def acquire_data(z_range):
""" micro-manager data acquisition. Creates acquisition events for z-stack.
This example: use custom events, not multi_d_acquisition because the
z-stage is not run from micro-manager but controlled via external DAQ."""
with Acquisition(directory=None,
name=None,
show_display=True,
image_process_fn=grab_image) as acq:
events = []
for index, z_um in enumerate(
np.linspace(z_range[0], z_range[1], z_range[2])):
evt = {"axes": {"z_ext": index}, "z_ext": z_um}
events.append(evt)
acq.acquire(events)
def acquire_multid(z_range):
""" micro-manager data acquisition. Creates acquisition events for z-stack.
This example: use multi_d_acquisition because the z-stage is run
from micro-manager.
Unless hardware triggering is set up in micro-manager, this will be fairly slow:
micro-manager does not sweep the z-stage, but acquires plane by plane. """
with Acquisition(directory=None,
name=None,
show_display=False,
image_process_fn=grab_image) as acq:
events = multi_d_acquisition_events(z_start=z_range[0],
z_end=z_range[1],
z_step=(z_range[1] - z_range[0]) /
(z_range[2] - 1))
acq.acquire(events)
def main():
bridge = Bridge()
mmc = bridge.get_core()
# Data set parameters
path = Path('E://20201023//')
name = 'test'
# z stack parameters
start_end_pos = -5
mid_pos = 5
step_size = .25
relative = True
# time series parameters
exposure_time = 200 # in milliseconds
num_z_positions = int(abs(mid_pos - start_end_pos)/step_size + 1)
z_idx = list(range(num_z_positions))
num_time_points = 10
# setup cameras
mmc.set_exposure(exposure_time)
# setup z stage
z_stage = mmc.get_focus_device()
z_pos, pos_sequence = upload_stage_sequence(bridge, start_end_pos, mid_pos, step_size, relative)
num_z_positions = len(pos_sequence)
print(pos_sequence)
# move to first position
mmc.set_position(z_stage, pos_sequence[0])
events = []
z_idx_ = z_idx.copy()
for i in range(num_time_points):
for j in z_idx_:
events.append({'axes': {'time':i, 'z': j}})
z_idx_.reverse()
with Acquisition(directory=path, name=name) as acq:
acq.acquire(events)
# turn off sequencing
mmc.set_property(z_stage, "UseFastSequence", "No")
mmc.set_property(z_stage, "UseSequence", "No")
# events = getattr(img_process_fn, 'events')
# if len(events) != 0:
# event_queue.put(events.pop(0))
# return image, metadata
# with Acquisition('/Users/henrypinkard/megllandump', 'pythonacqtest',
# image_process_fn=img_process_fn,
# post_hardware_hook_fn=hook_fn) as acq:
#
# acq.acquire(event_list[0])
# acq.await_completion()
# #magellan example
with Acquisition(magellan_acq_index=0,
post_hardware_hook_fn=hook_fn,
image_process_fn=img_process_fn,
debug=True) as acq:
pass
acq.await_completion()
from pycromanager import Acquisition, multi_d_acquisition_events
# with Acquisition(directory='/Users/henrypinkard/megllandump', name='tcz_acq') as acq:
# # Generate the events for a single z-stack
# events = multi_d_acquisition_events(
# num_time_points=3, time_interval_s=0,
# channel_group='channel', channels=['DAPI', 'FITC'],
# z_start=0, z_end=6, z_step=0.4,
# order='tcz')
# acq.acquire(events)
import numpy as np
from pycromanager import Acquisition, multi_d_acquisition_events
with Acquisition('/Users/henrypinkard/megllandump', 'l_axis') as acq:
#create one event for the image at each z-slice
for time in range(5):
z_stack = []
for index, z_um in enumerate(np.arange(start=0, stop=10, step=0.5)):
z_stack.append({
'axes': {
'z': index,
'time': time
},
'z': z_um,
'min_start_time': 5 * time
})
acq.acquire(z_stack, keep_shutter_open=True)
def acquireImage(channelGroup, channelName, hook):
x_array = []
y_array = []
z_array = []
for idx in range(pos_list.get_number_of_positions()):
pos = pos_list.get_position(idx)
#pos.go_to_position(pos, mmc)
x = pos_list.get_position(idx).get(0).x
y = pos_list.get_position(idx).get(0).y
z = pos_list.get_position(idx).get(1).x
x_array.append(x)
y_array.append(y)
z_array.append(z)
x_array = np.array(x_array)
y_array = np.array(y_array)
z_array = np.array(z_array)
with Acquisition(directory=directoryPATH,
name=nameofSAVEDFILE,
post_hardware_hook_fn=hook,
post_camera_hook_fn=hook_fn) as acq:
x = np.hstack([x_array[:, None]])
y = np.hstack([y_array[:, None]])
z = np.hstack([z_array[:, None]])
#Generate the events for a single z-stack
xyz = np.hstack([x_array[:, None], y_array[:, None], z_array[:, None]])
events = multi_d_acquisition_events(xyz_positions=xyz,
channel_group=channelGroup,
channels=[channelName])
acq.acquire(events)
#acquire a 2 x 1 grid
#acq.acquire({'row': 0, 'col': 0})
#acq.acquire({'row': 1, 'col': 0})
stackfolder = "**/*"
folder = Path(directoryPATH)
foldernames = []
for name in folder.glob('saving_name_*'):
print(name.stem)
foldernames.append(name.stem)
maximum = 1
for file in foldernames:
number = int(
re.search(nameofSAVEDFILE + "_" + '(\d*)',
file).group(1)) # assuming filename is "filexxx.txt"
