and load parameters.
mc = MotionCorrect(self.fnames,
dview=self.dview,
**self.opts.get_group('motion'))
# Do motion correction.
mc.motion_correct(save_movie=True)
# Plot motion correction statistics.
fname_mc = mc.fname_tot_els if self.opts.motion[
'pw_rigid'] else mc.fname_tot_rig
if self.opts.motion['pw_rigid']:
self.bord_px = np.ceil(
np.maximum(np.max(np.abs(mc.x_shifts_els)),
np.max(np.abs(mc.y_shifts_els)))).astype(np.int)
else:
self.bord_px = np.ceil(np.max(np.abs(mc.shifts_rig))).astype(
np.int)
plt.subplot(1, 2, 1)
plt.imshow(mc.total_template_rig) # % plot template
plt.subplot(1, 2, 2)
plt.plot(mc.shifts_rig) # % plot rigid shifts
plt.legend(['x shifts', 'y shifts'])
plt.xlabel('frames')
plt.ylabel('pixels')
# Save memory map.
self.bord_px = 0 if self.opts.motion[
'border_nan'] is 'copy' else self.bord_px
self.fname_new = cm.save_memmap(fname_mc,
base_name='full',
order='C',
border_to_0=self.bord_px)
def motion_corr(fnames, dview, opts, disp_movie, is_3d=False):
"""Perform motion correction"""
# Create a motion correction object with the parameters specified. Note
# that the file is not loaded in memory
mc = MotionCorrect(fnames, dview=dview, **opts.get_group('motion'))
# Run piecewise-rigid motion correction using NoRMCorre
mc.motion_correct(save_movie=True)
# Determine maximum shift to be used for trimming against NaNs
border_to_0 = 0 if mc.border_nan is 'copy' else mc.border_to_0
# Compare with original movie
if disp_movie and not is_3d:
m_els = cm.load(mc.fname_tot_els)
m_orig = cm.load_movie_chain(fnames)
ds_ratio = 0.2
cm.concatenate([
m_orig.resize(1, 1, ds_ratio) - mc.min_mov * mc.nonneg_movie,
m_els.resize(1, 1, ds_ratio)
],
axis=2).play(fr=60, gain=15, magnification=2,
offset=0) # press q to exit
return mc, border_to_0
def main():
pass # For compatibility between running under Spyder and the CLI
#%% Select file(s) to be processed (download if not present)
# fnames = [os.path.join(caiman_datadir(), 'example_movies/sampled3dMovieRigid.nwb')]
fnames = [
os.path.join(caiman_datadir(), 'example_movies/sampled3dMovie2.nwb')
]
# filename to be created or processed
# dataset dependent parameters
fr = 5 # imaging rate in frames per second
decay_time = 0.4 # length of a typical transient in seconds
starting_time = 0.
#%% load the file and save it in the NWB format (if it doesn't exist already)
if not os.path.exists(fnames[0]):
# fnames_orig = [os.path.join(caiman_datadir(), 'example_movies/sampled3dMovieRigid.h5')] # filename to be processed
fnames_orig = [
os.path.join(caiman_datadir(), 'example_movies/sampled3dMovie2.h5')
] # filename to be processed
orig_movie = cm.load(fnames_orig, fr=fr, is3D=True)
# orig_movie = cm.load_movie_chain(fnames_orig,fr=fr,is3D=True)
# save file in NWB format with various additional info
orig_movie.save(fnames[0],
sess_desc='test',
identifier='demo 3d',
exp_desc='demo movie',
imaging_plane_description='multi plane',
emission_lambda=520.0,
indicator='none',
location='visual cortex',
starting_time=starting_time,
experimenter='NAOMi',
lab_name='Tank Lab',
institution='Princeton U',
experiment_description='Experiment Description',
session_id='Session 1',
var_name_hdf5='TwoPhotonSeries')
#%% First setup some parameters for data and motion correction
# motion correction parameters
dxy = (1., 1., 5.) # spatial resolution in x, y, and z in (um per pixel)
# note the lower than usual spatial resolution here
max_shift_um = (10., 10., 10.) # maximum shift in um
patch_motion_um = (50., 50., 30.
) # patch size for non-rigid correction in um
# pw_rigid = False # flag to select rigid vs pw_rigid motion correction
niter_rig = 1
pw_rigid = True # flag to select rigid vs pw_rigid motion correction
# maximum allowed rigid shift in pixels
max_shifts = [int(a / b) for a, b in zip(max_shift_um, dxy)]
# start a new patch for pw-rigid motion correction every x pixels
strides = tuple([int(a / b) for a, b in zip(patch_motion_um, dxy)])
# overlap between pathes (size of patch in pixels: strides+overlaps)
overlaps = (24, 24, 4)
# maximum deviation allowed for patch with respect to rigid shifts
max_deviation_rigid = 3
is3D = True
mc_dict = {
'fnames': fnames,
'fr': fr,
'decay_time': decay_time,
'dxy': dxy,
'pw_rigid': pw_rigid,
'niter_rig': niter_rig,
'max_shifts': max_shifts,
'strides': strides,
'overlaps': overlaps,
'max_deviation_rigid': max_deviation_rigid,
'border_nan': 'copy',
'var_name_hdf5': 'acquisition/TwoPhotonSeries',
'is3D': is3D,
'splits_els': 12,
'splits_rig': 12
}
opts = params.CNMFParams(
params_dict=mc_dict
) #NOTE: default adjustments of parameters are not set yet, manually setting them now
# %% play the movie (optional)
# playing the movie using opencv. It requires loading the movie in memory.
# To close the video press q
display_images = True
if display_images:
m_orig = cm.load_movie_chain(fnames,
var_name_hdf5=opts.data['var_name_hdf5'],
is3D=True)
T, h, w, z = m_orig.shape # Time, plane, height, weight
m_orig = np.reshape(np.transpose(m_orig, (3, 0, 1, 2)), (T * z, h, w))
ds_ratio = 0.2
moviehandle = m_orig.resize(1, 1, ds_ratio)
moviehandle.play(q_max=99.5, fr=60, magnification=2)
# %% start a cluster for parallel processing
# NOTE: ignore dview right now for debugging purposes
# c, dview, n_processes = cm.cluster.setup_cluster(
# backend='local', n_processes=None, single_thread=False)
# %%% MOTION CORRECTION
# first we create a motion correction object with the specified parameters
mc = MotionCorrect(fnames,
dview=None,
var_name_hdf5=opts.data['var_name_hdf5'],
**opts.get_group('motion'))
# mc = MotionCorrect(fnames, dview=dview, var_name_hdf5=opts.data['var_name_hdf5'], **opts.get_group('motion'))
# note that the file is not loaded in memory
# %% Run (piecewise-rigid motion) correction using NoRMCorre
mc.motion_correct(save_movie=True)
# %% compare with original movie
if display_images:
m_orig = cm.load_movie_chain(fnames,
var_name_hdf5=opts.data['var_name_hdf5'],
is3D=True)
T, h, w, z = m_orig.shape # Time, plane, height, weight
m_orig = np.reshape(np.transpose(m_orig, (3, 0, 1, 2)), (T * z, h, w))
m_els = cm.load(mc.mmap_file, is3D=True)
m_els = np.reshape(np.transpose(m_els, (3, 0, 1, 2)), (T * z, h, w))
ds_ratio = 0.2
moviehandle = cm.concatenate([
m_orig.resize(1, 1, ds_ratio) - mc.min_mov * mc.nonneg_movie,
m_els.resize(1, 1, ds_ratio)
],
axis=2)
moviehandle.play(fr=60, q_max=99.5, magnification=2) # press q to exit
def run_volpy(fnames,
options=None,
do_motion_correction=True,
do_memory_mapping=True,
fr=400):
#pass # For compatibility between running under Spyder and the CLI
# %% Load demo movie and ROIs
file_dir = os.path.split(fnames)[0]
path_ROIs = [file for file in os.listdir(file_dir) if 'ROIs_gt' in file]
if len(path_ROIs) > 0:
path_ROIs = path_ROIs[0]
#path_ROIs = '/home/nel/NEL-LAB Dropbox/NEL/Datasets/voltage_lin/peyman_golshani/ROIs.hdf5'
#%% dataset dependent parameters
# dataset dependent parameters
fr = fr # sample rate of the movie
# motion correction parameters
pw_rigid = False # flag for pw-rigid motion correction
gSig_filt = (3, 3) # size of filter, in general gSig (see below),
# change this one if algorithm does not work
max_shifts = (5, 5) # maximum allowed rigid shift
strides = (
48, 48
) # start a new patch for pw-rigid motion correction every x pixels
overlaps = (24, 24
) # overlap between pathes (size of patch strides+overlaps)
max_deviation_rigid = 3 # maximum deviation allowed for patch with respect to rigid shifts
border_nan = 'copy'
opts_dict = {
'fnames': fnames,
'fr': fr,
'pw_rigid': pw_rigid,
'max_shifts': max_shifts,
'gSig_filt': gSig_filt,
'strides': strides,
'overlaps': overlaps,
'max_deviation_rigid': max_deviation_rigid,
'border_nan': border_nan
}
opts = volparams(params_dict=opts_dict)
# %% start a cluster for parallel processing
c, dview, n_processes = cm.cluster.setup_cluster(backend='local',
n_processes=None,
single_thread=False)
# %%% MOTION CORRECTION
# first we create a motion correction object with the specified parameters
mc = MotionCorrect(fnames, dview=dview, **opts.get_group('motion'))
# Run correction
do_motion_correction = do_motion_correction
if do_motion_correction:
mc.motion_correct(save_movie=True)
else:
mc_list = [
file for file in os.listdir(file_dir)
if (os.path.splitext(os.path.split(fnames)[-1])[0] in file
and '.mmap' in file)
]
mc.mmap_file = [os.path.join(file_dir, mc_list[0])]
print(f'reuse previously saved motion corrected file:{mc.mmap_file}')
# %% MEMORY MAPPING
do_memory_mapping = do_memory_mapping
if do_memory_mapping:
border_to_0 = 0 if mc.border_nan == 'copy' else mc.border_to_0
# you can include the boundaries of the FOV if you used the 'copy' option
# during motion correction, although be careful about the components near
# the boundaries
# memory map the file in order 'C'
fname_new = cm.save_memmap_join(
mc.mmap_file,
base_name='memmap_' +
os.path.splitext(os.path.split(fnames)[-1])[0],
add_to_mov=border_to_0,
dview=dview) # exclude border
else:
mmap_list = [
file for file in os.listdir(file_dir)
if ('memmap_' +
os.path.splitext(os.path.split(fnames)[-1])[0]) in file
]
fname_new = os.path.join(file_dir, mmap_list[0])
print(f'reuse previously saved memory mapping file:{fname_new}')
# %% SEGMENTATION
# create summary images
img = mean_image(mc.mmap_file[0], window=1000, dview=dview)
img = (img - np.mean(img)) / np.std(img)
gaussian_blur = False # Use gaussian blur when there is too much noise in the video
Cn = local_correlations_movie_offline(mc.mmap_file[0],
fr=fr,
window=fr * 4,
stride=fr * 4,
winSize_baseline=fr,
remove_baseline=True,
gaussian_blur=gaussian_blur,
dview=dview).max(axis=0)
img_corr = (Cn - np.mean(Cn)) / np.std(Cn)
summary_images = np.stack([img, img, img_corr], axis=0).astype(np.float32)
# ! save summary image, it is used in GUI
cm.movie(summary_images).save(fnames[:-5] + '_summary_images.tif')
#plt.imshow(summary_images[0])
#%% three methods for segmentation
methods_list = [
'manual_annotation', # manual annotation needs user to prepare annotated datasets same format as demo ROIs
'gui_annotation', # use gui to manually annotate neurons, but this is still under developing
'maskrcnn'
] # maskrcnn is a convolutional network trained for finding neurons using summary images
method = methods_list[0]
if method == 'manual_annotation':
#with h5py.File(path_ROIs, 'r') as fl:
# ROIs = fl['mov'][()]
ROIs = np.load(os.path.join(file_dir, path_ROIs))
elif method == 'gui_annotation':
# run volpy_gui file in the caiman/source_extraction/volpy folder
# load the summary images you have just saved
# save the ROIs to the video folder
path_ROIs = caiman_datadir() + '/example_movies/volpy/gui_roi.hdf5'
with h5py.File(path_ROIs, 'r') as fl:
ROIs = fl['mov'][()]
elif method == 'maskrcnn': # Important!! make sure install keras before using mask rcnn
weights_path = download_model('mask_rcnn')
weights_path = '/home/nel/Code/NEL_LAB/Mask_RCNN/logs/neurons20200824T1032/mask_rcnn_neurons_0040.h5'
ROIs = utils.mrcnn_inference(
img=summary_images.transpose([1, 2, 0]),
size_range=[5, 100],
weights_path=weights_path,
display_result=True
) # size parameter decides size range of masks to be selected
#np.save(os.path.join(file_dir, 'ROIs'), ROIs)
# %% restart cluster to clean up memory
cm.stop_server(dview=dview)
c, dview, n_processes = cm.cluster.setup_cluster(backend='local',
n_processes=None,
single_thread=False,
maxtasksperchild=1)
# %% parameters for trace denoising and spike extraction
ROIs = ROIs # region of interests
index = list(range(len(ROIs))) # index of neurons
weights = None # reuse spatial weights
context_size = 35 # number of pixels surrounding the ROI to censor from the background PCA
flip_signal = True # Important!! Flip signal or not, True for Voltron indicator, False for others
hp_freq_pb = 1 / 3 # parameter for high-pass filter to remove photobleaching
threshold_method = 'adaptive_threshold' # 'simple' or 'adaptive_threshold'
min_spikes = 30 # minimal spikes to be found
threshold = 4 # threshold for finding spikes, increase threshold to find less spikes
do_plot = False # plot detail of spikes, template for the last iteration
ridge_bg = 0.01 # ridge regression regularizer strength for background removement, larger value specifies stronger regularization
sub_freq = 20 # frequency for subthreshold extraction
weight_update = 'ridge' # 'ridge' or 'NMF' for weight update
n_iter = 2
opts_dict = {
'fnames': fname_new,
'ROIs': ROIs,
'index': index,
'weights': weights,
'context_size': context_size,
'flip_signal': flip_signal,
'hp_freq_pb': hp_freq_pb,
'threshold_method': threshold_method,
'min_spikes': min_spikes,
'threshold': threshold,
'do_plot': do_plot,
'ridge_bg': ridge_bg,
'sub_freq': sub_freq,
'weight_update': weight_update,
'n_iter': n_iter
}
opts.change_params(params_dict=opts_dict)
if options is not None:
print('using external options')
opts.change_params(params_dict=options)
else:
print('not using external options')
#%% TRACE DENOISING AND SPIKE DETECTION
vpy = VOLPY(n_processes=n_processes, dview=dview, params=opts)
vpy.fit(n_processes=n_processes, dview=dview)
#%% visualization
display_images = False
if display_images:
print(np.where(
vpy.estimates['locality'])[0]) # neurons that pass locality test
idx = np.where(vpy.estimates['locality'] > 0)[0]
utils.view_components(vpy.estimates, img_corr, idx)
#%% reconstructed movie
# note the negative spatial weights is cutoff
if display_images:
mv_all = utils.reconstructed_movie(vpy.estimates,
fnames=mc.mmap_file,
idx=idx,
scope=(0, 1000),
flip_signal=flip_signal)
mv_all.play(fr=40)
#%% save the result in .npy format
save_result = True
if save_result:
vpy.estimates['ROIs'] = ROIs
save_name = f'volpy_{os.path.split(fnames)[1][:-5]}_{opts.volspike["threshold_method"]}_{opts.volspike["threshold"]}_{opts.volspike["weight_update"]}_bg_{opts.volspike["ridge_bg"]}'
np.save(os.path.join(file_dir, save_name), vpy.estimates)
# %% STOP CLUSTER and clean up log files
cm.stop_server(dview=dview)
log_files = glob.glob('*_LOG_*')
for log_file in log_files:
os.remove(log_file)
def run_alignment(mouse, sessions, motion_correction_v, cropping_v, dview):
"""
This is the main function for the alignment step. It applies methods
from the CaImAn package used originally in motion correction
to do alignment.
"""
for session in sessions:
# Update the database
file_name = f"mouse_{mouse}_session_{session}_alignment"
sql1 = "UPDATE Analysis SET alignment_main=? WHERE mouse = ? AND session=? AND motion_correction_v =? AND cropping_v=? "
val1 = [file_name, mouse, session, motion_correction_v, cropping_v]
cursor.execute(sql1, val1)
# Determine the output .mmap file name
output_mmap_file_path = os.environ[
'DATA_DIR_LOCAL'] + f'data/interim/alignment/main/{file_name}.mmap'
sql = "SELECT motion_correction_main FROM Analysis WHERE mouse = ? AND session=? AND motion_correction_v =? AND cropping_v=? "
val = [mouse, session, motion_correction_v, cropping_v]
cursor.execute(sql, val)
result = cursor.fetchall()
input_mmap_file_list = []
inter = []
for x in result:
inter += x
for y in inter:
input_mmap_file_list.append(y)
sql = "SELECT motion_correction_cropping_points_x1 FROM Analysis WHERE mouse = ? AND session=?AND motion_correction_v =? AND cropping_v=? "
val = [mouse, session, motion_correction_v, cropping_v]
cursor.execute(sql, val)
result = cursor.fetchall()
x_ = []
inter = []
for i in result:
inter += i
for j in range(0, len(inter)):
x_.append(inter[j])
sql = "SELECT motion_correction_cropping_points_x2 FROM Analysis WHERE mouse = ? AND session=? AND motion_correction_v =? AND cropping_v=? "
val = [mouse, session, motion_correction_v, cropping_v]
cursor.execute(sql, val)
result = cursor.fetchall()
_x = []
inter = []
for i in result:
inter += i
for j in range(0, len(inter)):
_x.append(inter[j])
sql = "SELECT motion_correction_cropping_points_y1 FROM Analysis WHERE mouse = ? AND session=? AND motion_correction_v =? AND cropping_v=?"
val = [mouse, session, motion_correction_v, cropping_v]
cursor.execute(sql, val)
result = cursor.fetchall()
_y = []
inter = []
for i in result:
inter += i
for j in range(0, len(inter)):
_y.append(inter[j])
sql = "SELECT motion_correction_cropping_points_y2 FROM Analysis WHERE mouse = ? AND session=? AND motion_correction_v =? AND cropping_v=?"
val = [mouse, session, motion_correction_v, cropping_v]
cursor.execute(sql, val)
result = cursor.fetchall()
y_ = []
inter = []
for i in result:
inter += i
for j in range(0, len(inter)):
y_.append(inter[j])
new_x1 = max(x_)
new_x2 = max(_x)
new_y1 = max(y_)
new_y2 = max(_y)
m_list = []
for i in range(len(input_mmap_file_list)):
m = cm.load(input_mmap_file_list[i])
m = m.crop(new_x1 - x_[i], new_x2 - _x[i], new_y1 - y_[i],
new_y2 - _y[i], 0, 0)
m_list.append(m)
# Concatenate them using the concat function
m_concat = cm.concatenate(m_list, axis=0)
fname = m_concat.save(output_mmap_file_path, order='C')
# MOTION CORRECTING EACH INDIVIDUAL MOVIE WITH RESPECT TO A TEMPLATE MADE OF THE FIRST MOVIE
logging.info(
'Performing motion correction on all movies with respect to a template made of the first movie.'