# compare num to previous max, e.g.
maximum = number if number > maximum else maximum # set max = 0 before for-loop
print(number)
highest = nameofSAVEDFILE + "_" + str(maximum)
data_path = os.path.join(folder, highest)
dataset = Dataset(data_path)
#dataset = acq.get_dataset()
#dataset = acq.get_dataset()
#print(dataset)
#data_path=str(directoryPATH/saving_name)
dataset = Dataset(data_path)
print(dataset.axes)
print("data_path", data_path)
length = (len(xyz))
dataset_metadata = dataset.read_metadata(channel=0, position=1)
print(dataset_metadata)
pos = dataset_metadata["Axes"]["position"]
print(pos)
if (dataset):
sizeimg = dataset.read_image(channel=0, position=0)
sizeimg = cv2.cvtColor(sizeimg, cv2.COLOR_GRAY2RGB)
h, w, c = sizeimg.shape
length = int(
(sqrt(length)
)) #size of the grid (row or column should be same technically)
blank_image = np.zeros((h * (math.ceil(math.sqrt(length)) + 2), w *
(math.ceil(math.sqrt(length)) + 2), 3), np.uint16)
print("image size ", blank_image.shape)
pixelsizeinum = dataset_metadata["PixelSizeUm"] #get size of pixel in um
print(pixelsizeinum)
"""
for datarow in range(10):
for datacolumn in range(10):
metadata = dataset.read_metadata(row=datarow, col=datacolumn)
if(metadata["Axes"]["position"]>=0):
pos=metadata["Axes"]["position"]
#print(pos)
img = dataset.read_image(position=pos)
img = cv2.cvtColor(img,cv2.COLOR_GRAY2RGB)
cv2.imshow("test",img)
"""
xtotaloffset = 0
ytotaloffset = 0
for dataposition in range(
len(xyz)): #do range for all positions in micromanager
print(dataposition)
metadata = dataset.read_metadata(channel=0, position=dataposition)
img = dataset.read_image(channel=0, position=dataposition)
img = cv2.cvtColor(img, cv2.COLOR_GRAY2RGB)
img = cv2.flip(img, 1)
xoffset_um = metadata["XPosition_um_Intended"]
yoffset_um = metadata["YPosition_um_Intended"]
print("Intended location is : ", xoffset_um, yoffset_um)
# cv2.imshow("test",img)
# cv2.waitKey(0)
xoffset_px = (xoffset_um - dataset.read_metadata(
channel=0, position=0)['XPosition_um_Intended']) / pixelsizeinum
yoffset_px = (yoffset_um - dataset.read_metadata(
channel=0, position=0)['YPosition_um_Intended']) / pixelsizeinum
xoffset_px = int(xoffset_px)
print("Xoffset ", xoffset_px)
#print("img max X ",blank_image.shape[0])
yoffset_px = int(yoffset_px)
print("Yoffset ", yoffset_px)
#print("img max Y ",blank_image.shape[1])
alpha = 0
blank_image[xoffset_px:xoffset_px + (img.shape[1]),
yoffset_px:yoffset_px + (img.shape[0])] = cv2.addWeighted(
blank_image[xoffset_px:xoffset_px + (img.shape[1]),
yoffset_px:yoffset_px + (img.shape[0])],
alpha, img, 1 - alpha, 0)
#blank_image[:yoffset_px+img.shape[0], :xoffset_px+img.shape[1]] = img
#blank_image = cv2.addWeighted(blank_image[yoffset_px:yoffset_px+img.shape[0], xoffset_px:xoffset_px+img.shape[1]],img)
####################
#printout only ignore
####################
scale_percent = 5
width = int(blank_image.shape[1] * scale_percent / 100)
height = int(blank_image.shape[0] * scale_percent / 100)
dim = (width, height)
resized = cv2.resize(blank_image, dim, interpolation=cv2.INTER_AREA)
'''
#show image
winname = "test"
cv2.namedWindow(winname) # Create a named window
cv2.moveWindow(winname, 1000,1000) # Move it to (40,30)
cv2.imshow(winname, resized)
cv2.waitKey(0)
'''
blank_image = cv2.cvtColor(blank_image, cv2.COLOR_BGR2GRAY)
return blank_image, pixelsizeinum
from pycromanager import Acquisition, multi_d_acquisition_events
def storage_monitor_fn(axes):
dataset = acq.get_dataset()
pixels = dataset.read_image(**axes)
print(pixels)
dire = 'C:\\Users\\henry\\Desktop\\datadump'
with Acquisition(directory=dire,
name="tcz_acq",
debug=False,
storage_monitor_callback_fn=None) as acq:
events = multi_d_acquisition_events(
num_time_points=5,
time_interval_s=0,
order="tcz",
)
dataset = acq.get_dataset()
acq.acquire(events)
from pycromanager import Acquisition, multi_d_acquisition_events
with Acquisition(directory='/Users/henrypinkard/megllandump',
name='tcz_acq',
debug=True) as acq:
# Generate the events for a single z-stack
events = multi_d_acquisition_events(num_time_points=5,
time_interval_s=0,
channel_group='Channel',
channels=['DAPI', 'FITC'],
z_start=0,
z_end=6,
z_step=0.4,
order='tcz')
acq.acquire(events)
from pycromanager import Bridge, Acquisition
import numpy as np
bridge = Bridge()
# get object representing micro-magellan API
magellan = bridge.get_magellan()
def hook_fn(event):
coordinates = np.array([event["x"], event["y"], event["z"]])
return event
if __name__ == "__main__":
# magellan example
acq = Acquisition(magellan_acq_index=0, post_hardware_hook_fn=hook_fn)
acq.await_completion()
pass
def main():
bridge = Bridge()
core = bridge.get_core()
# FOV parameters
ROI = [1024, 0, 256, 1024] #unit: pixels
# camera exposure
exposure_ms = 5 #unit: ms
# set to high-res camera
core.set_config('Camera', 'HighRes')
# crop FOV
core.set_roi(*ROI)
# set exposure
core.set_exposure(exposure_ms)
# setup file name
save_directory = Path('C:/data/test/')
save_name = 'test_stages'
# get handle to xy and z stages
xy_stage = core.get_xy_stage_device()
z_stage = core.get_focus_device()
# move the stages to verify core can talk to them
# positions chosen at random
core.set_xy_position(100., 100.)
core.wait_for_device(xy_stage)
core.set_position(50.)
core.wait_for_device(z_stage)
# create events to hold all of the scan axis images during constant speed stage scan
channel_configs = ['DAPI', 'FITC', 'Rhodamine', 'Cy5']
events = []
for y in range(2):
for z in range(2):
for c in range(len(channel_configs)):
for x in range(2):
evt = {
'axes': {
'x': x,
'y': y,
'z': z
},
'x': 100,
'y': y * 1000,
'z': z * 100,
'channel': {
'group': 'Channel',
'config': channel_configs[c]
}
}
events.append(evt)
# run acquisition
# TO DO: properly handle an error here if camera driver fails to return expected number of images.
with Acquisition(directory=save_directory,
name=save_name,
post_hardware_hook_fn=setup_scan_fn,
post_camera_hook_fn=hook_fn,
show_display=False,
max_multi_res_index=0,
debug=False) as acq:
acq.acquire(events)
acq.acquire(None)
acq.await_completion()
"""
This simple example uses pycromanager to vary exposure times with three repetitions
and can be run with Micro-manager's virtual DemoCamera / DCam device. The resulting
dataset is saved to 'democam_X/Full Resolution/democam_MagellanStack.tif` within the
current folder; consecutively numbered `X` separate individual runs of this script.