)
t0 = datetime.datetime.today()
# parameters alignment
sql5 = "SELECT make_template_from_trial,gSig_filt,max_shifts,niter_rig,strides,overlaps,upsample_factor_grid,num_frames_split,max_deviation_rigid,shifts_opencv,use_conda,nonneg_movie, border_nan FROM Analysis WHERE alignment_main=? "
val5 = [
file_name,
]
cursor.execute(sql5, val5)
myresult = cursor.fetchall()
para = []
aux = []
for x in myresult:
aux = x
for y in aux:
para.append(y)
parameters = {
'make_template_from_trial': para[0],
'gSig_filt': (para[1], para[1]),
'max_shifts': (para[2], para[2]),
'niter_rig': para[3],
'strides': (para[4], para[4]),
'overlaps': (para[5], para[5]),
'upsample_factor_grid': para[6],
'num_frames_split': para[7],
'max_deviation_rigid': para[8],
'shifts_opencv': para[9],
'use_cuda': para[10],
'nonneg_movie': para[11],
'border_nan': para[12]
}
# Create a template of the first movie
template_index = parameters['make_template_from_trial']
m0 = cm.load(input_mmap_file_list[1])
[x1, x2, y1, y2] = [x_, _x, y_, _y]
for i in range(len(input_mmap_file_list)):
m0 = m0.crop(new_x1 - x_[i], new_x2 - _x[i], new_y1 - y_[i],
new_y2 - _y[i], 0, 0)
m0_filt = cm.movie(
np.array([
high_pass_filter_space(m_, parameters['gSig_filt'])
for m_ in m0
]))
template0 = cm.motion_correction.bin_median(
m0_filt.motion_correct(
5, 5, template=None)[0]) # may be improved in the future
# Setting the parameters
opts = params.CNMFParams(params_dict=parameters)
# Create a motion correction object
mc = MotionCorrect(fname, dview=dview, **opts.get_group('motion'))
# Perform non-rigid motion correction
mc.motion_correct(template=template0, save_movie=True)
# Cropping borders
x_ = math.ceil(
abs(np.array(mc.shifts_rig)[:, 1].max()
) if np.array(mc.shifts_rig)[:, 1].max() > 0 else 0)
_x = math.ceil(
abs(np.array(mc.shifts_rig)[:, 1].min()
) if np.array(mc.shifts_rig)[:, 1].min() < 0 else 0)
y_ = math.ceil(
abs(np.array(mc.shifts_rig)[:, 0].max()
) if np.array(mc.shifts_rig)[:, 0].max() > 0 else 0)
_y = math.ceil(
abs(np.array(mc.shifts_rig)[:, 0].min()
) if np.array(mc.shifts_rig)[:, 0].min() < 0 else 0)
# Load the motion corrected movie into memory
movie = cm.load(mc.fname_tot_rig[0])
# Crop all movies to those border pixels
movie.crop(x_, _x, y_, _y, 0, 0)
sql1 = "UPDATE Analysis SET alignment_x1=?, alignment_x2 =?, alignment_y1=?, alignment_y2=? WHERE mouse = ? AND session=? AND motion_correction_v =? AND cropping_v=?"
val1 = [
x_, _x, y_, _y, mouse, session, motion_correction_v, cropping_v
]
cursor.execute(sql1, val1)
# save motion corrected and cropped movie
output_mmap_file_path_tot = movie.save(
os.environ['DATA_DIR_LOCAL'] +
f'data/interim/alignment/main/{file_name}.mmap',
order='C')
logging.info(
f' Cropped and saved rigid movie as {output_mmap_file_path_tot}')
# Remove the remaining non-cropped movie
os.remove(mc.fname_tot_rig[0])
# Create a timeline and store it
sql = "SELECT trial FROM Analysis WHERE mouse = ? AND session=? AND motion_correction_v =? AND cropping_v=?"
val = [mouse, session, motion_correction_v, cropping_v]
cursor.execute(sql, val)
result = cursor.fetchall()
trial_index_list = []
inter = []
for i in result:
inter += i
for j in range(0, len(inter)):
trial_index_list.append(inter[j])
timeline = [[trial_index_list[0], 0]]
timepoints = [0]
for i in range(1, len(m_list)):
m = m_list[i]
timeline.append(
[trial_index_list[i], timeline[i - 1][1] + m.shape[0]])
timepoints.append(timepoints[i - 1] + m.shape[0])
timeline_pkl_file_path = os.environ[
'DATA_DIR'] + f'data/interim/alignment/meta/timeline/{file_name}.pkl'
with open(timeline_pkl_file_path, 'wb') as f:
pickle.dump(timeline, f)
sql1 = "UPDATE Analysis SET alignment_timeline=? WHERE mouse = ? AND session=?AND motion_correction_v =? AND cropping_v=? "
val1 = [
timeline_pkl_file_path, mouse, session, motion_correction_v,
cropping_v
]
cursor.execute(sql1, val1)
timepoints.append(movie.shape[0])
dt = int((datetime.datetime.today() - t0).seconds /
60) # timedelta in minutes
sql1 = "UPDATE Analysis SET alignment_duration_concatenation=? WHERE mouse = ? AND session=?AND motion_correction_v =? AND cropping_v=? "
val1 = [dt, mouse, session, motion_correction_v, cropping_v]
cursor.execute(sql1, val1)
logging.info(f' Performed concatenation. dt = {dt} min.')
## modify all motion correction file to the aligned version
data_dir = os.environ[
'DATA_DIR'] + 'data/interim/motion_correction/main/'
for i in range(len(input_mmap_file_list)):
aligned_movie = movie[timepoints[i]:timepoints[i + 1]]
motion_correction_output_aligned = aligned_movie.save(
data_dir + file_name + '_els' + '.mmap', order='C')
sql1 = "UPDATE Analysis SET motion_correct_align=? WHERE motion_correction_meta=? AND motion_correction_v"
val1 = [
motion_correction_output_aligned, input_mmap_file_list[i],
motion_correction_v
]
cursor.execute(sql1, val1)
database.commit()
return
def caimanAlign(fnames, frameRate=20):
"""
fnames: list of file path
"""
startSeconds = time.time()
opts = caimanOptions.createParams(fnames, frameRate=frameRate)
#start a cluster for parallel processing (if a cluster already exists it will be closed and a new session will be opened)
if 'dview' in locals():
cm.stop_server(dview=dview)
c, dview, n_processes = cm.cluster.setup_cluster(backend='local',
n_processes=None,
single_thread=False)
#
# create a motion correction object with the parameters specified.
#Note that the file is not loaded in memory
mc = MotionCorrect(fnames, dview=dview, **opts.get_group('motion'))
# set mmap_file
'''
tmpPath, tmpExt = os.path.splitext(fnames[0])
tmpPath += '.mmap'
print('setting mc.mmap_file to tmpPath:', tmpPath)
mc.mmap_file = tmpPath
'''
#print('mc.mmap_file:', mc.mmap_file)
#
# perform motion correction
# We will perform piecewise rigid motion correction using the NoRMCorre algorithm.
# This has already been selected by setting pw_rigid=True when defining the parameters object.
print(' performing motion correction ... please wait ...', fnames[0])
mc.motion_correct(save_movie=True)
m_els = cm.load(mc.fname_tot_els)
border_to_0 = 0 if mc.border_nan is 'copy' else mc.border_to_0
print(' border_to_0:', border_to_0)
# maximum shift to be used for trimming against NaNs
#
# memory mapping
print(' memory mapping')
print(' save_memmap mc.mmap_file:', mc.mmap_file)
fname_new = cm.save_memmap(mc.mmap_file,
base_name='memmap_',
order='C',
border_to_0=border_to_0,
dview=dview) # exclude borders
print(' mc.mmap_file:', mc.mmap_file)
print(' fname_new:', fname_new)
# make a copy of mmap file with a sane name
# does not work, seems you can not 'copy' a mmap file like a normal file
'''
tmpSavePath, tmpExt = os.path.splitext(fnames[0])
tmpSavePath += '.mmap' # need to be _aligned_ch1.tif
shutil.copyfile(fname_new, tmpSavePath)
'''
# save a _mmap.txt file with the name of the memory map file
'''
tmpFile, tmpFilename = os.path.splitext(fnames[0])
tmpFile += '_mmap.txt'
with open(tmpFile, 'w') as myTmpFile:
myTmpFile.write(fname_new)
'''
# now load the file
Yr, dims, T = cm.load_memmap(fname_new)
images = np.reshape(Yr.T, [T] + list(dims), order='F')
#load frames in python format (T x X x Y)
# save as .tif
images_8bit = ((images - images.min()) / (images.ptp() / 255.0)).astype(
np.uint8) # map the data range to 0 - 255
tmpSavePath, tmpExt = os.path.splitext(fnames[0])
tmpSavePath += '_aligned.tif' # need to be _aligned_ch1.tif
print(' saving aligned .tif', images_8bit.shape, images_8bit.dtype,
'tmpSavePath:', tmpSavePath)
tifffile.imsave(tmpSavePath, images_8bit, bigtiff=True)
# shut down the cluster
if 'dview' in locals():
cm.stop_server(dview=dview)
# remove mmap file fname_new
if os.path.isfile(fname_new):
print(' removing mmap file:', fname_new)
os.remove(fname_new)
oneFile = mc.mmap_file[0]
if os.path.isfile(oneFile):
print(' removing mmap file mc.mmap_file[0]:', oneFile)
os.remove(oneFile)
# done
stopSeconds = time.time()
elapsedSeconds = round(stopSeconds - startSeconds, 3)
elapsedMinutes = round(elapsedSeconds / 60, 3)
print(f' caimanAlign() done in {elapsedMinutes} minutes')
def run_alignmnet(states_df, parameters, dview):
'''
This is the main function for the alignment step. It applies methods
from the CaImAn package used originally in motion correction
to do alignment.
Args:
df: pd.DataFrame
A dataframe containing the analysis states you want to have aligned.
parameters: dict
The alignment parameters.
dview: object
The dview object
Returns:
df: pd.DataFrame
A dataframe containing the aligned analysis states.
'''
# Sort the dataframe correctly
df = states_df.copy()
df = df.sort_values(by=paths.multi_index_structure)
# Determine the mouse and session of the dataset
index = df.iloc[0].name
mouse, session, *r = index
#alignment_v = index[len(paths.data_structure) + step_index]
alignment_v = len(df)
alignment_index = (mouse, session, alignment_v)
# Determine the output .mmap file name
file_name = f'mouse_{mouse}_session_{session}_v{alignment_v}'
output_mmap_file_path = f'data/interim/alignment/main/{file_name}.mmap'
try:
df.reset_index()[['trial', 'is_rest']].set_index(['trial', 'is_rest'],
verify_integrity=True)
except ValueError:
logging.error(
'You passed multiple of the same trial in the dataframe df')
return df
output = {
'meta': {
'analysis': {
'analyst': os.environ['ANALYST'],
'date': datetime.datetime.today().strftime("%m-%d-%Y"),
'time': datetime.datetime.today().strftime("%H:%M:%S")
},
'duration': {}
}
}
# Get necessary parameters
motion_correction_parameters_list = []
motion_correction_output_list = []
input_mmap_file_list = []
trial_index_list = []
for idx, row in df.iterrows():
motion_correction_parameters_list.append(
eval(row.loc['motion_correction_parameters']))
motion_correction_output = eval(row.loc['motion_correction_output'])
motion_correction_output_list.append(motion_correction_output)
input_mmap_file_list.append(motion_correction_output['main'])
trial_index_list.append(db.get_trial_name(idx[2], idx[3]))
# MOTION CORRECTING EACH INDIVIDUAL MOVIE WITH RESPECT TO A TEMPLATE MADE OF THE FIRST MOVIE
logging.info(
f'{alignment_index} Performing motion correction on all movies with respect to a template made of \
the first movie.')
t0 = datetime.datetime.today()
# Create a template of the first movie
template_index = trial_index_list.index(
parameters['make_template_from_trial'])
m0 = cm.load(input_mmap_file_list[template_index])
m0_filt = cm.movie(
np.array([
high_pass_filter_space(m_, parameters['gSig_filt']) for m_ in m0
]))
template0 = cm.motion_correction.bin_median(
m0_filt.motion_correct(
5, 5, template=None)[0]) # may be improved in the future
# Setting the parameters
opts = params.CNMFParams(params_dict=parameters)
# Create a motion correction object
mc = MotionCorrect(input_mmap_file_list,
dview=dview,
**opts.get_group('motion'))
# Perform non-rigid motion correction
mc.motion_correct(template=template0, save_movie=True)
# Cropping borders
x_ = math.ceil(
abs(np.array(mc.shifts_rig)[:, 1].max()
) if np.array(mc.shifts_rig)[:, 1].max() > 0 else 0)
_x = math.ceil(
abs(np.array(mc.shifts_rig)[:, 1].min()
) if np.array(mc.shifts_rig)[:, 1].min() < 0 else 0)
y_ = math.ceil(
abs(np.array(mc.shifts_rig)[:, 0].max()
) if np.array(mc.shifts_rig)[:, 0].max() > 0 else 0)
_y = math.ceil(
abs(np.array(mc.shifts_rig)[:, 0].min()
) if np.array(mc.shifts_rig)[:, 0].min() < 0 else 0)
dt = int(
(datetime.datetime.today() - t0).seconds / 60) # timedelta in minutes
output['meta']['duration']['motion_correction'] = dt
logging.info(
f'{alignment_index} Performed motion correction. dt = {dt} min.')
# CONCATENATING ALL MOTION CORRECTED MOVIES
logging.info(
f'{alignment_index} Concatenating all motion corrected movies.')
# Load all movies into memory
m_list = [cm.load(fname) for fname in mc.fname_tot_rig]
# Crop all movies to those border pixels
for idx, m in enumerate(m_list):
m_list[idx] = m.crop(x_, _x, y_, _y, 0, 0)
output['meta']['cropping_points'] = [x_, _x, y_, _y]
# Concatenate them using the concat function
m_concat = cm.concatenate(m_list, axis=0)
# Create a timeline and store it
timeline = [[trial_index_list[0], 0]]
for i in range(1, len(m_list)):
m = m_list[i]
timeline.append([trial_index_list[i], timeline[i - 1][1] + m.shape[0]])
# timeline_pkl_file_path = f'data/interim/alignment/meta/timeline/{file_name}.pkl'
# with open(timeline_pkl_file_path,'wb') as f:
# pickle.dump(timeline,f)
# output['meta']['timeline'] = timeline_pkl_file_path
output['meta']['timeline'] = timeline
# Save the concatenated movie
output_mmap_file_path_tot = m_concat.save(output_mmap_file_path)
output['main'] = output_mmap_file_path_tot
# # Delete the motion corrected movies
# for fname in mc.fname_tot_rig:
# os.remove(fname)
dt = int(
(datetime.datetime.today() - t0).seconds / 60) # timedelta in minutes
output['meta']['duration']['concatenation'] = dt
logging.info(f'{alignment_index} Performed concatenation. dt = {dt} min.')
for idx, row in df.iterrows():
df.loc[idx, 'alignment_output'] = str(output)
df.loc[idx, 'alignment_parameters'] = str(parameters)
return df
def main():
pass # For compatibility between running under Spyder and the CLI
#%% Select file(s) to be processed (download if not present)
fnames = ['Sue_2x_3000_40_-46.tif'] # filename to be processed
if fnames[0] in ['Sue_2x_3000_40_-46.tif', 'demoMovie.tif']:
fnames = [download_demo(fnames[0])]
#%% First setup some parameters for data and motion correction
# dataset dependent parameters
fr = 30 # imaging rate in frames per second
decay_time = 0.4 # length of a typical transient in seconds
dxy = (2., 2.) # spatial resolution in x and y in (um per pixel)
# note the lower than usual spatial resolution here
max_shift_um = (12., 12.) # maximum shift in um
patch_motion_um = (100., 100.) # patch size for non-rigid correction in um
# motion correction parameters
pw_rigid = True # flag to select rigid vs pw_rigid motion correction
# maximum allowed rigid shift in pixels
max_shifts = [int(a/b) for a, b in zip(max_shift_um, dxy)]
# start a new patch for pw-rigid motion correction every x pixels
strides = tuple([int(a/b) for a, b in zip(patch_motion_um, dxy)])
# overlap between pathes (size of patch in pixels: strides+overlaps)
overlaps = (24, 24)
# maximum deviation allowed for patch with respect to rigid shifts
max_deviation_rigid = 3
mc_dict = {
'fnames': fnames,
'fr': fr,
'decay_time': decay_time,
'dxy': dxy,
'pw_rigid': pw_rigid,
'max_shifts': max_shifts,
'strides': strides,
'overlaps': overlaps,
'max_deviation_rigid': max_deviation_rigid,
'border_nan': 'copy'
}
opts = params.CNMFParams(params_dict=mc_dict)
# %% play the movie (optional)
# playing the movie using opencv. It requires loading the movie in memory.
# To close the video press q
display_images = True
if display_images:
m_orig = cm.load_movie_chain(fnames)
ds_ratio = 0.2
moviehandle = m_orig.resize(1, 1, ds_ratio)
moviehandle.play(q_max=99.5, fr=60, magnification=2)
# %% start a cluster for parallel processing
c, dview, n_processes = cm.cluster.setup_cluster(
backend='local', n_processes=None, single_thread=False)
# %%% MOTION CORRECTION
# first we create a motion correction object with the specified parameters
mc = MotionCorrect(fnames, dview=dview, **opts.get_group('motion'))
# note that the file is not loaded in memory
# %% Run (piecewise-rigid motion) correction using NoRMCorre
mc.motion_correct(save_movie=True)
# %% compare with original movie
if display_images:
m_orig = cm.load_movie_chain(fnames)
m_els = cm.load(mc.mmap_file)
ds_ratio = 0.2
moviehandle = cm.concatenate([m_orig.resize(1, 1, ds_ratio) - mc.min_mov*mc.nonneg_movie,
m_els.resize(1, 1, ds_ratio)], axis=2)
moviehandle.play(fr=60, q_max=99.5, magnification=2) # press q to exit
# %% MEMORY MAPPING
border_to_0 = 0 if mc.border_nan is 'copy' else mc.border_to_0
# you can include the boundaries of the FOV if you used the 'copy' option
# during motion correction, although be careful about the components near
# the boundaries
# memory map the file in order 'C'
fname_new = cm.save_memmap(mc.mmap_file, base_name='memmap_', order='C',
border_to_0=border_to_0) # exclude borders
# now load the file
Yr, dims, T = cm.load_memmap(fname_new)
images = np.reshape(Yr.T, [T] + list(dims), order='F')
# load frames in python format (T x X x Y)
# %% restart cluster to clean up memory
cm.stop_server(dview=dview)
c, dview, n_processes = cm.cluster.setup_cluster(
backend='local', n_processes=None, single_thread=False)
# %% parameters for source extraction and deconvolution
p = 1 # order of the autoregressive system
gnb = 2 # number of global background components
merge_thr = 0.85 # merging threshold, max correlation allowed
rf = 15
# half-size of the patches in pixels. e.g., if rf=25, patches are 50x50
stride_cnmf = 6 # amount of overlap between the patches in pixels
K = 4 # number of components per patch
gSig = [4, 4] # expected half size of neurons in pixels
# initialization method (if analyzing dendritic data using 'sparse_nmf')
method_init = 'greedy_roi'
ssub = 2 # spatial subsampling during initialization
tsub = 2 # temporal subsampling during intialization
# parameters for component evaluation
opts_dict = {'fnames': fnames,
'p': p,
'fr': fr,
'nb': gnb,
'rf': rf,
'K': K,
'gSig': gSig,
'stride': stride_cnmf,
'method_init': method_init,
'rolling_sum': True,
'merge_thr': merge_thr,
'n_processes': n_processes,
'only_init': True,
'ssub': ssub,
'tsub': tsub}
opts.change_params(params_dict=opts_dict);
# %% RUN CNMF ON PATCHES
# First extract spatial and temporal components on patches and combine them
# for this step deconvolution is turned off (p=0). If you want to have
# deconvolution within each patch change params.patch['p_patch'] to a
# nonzero value
#opts.change_params({'p': 0})
cnm = cnmf.CNMF(n_processes, params=opts, dview=dview)
cnm = cnm.fit(images)
# %% ALTERNATE WAY TO RUN THE PIPELINE AT ONCE
# you can also perform the motion correction plus cnmf fitting steps
# simultaneously after defining your parameters object using
# cnm1 = cnmf.CNMF(n_processes, params=opts, dview=dview)
# cnm1.fit_file(motion_correct=True)
# %% plot contours of found components
Cns = local_correlations_movie_offline(mc.mmap_file[0],
remove_baseline=True, window=1000, stride=1000,
winSize_baseline=100, quantil_min_baseline=10,
dview=dview)
Cn = Cns.max(axis=0)
Cn[np.isnan(Cn)] = 0
cnm.estimates.plot_contours(img=Cn)
plt.title('Contour plots of found components')
#%% save results
cnm.estimates.Cn = Cn
cnm.save(fname_new[:-5]+'_init.hdf5')
# %% RE-RUN seeded CNMF on accepted patches to refine and perform deconvolution
cnm2 = cnm.refit(images, dview=dview)
# %% COMPONENT EVALUATION
# the components are evaluated in three ways:
# a) the shape of each component must be correlated with the data
# b) a minimum peak SNR is required over the length of a transient
# c) each shape passes a CNN based classifier
min_SNR = 2 # signal to noise ratio for accepting a component
rval_thr = 0.85 # space correlation threshold for accepting a component
cnn_thr = 0.99 # threshold for CNN based classifier
cnn_lowest = 0.1 # neurons with cnn probability lower than this value are rejected
cnm2.params.set('quality', {'decay_time': decay_time,
'min_SNR': min_SNR,
'rval_thr': rval_thr,
'use_cnn': True,
'min_cnn_thr': cnn_thr,
'cnn_lowest': cnn_lowest})
cnm2.estimates.evaluate_components(images, cnm2.params, dview=dview)
# %% PLOT COMPONENTS
cnm2.estimates.plot_contours(img=Cn, idx=cnm2.estimates.idx_components)
# %% VIEW TRACES (accepted and rejected)
if display_images:
cnm2.estimates.view_components(images, img=Cn,
idx=cnm2.estimates.idx_components)
cnm2.estimates.view_components(images, img=Cn,
idx=cnm2.estimates.idx_components_bad)
#%% update object with selected components
cnm2.estimates.select_components(use_object=True)
#%% Extract DF/F values
cnm2.estimates.detrend_df_f(quantileMin=8, frames_window=250)
#%% Show final traces
cnm2.estimates.view_components(img=Cn)
#%%
cnm2.estimates.Cn = Cn
cnm2.save(cnm2.mmap_file[:-4] + 'hdf5')
#%% reconstruct denoised movie (press q to exit)
if display_images:
cnm2.estimates.play_movie(images, q_max=99.9, gain_res=2,
magnification=2,
bpx=border_to_0,
include_bck=False) # background not shown
#%% STOP CLUSTER and clean up log files
cm.stop_server(dview=dview)
log_files = glob.glob('*_LOG_*')
for log_file in log_files:
os.remove(log_file)
class MakeMasks3D:
"""Not yet fully working so dont use!"""