"""
from pycromanager import Acquisition
if __name__ == '__main__':
exposures = [100, 200, 300, 400]
with Acquisition(directory='.', name='democam') as acq:
events = []
for rep in range(3):
for idx, exposure in enumerate(exposures):
evt = {'axes': {'repetition': rep, 'exposure': idx},
'exposure': exposure}
events.append(evt)
acq.acquire(events)
import numpy as np
from pycromanager import Acquisition, multi_d_acquisition_events
if __name__ == "__main__":
with Acquisition("/Users/henrypinkard/megllandump", "l_axis") as acq:
# create one event for the image at each z-slice
events = []
for time in range(5):
for index, z_um in enumerate(np.arange(start=0, stop=10,
step=0.5)):
evt = {
#'axes' is required. It is used by the image viewer and data storage to
# identify the acquired image
"axes": {
"l": index,
"time": time
},
# the 'z' field provides the z position in µm
"z": z_um,
}
events.append(evt)
acq.acquire(events)
image2 = np.array(image, copy=True)
image2 = np.swapaxes(image2, 0, 1)
md_2 = copy.deepcopy(metadata)
image[250:350, 100:300] = np.random.randint(0, 4999)
if metadata["Channel"] == "DAPI":
image[:100, :100] = 0
image2[:100, :100] = 0
else:
image[-100:, -100:] = 0
image2[-100:, -100:] = 0
# metadata['Axes']['l'] = 0
md_2["Channel"] = "A_new_channel"
return [(image, metadata), (image2, md_2)]
with Acquisition(directory="/Users/henrypinkard/megllandump",
name="tcz_acq",
image_process_fn=img_process_fn) as acq:
# Generate the events for a single z-stack
events = multi_d_acquisition_events(
num_time_points=10,
time_interval_s=0,
channel_group="Channel",
channels=["DAPI", "FITC"],
order="tc",
)
acq.acquire(events)
from pycromanager import Acquisition, multi_d_acquisition_events
import numpy as np
def hook_fn(event):
return event
with Acquisition(
directory="/Users/henrypinkard/megllandump",
name="acquisition_name",
pre_hardware_hook_fn=hook_fn,
) as acq:
acq.acquire(multi_d_acquisition_events(10))
from pycromanager import Acquisition, multi_d_acquisition_events
import numpy as np
if __name__ == "__main__":
# this hook function can control the micro-manager core
def img_process_fn(image, metadata, bridge, event_queue):
if not hasattr(img_process_fn, "counter"):
img_process_fn.counter = 0
if img_process_fn.counter < 10:
evt = {"axes": {"time": 0, "n": img_process_fn.counter}}
img_process_fn.counter += 1
image[250:350, 100:300] = img_process_fn.counter * 10
else:
evt = None
event_queue.put(evt)
return image, metadata
acq = Acquisition(
directory="/Users/henrypinkard/megllandump",
name="acquisition_name",
image_process_fn=img_process_fn,
)
# kick it off with a single event
acq.acquire({"axes": {"time": 0}})
#this hook function can control the micro-manager core
def img_process_fn(image, metadata, bridge, event_queue):
if not hasattr(img_process_fn, 'counter'):
img_process_fn.counter = 0
if img_process_fn.counter < 10:
evt = {
'axes': {'time': 0, 'n': img_process_fn.counter}
}
img_process_fn.counter += 1
image[250:350, 100:300] = img_process_fn.counter * 10
else:
evt = None
event_queue.put(evt)
return image, metadata
acq = Acquisition(directory='/Users/henrypinkard/megllandump', name='acquisition_name',
image_process_fn=img_process_fn)
#kick it off with a single event
acq.acquire({'axes': {'time': 0}})
col = metadata["GridColumnIndex"]
channel = metadata["Channel"]
# other image axes (e.g. a z axis). 'position' axis is redundant to row and column indices
axes = metadata["Axes"]
# numpy array
image
# TODO: run callback function
return image, metadata
with Acquisition(
"/Users/henrypinkard/tmp",
"tiled",
tile_overlap=10,
image_process_fn=image_callback_fn,
pre_hardware_hook_fn=event_edit_fn,
debug=True,
) as acq:
# 10 pixel overlap between adjacent tiles
acq.acquire({
"row": 0,
"col": 0,
"Channel": {
"group": "channel",
"config": "DAPI"
}
})
acq.acquire({
"row": 0,
"col": 0,
from pycromanager import Bridge
from pycromanager import Acquisition, multi_d_acquisition_events
def img_process_fn(image, metadata):
# modify the pixels by setting a 100 pixel square at the top left to 0
image[:100, :100] = 0
# propogate the image and metadata to the default viewer and saving classes
return image, metadata
z_shg_center = 0
if __name__ == '__main__': # this is important, don't forget it
with Acquisition(directory='/Users/henrypinkard/megellandump/',
name='exp_2_mda',
image_process_fn=img_process_fn) as acq:
events = multi_d_acquisition_events(z_start=z_shg_center - 20,
z_end=z_shg_center + 20,
z_step=5)
acq.acquire(events)
# acq.acquire()
mm_app_path = '/Applications/Micro-Manager-2.0.0-gamma1'
# mm_app_path = 'C:/Program Files/Micro-Manager-2.0gamma'
config_file = mm_app_path + "/MMConfig_demo.cfg"
#Optional: specify your own version of java to run with
java_loc = "/Library/Internet Plug-Ins/JavaAppletPlugin.plugin/Contents/Home/bin/java"
# java_loc = None
start_headless(mm_app_path, config_file, java_loc=java_loc)
b = Bridge()
b.get_core().snap_image()
print(b.get_core().get_image())
save_dir = "/Users/henrypinkard/tmp"
# save_dir = "C:/Users/Henry Pinkard/Desktop/datadump"
with Acquisition(directory=save_dir, name="tcz_acq", debug=True) as acq:
# Generate the events for a single z-stack
events = multi_d_acquisition_events(
num_time_points=5,
time_interval_s=0,
channel_group="Channel",
channels=["DAPI", "FITC"],
z_start=0,
z_end=6,
z_step=0.4,
order="tcz",
)
acq.acquire(events)
# %% Imports
from pycromanager import Core
from pycromanager import Bridge
from pycromanager import Acquisition, multi_d_acquisition_events
import numpy as np
core = Core()
print(core)
core.get_version_info()
# %% Acquisiton
with Acquisition(directory=r'D:\Downloads', name='test') as acq:
events = multi_d_acquisition_events(channel_group='Channel',
channels=['DAPI', 'FITC'],
z_start=0,
z_end=6,
z_step=0.4,
order='tcz')
acq.acquire(events)
# %% Positions
import numpy as np
from pycromanager import multi_d_acquisition_events, Acquisition
def img_process_fn(image, metadata):
print(image)
pass # send them somewhere else, not default saving and display
with Acquisition(image_process_fn=img_process_fn) as acq:
# Generate the events for a single z-stack
events = multi_d_acquisition_events(
num_time_points=10,
time_interval_s=0,
channel_group="channel",
channels=["DAPI", "FITC"],
z_start=0,
z_end=6,
z_step=0.4,
order="tcz",
)
acq.acquire(events)
def img_process_fn(image, metadata):
image2 = np.array(image, copy=True)
image2 = np.swapaxes(image2, 0, 1)
md_2 = copy.deepcopy(metadata)
image[250:350, 100:300] = np.random.randint(0, 4999)
if metadata['Channel'] == 'DAPI':
image[:100, :100] = 0
image2[:100, :100] = 0
else:
image[-100:, -100:] = 0
image2[-100:, -100:] = 0
# metadata['Axes']['l'] = 0
md_2['Channel'] = 'A_new_channel'
return [(image, metadata), (image2, md_2)]
with Acquisition(directory='/Users/henrypinkard/megllandump',
name='tcz_acq',
image_process_fn=img_process_fn) as acq:
# Generate the events for a single z-stack
events = multi_d_acquisition_events(num_time_points=10,
time_interval_s=0,
channel_group='channel',
channels=['DAPI', 'FITC'],
order='tc')
acq.acquire(events)
def main():
#------------------------------------------------------------------------------------------------------------------------------------
#----------------------------------------------Begin setup of scan parameters--------------------------------------------------------
#------------------------------------------------------------------------------------------------------------------------------------
# lasers to use
# 0 -> inactive
# 1 -> active
state_405 = 1
state_488 = 0
state_561 = 0
state_635 = 1
state_730 = 0
# laser powers (0 -> 100%)
power_405 = 10
power_488 = 0
power_561 = 0
power_635 = 5
power_730 = 0
# exposure time
exposure_ms = 5.
# scan axis limits. Use stage positions reported by MM
scan_axis_start_um = 6000. #unit: um
scan_axis_end_um = 16000. #unit: um
# tile axis limits. Use stage positions reported by MM
tile_axis_start_um = -1300 #unit: um
tile_axis_end_um = -1350. #unit: um
# height axis limits. Use stage positions reported by MM
height_axis_start_um = 320. #unit: um
height_axis_end_um = 360. #unit: um
# FOV parameters
# ONLY MODIFY IF NECESSARY
ROI = [0, 1024, 1599, 255] #unit: pixels
# setup file name
save_directory = Path('E:/20201211/')
save_name = 'shaffer_lung'
#------------------------------------------------------------------------------------------------------------------------------------
#----------------------------------------------End setup of scan parameters----------------------------------------------------------
#------------------------------------------------------------------------------------------------------------------------------------
bridge = Bridge()
core = bridge.get_core()
# turn off lasers
core.set_config('Coherent-State', 'off')
core.wait_for_config('Coherent-State', 'off')
# set camera into 16bit readout mode
# give camera time to change modes if necessary
core.set_property('Camera', 'ReadoutRate', '100MHz 16bit')
time.sleep(1)
# set camera into low noise readout mode
# give camera time to change modes if necessary
core.set_property('Camera', 'Gain', '2-CMS')
time.sleep(1)