def __init__(self, tiff, channels, planes, x_start, x_end, mc_opts, use_green_ch=False,):
self.tiff = tiff
self.channels = channels
self.planes = planes
self.x_start = x_start
self.x_end = x_end
self.xslice = slice(x_start, x_end)
self.mc_opts = mc_opts
self.use_green_ch = use_green_ch
if self.use_green_ch == True:
self.channel2use = 0
else:
self.channel2use = 1
if self.mc_opts:
self.opts = params.CNMFParams(params_dict=mc_opts)
self.file_list = []
self.images = []
self.motion_corrected_images = []
self.masks = []
self.coords = None
self.corr_images = []
def crop_tiffs(self):
self.images = []
self.file_list = []
for plane in list(range(self.planes)):
time_slice = slice(plane*self.channels+self.channel2use, -1, self.channels*self.planes)
with ScanImageTiffReader(self.tiff) as reader:
data = reader.data()
data = data[time_slice, : , self.xslice]
self.images.append(data)
tif_name = self.tiff.split('.')[0] + '_template_plane' + str(plane) + '.tif'
self.file_list.append(tif_name)
tifffile.imsave(tif_name, data)
def view_planes(self):
fig, axes = plt.subplots(1, self.planes, constrained_layout=True)
for i,ax in enumerate(axes):
image = self.images[i].mean(axis=0)
ax.imshow(image)
ax.set_aspect('equal', 'box')
ax.axis('off')
ax.set_title(f'Plane {i}')
fig.suptitle('Original')
def view_corrected(self):
fig, axes = plt.subplots(1, self.planes, constrained_layout=True)
for i,ax in enumerate(axes):
image = self.motion_corrected_images[i]
ax.imshow(image)
ax.set_aspect('equal', 'box')
ax.axis('off')
ax.set_title(f'Plane {i}')
fig.suptitle('Corrected')
def view_corr(self):
self.corr_images = []
fig, axes = plt.subplots(1, self.planes, constrained_layout=True)
for i,ax in enumerate(axes):
image = self.motion_corrected_images[i]
corr_im = cm.local_correlations(image, swap_dim=False)
self.corr_images.append(corr_im)
ax.imshow(image)
ax.set_aspect('equal', 'box')
ax.axis('off')
ax.set_title(f'Plane {i}')
fig.suptitle('Correlation Image')
def motion_correct_red(self):
c, dview, n_processes = cm.cluster.setup_cluster(
backend='local', n_processes=None, single_thread=False)
self.motion_corrected_images = []
for plane in list(range(self.planes)):
print(f'Starting motion correction plane {plane}')
self.mc = MotionCorrect(self.file_list[plane], dview=dview, **self.opts.get_group('motion'))
self.mc.motion_correct()
self.motion_corrected_images.append(self.mc.total_template_els)
cm.stop_server(dview=dview)
def extract_masks(self, radius=7):
self.masks = []
self.coords = []
if len(self.motion_corrected_images) > 0:
image_source = self.motion_corrected_images
else:
image_source = self.images
for plane in range(self.planes):
self.masks.append(cm.base.rois.extract_binary_masks_from_structural_channel(image_source[plane])[0])
self.coords.append(cm.utils.visualization.get_contours(self.masks[plane],
image_source[plane].shape,
thr=0.99,
thr_method='max'))
def run(self):
self.crop_tiffs()
self.motion_correct_red()
self.extract_masks()
def main():
pass # For compatibility between running under Spyder and the CLI
# %% start the cluster
try:
cm.stop_server() # stop it if it was running
except ():
pass
c, dview, n_processes = cm.cluster.setup_cluster(
backend='local',
n_processes=
24, # number of process to use, if you go out of memory try to reduce this one
single_thread=False)
# %% First setup some parameters for motion correction
# dataset dependent parameters
fnames = ['data_endoscope.tif'] # filename to be processed
fnames = [download_demo(fnames[0])] # download file if not already present
filename_reorder = fnames
fr = 10 # movie frame rate
decay_time = 0.4 # length of a typical transient in seconds
# motion correction parameters
motion_correct = True # flag for motion correction
pw_rigid = False # flag for pw-rigid motion correction
gSig_filt = (3, 3) # size of filter, in general gSig (see below),
# change this one if algorithm does not work
max_shifts = (5, 5) # maximum allowed rigid shift
strides = (
48, 48
) # start a new patch for pw-rigid motion correction every x pixels
overlaps = (24, 24
) # overlap between pathes (size of patch strides+overlaps)
# maximum deviation allowed for patch with respect to rigid shifts
max_deviation_rigid = 3
border_nan = 'copy'
mc_dict = {
'fnames': fnames,
'fr': fr,
'decay_time': decay_time,
'pw_rigid': pw_rigid,
'max_shifts': max_shifts,
'gSig_filt': gSig_filt,
'strides': strides,
'overlaps': overlaps,
'max_deviation_rigid': max_deviation_rigid,
'border_nan': border_nan
}
opts = params.CNMFParams(params_dict=mc_dict)
# %% MOTION CORRECTION
# The pw_rigid flag set above, determines where to use rigid or pw-rigid
# motion correction
if motion_correct:
# do motion correction rigid
mc = MotionCorrect(fnames, dview=dview, **opts.get_group('motion'))
mc.motion_correct(save_movie=True)
fname_mc = mc.fname_tot_els if pw_rigid else mc.fname_tot_rig
if pw_rigid:
bord_px = np.ceil(
np.maximum(np.max(np.abs(mc.x_shifts_els)),
np.max(np.abs(mc.y_shifts_els)))).astype(np.int)
else:
bord_px = np.ceil(np.max(np.abs(mc.shifts_rig))).astype(np.int)
plt.subplot(1, 2, 1)
plt.imshow(mc.total_template_rig) # % plot template
plt.subplot(1, 2, 2)
plt.plot(mc.shifts_rig) # % plot rigid shifts
plt.legend(['x shifts', 'y shifts'])
plt.xlabel('frames')
plt.ylabel('pixels')
bord_px = 0 if border_nan == 'copy' else bord_px
fname_new = cm.save_memmap(fname_mc,
base_name='memmap_',
order='C',
border_to_0=bord_px)
else: # if no motion correction just memory map the file
fname_new = cm.save_memmap(filename_reorder,
base_name='memmap_',
order='C',
border_to_0=0,
dview=dview)
# load memory mappable file
Yr, dims, T = cm.load_memmap(fname_new)
images = Yr.T.reshape((T, ) + dims, order='F')
# %% Parameters for source extraction and deconvolution (CNMF-E algorithm)
p = 1 # order of the autoregressive system
K = None # upper bound on number of components per patch, in general None for 1p data
gSig = (
3, 3
) # gaussian width of a 2D gaussian kernel, which approximates a neuron
gSiz = (13, 13) # average diameter of a neuron, in general 4*gSig+1
Ain = None # possibility to seed with predetermined binary masks
merge_thr = .7 # merging threshold, max correlation allowed
rf = 40 # half-size of the patches in pixels. e.g., if rf=40, patches are 80x80
stride_cnmf = 20 # amount of overlap between the patches in pixels
# (keep it at least large as gSiz, i.e 4 times the neuron size gSig)
tsub = 2 # downsampling factor in time for initialization,
# increase if you have memory problems
ssub = 1 # downsampling factor in space for initialization,
# increase if you have memory problems
# you can pass them here as boolean vectors
low_rank_background = None # None leaves background of each patch intact,
# True performs global low-rank approximation if gnb>0
gnb = 0 # number of background components (rank) if positive,
# else exact ring model with following settings
# gnb= 0: Return background as b and W
# gnb=-1: Return full rank background B
# gnb<-1: Don't return background
nb_patch = 0 # number of background components (rank) per patch if gnb>0,
# else it is set automatically
min_corr = .8 # min peak value from correlation image
min_pnr = 10 # min peak to noise ration from PNR image
ssub_B = 2 # additional downsampling factor in space for background
ring_size_factor = 1.4 # radius of ring is gSiz*ring_size_factor
opts.change_params(
params_dict={
'dims': dims,
'method_init': 'corr_pnr', # use this for 1 photon
'K': K,
'gSig': gSig,
'gSiz': gSiz,
'merge_thr': merge_thr,
'p': p,
'tsub': tsub,
'ssub': ssub,
'rf': rf,
'stride': stride_cnmf,
'only_init': True, # set it to True to run CNMF-E
'nb': gnb,
'nb_patch': nb_patch,
'method_deconvolution': 'oasis', # could use 'cvxpy' alternatively
'low_rank_background': low_rank_background,
'update_background_components':
True, # sometimes setting to False improve the results
'min_corr': min_corr,
'min_pnr': min_pnr,
'normalize_init': False, # just leave as is
'center_psf': True, # leave as is for 1 photon
'ssub_B': ssub_B,
'ring_size_factor': ring_size_factor,
'del_duplicates':
True, # whether to remove duplicates from initialization
'border_pix': bord_px
}) # number of pixels to not consider in the borders)
# %% compute some summary images (correlation and peak to noise)
# change swap dim if output looks weird, it is a problem with tiffile
cn_filter, pnr = cm.summary_images.correlation_pnr(images[::1],
gSig=gSig[0],
swap_dim=False)
# if your images file is too long this computation will take unnecessarily
# long time and consume a lot of memory. Consider changing images[::1] to
# images[::5] or something similar to compute on a subset of the data
# inspect the summary images and set the parameters
inspect_correlation_pnr(cn_filter, pnr)
# print parameters set above, modify them if necessary based on summary images
print(min_corr) # min correlation of peak (from correlation image)
print(min_pnr) # min peak to noise ratio
# %% RUN CNMF ON PATCHES
cnm = cnmf.CNMF(n_processes=n_processes, dview=dview, Ain=Ain, params=opts)
cnm.fit(images)
# %% ALTERNATE WAY TO RUN THE PIPELINE AT ONCE
# you can also perform the motion correction plus cnmf fitting steps
# simultaneously after defining your parameters object using
# cnm1 = cnmf.CNMF(n_processes, params=opts, dview=dview)
# cnm1.fit_file(motion_correct=True)
# %% DISCARD LOW QUALITY COMPONENTS
min_SNR = 2.5 # adaptive way to set threshold on the transient size
r_values_min = 0.85 # threshold on space consistency (if you lower more components
# will be accepted, potentially with worst quality)
cnm.params.set('quality', {
'min_SNR': min_SNR,
'rval_thr': r_values_min,
'use_cnn': False
})
cnm.estimates.evaluate_components(images, cnm.params, dview=dview)
print(' ***** ')
print('Number of total components: ', len(cnm.estimates.C))
print('Number of accepted components: ', len(cnm.estimates.idx_components))
# %% PLOT COMPONENTS
cnm.dims = dims
display_images = True # Set to true to show movies and images
if display_images:
cnm.estimates.plot_contours(img=cn_filter,
idx=cnm.estimates.idx_components)
cnm.estimates.view_components(images, idx=cnm.estimates.idx_components)
# %% MOVIES
display_images = False # Set to true to show movies and images
if display_images:
# fully reconstructed movie
cnm.estimates.play_movie(images,
q_max=99.5,
magnification=2,
include_bck=True,
gain_res=10,
bpx=bord_px)
# movie without background
cnm.estimates.play_movie(images,
q_max=99.9,
magnification=2,
include_bck=False,
gain_res=4,
bpx=bord_px)
# %% STOP SERVER
cm.stop_server(dview=dview)
def run(
file_path,
n_cpus,
motion_correct: bool = True,
mc_settings: dict = {},
job_name: str = "job",
output_directory: str = "",
):
mkl = os.environ.get("MKL_NUM_THREADS")
blas = os.environ.get("OPENBLAS_NUM_THREADS")
vec = os.environ.get("VECLIB_MAXIMUM_THREADS")
print(f"MKL: {mkl}")
print(f"blas: {blas}")
print(f"vec: {vec}")
# we import the pipeline upon running so they aren't required for all installs
import caiman as cm
from caiman.motion_correction import MotionCorrect
from caiman.source_extraction.cnmf import params as params
# print the directory caiman is imported from
caiman_path = os.path.abspath(cm.__file__)
print(f"caiman path: {caiman_path}")
sys.stdout.flush()
# setup the logger
logger_file = os.path.join(output_directory, "caiman.log")
logging.basicConfig(
format=LOGGER_FORMAT,
filename=logger_file,
filemode="w",
level=logging.DEBUG,
)
# if indices to perform mcorr are set, format them
if "indices" in mc_settings:
indices = mc_settings["indices"]
indices_formatted = ()
for axis_slice in indices:
start = axis_slice[0]
stop = axis_slice[1]
if len(axis_slice) == 3:
step = axis_slice[2]
else:
step = 1
indices_formatted += (slice(start, stop, step), )
mc_settings["indices"] = indices_formatted
# load and update the pipeline settings
mc_parameters = DEFAULT_MCORR_SETTINGS
for k, v in mc_settings.items():
mc_parameters[k] = v
opts = params.CNMFParams(params_dict=mc_parameters)
# get the filenames
if os.path.isfile(file_path):
print(file_path)
fnames = [file_path]
else:
file_pattern = os.path.join(file_path, "*.tif*")
fnames = sorted(glob.glob(file_pattern))
print(fnames)
opts.set("data", {"fnames": fnames})
if n_cpus > 1:
print("starting server")
# start the server
n_proc = np.max([(n_cpus - 1), 1])
c, dview, n_processes = cm.cluster.setup_cluster(backend="local",
n_processes=n_proc,
single_thread=False)
sleep(30)
else:
print("multiprocessing disabled")
dview = None
n_processes = 1
print(n_processes)
print("starting motion correction")
sys.stdout.flush()
mc = MotionCorrect(fnames, dview=dview, **opts.get_group("motion"))
mc.motion_correct(save_movie=True)
pw_rigid = mc_parameters["pw_rigid"]
fname_mc = mc.fname_tot_els if pw_rigid else mc.fname_tot_rig
if pw_rigid:
bord_px = np.ceil(
np.maximum(np.max(np.abs(mc.x_shifts_els)),
np.max(np.abs(mc.y_shifts_els)))).astype(np.int)
else:
bord_px = np.ceil(np.max(np.abs(mc.shifts_rig))).astype(np.int)
border_nan = mc_parameters["border_nan"]
bord_px = 0 if border_nan == "copy" else bord_px
_ = cm.save_memmap(fname_mc,
base_name="memmap_",
order="C",
border_to_0=bord_px)
# if motion correction was performed, save the file
# we save as hdf5 for better reading performance
# downstream
if motion_correct:
print("saving motion corrected file")
results_filebase = os.path.join(output_directory, job_name)
mcorr_fname = results_filebase + "_mcorr.hdf5"
dataset_name = opts.data["var_name_hdf5"]
# fnames = opts.data["fnames"]
# memmap_files = []
# for f in fnames:
# if isinstance(f, bytes):
# f = f.decode()
# base_file = os.path.splitext(f)[0]
# if pw_rigid:
# memmap_pattern = base_file + "*_els_*"
# else:
# memmap_pattern = base_file + "*_rig_*"
# memmap_files += sorted(glob.glob(memmap_pattern))
if pw_rigid:
memmap_files = mc.fname_tot_els
else:
memmap_files = mc.fname_tot_rig
# get the frame shape
mov = cm.load(memmap_files[0])
frame_shape = mov.shape[-2::]
write_hdf5_movie(
movie_name=mcorr_fname,
memmap_files=memmap_files,
frame_shape=frame_shape,
dataset_name=dataset_name,
compression="gzip",
)
# save the parameters in the same dir as the results
final_params = opts.to_dict()
params_file = os.path.join(output_directory, "all_mcorr_parameters.pkl")
with open(params_file, "wb") as fp:
pickle.dump(final_params, fp)
# deleting mcorr unused memmap files
if pw_rigid:
memmap_files = mc.fname_tot_els
else:
memmap_files = mc.fname_tot_rig
print(f"deleting {memmap_files}")
for f in memmap_files:
os.remove(f)
print("stopping server")
cm.stop_server(dview=dview)
def main():
pass # For compatibility between running under Spyder and the CLI
# %% Please download the .npz file manually to your file path.
url = 'https://www.dropbox.com/s/tuv1bx9w6wdywnm/demo_voltage_imaging.npz?dl=0'
# %% Load demo movie and ROIs
file_path = '/home/nel/Code/Voltage_imaging/exampledata/403106_3min/demo_voltage_imaging.npz'
m = np.load(file_path)
mv = cm.movie(m.f.arr_0)
ROIs = m.f.arr_1
# %% Save the movie
fnames = '/home/nel/Code/Voltage_imaging/exampledata/403106_3min/demomovie_voltage_imaging.hdf5'
mv.save(fnames) # neglect the warning
# %% Setup some parameters for data and motion correction
# dataset parameters
fr = 400 # sample rate of the movie
index = list(range(ROIs.shape[0])) # index of neurons
weights = None # reuse spatial weights by
# opts.change_params(params_dict={'weights':vpy.estimates['weights']})
# motion correction parameters
motion_correct = True # flag for motion correction
pw_rigid = True # flag for pw-rigid motion correction
gSig_filt = (3, 3) # size of filter, in general gSig (see below),
# change this one if algorithm does not work
max_shifts = (5, 5) # maximum allowed rigid shift
strides = (48, 48) # start a new patch for pw-rigid motion correction every x pixels
overlaps = (24, 24) # overlap between pathes (size of patch strides+overlaps)
max_deviation_rigid = 3 # maximum deviation allowed for patch with respect to rigid shifts
border_nan = 'copy'
mc_dict = {
'fnames': fnames,
'fr': fr,
'index': index,
'ROIs':ROIs,
'weights':weights,
'pw_rigid': pw_rigid,
'max_shifts': max_shifts,
'gSig_filt': gSig_filt,
'strides': strides,
'overlaps': overlaps,
'max_deviation_rigid': max_deviation_rigid,
'border_nan': border_nan
}
opts = volparams(params_dict=mc_dict)