# set camera to trigger first mode
# give camera time to change modes if necessary
core.set_property('Camera', 'Trigger Timeout (secs)', 300)
time.sleep(1)
# set camera to internal trigger
# give camera time to change modes if necessary
core.set_property('Camera', 'TriggerMode', 'Internal Trigger')
time.sleep(1)
# change core timeout for long stage moves
time.sleep(1)
# crop FOV
#core.set_roi(*ROI)
# set exposure
core.set_exposure(exposure_ms)
# get actual framerate from micromanager properties
# TO DO: fix need for user to have manually run an exposure with correct crop to get this value
actual_readout_ms = float(core.get_property(
'Camera', 'ActualInterval-ms')) #unit: ms
# camera pixel size
pixel_size_um = .115 # unit: um
# scan axis setup
scan_axis_step_um = 0.2 # unit: um
scan_axis_step_mm = scan_axis_step_um / 1000. #unit: mm
scan_axis_start_mm = scan_axis_start_um / 1000. #unit: mm
scan_axis_end_mm = scan_axis_end_um / 1000. #unit: mm
scan_axis_range_um = np.abs(scan_axis_end_um -
scan_axis_start_um) # unit: um
scan_axis_range_mm = scan_axis_range_um / 1000 #unit: mm
actual_exposure_s = actual_readout_ms / 1000. #unit: s
scan_axis_speed = np.round(scan_axis_step_mm / actual_exposure_s,
2) #unit: mm/s
scan_axis_positions = np.rint(scan_axis_range_mm /
scan_axis_step_mm).astype(
int) #unit: number of positions
# tile axis setup
tile_axis_overlap = 0.2 #unit: percentage
tile_axis_range_um = np.abs(tile_axis_end_um -
tile_axis_start_um) #unit: um
tile_axis_range_mm = tile_axis_range_um / 1000 #unit: mm
tile_axis_ROI = ROI[2] * pixel_size_um #unit: um
tile_axis_step_um = np.round((tile_axis_ROI) * (1 - tile_axis_overlap),
2) #unit: um
tile_axis_step_mm = tile_axis_step_um / 1000 #unit: mm
tile_axis_positions = np.rint(tile_axis_range_mm /
tile_axis_step_mm).astype(
int) #unit: number of positions
# if tile_axis_positions rounded to zero, make sure we acquire at least one position
if tile_axis_positions == 0:
tile_axis_positions = 1
# height axis setup
height_axis_overlap = 0.2 #unit: percentage
height_axis_range_um = np.abs(height_axis_end_um -
height_axis_start_um) #unit: um
height_axis_range_mm = height_axis_range_um / 1000 #unit: mm
#height_axis_ROI = ROI[3]*pixel_size_um*np.sin(30*(np.pi/180.)) #unit: um TO DO: Why is overlap so large when using oblique pixel height??
height_axis_ROI = ROI[3] * pixel_size_um #unit: um
height_axis_step_um = np.round(
(height_axis_ROI) * (1 - height_axis_overlap), 2) #unit: um
height_axis_step_mm = height_axis_step_um / 1000 #unit: mm
height_axis_positions = np.rint(height_axis_range_mm /
height_axis_step_mm).astype(
int) #unit: number of positions
# if height_axis_positions rounded to zero, make sure we acquire at least one position
if height_axis_positions == 0:
height_axis_positions = 1
# get handle to xy and z stages
xy_stage = core.get_xy_stage_device()
z_stage = core.get_focus_device()
# Setup Tiger controller to pass signal when the scan stage cross the start position to the PLC
plcName = 'PLogic:E:36'
propPosition = 'PointerPosition'
propCellConfig = 'EditCellConfig'
#addrOutputBNC3 = 35 # BNC3 on the PLC front panel
addrOutputBNC1 = 33 # BNC1 on the PLC front panel
addrStageSync = 46 # TTL5 on Tiger backplane = stage sync signal
# connect stage sync signal to BNC output
core.set_property(plcName, propPosition, addrOutputBNC1)
core.set_property(plcName, propCellConfig, addrStageSync)
# turn on 'transmit repeated commands' for Tiger
core.set_property('TigerCommHub', 'OnlySendSerialCommandOnChange', 'No')
# set tile axis speed for all moves
command = 'SPEED Y=.1'
core.set_property('TigerCommHub', 'SerialCommand', command)
# check to make sure Tiger is not busy
ready = 'B'
while (ready != 'N'):
command = 'STATUS'
core.set_property('TigerCommHub', 'SerialCommand', command)
ready = core.get_property('TigerCommHub', 'SerialResponse')
time.sleep(.500)
# set scan axis speed for large move to initial position
command = 'SPEED X=.1'
core.set_property('TigerCommHub', 'SerialCommand', command)
# check to make sure Tiger is not busy
ready = 'B'
while (ready != 'N'):
command = 'STATUS'
core.set_property('TigerCommHub', 'SerialCommand', command)
ready = core.get_property('TigerCommHub', 'SerialResponse')
time.sleep(.500)
# turn off 'transmit repeated commands' for Tiger
core.set_property('TigerCommHub', 'OnlySendSerialCommandOnChange', 'Yes')
# move scan scan stage to initial position
core.set_xy_position(scan_axis_start_um, tile_axis_start_um)
core.wait_for_device(xy_stage)
core.set_position(height_axis_start_um)
core.wait_for_device(z_stage)