# %% play the movie (optional)
# playing the movie using opencv. It requires loading the movie in memory.
# To close the video press q
display_images = True
if display_images:
m_orig = cm.load(fnames)
ds_ratio = 0.2
moviehandle = m_orig.resize(1, 1, ds_ratio)
moviehandle.play(q_max=99.5, fr=60, magnification=2)
# %% start a cluster for parallel processing
c, dview, n_processes = cm.cluster.setup_cluster(
backend='local', n_processes=12, single_thread=False)
# %%% MOTION CORRECTION
# Create a motion correction object with the specified parameters
mc_pw = MotionCorrect(fnames, dview=dview, **opts.get_group('motion'))
# Run piecewise rigid motion correction
mc_pw.motion_correct(save_movie=True)
# %% motion correction compared with original movie
display_images = True
if display_images:
m_orig = cm.load(fnames)
m_rig = cm.load(mc_pw.mmap_file)
ds_ratio = 0.2
moviehandle = cm.concatenate([m_orig.resize(1, 1, ds_ratio) - mc_pw.min_mov * mc_pw.nonneg_movie,
m_rig.resize(1, 1, ds_ratio)], axis=2)
moviehandle.play(fr=60, q_max=99.5, magnification=2) # press q to exit
# %% movie subtracted from the mean
m_orig2 = (m_orig - np.mean(m_orig, axis=0))
m_rig2 = (m_rig - np.mean(m_rig, axis=0))
moviehandle1 = cm.concatenate([m_orig2.resize(1, 1, ds_ratio),
m_rig2.resize(1, 1, ds_ratio)], axis=2)
moviehandle1.play(fr=60, q_max=99.5, magnification=2)
# %% change fnames to the new motion corrected one
fname_new = mc_pw.mmap_file[0] # memory map file name
opts.change_params(params_dict={'fnames':fname_new})
# %% restart cluster to clean up memory
cm.stop_server(dview=dview)
c, dview, n_processes = cm.cluster.setup_cluster(
backend='local', n_processes=12, single_thread=False)
# %% process cells using volspike function
vpy = VOLPY(n_processes=n_processes, dview=dview, params=opts)
vpy.fit()
# %% some visualization
vpy.estimates['cellN']
n = 0 # cell you are interested in
plt.figure()
plt.plot(vpy.estimates['trace'][n])
plt.plot(vpy.estimates['spikeTimes'][n], np.max(vpy.estimates['trace'][n]) * 1 * np.ones(vpy.estimates['spikeTimes'][n].shape),
color='g', marker='o', fillstyle='none', linestyle='none')
plt.title('signal and spike times')
plt.show()
plt.figure()
plt.imshow(vpy.estimates['spatialFilter'][n])
plt.colorbar()
plt.title('spatial filter')
plt.show()
# %% STOP CLUSTER and clean up log files
cm.stop_server(dview=dview)
log_files = glob.glob('*_LOG_*')
for log_file in log_files:
os.remove(log_file)
def piecewise_rigid_registration(fnames,
max_shifts=(6, 6),
strides=(48, 48),
overlaps=(24, 24),
num_frames_split=100,
max_deviation_rigid=3,
pw_rigid=False,
shifts_opencv=True,
border_nan='copy',
highPassFilter=False,
sigma=7):
'''Apply CaImAn piecewise rigid registration on raw imaging data
:param fnames: full path filename for motion correction, possible extensions are tif, avi, npy
:param max_shifts: maximum allowed rigid shift in pixels (view the movie to get a sense of motion)
:param strides: create a new patch every x pixels for pw-rigid correction
:param overlaps: overlap between pathes (size of patch strides+overlaps)
:param num_frames_split: length in frames of each chunk of the movie (to be processed in parallel)
:param max_deviation_rigid: maximum deviation allowed for patch with respect to rigid shifts
:param pw_rigid: flag for performing rigid or piecewise rigid motion correction
:param shifts_opencv: flag for correcting motion using bicubic interpolation (otherwise FFT interpolation is used)
:param border_nan: replicate values along the boundary (if True, fill in with NaN)
:param highPassFilter: whether applying high pass filter
:param sigma: lowpass gaussian filter sigma value
:return: motion corrected image in numpy
'''
fnames = [fnames]
m_orig = cm.load_movie_chain(fnames)
if 'dview' in locals():
cm.stop_server(dview=dview)
c, dview, n_processes = cm.cluster.setup_cluster(backend='local',
n_processes=None,
single_thread=False)
# create a motion correction object
mc = MotionCorrect(fnames,
dview=dview,
max_shifts=max_shifts,
strides=strides,
overlaps=overlaps,
max_deviation_rigid=max_deviation_rigid,
shifts_opencv=shifts_opencv,
nonneg_movie=True,
border_nan=border_nan)
# correct for rigid motion correction and save the file (in memory mapped form)
mc.motion_correct(save_movie=True)
# load motion corrected movie and apply piece-wise rigid registration
m_rig = cm.load(mc.mmap_file)
mc.pw_rigid = True # turn the flag to True for pw-rigid motion correction
mc.template = mc.mmap_file # use the template obtained before to save in computation (optional)
mc.motion_correct(save_movie=True, template=mc.total_template_rig)
cm.stop_server(dview=dview) # stop the server
m_els = cm.load(mc.fname_tot_els)
Y = np.array(m_els.tolist())
if highPassFilter:
Y = high_pass_filtering(Y, sigma)
return Y
def main():
pass # For compatibility between running under Spyder and the CLI
# %% Load demo movie and ROIs
fnames = download_demo(
'demo_voltage_imaging.hdf5',
'volpy') # file path to movie file (will download if not present)
path_ROIs = download_demo(
'demo_voltage_imaging_ROIs.hdf5',
'volpy') # file path to ROIs file (will download if not present)
file_dir = os.path.split(fnames)[0]
#%% dataset dependent parameters
# dataset dependent parameters
fr = 400 # sample rate of the movie
# motion correction parameters
pw_rigid = False # flag for pw-rigid motion correction
gSig_filt = (3, 3) # size of filter, in general gSig (see below),
# change this one if algorithm does not work
max_shifts = (5, 5) # maximum allowed rigid shift
strides = (
48, 48
) # start a new patch for pw-rigid motion correction every x pixels
overlaps = (24, 24
) # overlap between pathes (size of patch strides+overlaps)
max_deviation_rigid = 3 # maximum deviation allowed for patch with respect to rigid shifts
border_nan = 'copy'
opts_dict = {
'fnames': fnames,
'fr': fr,
'pw_rigid': pw_rigid,
'max_shifts': max_shifts,
'gSig_filt': gSig_filt,
'strides': strides,
'overlaps': overlaps,
'max_deviation_rigid': max_deviation_rigid,
'border_nan': border_nan
}
opts = volparams(params_dict=opts_dict)
# %% play the movie (optional)
# playing the movie using opencv. It requires loading the movie in memory.
# To close the movie press q
display_images = False
if display_images:
m_orig = cm.load(fnames)
ds_ratio = 0.2
moviehandle = m_orig.resize(1, 1, ds_ratio)
moviehandle.play(q_max=99.5, fr=40, magnification=4)
# %% start a cluster for parallel processing
c, dview, n_processes = cm.cluster.setup_cluster(backend='local',
n_processes=None,
single_thread=False)
# %%% MOTION CORRECTION
# first we create a motion correction object with the specified parameters
mc = MotionCorrect(fnames, dview=dview, **opts.get_group('motion'))
# Run correction
do_motion_correction = True
if do_motion_correction:
mc.motion_correct(save_movie=True)
else:
mc_list = [
file for file in os.listdir(file_dir)
if (os.path.splitext(os.path.split(fnames)[-1])[0] in file
and '.mmap' in file)
]
mc.mmap_file = [os.path.join(file_dir, mc_list[0])]
print(f'reuse previously saved motion corrected file:{mc.mmap_file}')
# %% compare with original movie
if display_images:
m_orig = cm.load(fnames)
m_rig = cm.load(mc.mmap_file)
ds_ratio = 0.2
moviehandle = cm.concatenate(
[m_orig.resize(1, 1, ds_ratio),
m_rig.resize(1, 1, ds_ratio)],
axis=2)
moviehandle.play(fr=40, q_max=99.5, magnification=4) # press q to exit
# %% MEMORY MAPPING
do_memory_mapping = True
if do_memory_mapping:
border_to_0 = 0 if mc.border_nan == 'copy' else mc.border_to_0
# you can include the boundaries of the FOV if you used the 'copy' option
# during motion correction, although be careful about the components near
# the boundaries
# memory map the file in order 'C'
fname_new = cm.save_memmap_join(
mc.mmap_file,
base_name='memmap_' +
os.path.splitext(os.path.split(fnames)[-1])[0],
add_to_mov=border_to_0,
dview=dview) # exclude border
else:
mmap_list = [
file for file in os.listdir(file_dir)
if ('memmap_' +
os.path.splitext(os.path.split(fnames)[-1])[0]) in file
]
fname_new = os.path.join(file_dir, mmap_list[0])
print(f'reuse previously saved memory mapping file:{fname_new}')
# %% SEGMENTATION
# create summary images
img = mean_image(mc.mmap_file[0], window=1000, dview=dview)
img = (img - np.mean(img)) / np.std(img)
gaussian_blur = False # Use gaussian blur when there is too much noise in the video
Cn = local_correlations_movie_offline(mc.mmap_file[0],
fr=fr,
window=fr * 4,
stride=fr * 4,
winSize_baseline=fr,
remove_baseline=True,
gaussian_blur=gaussian_blur,
dview=dview).max(axis=0)
img_corr = (Cn - np.mean(Cn)) / np.std(Cn)
summary_images = np.stack([img, img, img_corr], axis=0).astype(np.float32)
# save summary images which are used in the VolPy GUI
cm.movie(summary_images).save(fnames[:-5] + '_summary_images.tif')
fig, axs = plt.subplots(1, 2)
axs[0].imshow(summary_images[0])
axs[1].imshow(summary_images[2])
axs[0].set_title('mean image')
axs[1].set_title('corr image')
#%% methods for segmentation
methods_list = [
'manual_annotation', # manual annotations need prepared annotated datasets in the same format as demo_voltage_imaging_ROIs.hdf5
'maskrcnn', # Mask R-CNN is a convolutional neural network trained for detecting neurons in summary images
'gui_annotation'
] # use VolPy GUI to correct outputs of Mask R-CNN or annotate new datasets
method = methods_list[0]
if method == 'manual_annotation':
with h5py.File(path_ROIs, 'r') as fl:
ROIs = fl['mov'][()]
elif method == 'maskrcnn': # Important!! Make sure install keras before using mask rcnn.
weights_path = download_model(
'mask_rcnn'
) # also make sure you have downloaded the new weight. The weight was updated on Dec 1st 2020.
ROIs = utils.mrcnn_inference(
img=summary_images.transpose([1, 2, 0]),
size_range=[5, 22],
weights_path=weights_path,
display_result=True
) # size parameter decides size range of masks to be selected
cm.movie(ROIs).save(fnames[:-5] + 'mrcnn_ROIs.hdf5')
elif method == 'gui_annotation':
# run volpy_gui.py file in the caiman/source_extraction/volpy folder
gui_ROIs = caiman_datadir() + '/example_movies/volpy/gui_roi.hdf5'
with h5py.File(gui_ROIs, 'r') as fl:
ROIs = fl['mov'][()]
fig, axs = plt.subplots(1, 2)
axs[0].imshow(summary_images[0])
axs[1].imshow(ROIs.sum(0))
axs[0].set_title('mean image')
axs[1].set_title('masks')
# %% restart cluster to clean up memory
cm.stop_server(dview=dview)
c, dview, n_processes = cm.cluster.setup_cluster(backend='local',
n_processes=None,
single_thread=False,
maxtasksperchild=1)
# %% parameters for trace denoising and spike extraction
ROIs = ROIs # region of interests
index = list(range(len(ROIs))) # index of neurons
weights = None # reuse spatial weights
context_size = 35 # number of pixels surrounding the ROI to censor from the background PCA
visualize_ROI = False # whether to visualize the region of interest inside the context region
flip_signal = True # Important!! Flip signal or not, True for Voltron indicator, False for others
hp_freq_pb = 1 / 3 # parameter for high-pass filter to remove photobleaching
clip = 100 # maximum number of spikes to form spike template
threshold_method = 'adaptive_threshold' # adaptive_threshold or simple
min_spikes = 10 # minimal spikes to be found
pnorm = 0.5 # a variable deciding the amount of spikes chosen for adaptive threshold method
threshold = 3 # threshold for finding spikes only used in simple threshold method, Increase the threshold to find less spikes
do_plot = False # plot detail of spikes, template for the last iteration
ridge_bg = 0.01 # ridge regression regularizer strength for background removement, larger value specifies stronger regularization
sub_freq = 20 # frequency for subthreshold extraction
weight_update = 'ridge' # ridge or NMF for weight update
n_iter = 2 # number of iterations alternating between estimating spike times and spatial filters
opts_dict = {
'fnames': fname_new,
'ROIs': ROIs,
'index': index,
'weights': weights,
'context_size': context_size,
'visualize_ROI': visualize_ROI,
'flip_signal': flip_signal,
'hp_freq_pb': hp_freq_pb,
'clip': clip,
'threshold_method': threshold_method,
'min_spikes': min_spikes,
'pnorm': pnorm,
'threshold': threshold,
'do_plot': do_plot,
'ridge_bg': ridge_bg,
'sub_freq': sub_freq,
'weight_update': weight_update,
'n_iter': n_iter
}
opts.change_params(params_dict=opts_dict)
#%% TRACE DENOISING AND SPIKE DETECTION
vpy = VOLPY(n_processes=n_processes, dview=dview, params=opts)
vpy.fit(n_processes=n_processes, dview=dview)
#%% visualization
display_images = True
if display_images:
print(np.where(
vpy.estimates['locality'])[0]) # neurons that pass locality test
idx = np.where(vpy.estimates['locality'] > 0)[0]
utils.view_components(vpy.estimates, img_corr, idx)
#%% reconstructed movie
# note the negative spatial weights is cutoff
if display_images:
mv_all = utils.reconstructed_movie(vpy.estimates.copy(),
fnames=mc.mmap_file,
idx=idx,
scope=(0, 1000),
flip_signal=flip_signal)
mv_all.play(fr=40)
#%% save the result in .npy format
save_result = True
if save_result:
vpy.estimates['ROIs'] = ROIs
vpy.estimates['params'] = opts
save_name = f'volpy_{os.path.split(fnames)[1][:-5]}_{threshold_method}'
np.save(os.path.join(file_dir, save_name), vpy.estimates)
# %% STOP CLUSTER and clean up log files
cm.stop_server(dview=dview)
log_files = glob.glob('*_LOG_*')
for log_file in log_files:
os.remove(log_file)
def main():
#%% First setup some parameters for data and motion correction
# dataset dependent parameters
fr = 15.49 # imaging rate in frames per second
decay_time = 0.9 # length of a typical transient in seconds
dxy = (1.4, 1.4) # spatial resolution in x and y in (um per pixel)
# note the lower than usual spatial resolution here
max_shift_um = (12., 12.) # maximum shift in um
patch_motion_um = (100., 100.) # patch size for non-rigid correction in um
# motion correction parameters
pw_rigid = True # flag to select rigid vs pw_rigid motion correction
# maximum allowed rigid shift in pixels
max_shifts = [int(a/b) for a, b in zip(max_shift_um, dxy)]
# start a new patch for pw-rigid motion correction every x pixels
strides = tuple([int(a/b) for a, b in zip(patch_motion_um, dxy)])
# overlap between pathes (size of patch in pixels: strides+overlaps)
overlaps = (24, 24)
# maximum deviation allowed for patch with respect to rigid shifts
max_deviation_rigid = 3
mc_dict = {
'fnames': fnames,
'fr': fr,
'decay_time': decay_time,
'dxy': dxy,
'pw_rigid': pw_rigid,
'max_shifts': max_shifts,
'strides': strides,
'overlaps': overlaps,
'max_deviation_rigid': max_deviation_rigid,
'border_nan': 'copy'
}
opts = params.CNMFParams(params_dict=mc_dict)
# %% start a cluster for parallel processing
c, dview, n_processes = cm.cluster.setup_cluster(
backend='local', n_processes=None, single_thread=False)
# %%% MOTION CORRECTION
# first we create a motion correction object with the specified parameters
mc = MotionCorrect(fnames, dview=dview, **opts.get_group('motion'))
# note that the file is not loaded in memory
# %% Run (piecewise-rigid motion) correction using NoRMCorre
mc.motion_correct(save_movie=True)
# %% MEMORY MAPPING
border_to_0 = 0 if mc.border_nan is 'copy' else mc.border_to_0
# you can include the boundaries of the FOV if you used the 'copy' option
# during motion correction, although be careful about the components near
# the boundaries
# memory map the file in order 'C'
fname_new = cm.save_memmap(mc.mmap_file, base_name='_memmap_', order='C',
border_to_0=border_to_0) # exclude borders
# now load the file
Yr, dims, T = cm.load_memmap(fname_new)
images = np.reshape(Yr.T, [T] + list(dims), order='F')
# load frames in python format (T x X x Y)
# %% restart cluster to clean up memory
cm.stop_server(dview=dview)
c, dview, n_processes = cm.cluster.setup_cluster(
backend='local', n_processes=None, single_thread=False)
# %% parameters for source extraction and deconvolution
p = 1 # order of the autoregressive system
gnb = 3 # number of global background components
merge_thr = 0.85 # merging threshold, max correlation allowed
rf = 20 # half-size of the patches in pixels. e.g., if rf=25, patches are 50x50
stride_cnmf = 6 # amount of overlap between the patches in pixels
K = 3 # number of components per patch
gSig = [7, 7] # expected half size of neurons in pixels
method_init = 'greedy_roi' # initialization method (if analyzing dendritic data using 'sparse_nmf')
ssub = 2 # spatial subsampling during initialization
tsub = 2 # temporal subsampling during intialization
s_min = -20 # minimum signal amplitude needed in order for a transient to be considered as activity
# parameters for component evaluation
opts_dict = {'fnames': fnames,
'fr': fr,
'nb': gnb,
'rf': rf,
'K': K,
'gSig': gSig,
'stride': stride_cnmf,
'method_init': method_init,
'rolling_sum': True,
'merge_thr': merge_thr,
'n_processes': n_processes,
'only_init': True,
'ssub': ssub,
'tsub': tsub,
's_min': s_min}
opts.change_params(params_dict=opts_dict)
# %% RUN CNMF ON PATCHES
# First extract spatial and temporal components on patches and combine them
# for this step deconvolution is turned off (p=0)
opts.change_params({'p': 0})
cnm = cnmf.CNMF(n_processes, params=opts, dview=dview)
cnm = cnm.fit(images)
# %% ALTERNATE WAY TO RUN THE PIPELINE AT ONCE
# you can also perform the motion correction plus cnmf fitting steps
# simultaneously after defining your parameters object using
# cnm1 = cnmf.CNMF(n_processes, params=opts, dview=dview)
# cnm1.fit_file(motion_correct=True)
# %% RE-RUN seeded CNMF on accepted patches to refine and perform deconvolution
cnm.params.change_params({'p': p})
cnm2 = cnm.refit(images, dview=dview)
# %% COMPONENT EVALUATION
# the components are evaluated in three ways:
# a) the shape of each component must be correlated with the data
# b) a minimum peak SNR is required over the length of a transient
# c) each shape passes a CNN based classifier
min_SNR = 2 # Overall minimum signal to noise ratio for accepting a component
rval_thr = 0.85 # space correlation threshold for accepting a component
cnn_thr = 0.99 # threshold for CNN based classifier
cnn_lowest = 0.15 # neurons with cnn probability lower than this value are rejected
min_size_neuro = 0.1*gSig[0]*np.pi**2
max_size_neuro = 2.5*gSig[0]*np.pi**2
cnm2.params.set('quality', {'decay_time': decay_time,
'min_SNR': min_SNR,
'rval_thr': rval_thr,
'use_cnn': True,
'min_cnn_thr': cnn_thr,
'cnn_lowest': cnn_lowest,
'min_size_neuro': min_size_neuro,
'max_size_neuro': max_size_neuro,})
cnm2.estimates.evaluate_components(images, cnm2.params, dview=dview)
#%% update object with selected components
cnm2.estimates.select_components(use_object=True)
#%% Extract DF/F values
cnm2.estimates.detrend_df_f(quantileMin=8, frames_window=250)
#cnm2.estimates.threshold_spatial_components(maxthr=0.85)
#cnm2.estimates.remove_small_large_neurons(min_size_neuro=min_size_neuro, max_size_neuro=max_size_neuro)
# %% Generate heat image of video and save data
Cn = cm.local_correlations(images, swap_dim=False)
Cn[np.isnan(Cn)] = 0
cnm2.estimates.Cn = Cn
cnm2.save(cnm2.mmap_file[:-4] + 'hdf5')
#%% STOP CLUSTER and clean up log files
cm.stop_server(dview=dview)
#Create denoised video as matrix
denoised = cm.movie(cnm2.estimates.A.dot(cnm2.estimates.C) + \
cnm2.estimates.b.dot(cnm2.estimates.f)).reshape(dims + (-1,), order='F').transpose([2, 0, 1])
#Normalizing denoised matrix to the [0,255] range prior to type conversion
max_pixel = np.nanmax(denoised)
norm_denoised = (denoised/max_pixel)*255
#Type conversion so imageio doesn't get mad
norm_denoised = np.uint8(norm_denoised)
#Saving a denoised avi of the analyzed video
sname = Path(args.in_file).stem + '.avi'
imageio.mimwrite(sname, norm_denoised, fps=15.49)
def main():
pass # For compatibility between running under Spyder and the CLI
# %% Load demo movie and ROIs
fnames = download_demo(
'demo_voltage_imaging.hdf5',
'volpy') # file path to movie file (will download if not present)
path_ROIs = download_demo(
'demo_voltage_imaging_ROIs.hdf5',
'volpy') # file path to ROIs file (will download if not present)
#%% dataset dependent parameters
# dataset dependent parameters
fr = 400 # sample rate of the movie
# motion correction parameters
pw_rigid = False # flag for pw-rigid motion correction
gSig_filt = (3, 3) # size of filter, in general gSig (see below),
# change this one if algorithm does not work
max_shifts = (5, 5) # maximum allowed rigid shift
strides = (
48, 48
) # start a new patch for pw-rigid motion correction every x pixels
overlaps = (24, 24
) # overlap between pathes (size of patch strides+overlaps)
max_deviation_rigid = 3 # maximum deviation allowed for patch with respect to rigid shifts
border_nan = 'copy'
opts_dict = {
'fnames': fnames,
'fr': fr,
'pw_rigid': pw_rigid,
'max_shifts': max_shifts,
'gSig_filt': gSig_filt,
'strides': strides,
'overlaps': overlaps,
'max_deviation_rigid': max_deviation_rigid,
'border_nan': border_nan
}
opts = volparams(params_dict=opts_dict)
# %% play the movie (optional)
# playing the movie using opencv. It requires loading the movie in memory.
# To close the movie press q
display_images = False
if display_images:
m_orig = cm.load(fnames)
ds_ratio = 0.2
moviehandle = m_orig.resize(1, 1, ds_ratio)
moviehandle.play(q_max=99.5, fr=40, magnification=6)
# %% start a cluster for parallel processing
c, dview, n_processes = cm.cluster.setup_cluster(backend='local',
n_processes=None,
single_thread=False)
# %%% MOTION CORRECTION
# first we create a motion correction object with the specified parameters
mc = MotionCorrect(fnames, dview=dview, **opts.get_group('motion'))
# Run correction
mc.motion_correct(save_movie=True)
# %% compare with original movie
if display_images:
m_orig = cm.load(fnames)
m_rig = cm.load(mc.mmap_file)
ds_ratio = 0.2
moviehandle = cm.concatenate([
m_orig.resize(1, 1, ds_ratio) - mc.min_mov * mc.nonneg_movie,
m_rig.resize(1, 1, ds_ratio)
],
axis=2)
moviehandle.play(fr=60, q_max=99.5, magnification=4) # press q to exit
# %% MEMORY MAPPING
border_to_0 = 0 if mc.border_nan == 'copy' else mc.border_to_0
# you can include the boundaries of the FOV if you used the 'copy' option
# during motion correction, although be careful about the components near
# the boundaries
# memory map the file in order 'C'
fname_new = cm.save_memmap_join(mc.mmap_file,
base_name='memmap_',
add_to_mov=border_to_0,
dview=dview) # exclude border
# %% SEGMENTATION
# create summary images
img = mean_image(mc.mmap_file[0], window=1000, dview=dview)
img = (img - np.mean(img)) / np.std(img)
gaussian_blur = False # Use gaussian blur when the quality of corr image(Cn) is bad
Cn = local_correlations_movie_offline(mc.mmap_file[0],
fr=fr,
window=fr * 4,
stride=fr * 4,
winSize_baseline=fr,
remove_baseline=True,
gaussian_blur=gaussian_blur,
dview=dview).max(axis=0)
img_corr = (Cn - np.mean(Cn)) / np.std(Cn)
summary_image = np.stack([img, img, img_corr], axis=2).astype(np.float32)
#%% three methods for segmentation
methods_list = [
'manual_annotation', # manual annotation needs user to prepare annotated datasets same format as demo ROIs
'quick_annotation', # quick annotation annotates data with simple interface in python
'maskrcnn'
] # maskrcnn is a convolutional network trained for finding neurons using summary images
method = methods_list[0]
if method == 'manual_annotation':
with h5py.File(path_ROIs, 'r') as fl:
ROIs = fl['mov'][()]
elif method == 'quick_annotation':
ROIs = utils.quick_annotation(img, min_radius=4, max_radius=8)
elif method == 'maskrcnn': # Important!! make sure install keras before using mask rcnn
weights_path = download_model('mask_rcnn')
ROIs = utils.mrcnn_inference(img=summary_image,
weights_path=weights_path,
display_result=True)
# %% restart cluster to clean up memory
cm.stop_server(dview=dview)
c, dview, n_processes = cm.cluster.setup_cluster(backend='local',
n_processes=None,
single_thread=False,
maxtasksperchild=1)
# %% parameters for trace denoising and spike extraction
ROIs = ROIs # region of interests
index = list(range(len(ROIs))) # index of neurons
weights = None # reuse spatial weights
context_size = 35 # number of pixels surrounding the ROI to censor from the background PCA
flip_signal = True # Important!! Flip signal or not, True for Voltron indicator, False for others
hp_freq_pb = 1 / 3 # parameter for high-pass filter to remove photobleaching
threshold_method = 'simple' # 'simple' or 'adaptive_threshold'
min_spikes = 10 # minimal spikes to be found
threshold = 3.5 # threshold for finding spikes, increase threshold to find less spikes
do_plot = False # plot detail of spikes, template for the last iteration
ridge_bg = 0.001 # ridge regression regularizer strength for background removement
sub_freq = 20 # frequency for subthreshold extraction
weight_update = 'ridge' # 'ridge' or 'NMF' for weight update
opts_dict = {
'fnames': fname_new,
'ROIs': ROIs,
'index': index,
'weights': weights,
'context_size': context_size,
'flip_signal': flip_signal,
'hp_freq_pb': hp_freq_pb,
'threshold_method': threshold_method,
'min_spikes': min_spikes,
'threshold': threshold,
'do_plot': do_plot,
'ridge_bg': ridge_bg,
'sub_freq': sub_freq,
'weight_update': weight_update
}
opts.change_params(params_dict=opts_dict)
#%% TRACE DENOISING AND SPIKE DETECTION
vpy = VOLPY(n_processes=n_processes, dview=dview, params=opts)
vpy.fit(n_processes=n_processes, dview=dview)
#%% visualization
if display_images:
print(np.where(
vpy.estimates['locality'])[0]) # neurons that pass locality test
idx = np.where(vpy.estimates['locality'] > 0)[0]
utils.view_components(vpy.estimates, img_corr, idx)
#%% reconstructed movie
# note the negative spatial weights is cutoff
if display_images:
mv_all = utils.reconstructed_movie(vpy.estimates,
fnames=mc.mmap_file,
idx=idx,
scope=(0, 1000),
flip_signal=flip_signal)
mv_all.play(fr=40)
# %% STOP CLUSTER and clean up log files
cm.stop_server(dview=dview)
log_files = glob.glob('*_LOG_*')
for log_file in log_files:
os.remove(log_file)
def run_caiman_pipeline(movie, fr, fnames, savedir, usematlabroi):
#%%
cpu_num = 7
cpu_num_spikepursuit = 2
#gsig_filt_micron = (4, 4)
#max_shifts_micron = (6,6)
#strides_micron = (60,60)
#overlaps_micron = (30, 30)
gsig_filt_micron = (4, 4)
max_shifts_micron = (6, 6)
strides_micron = (30, 30)
overlaps_micron = (15, 15)
max_deviation_rigid_micron = 4
pixel_size = movie['movie_pixel_size']
ROIs = None # Region of interests
index = None # index of neurons
weights = None # reuse spatial weights by
# opts.change_params(params_dict={'weights':vpy.estimates['weights']})
# motion correction parameters
pw_rigid = False # flag for pw-rigid motion correction
gSig_filt = tuple(
np.asarray(np.round(np.asarray(gsig_filt_micron) / float(pixel_size)),
int)) # size of filter, in general gSig (see below),
# change this one if algorithm does not work
max_shifts = tuple(
np.asarray(np.round(np.asarray(max_shifts_micron) / float(pixel_size)),
int))
strides = tuple(
np.asarray(np.round(np.asarray(strides_micron) / float(pixel_size)),
int)
) # start a new patch for pw-rigid motion correction every x pixels
overlaps = tuple(
np.asarray(np.round(np.asarray(overlaps_micron) / float(pixel_size)),
int)
) # start a new patch for pw-rigid motion correction every x pixels
# overlap between pathes (size of patch strides+overlaps)
max_deviation_rigid = int(
round(max_deviation_rigid_micron / pixel_size)
) # maximum deviation allowed for patch with respect to rigid shifts
border_nan = 'copy'
opts_dict = {
'fnames': fnames,
'fr': fr,
'index': index,
'ROIs': ROIs,
'weights': weights,
'pw_rigid': pw_rigid,
'max_shifts': max_shifts,
'gSig_filt': gSig_filt,
'strides': strides,
'overlaps': overlaps,
'max_deviation_rigid': max_deviation_rigid,
'border_nan': border_nan
}
opts = volparams(params_dict=opts_dict)
# %% play the movie (optional)
# playing the movie using opencv. It requires loading the movie in memory.
# To close the video press q
display_images = False
if display_images:
m_orig = cm.load(fnames)
ds_ratio = 0.2
moviehandle = m_orig.resize(1, 1, ds_ratio)
moviehandle.play(q_max=99.5, fr=60, magnification=2)
# %% start a cluster for parallel processing
c, dview, n_processes = cm.cluster.setup_cluster(backend='local',
n_processes=cpu_num,
single_thread=False)
# % MOTION CORRECTION
# Create a motion correction object with the specified parameters
mcrig = MotionCorrect(fnames, dview=dview, **opts.get_group('motion'))
# Run piecewise rigid motion correction
#%
mcrig.motion_correct(save_movie=True)
dview.terminate()
# % MOTION CORRECTION2
opts.change_params({'pw_rigid': True})
c, dview, n_processes = cm.cluster.setup_cluster(backend='local',
n_processes=cpu_num,
single_thread=False)
# Create a motion correction object with the specified parameters
mc = MotionCorrect(mcrig.mmap_file,
dview=dview,
**opts.get_group('motion'))
# Run piecewise rigid motion correction
mc.motion_correct(save_movie=True)
dview.terminate()
# %% motion correction compared with original movie
display_images = False
if display_images:
m_orig = cm.load(fnames)
m_rig = cm.load(mcrig.mmap_file)
m_pwrig = cm.load(mc.mmap_file)
ds_ratio = 0.2
moviehandle = cm.concatenate([
m_orig.resize(1, 1, ds_ratio) - mc.min_mov * mc.nonneg_movie,
m_rig.resize(1, 1, ds_ratio),
m_pwrig.resize(1, 1, ds_ratio)
],
axis=2)
moviehandle.play(fr=60, q_max=99.5, magnification=2) # press q to exit
# % movie subtracted from the mean
m_orig2 = (m_orig - np.mean(m_orig, axis=0))
m_rig2 = (m_rig - np.mean(m_rig, axis=0))
m_pwrig2 = (m_pwrig - np.mean(m_pwrig, axis=0))
moviehandle1 = cm.concatenate([
m_orig2.resize(1, 1, ds_ratio),
m_rig2.resize(1, 1, ds_ratio),
m_pwrig2.resize(1, 1, ds_ratio)
],
axis=2)
moviehandle1.play(fr=60, q_max=99.5, magnification=2)
# %% Memory Mapping
c, dview, n_processes = cm.cluster.setup_cluster(backend='local',
n_processes=cpu_num,
single_thread=False)
border_to_0 = 0 if mc.border_nan == 'copy' else mc.border_to_0
fname_new = cm.save_memmap_join(mc.mmap_file,
base_name='memmap_',
add_to_mov=border_to_0,
dview=dview,
n_chunks=10)
dview.terminate()
# %% change fnames to the new motion corrected one
opts.change_params(params_dict={'fnames': fname_new})
# %% SEGMENTATION
roidir = savedir[:savedir.find('VolPy')] + 'Spikepursuit' + savedir[
savedir.find('VolPy') + len('Volpy'):]
try:
files = os.listdir(roidir)
except:
files = []
if usematlabroi and 'ROIs.mat' in files:
ROIs = loadmat(os.path.join(roidir, 'ROIs.mat'))['ROIs']
if len(np.shape(ROIs)) == 3:
ROIs = np.moveaxis(np.asarray(ROIs, bool), 2, 0)
else:
ROIs = np.asarray([ROIs])
all_rois = ROIs
opts.change_params(
params_dict={
'ROIs': ROIs,
'index': list(range(ROIs.shape[0])),
'method': 'SpikePursuit'
})
else:
#%
print('WTF')
# Create mean and correlation image
use_maskrcnn = True # set to True to predict the ROIs using the mask R-CNN
if not use_maskrcnn: # use manual annotations
with h5py.File(path_ROIs, 'r') as fl:
ROIs = fl['mov'][()] # load ROIs
opts.change_params(
params_dict={
'ROIs': ROIs,
'index': list(range(ROIs.shape[0])),
'method': 'SpikePursuit'
})
else:
try:
m = cm.load(mc.mmap_file[0], subindices=slice(0, 20000))
except:
m = cm.load(
'/home/rozmar/Data/Voltage_imaging/Voltage_rig_1P/rozsam/20200120/40x_1xtube_10A_7_000_rig__d1_128_d2_512_d3_1_order_F_frames_2273_._els__d1_128_d2_512_d3_1_order_F_frames_2273_.mmap',
subindices=slice(0, 20000))
m.fr = fr
img = m.mean(axis=0)
img = (img - np.mean(img)) / np.std(img)
m1 = m.computeDFF(secsWindow=1, in_place=True)[0]
m = m - m1
Cn = m.local_correlations(swap_dim=False, eight_neighbours=True)
img_corr = (Cn - np.mean(Cn)) / np.std(Cn)
summary_image = np.stack([img, img, img_corr],
axis=2).astype(np.float32)
del m
del m1
# %
# Mask R-CNN
config = neurons.NeuronsConfig()
class InferenceConfig(config.__class__):
# Run detection on one image at a time
GPU_COUNT = 1
IMAGES_PER_GPU = 1
DETECTION_MIN_CONFIDENCE = 0.7
IMAGE_RESIZE_MODE = "pad64"
IMAGE_MAX_DIM = 512
RPN_NMS_THRESHOLD = 0.7
POST_NMS_ROIS_INFERENCE = 1000
config = InferenceConfig()
config.display()
model_dir = os.path.join(caiman_datadir(), 'model')
DEVICE = "/cpu:0" # /cpu:0 or /gpu:0
with tf.device(DEVICE):
model = modellib.MaskRCNN(mode="inference",
model_dir=model_dir,
config=config)
weights_path = download_model('mask_rcnn')
model.load_weights(weights_path, by_name=True)
results = model.detect([summary_image], verbose=1)
r = results[0]
ROIs_mrcnn = r['masks'].transpose([2, 0, 1])
# %% visualize the result
display_result = False
if display_result:
_, ax = plt.subplots(1, 1, figsize=(16, 16))
visualize.display_instances(summary_image,
r['rois'],
r['masks'],
r['class_ids'], ['BG', 'neurons'],
r['scores'],
ax=ax,
title="Predictions")
# %% set rois
opts.change_params(
params_dict={
'ROIs': ROIs_mrcnn,
'index': list(range(ROIs_mrcnn.shape[0])),
'method': 'SpikePursuit'
})
#all_rois = ROIs_mrcnn
# %% Trace Denoising and Spike Extraction
c, dview, n_processes = cm.cluster.setup_cluster(
backend='local',
n_processes=cpu_num_spikepursuit,
single_thread=False,
maxtasksperchild=1)
#dview=None
vpy = VOLPY(n_processes=n_processes, dview=dview, params=opts)
vpy.fit(n_processes=n_processes, dview=dview)
#%%
print('saving parameters')
parameters = dict()
parameters['motion'] = opts.motion
parameters['data'] = opts.data
parameters['volspike'] = opts.volspike
with open(os.path.join(savedir, 'parameters.pickle'), 'wb') as outfile:
pickle.dump(parameters, outfile)
#%%
volspikedata = dict()
volspikedata['estimates'] = vpy.estimates
volspikedata['params'] = vpy.params.data
with open(os.path.join(savedir, 'spikepursuit.pickle'), 'wb') as outfile:
pickle.dump(volspikedata, outfile)
#%%
for mcidx, mc_now in enumerate([mcrig, mc]):
motioncorr = dict()
motioncorr['fname'] = mc_now.fname
motioncorr['fname_tot_rig'] = mc_now.fname_tot_rig
motioncorr['mmap_file'] = mc_now.mmap_file
motioncorr['min_mov'] = mc_now.min_mov
motioncorr['shifts_rig'] = mc_now.shifts_rig
motioncorr['shifts_opencv'] = mc_now.shifts_opencv
motioncorr['niter_rig'] = mc_now.niter_rig
motioncorr['min_mov'] = mc_now.min_mov
motioncorr['templates_rig'] = mc_now.templates_rig
motioncorr['total_template_rig'] = mc_now.total_template_rig
try:
motioncorr['x_shifts_els'] = mc_now.x_shifts_els
motioncorr['y_shifts_els'] = mc_now.y_shifts_els
except:
pass
with open(
os.path.join(savedir, 'motion_corr_' + str(mcidx) + '.pickle'),
'wb') as outfile:
pickle.dump(motioncorr, outfile)
#%% saving stuff
print('moving files')
for mmap_file in mcrig.mmap_file:
fname = pathlib.Path(mmap_file).name
os.remove(mmap_file)
#shutil.move(mmap_file, os.path.join(savedir,fname))
for mmap_file in mc.mmap_file:
fname = pathlib.Path(mmap_file).name
os.remove(mmap_file)
#shutil.move(mmap_file, os.path.join(savedir,fname))
fname = pathlib.Path(fname_new).name
shutil.move(fname_new, os.path.join(savedir, fname))
#print('waiting')
#time.sleep(1000)
# %% some visualization
plotstuff = False
if plotstuff:
print(np.where(vpy.estimates['passedLocalityTest'])
[0]) # neurons that pass locality test
n = 0
# Processed signal and spikes of neurons
plt.figure()
plt.plot(vpy.estimates['trace'][n])
plt.plot(vpy.estimates['spikeTimes'][n],
np.max(vpy.estimates['trace'][n]) *
np.ones(vpy.estimates['spikeTimes'][n].shape),
color='g',
marker='o',
fillstyle='none',
linestyle='none')
plt.title('signal and spike times')
plt.show()
# Location of neurons by Mask R-CNN or manual annotation
plt.figure()
if use_maskrcnn:
plt.imshow(ROIs_mrcnn[n])
else:
plt.imshow(ROIs[n])
mv = cm.load(fname_new)
plt.imshow(mv.mean(axis=0), alpha=0.5)
# Spatial filter created by algorithm
plt.figure()
plt.imshow(vpy.estimates['spatialFilter'][n])
plt.colorbar()
plt.title('spatial filter')
plt.show()
# %% STOP CLUSTER and clean up log files
cm.stop_server(dview=dview)
log_files = glob.glob('*_LOG_*')
for log_file in log_files:
os.remove(log_file)
print('checkpoint 1: mcorrect', flush=True)
# %%% MOTION CORRECTION
# first we create a motion correction object with the specified parameters
if nomc:
print('Skipping motion correction due to -nomc flag', flush=True)
print('saving movies as mmap file', flush=True)
fname_new = cm.save_memmap(fname, base_name='memmap_{}_full_'.format(slurmid), order='C',
border_to_0=0)
else:
mc = MotionCorrect(fnames, dview=dview, **opts.get_group('motion'))
# note that the file is not loaded in memory
# %% Run (piecewise-rigid motion) correction using NoRMCorre
mc.motion_correct(save_movie=True, template=mc_temp)
# %% MEMORY MAPPING
border_to_0 = 0 if mc.border_nan is 'copy' else mc.border_to_0
# you can include the boundaries of the FOV if you used the 'copy' option
# during motion correction, although be careful about the components near
# the boundaries
# memory map the file in order 'C'
save_name = mc.fname_tot_els if opts.get('motion','pw_rigid') else mc.fname_tot_rig
print('saving movies as mmap file', flush=True)
fname_new = cm.save_memmap(save_name, base_name='memmap_{}_full_'.format(slurmid), order='C',
border_to_0=border_to_0) # exclude borders
def main():
pass # For compatibility between running under Spyder and the CLI
#%%
"""
General parameters
"""
play_movie = 1
plot_extras = 1
plot_extras_cell = 1
compute_mc_metrics = 1
#%% Select file(s) to be processed (download if not present)
"""
Load file
"""
#fnames = ['Sue_2x_3000_40_-46.tif'] # filename to be processed
#if fnames[0] in ['Sue_2x_3000_40_-46.tif', 'demoMovie.tif']:
# fnames = [download_demo(fnames[0])]
#fnames = ['/home/yuriy/Desktop/Data/rest1_5_9_19_cut.tif']
#f_dir = 'C:\\Users\\rylab_dataPC\\Desktop\\Yuriy\\caiman_data\\short\\'
f_dir = 'G:\\analysis\\190828-calcium_voltage\\soma_dendrites\\pCAG_jREGECO1a_ASAP3_anesth_001\\'
f_name = 'Ch1'
f_ext = 'tif'
fnames = [f_dir + f_name + '.' + f_ext]
#fnames = ['C:/Users/rylab_dataPC/Desktop/Yuriy/caiman_data/rest1_5_9_19_2_cut_ca.hdf5']
#%% First setup some parameters for data and motion correction
"""
Parameters
"""
# dataset dependent parameters
fr = 30 # imaging rate in frames per second
decay_time = 1
#0.4 # length of a typical transient in seconds
dxy = (2., 2.) # spatial resolution in x and y in (um per pixel)
# note the lower than usual spatial resolution here
max_shift_um = (12., 12.) # maximum shift in um
patch_motion_um = (100., 100.) # patch size for non-rigid correction in um
# motion correction parameters
pw_rigid = True # flag to select rigid vs pw_rigid motion correction
# maximum allowed rigid shift in pixels
#max_shifts = [int(a/b) for a, b in zip(max_shift_um, dxy)]
max_shifts = [6, 6]
# start a new patch for pw-rigid motion correction every x pixels
#strides = tuple([int(a/b) for a, b in zip(patch_motion_um, dxy)])
strides = [48, 48]
# overlap between pathes (size of patch in pixels: strides+overlaps)
overlaps = (24, 24)
# maximum deviation allowed for patch with respect to rigid shifts
max_deviation_rigid = 3
mc_dict = {
'fnames': fnames,
'fr': fr,
'decay_time': decay_time,
'dxy': dxy,
'pw_rigid': pw_rigid,
'max_shifts': max_shifts,
'strides': strides,
'overlaps': overlaps,
'max_deviation_rigid': max_deviation_rigid,
'border_nan': 'copy'
}
opts = params.CNMFParams(params_dict=mc_dict)
# %% play the movie (optional)
# playing the movie using opencv. It requires loading the movie in memory.
# To close the video press q
if play_movie:
m_orig = cm.load_movie_chain(fnames)
ds_ratio = 0.2
moviehandle = m_orig.resize(1, 1, ds_ratio)
moviehandle.play(q_max=99.5, fr=60, magnification=2)
# %% start a cluster for parallel processing
c, dview, n_processes = cm.cluster.setup_cluster(backend='local',
n_processes=None,
single_thread=False)
# %%% MOTION CORRECTION
# first we create a motion correction object with the specified parameters
mc = MotionCorrect(fnames, dview=dview, **opts.get_group('motion'))
# note that the file is not loaded in memory
# %% Run (piecewise-rigid motion) correction using NoRMCorre
mc.motion_correct(save_movie=True)
# type "mc."and press TAB to see all interesting associated variables and self. outputs
# interesting outputs
# saved file is mc.fname_tot_els / mc.fname_tot_rig
# mc.x_shifts_els / mc.y_shifts_els: shifts in x/y per frame per patch
# mc.coord_shifts_els: coordinates associated to patches with shifts
# mc.total_template_els: updated template for pw
# mc.total_template_rig: updated template for rigid
# mc.templates_rig: templates for each iteration in rig
#%% # compute metrics for the results (TAKES TIME!!)