# turn on 'transmit repeated commands' for Tiger
core.set_property('TigerCommHub', 'OnlySendSerialCommandOnChange', 'No')
# set scan axis speed to correct speed for continuous stage scan
# expects mm/s
command = 'SPEED X=' + str(scan_axis_speed)
core.set_property('TigerCommHub', 'SerialCommand', command)
# check to make sure Tiger is not busy
ready = 'B'
while (ready != 'N'):
command = 'STATUS'
core.set_property('TigerCommHub', 'SerialCommand', command)
ready = core.get_property('TigerCommHub', 'SerialResponse')
time.sleep(.500)
# set scan axis to true 1D scan with no backlash
command = '1SCAN X? Y=0 Z=9 F=0'
core.set_property('TigerCommHub', 'SerialCommand', command)
# check to make sure Tiger is not busy
ready = 'B'
while (ready != 'N'):
command = 'STATUS'
core.set_property('TigerCommHub', 'SerialCommand', command)
ready = core.get_property('TigerCommHub', 'SerialResponse')
time.sleep(.500)
# set range and return speed (5% of max) for scan axis
# expects mm
command = '1SCANR X=' + str(scan_axis_start_mm) + ' Y=' + str(
scan_axis_end_mm) + ' R=10'
core.set_property('TigerCommHub', 'SerialCommand', command)
# check to make sure Tiger is not busy
ready = 'B'
while (ready != 'N'):
command = 'STATUS'
core.set_property('TigerCommHub', 'SerialCommand', command)
ready = core.get_property('TigerCommHub', 'SerialResponse')
time.sleep(.500)
# turn off 'transmit repeated commands' for Tiger
core.set_property('TigerCommHub', 'OnlySendSerialCommandOnChange', 'Yes')
# construct boolean array for lasers to use
channel_states = [state_405, state_488, state_561, state_635, state_730]
channel_powers = [power_405, power_488, power_561, power_635, power_730]
# set lasers to user defined power
core.set_property('Coherent-Scientific Remote',
'Laser 405-100C - PowerSetpoint (%)', channel_powers[0])
core.set_property('Coherent-Scientific Remote',
'Laser 488-150C - PowerSetpoint (%)', channel_powers[1])
core.set_property('Coherent-Scientific Remote',
'Laser OBIS LS 561-150 - PowerSetpoint (%)',
channel_powers[2])
core.set_property('Coherent-Scientific Remote',
'Laser 637-140C - PowerSetpoint (%)', channel_powers[3])
core.set_property('Coherent-Scientific Remote',
'Laser 730-30C - PowerSetpoint (%)', channel_powers[4])
print('Number of X positions: ' + str(scan_axis_positions))
print('Number of Y tiles: ' + str(tile_axis_positions))
print('Number of Z slabs: ' + str(height_axis_positions))
#time.sleep(10)
for y in range(tile_axis_positions):
# calculate tile axis position
tile_position_um = tile_axis_start_um + (tile_axis_step_um * y)
# move XY stage to new tile axis position
core.set_xy_position(scan_axis_start_um, tile_position_um)
core.wait_for_device(xy_stage)
for z in range(height_axis_positions):
# calculate height axis position
height_position_um = height_axis_start_um + (height_axis_step_um *
z)
# move Z stage to new height axis position
core.set_position(height_position_um)
core.wait_for_device(z_stage)
# create events to execute scan across this z plane
events = []
for c in range(len(channel_states)):
for x in range(
scan_axis_positions + 10
): #TO DO: Fix need for extra frames in ASI setup, not here.
if channel_states[c] == 1:
if (c == 0):
evt = {
'axes': {
'z': x
},
'channel': {
'group': 'Coherent-State',
'config': '405nm'
}
}
elif (c == 1):
evt = {
'axes': {
'z': x
},
'channel': {
'group': 'Coherent-State',
'config': '488nm'
}
}
elif (c == 2):
evt = {
'axes': {
'z': x
},
'channel': {
'group': 'Coherent-State',
'config': '561nm'
}
}
elif (c == 3):
evt = {
'axes': {
'z': x
},
'channel': {
'group': 'Coherent-State',
'config': '637nm'
}
}
elif (c == 4):
evt = {
'axes': {
'z': x
},
'channel': {
'group': 'Coherent-State',
'config': '730nm'
}
}
events.append(evt)
# set camera to trigger first mode for stage synchronization
# give camera time to change modes
core.set_property('Camera', 'TriggerMode', 'Trigger first')
time.sleep(1)
# update save_name with current Z plane
save_name_z = save_name + '_y' + str(y).zfill(4) + '_z' + str(
z).zfill(4)
# run acquisition at this Z plane
with Acquisition(directory=save_directory,
name=save_name_z,
post_hardware_hook_fn=post_hook_fn,
post_camera_hook_fn=camera_hook_fn,
show_display=False,
max_multi_res_index=0) as acq:
acq.acquire(events)
# added this code in an attempt to clean up resources, given the ZMQ error we are getting when using two hooks
acq.acquire(None)
acq.await_completion()
# try to clean up acquisition so that AcqEngJ releases directory. This way we can move it to the network storage
# in the background.
acq = None
# turn off lasers
core.set_config('Coherent-State', 'off')
core.wait_for_config('Coherent-State', 'off')