if compute_mc_metrics:
# not finished
bord_px_els = np.ceil(
np.maximum(np.max(np.abs(mc.x_shifts_els)),
np.max(np.abs(mc.y_shifts_els)))).astype(np.int)
final_size = np.subtract(
mc.total_template_els.shape,
2 * bord_px_els) # remove pixels in the boundaries
winsize = 100
swap_dim = False
resize_fact_flow = .2 # downsample for computing ROF
tmpl_rig, correlations_orig, flows_orig, norms_orig, crispness_orig = cm.motion_correction.compute_metrics_motion_correction(
fnames[0],
final_size[0],
final_size[1],
swap_dim,
winsize=winsize,
play_flow=False,
resize_fact_flow=resize_fact_flow)
plt.figure()
plt.plot(correlations_orig)
# %% compare with original movie
if play_movie:
m_orig = cm.load_movie_chain(fnames)
m_els = cm.load(mc.mmap_file)
ds_ratio = 0.2
moviehandle = cm.concatenate([
m_orig.resize(1, 1, ds_ratio) - mc.min_mov * mc.nonneg_movie,
m_els.resize(1, 1, ds_ratio)
],
axis=2)
moviehandle.play(fr=60, q_max=99.5, magnification=2) # press q to exit
del m_orig
del m_els
if plot_extras:
# plot total template
plt.figure()
plt.imshow(mc.total_template_els)
plt.title('Template after iteration')
# plot x and y corrections
plt.figure()
plt.plot(mc.shifts_rig)
plt.title('Rigid motion correction xy movement')
plt.legend(['x shift', 'y shift'])
plt.xlabel('frames')
# %% MEMORY MAPPING
border_to_0 = 0 if mc.border_nan is 'copy' else mc.border_to_0
# you can include the boundaries of the FOV if you used the 'copy' option
# during motion correction, although be careful about the components near
# the boundaries
# memory map the file in order 'C'
fname_new = cm.save_memmap(mc.mmap_file,
base_name='memmap_',
order='C',
border_to_0=border_to_0) # exclude borders
# now load the file
Yr, dims, T = cm.load_memmap(fname_new)
images = np.reshape(Yr.T, [T] + list(dims), order='F')
# load frames in python format (T x X x Y)
# %% restart cluster to clean up memory
cm.stop_server(dview=dview)
c, dview, n_processes = cm.cluster.setup_cluster(backend='local',
n_processes=None,
single_thread=False)
# %% parameters for source extraction and deconvolution
p = 2 # order of the autoregressive system
gnb = 2 # number of global background components
merge_thr = 0.85 # merging threshold, max correlation allowed
rf = 15 # half-size of the patches in pixels. e.g., if rf=25, patches are 50x50
stride_cnmf = 6 # amount of overlap between the patches in pixels
K = 2 # number of components per patch
gSig = [15, 15] # expected half size of neurons in pixels
# initialization method (if analyzing dendritic data using 'sparse_nmf')
method_init = 'greedy_roi'
ssub = 1 # spatial subsampling during initialization
tsub = 1 # temporal subsampling during intialization
# parameters for component evaluation
opts_dict = {
'fnames': fnames,
'fr': fr,
'nb': gnb,
'rf': rf,
'K': K,
'gSig': gSig,
'stride': stride_cnmf,
'method_init': method_init,
'rolling_sum': True,
'merge_thr': merge_thr,
'n_processes': n_processes,
'only_init': True,
'ssub': ssub,
'tsub': tsub
}
opts.change_params(params_dict=opts_dict)
# %% RUN CNMF ON PATCHES
# First extract spatial and temporal components on patches and combine them
# for this step deconvolution is turned off (p=0)
opts.change_params({'p': 0})
cnm = cnmf.CNMF(n_processes, params=opts, dview=dview)
cnm = cnm.fit(images)
if plot_extras_cell:
num_cell_plot = 51
plt.figure()
plt.plot(cnm.estimates.C[num_cell_plot, :])
plt.title('Temporal component')
plt.legend(['Cell ' + str(num_cell_plot)])
# plot component sptial profile A
# first convert back to dense components
plot_spat_A = cnm.estimates.A[:, num_cell_plot].toarray().reshape(
list(dims))
plt.figure()
plt.imshow(plot_spat_A)
plt.title('Spatial component cell ' + str(num_cell_plot))
# %% ALTERNATE WAY TO RUN THE PIPELINE AT ONCE
# you can also perform the motion correction plus cnmf fitting steps
# simultaneously after defining your parameters object using
# cnm1 = cnmf.CNMF(n_processes, params=opts, dview=dview)
# cnm1.fit_file(motion_correct=True)
# %% plot contours of found components
Cn = cm.local_correlations(images, swap_dim=False)
Cn[np.isnan(Cn)] = 0
cnm.estimates.plot_contours(img=Cn)
plt.title('Contour plots of found components')
if plot_extras:
plt.figure()
plt.imshow(Cn)
plt.title('Local correlations')
# %% RE-RUN seeded CNMF on accepted patches to refine and perform deconvolution
cnm.params.change_params({'p': p})
cnm2 = cnm.refit(images, dview=dview)
# %% COMPONENT EVALUATION
# the components are evaluated in three ways:
# a) the shape of each component must be correlated with the data
# b) a minimum peak SNR is required over the length of a transient
# c) each shape passes a CNN based classifier
min_SNR = 2 # signal to noise ratio for accepting a component
rval_thr = 0.90 # space correlation threshold for accepting a component
cnn_thr = 0.99 # threshold for CNN based classifier
cnn_lowest = 0.1 # neurons with cnn probability lower than this value are rejected
cnm2.params.set(
'quality', {
'decay_time': decay_time,
'min_SNR': min_SNR,
'rval_thr': rval_thr,
'use_cnn': True,
'min_cnn_thr': cnn_thr,
'cnn_lowest': cnn_lowest
})
cnm2.estimates.evaluate_components(images, cnm2.params, dview=dview)
# %% PLOT COMPONENTS
cnm2.estimates.plot_contours(img=Cn, idx=cnm2.estimates.idx_components)
plt.suptitle('Component selection: min_SNR=' + str(min_SNR) +
'; rval_thr=' + str(rval_thr) + '; cnn prob range=[' +
str(cnn_lowest) + ' ' + str(cnn_thr) + ']')
# %% VIEW TRACES (accepted and rejected)
if plot_extras:
cnm2.estimates.view_components(images,
img=Cn,
idx=cnm2.estimates.idx_components)
plt.suptitle('Accepted')
cnm2.estimates.view_components(images,
img=Cn,
idx=cnm2.estimates.idx_components_bad)
plt.suptitle('Rejected')
#plt.figure();
#plt.plot(cnm2.estimates.YrA[0,:]+cnm2.estimates.C[0,:])
#
#
#
#
#plt.figure();
#plt.plot(cnm2.estimates.R[0,:]-cnm2.estimates.YrA[0,:]);
#plt.plot();
#plt.show();
#
#
#plt.figure();
#plt.plot(cnm2.estimates.detrend_df_f[1,:])
# these store the good and bad components, and next step sorts them
# cnm2.estimates.idx_components
# cnm2.estimates.idx_components_bad
#%% update object with selected components
#cnm2.estimates.select_components(use_object=True)
#%% Extract DF/F values
cnm2.estimates.detrend_df_f(quantileMin=8, frames_window=250)
#%% Show final traces
cnm2.estimates.view_components(img=Cn)
plt.suptitle("Final results")
#%% Save the mc data as in cmn struct as well
##
#mc_out = dict(
# pw_rigid = mc.pw_rigid,
# fname = mc.fname,
# mmap_file = mc.mmap_file,
# total_template_els = mc.total_template_els,
# total_template_rig = mc.total_template_rig,
# border_nan = mc.border_nan,
# border_to_0 = mc.border_to_0,
# x_shifts_els = mc.x_shifts_els,
# y_shifts_els = mc.y_shifts_els,
# Cn = Cn
# )
#
#
#deepdish.io.save(fnames[0] + '_mc_data.hdf5', mc_out)
#%% reconstruct denoised movie (press q to exit)
if play_movie:
cnm2.estimates.play_movie(images,
q_max=99.9,
gain_res=2,
magnification=2,
bpx=border_to_0,
include_bck=False) # background not shown
#%% STOP CLUSTER and clean up log files
cm.stop_server(dview=dview)
log_files = glob.glob('*_LOG_*')
for log_file in log_files:
os.remove(log_file)
save_results = True
if save_results:
cnm2.save(fnames[0][:-4] + '_results.hdf5')
def main():
pass # For compatibility between running under Spyder and the CLI
# %% Load demo movie and ROIs
fnames = download_demo('demo_voltage_imaging.hdf5', 'volpy') # file path to movie file (will download if not present)
path_ROIs = download_demo('demo_voltage_imaging_ROIs.hdf5', 'volpy') # file path to ROIs file (will download if not present)
# %% Setup some parameters for data and motion correction
# dataset parameters
fr = 400 # sample rate of the movie
ROIs = None # Region of interests
index = None # index of neurons
weights = None # reuse spatial weights by
# opts.change_params(params_dict={'weights':vpy.estimates['weights']})
# motion correction parameters
pw_rigid = False # flag for pw-rigid motion correction
gSig_filt = (3, 3) # size of filter, in general gSig (see below),
# change this one if algorithm does not work
max_shifts = (5, 5) # maximum allowed rigid shift
strides = (48, 48) # start a new patch for pw-rigid motion correction every x pixels
overlaps = (24, 24) # overlap between pathes (size of patch strides+overlaps)
max_deviation_rigid = 3 # maximum deviation allowed for patch with respect to rigid shifts
border_nan = 'copy'
opts_dict = {
'fnames': fnames,
'fr': fr,
'index': index,
'ROIs': ROIs,
'weights': weights,
'pw_rigid': pw_rigid,
'max_shifts': max_shifts,
'gSig_filt': gSig_filt,
'strides': strides,
'overlaps': overlaps,
'max_deviation_rigid': max_deviation_rigid,
'border_nan': border_nan
}
opts = volparams(params_dict=opts_dict)
# %% play the movie (optional)
# playing the movie using opencv. It requires loading the movie in memory.
# To close the video press q
display_images = False
if display_images:
m_orig = cm.load(fnames)
ds_ratio = 0.2
moviehandle = m_orig.resize(1, 1, ds_ratio)
moviehandle.play(q_max=99.5, fr=60, magnification=2)
# %% start a cluster for parallel processing
c, dview, n_processes = cm.cluster.setup_cluster(
backend='local', n_processes=None, single_thread=False)
# %%% MOTION CORRECTION
# Create a motion correction object with the specified parameters
mc = MotionCorrect(fnames, dview=dview, **opts.get_group('motion'))
# Run piecewise rigid motion correction
mc.motion_correct(save_movie=True)
dview.terminate()
# %% motion correction compared with original movie
display_images = False
if display_images:
m_orig = cm.load(fnames)
m_rig = cm.load(mc.mmap_file)
ds_ratio = 0.2
moviehandle = cm.concatenate([m_orig.resize(1, 1, ds_ratio) - mc.min_mov * mc.nonneg_movie,
m_rig.resize(1, 1, ds_ratio)], axis=2)
moviehandle.play(fr=60, q_max=99.5, magnification=2) # press q to exit
# % movie subtracted from the mean
m_orig2 = (m_orig - np.mean(m_orig, axis=0))
m_rig2 = (m_rig - np.mean(m_rig, axis=0))
moviehandle1 = cm.concatenate([m_orig2.resize(1, 1, ds_ratio),
m_rig2.resize(1, 1, ds_ratio)], axis=2)
moviehandle1.play(fr=60, q_max=99.5, magnification=2)
# %% Memory Mapping
c, dview, n_processes = cm.cluster.setup_cluster(
backend='local', n_processes=None, single_thread=False)
border_to_0 = 0 if mc.border_nan == 'copy' else mc.border_to_0
fname_new = cm.save_memmap_join(mc.mmap_file, base_name='memmap_',
add_to_mov=border_to_0, dview=dview, n_chunks=10)
dview.terminate()
# %% change fnames to the new motion corrected one
opts.change_params(params_dict={'fnames': fname_new})
# %% SEGMENTATION
# Create mean and correlation image
use_maskrcnn = True # set to True to predict the ROIs using the mask R-CNN
if not use_maskrcnn: # use manual annotations
with h5py.File(path_ROIs, 'r') as fl:
ROIs = fl['mov'][()] # load ROIs
opts.change_params(params_dict={'ROIs': ROIs,
'index': list(range(ROIs.shape[0])),
'method': 'SpikePursuit'})
else:
m = cm.load(mc.mmap_file[0], subindices=slice(0, 20000))
m.fr = fr
img = m.mean(axis=0)
img = (img-np.mean(img))/np.std(img)
m1 = m.computeDFF(secsWindow=1, in_place=True)[0]
m = m - m1
Cn = m.local_correlations(swap_dim=False, eight_neighbours=True)
img_corr = (Cn-np.mean(Cn))/np.std(Cn)
summary_image = np.stack([img, img, img_corr], axis=2).astype(np.float32)
del m
del m1
# %%
# Mask R-CNN
config = neurons.NeuronsConfig()
class InferenceConfig(config.__class__):
# Run detection on one image at a time
GPU_COUNT = 1
IMAGES_PER_GPU = 1
DETECTION_MIN_CONFIDENCE = 0.7
IMAGE_RESIZE_MODE = "pad64"
IMAGE_MAX_DIM = 512
RPN_NMS_THRESHOLD = 0.7
POST_NMS_ROIS_INFERENCE = 1000
config = InferenceConfig()
config.display()
model_dir = os.path.join(caiman_datadir(), 'model')
DEVICE = "/cpu:0" # /cpu:0 or /gpu:0
with tf.device(DEVICE):
model = modellib.MaskRCNN(mode="inference", model_dir=model_dir,
config=config)
weights_path = download_model('mask_rcnn')
model.load_weights(weights_path, by_name=True)
results = model.detect([summary_image], verbose=1)
r = results[0]
ROIs_mrcnn = r['masks'].transpose([2, 0, 1])
# %% visualize the result
display_result = False
if display_result:
_, ax = plt.subplots(1,1, figsize=(16,16))
visualize.display_instances(summary_image, r['rois'], r['masks'], r['class_ids'],
['BG', 'neurons'], r['scores'], ax=ax,
title="Predictions")
# %% set rois
opts.change_params(params_dict={'ROIs':ROIs_mrcnn,
'index':list(range(ROIs_mrcnn.shape[0])),
'method':'SpikePursuit'})
# %% Trace Denoising and Spike Extraction
c, dview, n_processes = cm.cluster.setup_cluster(
backend='local', n_processes=None, single_thread=False, maxtasksperchild=1)
vpy = VOLPY(n_processes=n_processes, dview=dview, params=opts)
vpy.fit(n_processes=n_processes, dview=dview)
# %% some visualization
print(np.where(vpy.estimates['passedLocalityTest'])[0]) # neurons that pass locality test
n = 0
# Processed signal and spikes of neurons
plt.figure()
plt.plot(vpy.estimates['trace'][n])
plt.plot(vpy.estimates['spikeTimes'][n],
np.max(vpy.estimates['trace'][n]) * np.ones(vpy.estimates['spikeTimes'][n].shape),
color='g', marker='o', fillstyle='none', linestyle='none')
plt.title('signal and spike times')
plt.show()
# Location of neurons by Mask R-CNN or manual annotation
plt.figure()
if use_maskrcnn:
plt.imshow(ROIs_mrcnn[n])
else:
plt.imshow(ROIs[n])
mv = cm.load(fname_new)
plt.imshow(mv.mean(axis=0),alpha=0.5)
# Spatial filter created by algorithm
plt.figure()
plt.imshow(vpy.estimates['spatialFilter'][n])
plt.colorbar()
plt.title('spatial filter')
plt.show()
# %% STOP CLUSTER and clean up log files
cm.stop_server(dview=dview)
log_files = glob.glob('*_LOG_*')
for log_file in log_files:
os.remove(log_file)
def run_caiman_pipeline(movie, fr, fnames, savedir, caiman_parameters):
#%%
usematlabroi = caiman_parameters['usematlabroi']
cpu_num = 16
cpu_num_spikepursuit = 3
#gsig_filt_micron = (4, 4)
#max_shifts_micron = (6,6)
#strides_micron = (60,60)
#overlaps_micron = (30, 30)
gsig_filt_micron = (4, 4)
max_shifts_micron = (6, 6)
strides_micron = (30, 30)
overlaps_micron = (15, 15)
max_deviation_rigid_micron = 4
pixel_size = movie['movie_pixel_size']
ROIs = None # Region of interests
index = None # index of neurons
weights = None # reuse spatial weights by
# opts.change_params(params_dict={'weights':vpy.estimates['weights']})
# motion correction parameters
pw_rigid = False # flag for pw-rigid motion correction
gSig_filt = tuple(
np.asarray(np.round(np.asarray(gsig_filt_micron) / float(pixel_size)),
int)) # size of filter, in general gSig (see below),
# change this one if algorithm does not work
max_shifts = tuple(
np.asarray(np.round(np.asarray(max_shifts_micron) / float(pixel_size)),
int))
strides = tuple(
np.asarray(np.round(np.asarray(strides_micron) / float(pixel_size)),
int)
) # start a new patch for pw-rigid motion correction every x pixels
overlaps = tuple(
np.asarray(np.round(np.asarray(overlaps_micron) / float(pixel_size)),
int)
) # start a new patch for pw-rigid motion correction every x pixels
# overlap between pathes (size of patch strides+overlaps)
max_deviation_rigid = int(
round(max_deviation_rigid_micron / pixel_size)
) # maximum deviation allowed for patch with respect to rigid shifts
border_nan = 'copy'
opts_dict = {
'fnames': fnames,
'fr': fr,
'index': index,
'ROIs': ROIs,
'weights': weights,
'pw_rigid': pw_rigid,
'max_shifts': max_shifts,
'gSig_filt': gSig_filt,
'strides': strides,
'overlaps': overlaps,
'max_deviation_rigid': max_deviation_rigid,
'border_nan': border_nan
}
opts = volparams(params_dict=opts_dict)
#%%
if not caiman_parameters['motion_correction_already_done']:
# %% play the movie (optional)
# playing the movie using opencv. It requires loading the movie in memory.
# To close the video press q
display_images = False
if display_images:
m_orig = cm.load(fnames)
ds_ratio = 0.2
moviehandle = m_orig.resize(1, 1, ds_ratio)
moviehandle.play(q_max=99.5, fr=60, magnification=2)
# %% start a cluster for parallel processing
c, dview, n_processes = cm.cluster.setup_cluster(backend='local',
n_processes=cpu_num,
single_thread=False)
# % MOTION CORRECTION
# Create a motion correction object with the specified parameters
mcrig = MotionCorrect(fnames, dview=dview, **opts.get_group('motion'))
# Run piecewise rigid motion correction
#%
mcrig.motion_correct(save_movie=True)
dview.terminate()
# % MOTION CORRECTION2
opts.change_params({'pw_rigid': True})
c, dview, n_processes = cm.cluster.setup_cluster(backend='local',
n_processes=cpu_num,
single_thread=False)
# Create a motion correction object with the specified parameters
mc = MotionCorrect(mcrig.mmap_file,
dview=dview,
**opts.get_group('motion'))
# Run piecewise rigid motion correction
mc.motion_correct(save_movie=True)
dview.terminate()
# %% motion correction compared with original movie
display_images = False
if display_images:
m_orig = cm.load(fnames)
m_rig = cm.load(mcrig.mmap_file)
m_pwrig = cm.load(mc.mmap_file)
ds_ratio = 0.2
moviehandle = cm.concatenate([
m_orig.resize(1, 1, ds_ratio) - mc.min_mov * mc.nonneg_movie,
m_rig.resize(1, 1, ds_ratio),
m_pwrig.resize(1, 1, ds_ratio)
],
axis=2)
moviehandle.play(fr=60, q_max=99.5,
magnification=2) # press q to exit
# % movie subtracted from the mean
m_orig2 = (m_orig - np.mean(m_orig, axis=0))
m_rig2 = (m_rig - np.mean(m_rig, axis=0))
m_pwrig2 = (m_pwrig - np.mean(m_pwrig, axis=0))
moviehandle1 = cm.concatenate([
m_orig2.resize(1, 1, ds_ratio),
m_rig2.resize(1, 1, ds_ratio),
m_pwrig2.resize(1, 1, ds_ratio)
],
axis=2)
moviehandle1.play(fr=60, q_max=99.5, magnification=2)
# %% Memory Mapping
c, dview, n_processes = cm.cluster.setup_cluster(backend='local',
n_processes=cpu_num,
single_thread=False)
border_to_0 = 0 if mc.border_nan == 'copy' else mc.border_to_0
fname_new = cm.save_memmap_join(mc.mmap_file,
base_name='memmap_',
add_to_mov=border_to_0,
dview=dview,
n_chunks=10)
dview.terminate()
# %% change fnames to the new motion corrected one
opts.change_params(params_dict={'fnames': fname_new})
else:
#%%
files = os.listdir(savedir)
for file in files:
if '.mmap' in file:
break
fname_new = os.path.join(savedir, file)
opts.change_params(params_dict={'fnames': fname_new})
# %% SEGMENTATION
if caiman_parameters['dospikepursuit']:
roidir_matlab = savedir[:savedir.find(
'VolPy')] + 'Spikepursuit' + savedir[savedir.find('VolPy') +
len('Volpy'):]
try:
files_matlab = os.listdir(roidir)
except:
files_matlab = []
roidir_volpy = savedir[:savedir.find('VolPy')] + 'ROI' + savedir[
savedir.find('VolPy') + len('Volpy'):]
try:
files_volpy = os.listdir(roidir_volpy)
except:
files_volpy = []
if usematlabroi and 'ROIs.mat' in files_matlab:
ROIs = loadmat(os.path.join(roidir_matlab, 'ROIs.mat'))['ROIs']
if len(np.shape(ROIs)) == 3:
ROIs = np.moveaxis(np.asarray(ROIs, bool), 2, 0)
else:
ROIs = np.asarray([ROIs])
all_rois = ROIs
opts.change_params(
params_dict={
'ROIs': ROIs,
'index': list(range(ROIs.shape[0])),
'method': 'SpikePursuit'
})
elif caiman_parameters['usevolpyroi'] and 'VolPy.npy' in files_volpy:
ROIs_original = np.load(os.path.join(roidir_volpy, 'VolPy.npy'))
roinum = int(np.max(np.unique(ROIs_original)))
ROIs = np.asarray(
np.zeros([
int(roinum), ROIs_original.shape[0], ROIs_original.shape[1]
]), bool)
for i in range(1, roinum + 1):
ROIs[i - 1, ROIs_original == i] = True
all_rois = ROIs
opts.change_params(
params_dict={
'ROIs': ROIs,
'index': list(range(ROIs.shape[0])),
'method': 'SpikePursuit'
})
else:
#%
print('WTF')
# %% Trace Denoising and Spike Extraction
c, dview, n_processes = cm.cluster.setup_cluster(
backend='local',
n_processes=cpu_num_spikepursuit,
single_thread=False,
maxtasksperchild=1)
#dview=None
vpy = VOLPY(n_processes=n_processes, dview=dview, params=opts)
vpy.fit(n_processes=n_processes, dview=dview)
#%%
if caiman_parameters['dospikepursuit']:
volspikedata = dict()
volspikedata['estimates'] = vpy.estimates
volspikedata['params'] = vpy.params.data
with open(os.path.join(savedir, 'spikepursuit.pickle'),
'wb') as outfile:
pickle.dump(volspikedata, outfile)
if not caiman_parameters['motion_correction_already_done']:
print('saving parameters')
parameters = dict()
parameters['motion'] = opts.motion
parameters['data'] = opts.data
if caiman_parameters['dospikepursuit']:
parameters['volspike'] = opts.volspike
with open(os.path.join(savedir, 'parameters.pickle'), 'wb') as outfile:
pickle.dump(parameters, outfile)
#%
#%
for mcidx, mc_now in enumerate([mcrig, mc]):
motioncorr = dict()
motioncorr['fname'] = mc_now.fname
motioncorr['fname_tot_rig'] = mc_now.fname_tot_rig
motioncorr['mmap_file'] = mc_now.mmap_file
motioncorr['min_mov'] = mc_now.min_mov
motioncorr['shifts_rig'] = mc_now.shifts_rig
motioncorr['shifts_opencv'] = mc_now.shifts_opencv
motioncorr['niter_rig'] = mc_now.niter_rig
motioncorr['min_mov'] = mc_now.min_mov
motioncorr['templates_rig'] = mc_now.templates_rig
motioncorr['total_template_rig'] = mc_now.total_template_rig
try:
motioncorr['x_shifts_els'] = mc_now.x_shifts_els
motioncorr['y_shifts_els'] = mc_now.y_shifts_els
except:
pass
with open(
os.path.join(savedir,
'motion_corr_' + str(mcidx) + '.pickle'),
'wb') as outfile:
pickle.dump(motioncorr, outfile)
#%saving stuff
print('moving files')
for mmap_file in mcrig.mmap_file:
fname = pathlib.Path(mmap_file).name
os.remove(mmap_file)
#shutil.move(mmap_file, os.path.join(savedir,fname))
for mmap_file in mc.mmap_file:
fname = pathlib.Path(mmap_file).name
os.remove(mmap_file)
#shutil.move(mmap_file, os.path.join(savedir,fname))
fname = pathlib.Path(fname_new).name
#Thread(target=shutil.copy, args=[fname_new, os.path.join(savedir,fname)]).start()
shutil.move(fname_new, os.path.join(savedir, fname))
#print('waiting')
#time.sleep(1000)
# %% some visualization
plotstuff = False
if plotstuff:
print(np.where(vpy.estimates['passedLocalityTest'])
[0]) # neurons that pass locality test
n = 0
# Processed signal and spikes of neurons
plt.figure()
plt.plot(vpy.estimates['trace'][n])
plt.plot(vpy.estimates['spikeTimes'][n],
np.max(vpy.estimates['trace'][n]) *
np.ones(vpy.estimates['spikeTimes'][n].shape),
color='g',
marker='o',
fillstyle='none',
linestyle='none')
plt.title('signal and spike times')
plt.show()
# Location of neurons by Mask R-CNN or manual annotation
plt.figure()
if use_maskrcnn:
plt.imshow(ROIs_mrcnn[n])
else:
plt.imshow(ROIs[n])
mv = cm.load(fname_new)
plt.imshow(mv.mean(axis=0), alpha=0.5)
# Spatial filter created by algorithm
plt.figure()
plt.imshow(vpy.estimates['spatialFilter'][n])
plt.colorbar()
plt.title('spatial filter')
plt.show()
# %% STOP CLUSTER and clean up log files
cm.stop_server(dview=dview)
log_files = glob.glob('*_LOG_*')
for log_file in log_files:
os.remove(log_file)
def run_alignmnet(selected_rows, parameters, dview):
'''
This is the main function for the alignment step. It applies methods
from the CaImAn package used originally in motion correction
to do alignment.
Args:
df: pd.DataFrame
A dataframe containing the analysis states you want to have aligned.
parameters: dict
The alignment parameters.
dview: object
The dview object
Returns:
df: pd.DataFrame
A dataframe containing the aligned analysis states.
'''
# Sort the dataframe correctly
df = selected_rows.copy()
df = df.sort_values(by=paths.multi_index_structure)
# Determine the mouse and session of the dataset
index = df.iloc[0].name
mouse, session, *r = index
# alignment_v = index[len(paths.data_structure) + step_index]
alignment_v = len(df)
alignment_index = (mouse, session, alignment_v)
# Determine the output .mmap file name
file_name = f'mouse_{mouse}_session_{session}_v{alignment_v}'
output_mmap_file_path = os.environ['DATA_DIR'] + f'data/interim/alignment/main/{file_name}.mmap'
try:
df.reset_index()[['session','trial', 'is_rest']].set_index(['session','trial', 'is_rest'], verify_integrity=True)
except ValueError:
logging.error('You passed multiple of the same trial in the dataframe df')
return df
output = {
'meta': {
'analysis': {
'analyst': os.environ['ANALYST'],
'date': datetime.datetime.today().strftime("%m-%d-%Y"),
'time': datetime.datetime.today().strftime("%H:%M:%S")
},
'duration': {}
}
}
# Get necessary parameters
motion_correction_parameters_list = []
motion_correction_output_list = []
input_mmap_file_list = []
trial_index_list = []
x_ = []
_x = []
y_ = []
_y = []
for idx, row in df.iterrows():
motion_correction_parameters_list.append(eval(row.loc['motion_correction_parameters']))
motion_correction_output = eval(row.loc['motion_correction_output'])
motion_correction_output_list.append(motion_correction_output)
input_mmap_file_list.append(motion_correction_output['main'])
trial_index_list.append(db.get_trial_name(idx[2], idx[3]))
[x1,x2,y1,y2] = motion_correction_output['meta']['cropping_points']
x_.append(x1)
_x.append(x2)
y_.append(y1)
_y.append(y2)
new_x1 = max(x_)
new_x2 = max(_x)
new_y1 = max(y_)
new_y2 = max(_y)
m_list = []
for i in range(len(input_mmap_file_list)):
m = cm.load(input_mmap_file_list[i])
motion_correction_output = eval(df.iloc[i].loc['motion_correction_output'])
[x1,x2,y1,y2] = motion_correction_output['meta']['cropping_points']
m = m.crop(new_x1 - x1, new_x2 - x2, new_y1 - y1, new_y2 - y2, 0, 0)
m_list.append(m)
# Concatenate them using the concat function
m_concat = cm.concatenate(m_list, axis=0)
data_dir = os.environ['DATA_DIR'] + 'data/interim/alignment/main/'
file_name = db.create_file_name(step_index, index)
fname= m_concat.save(data_dir + file_name + '.mmap', order='C')
#meta_pkl_dict['pw_rigid']['cropping_points'] = [x_, _x, y_, _y]
#output['meta']['cropping_points'] = [x_, _x, y_, _y]
# Save the movie
#fname_tot_els = m_els.save(data_dir + 'main/' + file_name + '_els' + '.mmap', order='C')
#logging.info(f'{index} Cropped and saved rigid movie as {fname_tot_els}')
# MOTION CORRECTING EACH INDIVIDUAL MOVIE WITH RESPECT TO A TEMPLATE MADE OF THE FIRST MOVIE
logging.info(f'{alignment_index} Performing motion correction on all movies with respect to a template made of \
the first movie.')
t0 = datetime.datetime.today()
# Create a template of the first movie
template_index = trial_index_list.index(parameters['make_template_from_trial'])
m0 = cm.load(input_mmap_file_list[template_index ])
[x1, x2, y1, y2] = motion_correction_output_list[template_index]['meta']['cropping_points']
m0 = m0.crop(new_x1 - x1, new_x2 - x2, new_y1 - y1, new_y2 - y2, 0, 0)
m0_filt = cm.movie(
np.array([high_pass_filter_space(m_, parameters['gSig_filt']) for m_ in m0]))
template0 = cm.motion_correction.bin_median(
m0_filt.motion_correct(5, 5, template=None)[0]) # may be improved in the future
# Setting the parameters
opts = params.CNMFParams(params_dict=parameters)
# Create a motion correction object
mc = MotionCorrect(fname, dview=dview, **opts.get_group('motion'))
# Perform non-rigid motion correction
mc.motion_correct(template=template0, save_movie=True)
# Cropping borders
x_ = math.ceil(abs(np.array(mc.shifts_rig)[:, 1].max()) if np.array(mc.shifts_rig)[:, 1].max() > 0 else 0)
_x = math.ceil(abs(np.array(mc.shifts_rig)[:, 1].min()) if np.array(mc.shifts_rig)[:, 1].min() < 0 else 0)
y_ = math.ceil(abs(np.array(mc.shifts_rig)[:, 0].max()) if np.array(mc.shifts_rig)[:, 0].max() > 0 else 0)
_y = math.ceil(abs(np.array(mc.shifts_rig)[:, 0].min()) if np.array(mc.shifts_rig)[:, 0].min() < 0 else 0)
# Load the motion corrected movie into memory
movie= cm.load(mc.fname_tot_rig[0])
# Crop all movies to those border pixels
movie.crop(x_, _x, y_, _y, 0, 0)
output['meta']['cropping_points'] = [x_, _x, y_, _y]
#save motion corrected and cropped movie
output_mmap_file_path_tot = movie.save(data_dir + file_name + '.mmap', order='C')
logging.info(f'{index} Cropped and saved rigid movie as {output_mmap_file_path_tot}')
# Save the path in teh output dictionary
output['main'] = output_mmap_file_path_tot
# Remove the remaining non-cropped movie
os.remove(mc.fname_tot_rig[0])
# Create a timeline and store it
timeline = [[trial_index_list[0], 0]]
timepoints = [0]
for i in range(1, len(m_list)):
m = m_list[i]
timeline.append([trial_index_list[i], timeline[i - 1][1] + m.shape[0]])
timepoints.append(timepoints[i-1]+ m.shape[0])
timeline_pkl_file_path = os.environ['DATA_DIR'] + f'data/interim/alignment/meta/timeline/{file_name}.pkl'
with open(timeline_pkl_file_path,'wb') as f:
pickle.dump(timeline,f)
output['meta']['timeline'] = timeline_pkl_file_path
timepoints.append(movie.shape[0])
dt = int((datetime.datetime.today() - t0).seconds / 60) # timedelta in minutes
output['meta']['duration']['concatenation'] = dt
logging.info(f'{alignment_index} Performed concatenation. dt = {dt} min.')
for idx, row in df.iterrows():
df.loc[idx, 'alignment_output'] = str(output)
df.loc[idx, 'alignment_parameters'] = str(parameters)
## modify all motion correction file to the aligned version
data_dir = os.environ['DATA_DIR'] + 'data/interim/motion_correction/main/'
for i in range(len(input_mmap_file_list)):
row = df.iloc[i].copy()
motion_correction_output_list.append(motion_correction_output)
aligned_movie = movie[timepoints[i]:timepoints[i+1]]
file_name = db.create_file_name(2, selected_rows.iloc[i].name)
motion_correction_output_aligned = aligned_movie.save(data_dir + file_name + '_els' + '.mmap', order='C')
new_output= {'main' : motion_correction_output_aligned }
new_dict = eval(row['motion_correction_output'])
new_dict.update(new_output)
row['motion_correction_output'] = str(new_dict)
df = db.append_to_or_merge_with_states_df(df, row)
# # Delete the motion corrected movies
# for fname in mc.fname_tot_rig:
# os.remove(fname)
return df
def main():
pass # For compatibility between running under Spyder and the CLI
#%% Select file(s) to be processed (download if not present)
root = '/Users/hheiser/Desktop/testing data/chronic_M2N3/0d_baseline/channel1'
fnames = [r'W:\Neurophysiology-Storage1\Wahl\Hendrik\PhD\Data\Batch2\M22\20191208\N1\1\file_00003.tif',
r'W:\Neurophysiology-Storage1\Wahl\Hendrik\PhD\Data\Batch2\M22\20191208\N1\2\file_00004.tif',
r'W:\Neurophysiology-Storage1\Wahl\Hendrik\PhD\Data\Batch2\M22\20191208\N1\3\file_00005.tif',
r'W:\Neurophysiology-Storage1\Wahl\Hendrik\PhD\Data\Batch2\M22\20191208\N1\4\file_00006.tif',
r'W:\Neurophysiology-Storage1\Wahl\Hendrik\PhD\Data\Batch2\M22\20191208\N1\5\file_00007.tif',
r'W:\Neurophysiology-Storage1\Wahl\Hendrik\PhD\Data\Batch2\M22\20191208\N1\6\file_00008.tif']
fnames = ['Sue_2x_3000_40_-46.tif'] # filename to be processed
if fnames[0] in ['Sue_2x_3000_40_-46.tif', 'demoMovie.tif']:
fnames = [download_demo(fnames[0])]
#%% First setup some parameters for data and motion correction
# dataset dependent parameters
fr = 30 # imaging rate in frames per second
decay_time = 0.4 # length of a typical transient in seconds
dxy = (2., 2.) # spatial resolution in x and y in (um per pixel)
# note the lower than usual spatial resolution here
max_shift_um = (12., 12.) # maximum shift in um
patch_motion_um = (100., 100.) # patch size for non-rigid correction in um
# motion correction parameters
pw_rigid = True # flag to select rigid vs pw_rigid motion correction
# maximum allowed rigid shift in pixels
max_shifts = [int(a/b) for a, b in zip(max_shift_um, dxy)]
# start a new patch for pw-rigid motion correction every x pixels
strides = tuple([int(a/b) for a, b in zip(patch_motion_um, dxy)])
# overlap between pathes (size of patch in pixels: strides+overlaps)
overlaps = (24, 24)
# maximum deviation allowed for patch with respect to rigid shifts
max_deviation_rigid = 3
mc_dict = {
'fnames': fnames,
'fr': fr,
'decay_time': decay_time,
'dxy': dxy,
'pw_rigid': pw_rigid,
'max_shifts': max_shifts,
'strides': strides,
'overlaps': overlaps,
'max_deviation_rigid': max_deviation_rigid,
'border_nan': 'copy'
}
opts = params.CNMFParams(params_dict=mc_dict)
#%% play the movie (optional)
# playing the movie using opencv. It requires loading the movie in memory.
# To close the video press q
display_images = True
if display_images:
m_orig = cm.load_movie_chain(fnames)
ds_ratio = 0.2
moviehandle = m_orig.resize(1, 1, ds_ratio)
moviehandle.play(q_max=99.5, fr=30, magnification=1, do_loop=False)
#%% start a cluster for parallel processing
c, dview, n_processes = cm.cluster.setup_cluster(
backend='local', n_processes=None, single_thread=False)
#%% MOTION CORRECTION
# first we create a motion correction object with the specified parameters
mc = MotionCorrect(fnames, dview=dview, **opts.get_group('motion'))
# note that the file is not loaded in memory
#%% Run (piecewise-rigid motion) correction using NoRMCorre
mc.motion_correct(save_movie=True)
#%% compare with original movie
if display_images:
m_orig = cm.load_movie_chain(fnames[:3])
m_els = cm.load(mmap_file[:3])
ds_ratio = 0.2
moviehandle = cm.concatenate([m_orig.resize(1, 1, ds_ratio) - mc.min_mov*mc.nonneg_movie,
m_els.resize(1, 1, ds_ratio)], axis=2)
moviehandle.play(fr=15, q_max=99.5, magnification=2) # press q to exit
#%% MEMORY MAPPING
border_to_0 = 0 if mc.border_nan == 'copy' else mc.border_to_0
# you can include the boundaries of the FOV if you used the 'copy' option
# during motion correction, although be careful about the components near
# the boundaries
mmap_file = [r'W:\Neurophysiology-Storage1\Wahl\Hendrik\PhD\Data\Batch2\M19\20191219\N2\1\file_00001_els__d1_512_d2_512_d3_1_order_F_frames_1147_.mmap',
r'W:\Neurophysiology-Storage1\Wahl\Hendrik\PhD\Data\Batch2\M19\20191219\N2\2\file_00002_els__d1_512_d2_512_d3_1_order_F_frames_2520_.mmap',
r'W:\Neurophysiology-Storage1\Wahl\Hendrik\PhD\Data\Batch2\M19\20191219\N2\3\file_00003_els__d1_512_d2_512_d3_1_order_F_frames_3814_.mmap',
r'W:\Neurophysiology-Storage1\Wahl\Hendrik\PhD\Data\Batch2\M19\20191219\N2\4\file_00004_els__d1_512_d2_512_d3_1_order_F_frames_5154_.mmap',
r'W:\Neurophysiology-Storage1\Wahl\Hendrik\PhD\Data\Batch2\M19\20191219\N2\5\file_00005_els__d1_512_d2_512_d3_1_order_F_frames_2677_.mmap',
r'W:\Neurophysiology-Storage1\Wahl\Hendrik\PhD\Data\Batch2\M19\20191219\N2\6\file_00006_els__d1_512_d2_512_d3_1_order_F_frames_3685_.mmap']
fname_new = r'E:\PhD\Data\DG\M14_20191014\N1\memmap__d1_512_d2_512_d3_1_order_C_frames_34939_.mmap'
# memory map the file in order 'C'
fname_new = cm.save_memmap(mc.mmap_file, base_name='memmap_', order='C',
border_to_0=0) # exclude borders
# now load the file
Yr, dims, T = cm.load_memmap(fname_new)
images = np.reshape(Yr.T, [T] + list(dims), order='F')
# load frames in python format (T x X x Y)
#%% restart cluster to clean up memory
cm.stop_server(dview=dview)
c, dview, n_processes = cm.cluster.setup_cluster(
backend='local', n_processes=None, single_thread=False)
#%% parameters for source extraction and deconvolution
p = 1 # order of the autoregressive system
gnb = 2 # number of global background components
merge_thr = 0.85 # merging threshold, max correlation allowed
rf = 15
# half-size of the patches in pixels. e.g., if rf=25, patches are 50x50
stride_cnmf = 6 # amount of overlap between the patches in pixels
K = 4 # number of components per patch
gSig = [4, 4] # expected half size of neurons in pixels
# initialization method (if analyzing dendritic data using 'sparse_nmf')
method_init = 'greedy_roi'
ssub = 2 # spatial subsampling during initialization
tsub = 2 # temporal subsampling during intialization
# parameters for component evaluation
opts_dict = {'fnames': fnames,
'fr': fr,
'nb': gnb,
'rf': rf,
'K': K,
'gSig': gSig,
'stride': stride_cnmf,
'method_init': method_init,
'rolling_sum': True,
'merge_thr': merge_thr,
'n_processes': n_processes,
'only_init': True,
'ssub': ssub,
'tsub': tsub}
opts.change_params(params_dict=opts_dict)
#%% RUN CNMF ON PATCHES
# First extract spatial and temporal components on patches and combine them
# for this step deconvolution is turned off (p=0)
#opts.change_params({'p': 1,'rf':None, 'only_init':False})
opts.change_params({'p': 0})
cnm = cnmf.CNMF(n_processes, params=opts, dview=dview)
cnm = cnm.fit(images)
#%% RUN CNMF SEEDED WITH MANUAL MASK
# load mask
mask = np.asarray(imageio.imread('/Users/hheiser/Desktop/testing data/file_00020_no_motion/avg_mask_fixed.png'), dtype=bool)
# get component ROIs from the mask and plot them
Ain, labels, mR = cm.base.rois.extract_binary_masks(mask)
# plot original mask and extracted labels to check mask
fig, ax = plt.subplots(1,2)
ax[0].imshow(-mR,cmap='binary')
ax[0].set_title('Original mask')
ax[1].imshow(labels)
ax[1].set_title('Extracted labelled ROIs')
""""
plt.figure()
crd = cm.utils.visualization.plot_contours(
Ain.astype('float32'), mR, thr=0.99, display_numbers=True) # todo check if this is important for the pipeline
plt.title('Contour plots of detected ROIs in the structural channel')
"""
opts.change_params({'rf': None, 'only_init': False})
# run CNMF seeded with this mask
cnm_corr = cnmf.CNMF(n_processes, params=opts, dview=dview, Ain=Ain)
cnm_corr_seed = cnm_corr_seed.fit(images)
#cnm_seed = cnm_seed.fit_file(motion_correct=False)
#%% ALTERNATE WAY TO RUN THE PIPELINE AT ONCE
# you can also perform the motion correction plus cnmf fitting steps
# simultaneously after defining your parameters object using
# cnm1 = cnmf.CNMF(n_processes, params=opts, dview=dview)
# cnm1.fit_file(motion_correct=True)
#%% plot contours of found components
Cn = cm.local_correlations(images, swap_dim=False)
Cn[np.isnan(Cn)] = 0
cnm.estimates.plot_contours(img=Cn)
plt.title('Contour plots of found components')
#%% RE-RUN seeded CNMF on accepted patches to refine and perform deconvolution
cnm.params.change_params({'p': p})
cnm2 = cnm.refit(images, dview=dview)
#%% COMPONENT EVALUATION
# the components are evaluated in three ways:
# a) the shape of each component must be correlated with the data
# b) a minimum peak SNR is required over the length of a transient
# c) each shape passes a CNN based classifier
min_SNR = 2 # signal to noise ratio for accepting a component
rval_thr = 0.85 # space correlation threshold for accepting a component
cnn_thr = 0.99 # threshold for CNN based classifier
cnn_lowest = 0.1 # neurons with cnn probability lower than this value are rejected
cnm2.params.set('quality', {'decay_time': decay_time,
'min_SNR': min_SNR,
'rval_thr': rval_thr,
'use_cnn': True,
'min_cnn_thr': cnn_thr,
'cnn_lowest': cnn_lowest})
cnm2.estimates.evaluate_components(images, params=cnm2.params, dview=dview)
#%% PLOT COMPONENTS
cnm2.estimates.plot_contours(img=Cn, idx=cnm2.estimates.idx_components)
#%% VIEW TRACES (accepted and rejected)
if display_images:
cnm2.estimates.view_components(images, img=Cn,
idx=cnm2.estimates.idx_components)
cnm2.estimates.view_components(images, img=Cn,
idx=cnm2.estimates.idx_components_bad)
#%% update object with selected components
#### -> will delete rejected components!
cnm2.estimates.select_components(use_object=True)
#%% Extract DF/F values
cnm2.estimates.detrend_df_f(quantileMin=8, frames_window=250)
#%% Show final traces
cnm2.estimates.view_components(img=Cn)
#%% reconstruct denoised movie (press q to exit)
if display_images:
cnm2.estimates.play_movie(images, q_max=99.9, gain_res=2,
magnification=1,
bpx=border_to_0,
include_bck=True) # background not shown
#%% STOP CLUSTER and clean up log files
cm.stop_server(dview=dview)
log_files = glob.glob('*_LOG_*')
for log_file in log_files:
os.remove(log_file)
#%% save results
dirname = fnames[0][:-4] + "_results.hdf5"
cnm2.estimates.Cn = Cn
cnm2.save(dirname)
#load results
cnm2 = cnmf.load_CNMF(dirname)
mov_name = fnames[0][:-4] + "_movie_restored_2_gain.tif"
helper.save_movie(cnm2.estimates,images,mov_name,frame_range=range(200),include_bck=True)
def test_computational_performance(fnames, path_ROIs, n_processes):
import os
import cv2
import glob
import logging
import matplotlib.pyplot as plt
import numpy as np
import tensorflow as tf
import h5py
from time import time
try:
cv2.setNumThreads(0)
except:
pass
try:
if __IPYTHON__:
# this is used for debugging purposes only. allows to reload classes
# when changed
get_ipython().magic('load_ext autoreload')
get_ipython().magic('autoreload 2')
except NameError:
pass
import caiman as cm
from caiman.motion_correction import MotionCorrect
from caiman.utils.utils import download_demo, download_model
from caiman.source_extraction.volpy.volparams import volparams
from caiman.source_extraction.volpy.volpy import VOLPY
from caiman.source_extraction.volpy.mrcnn import visualize, neurons
import caiman.source_extraction.volpy.mrcnn.model as modellib
from caiman.paths import caiman_datadir
from caiman.summary_images import local_correlations_movie_offline
from caiman.summary_images import mean_image
from caiman.source_extraction.volpy.utils import quick_annotation
from multiprocessing import Pool
time_start = time()
print('Start MOTION CORRECTION')
# %% Load demo movie and ROIs
fnames = fnames
path_ROIs = path_ROIs
#%% dataset dependent parameters
# dataset dependent parameters
fr = 400 # sample rate of the movie
# motion correction parameters
pw_rigid = False # flag for pw-rigid motion correction
gSig_filt = (3, 3) # size of filter, in general gSig (see below),
# change this one if algorithm does not work
max_shifts = (5, 5) # maximum allowed rigid shift
strides = (
48, 48
) # start a new patch for pw-rigid motion correction every x pixels
overlaps = (24, 24
) # overlap between pathes (size of patch strides+overlaps)
max_deviation_rigid = 3 # maximum deviation allowed for patch with respect to rigid shifts
border_nan = 'copy'
opts_dict = {
'fnames': fnames,
'fr': fr,
'pw_rigid': pw_rigid,
'max_shifts': max_shifts,
'gSig_filt': gSig_filt,
'strides': strides,
'overlaps': overlaps,
'max_deviation_rigid': max_deviation_rigid,
'border_nan': border_nan
}
opts = volparams(params_dict=opts_dict)
# %% start a cluster for parallel processing
dview = Pool(n_processes)
#dview = None
# %%% MOTION CORRECTION
# first we create a motion correction object with the specified parameters
mc = MotionCorrect(fnames, dview=dview, **opts.get_group('motion'))
# Run correction
mc.motion_correct(save_movie=True)
time_mc = time() - time_start
print(time_mc)
print('START MEMORY MAPPING')
# %% restart cluster to clean up memory
dview.terminate()
dview = Pool(n_processes)
# %% MEMORY MAPPING
border_to_0 = 0 if mc.border_nan == 'copy' else mc.border_to_0
# you can include the boundaries of the FOV if you used the 'copy' option
# during motion correction, although be careful about the components near
# the boundaries
# memory map the file in order 'C'
fname_new = cm.save_memmap_join(mc.mmap_file,
base_name='memmap_',
add_to_mov=border_to_0,
dview=dview,
n_chunks=1000) # exclude border
time_mmap = time() - time_start - time_mc
print('Start Segmentation')
# %% SEGMENTATION
# create summary images
img = mean_image(mc.mmap_file[0], window=1000, dview=dview)
img = (img - np.mean(img)) / np.std(img)
Cn = local_correlations_movie_offline(mc.mmap_file[0],
fr=fr,
window=1500,
stride=1500,
winSize_baseline=400,
remove_baseline=True,
dview=dview).max(axis=0)
img_corr = (Cn - np.mean(Cn)) / np.std(Cn)
summary_image = np.stack([img, img, img_corr], axis=2).astype(np.float32)
#%% three methods for segmentation
methods_list = [
'manual_annotation', # manual annotation needs user to prepare annotated datasets same format as demo ROIs
'quick_annotation', # quick annotation annotates data with simple interface in python
'maskrcnn'
] # maskrcnn is a convolutional network trained for finding neurons using summary images
method = methods_list[0]
if method == 'manual_annotation':
with h5py.File(path_ROIs, 'r') as fl:
ROIs = fl['mov'][()] # load ROIs
elif method == 'quick_annotation':
ROIs = quick_annotation(img_corr, min_radius=4, max_radius=10)
elif method == 'maskrcnn':
config = neurons.NeuronsConfig()
class InferenceConfig(config.__class__):
# Run detection on one image at a time
GPU_COUNT = 1
IMAGES_PER_GPU = 1
DETECTION_MIN_CONFIDENCE = 0.7
IMAGE_RESIZE_MODE = "pad64"
IMAGE_MAX_DIM = 512
RPN_NMS_THRESHOLD = 0.7
POST_NMS_ROIS_INFERENCE = 1000
config = InferenceConfig()
config.display()
model_dir = os.path.join(caiman_datadir(), 'model')
DEVICE = "/cpu:0" # /cpu:0 or /gpu:0
with tf.device(DEVICE):
model = modellib.MaskRCNN(mode="inference",
model_dir=model_dir,
config=config)
weights_path = download_model('mask_rcnn')
model.load_weights(weights_path, by_name=True)
results = model.detect([summary_image], verbose=1)
r = results[0]
ROIs = r['masks'].transpose([2, 0, 1])
display_result = False
if display_result:
_, ax = plt.subplots(1, 1, figsize=(16, 16))
visualize.display_instances(summary_image,
r['rois'],
r['masks'],
r['class_ids'], ['BG', 'neurons'],
r['scores'],
ax=ax,
title="Predictions")
time_seg = time() - time_mmap - time_mc - time_start
print('Start SPIKE EXTRACTION')
# %% restart cluster to clean up memory
dview.terminate()
dview = Pool(n_processes, maxtasksperchild=1)
# %% parameters for trace denoising and spike extraction
fnames = fname_new # change file
ROIs = ROIs # region of interests
index = list(range(len(ROIs))) # index of neurons
weights = None # reuse spatial weights
tau_lp = 5 # parameter for high-pass filter to remove photobleaching
threshold = 4 # threshold for finding spikes, increase threshold to find less spikes
contextSize = 35 # number of pixels surrounding the ROI to censor from the background PCA
flip_signal = True # Important! Flip signal or not, True for Voltron indicator, False for others
opts_dict = {
'fnames': fnames,
'ROIs': ROIs,
'index': index,
'weights': weights,
'tau_lp': tau_lp,
'threshold': threshold,
'contextSize': contextSize,
'flip_signal': flip_signal
}
opts.change_params(params_dict=opts_dict)
#%% Trace Denoising and Spike Extraction
vpy = VOLPY(n_processes=n_processes, dview=dview, params=opts)
vpy.fit(n_processes=n_processes, dview=dview)
# %% STOP CLUSTER and clean up log files
#dview.terminate()
log_files = glob.glob('*_LOG_*')
for log_file in log_files:
os.remove(log_file)
time_ext = time() - time_mmap - time_mc - time_start - time_seg
#%%
print('file:' + fnames)
print('number of processes' + str(n_processes))
print(time_mc)
print(time_mmap)
print(time_seg)
print(time_ext)
time_list = [time_mc, time_mmap, time_seg, time_ext]
return time_list
def motion_correct(
self, fname: str,
co_fname: str) -> Tuple[np.ndarray, dict, Optional[np.ndarray]]:
"""
Uses caiman non-rigid motion correction to remove/reduce motion artefacts
Note: ALways saves an intermediate mem-map representation in order C of the corrected 32-bit stack
:param fname: The filename of the source file
:param co_fname: Filename of a stack that should be co-aligned or None
:return:
[0]: Corrected stack as a memmap
[1]: Wrapped CaImAn parameter dictionary
[2]: Corrected co-stack as a memmap or None
"""
cont_folder = path.dirname(fname) # the containing folder
stack_name = path.split(fname)[1]
save_dir = cont_folder + f"/{self.ana_dir}"
if not path.exists(save_dir):
makedirs(save_dir)
print("Created analysis directory", flush=True)
out_name = save_dir + '/' + stack_name
if co_fname is not None:
co_stack_name = path.split(co_fname)[1]
co_out_name = save_dir + '/' + co_stack_name
else:
co_out_name = None
test_image = imread(
fname, key=0) # load first frame of stack to compute resolution
assert test_image.shape[0] == test_image.shape[1]
resolution = self.fov_um / test_image.shape[0]
fr = 1 / self.time_per_frame # frame-rate
dxy = (resolution, resolution) # spatial resolution in um/pixel
max_shift_um = (self.neuron_radius * 4, self.neuron_radius * 4
) # maximally allow shift by ~2 cell diameters
# maximum allowed rigid shift in pixels
max_shifts = [int(a / b) for a, b in zip(max_shift_um, dxy)]
# start a new patch for pw-rigid motion correction every x pixels
strides = tuple(
[int(a / b) for a, b in zip(self.patch_motion_um, dxy)])
# overlap between patches (size of patch in pixels: strides+overlaps)
overlaps = (24, 24)
# maximum deviation allowed for patch with respect to rigid shifts (unit unclear - likely pixels as it is int)
max_deviation_rigid = 3
# create parameter dictionary
mc_dict = {
'fnames': [fname],
'fr': fr,
'decay_time': self.decay_time,
'dxy': dxy,
'pw_rigid': self.pw_rigid,
'max_shifts': max_shifts,
'strides': strides,
'overlaps': overlaps,
'max_deviation_rigid': max_deviation_rigid,
'border_nan': 'copy'
}
opts = params.CNMFParams(params_dict=mc_dict)
# start a cluster for parallel processing
c, dview, n_processes = cm.cluster.setup_cluster(backend='local',
n_processes=None,
single_thread=False)
try:
# motion correction
mc = MotionCorrect(fname, dview=dview, **opts.get_group('motion'))
# Run (piecewise-rigid motion) correction using NoRMCorre
# if we don't save the movie into a memmap, there doesn't seem to be
# any possibility to get at the corrected data later???
mc.motion_correct(save_movie=True)
# memory mapping
border_to_0 = 0 if mc.border_nan is 'copy' else mc.border_to_0
# memory map the file in order 'C'
fname_new = cm.save_memmap(mc.mmap_file,
base_name=out_name,
order='C',
border_to_0=border_to_0)
# delete the original mem-map
if mc.fname_tot_els is None:
[remove(fn) for fn in mc.fname_tot_rig]
else:
[remove(fn) for fn in mc.fname_tot_els]
# now load the new file and transform output into [z,y,x] stack
yr, dims, n_t = cm.load_memmap(fname_new)
images = np.reshape(yr.T, [n_t] + list(dims), order='F')
if self.save_projection:
# save anatomical projection as 16bit tif
anat_projection = images.copy()
anat_projection = np.sum(anat_projection, 0)
anat_projection -= np.min(anat_projection)
anat_projection /= np.max(anat_projection)
anat_projection *= (2**16 - 1)
anat_projection[anat_projection < 0] = 0
anat_projection[anat_projection > (2**16 - 1)] = (2**16 - 1)
anat_projection = anat_projection.astype(np.uint16)
imsave(out_name,
anat_projection,
imagej=True,
resolution=(1 / dxy[0], 1 / dxy[1]),
metadata={
'axes': 'YX',
'unit': 'um'
})
# Repeat for the co-stack if present
if co_fname is not None:
co_aligned_images = np.array(mc.apply_shifts_movie(co_fname))
if self.save_projection:
# save anatomical projection as 16bit tif
anat_projection = np.sum(co_aligned_images, 0)
anat_projection -= np.min(anat_projection)
anat_projection /= np.max(anat_projection)
anat_projection *= (2**16 - 1)
anat_projection[anat_projection < 0] = 0
anat_projection[anat_projection > (2**16 - 1)] = (2**16 -
1)
anat_projection = anat_projection.astype(np.uint16)
imsave(co_out_name,
anat_projection,
imagej=True,
resolution=(1 / dxy[0], 1 / dxy[1]),
metadata={
'axes': 'YX',
'unit': 'um'
})
else:
co_aligned_images = None
finally:
cm.stop_server(dview=dview)
return images, {"Motion Correction": mc_dict}, co_aligned_images
'decay_time': decay_time,
'pw_rigid': pw_rigid,
'max_shifts': max_shifts,
'gSig_filt': gSig_filt,
'strides': strides,
'overlaps': overlaps,
'max_deviation_rigid': max_deviation_rigid,
'border_nan': border_nan
}
opts = params.CNMFParams(params_dict=mc_dict)
if motion_correct:
# do motion correction rigid
mc = MotionCorrect(fnames, dview=dview, **opts.get_group('motion'))
mc.motion_correct(save_movie=True)
fname_mc = mc.fname_tot_els if pw_rigid else mc.fname_tot_rig
if pw_rigid:
bord_px = np.ceil(np.maximum(np.max(np.abs(mc.x_shifts_els)),
np.max(np.abs(mc.y_shifts_els)))).astype(np.int)
else:
bord_px = np.ceil(np.max(np.abs(mc.shifts_rig))).astype(np.int)
plt.subplot(1, 2, 1); plt.imshow(mc.total_template_rig) # % plot template
plt.subplot(1, 2, 2); plt.plot(mc.shifts_rig) # % plot rigid shifts
plt.legend(['x shifts', 'y shifts'])
plt.xlabel('frames')
plt.ylabel('pixels');plt.savefig(newpath + r'/' + "shift.pdf",edgecolor='w',format='pdf',transparent=True)
bord_px = 0 if border_nan is 'copy' else bord_px
fname_new = cm.save_memmap(fname_mc, base_name='memmap_', order='C',
border_to_0=bord_px)
class MakeMasks3D:
def __init__(self, mc_opts, tiff, channels, planes, x_start, x_end):
self.tiff = tiff
self.channels = channels
self.planes = planes
self.x_start = x_start
self.x_end = x_end
self.xslice = slice(x_start, x_end)
self.mc_opts = mc_opts
self.opts = params.CNMFParams(params_dict=mc_opts)
self.c, self.dview, self.n_processes = cm.cluster.setup_cluster(
backend='local', n_processes=None, single_thread=False)
self.file_list = []
self.images = []
self.motion_corrected_images = []
self.masks = []
def crop_tiffs(self):
self.images = []
self.file_list = []
for plane in list(range(self.planes)):
time_slice = slice(plane * self.channels + 1, -1,
self.channels * self.planes)
with ScanImageTiffReader(self.tiff) as reader:
data = reader.data()
data = data[time_slice, :, self.xslice]
self.images.append(data)
tif_name = self.tiff.split('.')[0] + '_template_plane' + str(
plane) + '.tif'
self.file_list.append(tif_name)
tifffile.imsave(tif_name, data)
def view_planes(self):
fig, axes = plt.subplots(1, self.planes, constrained_layout=True)
for i, ax in enumerate(axes):
image = self.images[i].mean(axis=0)
ax.imshow(image)
ax.set_aspect('equal', 'box')
ax.axis('off')
ax.set_title(f'Plane {i}')
fig.suptitle('Original')
def view_corrected(self):
fig, axes = plt.subplots(1, self.planes, constrained_layout=True)
for i, ax in enumerate(axes):
image = self.motion_corrected_images[i]
ax.imshow(image)
ax.set_aspect('equal', 'box')
ax.axis('off')
ax.set_title(f'Plane {i}')
fig.suptitle('Corrected')
# def view_sources(self):
# if len(self.motion_corrected_images) > 0:
# image_source = self.motion_corrected_images[0]
# else:
# image_source = self.images[0]
# cm.utils.visualization.nb_plot_contour(image_source, self.masks[0].astype('float32'),
# image_source.shape[0], image_source.shape[1])
def motion_correct_red(self):
self.motion_corrected_images = []
for plane in list(range(self.planes)):
print(f'Starting motion correction plane {plane}')
self.mc = MotionCorrect(self.file_list[plane],
dview=self.dview,
**self.opts.get_group('motion'))
self.mc.motion_correct()
self.motion_corrected_images.append(self.mc.total_template_els)
def extract_masks(self, radius=7):
self.masks = []
if len(self.motion_corrected_images) > 0:
image_source = self.motion_corrected_images
else:
image_source = self.images
for plane in list(range(self.planes)):
self.masks.append(
cm.base.rois.extract_binary_masks_from_structural_channel(
image_source[plane])[0])
def run(self):
self.crop_tiffs()
self.motion_correct_red()