# set camera to internal trigger
# this is necessary to avoid PVCAM driver issues that we keep having for long acquisitions.
# give camera time to change modes
core.set_property('Camera', 'TriggerMode', 'Internal Trigger')
time.sleep(1)
import numpy as np
from pycromanager import Acquisition, multi_d_acquisition_events
with Acquisition("/Users/henrypinkard/tmp", "l_axis") as acq:
# create one event for the image at each z-slice
events = []
for time in range(5):
for index, z_um in enumerate(np.arange(start=0, stop=10, step=0.5)):
evt = {
#'axes' is required. It is used by the image viewer and data storage to
# identify the acquired image
"axes": {
"l": index,
"time": time
},
# the 'z' field provides the z position in µm
"z": z_um,
}
events.append(evt)
acq.acquire(events)
mm_app_path = '/Applications/Micro-Manager-2.0.0-gamma1'
# mm_app_path = 'C:/Program Files/Micro-Manager-2.0gamma'
config_file = mm_app_path + "/MMConfig_demo.cfg"
#Optional: specify your own version of java to run with
java_loc = "/Library/Internet Plug-Ins/JavaAppletPlugin.plugin/Contents/Home/bin/java"
# java_loc = None
start_headless(mm_app_path, config_file, java_loc=java_loc, port=5000)
with Bridge(port=5000) as b:
b.get_core().snap_image()
print(b.get_core().get_image())
save_dir = "/Users/henrypinkard/tmp"
# save_dir = "C:/Users/Henry Pinkard/Desktop/datadump"
with Acquisition(directory=save_dir, name="tcz_acq", port=5000) as acq:
# Generate the events for a single z-stack
events = multi_d_acquisition_events(
num_time_points=5,
time_interval_s=0,
channel_group="Channel",
channels=["DAPI", "FITC"],
z_start=0,
z_end=6,
z_step=0.4,
order="tcz",
)
acq.acquire(events)
def image_callback_fn(image, metadata):
row = metadata['GridRowIndex']
col = metadata['GridColumnIndex']
channel = metadata['Channel']
# other image axes (e.g. a z axis). 'position' axis is redundant to row and column indices
axes = metadata['Axes']
# numpy array
image
# TODO: run callback function
return image, metadata
with Acquisition('/Users/henrypinkard/megllandump',
'tiled',
tile_overlap=10,
image_process_fn=image_callback_fn,
pre_hardware_hook_fn=event_edit_fn) as acq:
# 10 pixel overlap between adjacent tiles
acq.acquire({
'row': 0,
'col': 0,
'channel': {
'group': 'channel',
'config': 'DAPI'
}
})
acq.acquire({
'row': 0,
'col': 0,
import numpy as np
from pycromanager import multi_d_acquisition_events, Acquisition
if __name__ == '__main__':
def external_trigger_fn(event):
#TODO: send signal to external device here
return event
with Acquisition(directory='/Users/henrypinkard/megllandump',
name='tcz_acq',
post_camera_hook_fn=external_trigger_fn) as acq:
# Generate the events for a single z-stack
events = multi_d_acquisition_events(num_time_points=10,
time_interval_s=0,
channel_group='channel',
channels=['DAPI', 'FITC'],
z_start=0,
z_end=6,
z_step=0.4,
order='tcz')
acq.acquire(events)
from pycromanager import Acquisition, multi_d_acquisition_events, Bridge
import numpy as np
if __name__ == "__main__":
def hook_fn(event):
# if np.random.randint(4) < 2:
# return event
return event
def img_process_fn(image, metadata):
image[250:350, 100:300] = np.random.randint(0, 4999)
return image, metadata
# magellan example
acq = Acquisition(
magellan_acq_index=0,
post_hardware_hook_fn=hook_fn,
image_process_fn=img_process_fn,
debug=True,
)
acq.await_completion()
from pycromanager import Acquisition, multi_d_acquisition_events
with Acquisition(directory="/Users/henrypinkard/tmp", name="tcz_acq", debug=True) as acq:
# Generate the events for a single z-stack
events = multi_d_acquisition_events(
num_time_points=5,
time_interval_s=0,
channel_group="Channel",
channels=["DAPI", "FITC"],
z_start=0,
z_end=6,
z_step=0.4,
order="tcz",
)
acq.acquire(events)
from pycromanager import Acquisition
if __name__ == '__main__':
with Acquisition('/Users/henrypinkard/megllandump', 'tiled', tile_overlap=10) as acq:
#10 pixel overlap between adjacent tiles
acq.acquire({'row': 0, 'col': 0})
acq.acquire({'row': 1, 'col': 0})
acq.acquire({'row': 0, 'col': 1})
dataset = acq.get_dataset()
dataset.has_image(row=0, col=0, resolution_level=1)
dataset.read_image(row=0, col=0, resolution_level=1)