def test_mspb_3():
"""
Test (single) PSF measurement with drift.
"""
# Make test movie.
im_max = 1000.0
n_pts = 10
x = 7.0
y = 11.0
drift_xy = numpy.random.uniform(size=(n_pts, 2))
psf_movie = storm_analysis.getPathOutputTest("psf_movie.tif")
with tifffile.TiffWriter(psf_movie) as tf:
for i in range(n_pts):
image = dg.drawGaussiansXY((20, 20),
numpy.array([x + drift_xy[i][0]]),
numpy.array([y + drift_xy[i][1]]))
image = image * im_max
tf.save(image.astype(numpy.float32))
# Parameters.
p = params.ParametersDAO()
p.changeAttr("camera_gain", 1.0)
p.changeAttr("camera_offset", 0.0)
# Frame reader.
frdr = analysisIO.FrameReaderStd(movie_file=psf_movie, parameters=p)
z_index = numpy.zeros(n_pts).astype(numpy.int) - 1
z_index[0] = 0
[psf0, samples] = mPSFUtils.measureSinglePSFBeads(frdr,
z_index,
6,
x + drift_xy[0][0],
y + drift_xy[0][1],
zoom=2)
for i in range(1, n_pts):
z_index = numpy.zeros(n_pts).astype(numpy.int) - 1
z_index[i] = 0
[psf, samples] = mPSFUtils.measureSinglePSFBeads(frdr,
z_index,
6,
x,
y,
drift_xy=drift_xy,
zoom=2)
assert (numpy.max(numpy.abs(psf0 - psf) / numpy.max(psf)) < 0.05)
def test_mspb_2():
"""
Test (single) PSF measurement, no drift, recentering.
The maximum relative difference is typically on the order of 2%.
"""
# Make test movie.
im_max = 1000.0
x = 7.0 + numpy.random.uniform(size=10)
y = 11.0 + numpy.random.uniform(size=10)
psf_movie = storm_analysis.getPathOutputTest("psf_movie.tif")
with tifffile.TiffWriter(psf_movie) as tf:
for i in range(x.size):
image = dg.drawGaussiansXY((20, 20), numpy.array([x[i]]),
numpy.array([y[i]]))
image = image * im_max
tf.save(image.astype(numpy.float32))
# Parameters.
p = params.ParametersDAO()
p.changeAttr("camera_gain", 1.0)
p.changeAttr("camera_offset", 0.0)
# Frame reader.
frdr = analysisIO.FrameReaderStd(movie_file=psf_movie, parameters=p)
z_index = numpy.zeros(x.size).astype(numpy.int) - 1
z_index[0] = 0
[psf0, samples] = mPSFUtils.measureSinglePSFBeads(frdr,
z_index,
6,
x[0],
y[0],
zoom=2)
for i in range(1, x.size):
z_index = numpy.zeros(x.size).astype(numpy.int) - 1
z_index[i] = 0
[psf, samples] = mPSFUtils.measureSinglePSFBeads(frdr,
z_index,
6,
x[i],
y[i],
zoom=2)
assert (numpy.max(numpy.abs(psf0 - psf) / numpy.max(psf)) < 0.05)
def test_mspb_1():
"""
Test (single) PSF measurement, no drift.
"""
# Make test movie.
x = 7.2
y = 9.8
psf_movie = storm_analysis.getPathOutputTest("psf_movie.tif")
image = 1000.0 * dg.drawGaussiansXY(
(20, 20), numpy.array([x]), numpy.array([y]))
with tifffile.TiffWriter(psf_movie) as tf:
for i in range(6):
tf.save(image.astype(numpy.float32))
# Parameters.
p = params.ParametersDAO()
p.changeAttr("camera_gain", 1.0)
p.changeAttr("camera_offset", 0.0)
# Frame reader.
frdr = analysisIO.FrameReaderStd(movie_file=psf_movie, parameters=p)
z_index = numpy.array([0, 1, 2, 2, -1, -1])
[psf, samples] = mPSFUtils.measureSinglePSFBeads(frdr,
z_index,
6,
x,
y,
zoom=2)
assert (numpy.allclose(samples, numpy.array([1, 1, 2])))
for i in range(1, psf.shape[0]):
assert (numpy.allclose(psf[0, :, :], psf[i, :, :] / samples[i]))
if False:
with tifffile.TiffWriter("psf.tif") as tf:
for i in range(psf.shape[0]):
tf.save(psf[i, :, :].astype(numpy.float32))
def measurePSFBeads(movie_name,
zfile_name,
beads_file,
psf_name,
aoi_size=12,
pixel_size=0.1,
refine=False,
z_range=0.6,
z_step=0.05):
"""
movie_name - The name of the movie, presumably a z stack for PSF measurement.
zfile_name - The text file containing the z offsets (in microns) for each frame.
beads_file - The text file containing the locations of the beads.
psf_name - The name of the file to save the measured PSF in (as a pickled Python dictionary).
aoi_size - The AOI of interest in pixels. The final AOI is 2x this number.
pixel_size - The pixel size in microns.
refine - Align the measured PSF for each bead to the average PSF.
z_range - The range the PSF should cover in microns.
z_step - The z step size of the PSF.
"""
# Load the z-offset information for the dax file.
#
# This is a text file with one line per frame that contains the
# z-offset (in microns) for that frame. Each line is a space separated
# valid, z_pos pair. If valid if 0 the frame will be ignored,
# otherwise it will be used.
#
z_offsets = numpy.loadtxt(zfile_name)
# Create array specifying what frame corresponds to what
# Z slice in the PSF.
#
z_index = measurePSFUtils.makeZIndexArray(z_offsets, z_range, z_step)
# Load the locations of the beads.
#
# This is a text file the contains the locations of the beads that
# will be used to construct the PSF. Each line is a space separated
# x, y pair of bead locations (in pixels).
#
# One way to create this file is to look at the bead movie with
# visualizer.py and record the center positions of several beads.
#
data = numpy.loadtxt(beads_file, ndmin=2)
bead_x = data[:, 1] + 1
bead_y = data[:, 0] + 1
# Create a reader of the movie.
#
# We assume that the bead stack was measured with a camera
# that does not have a large pixel to pixel variation in
# gain and offset. The offset and magnitude are not that
# important at we will estimate and subtract the offset
# and normalize 1.
#
# Movie (frame) reader.
frame_reader = analysisIO.FrameReaderStd(movie_file=movie_name,
camera_gain=1.0,
camera_offset=0.0)
# Measure PSFs for each bead.
#
total_samples = None
psfs = []
for i in range(bead_x.size):
[psf, samples] = measurePSFUtils.measureSinglePSFBeads(frame_reader,
z_index,
aoi_size,
bead_x[i],
bead_y[i],
zoom=2)
# Verify that we have at least one sample per section, because if
# we don't this almost surely means something is wrong.
if (i == 0):
for j in range(samples.size):
assert (samples[i] > 0), "No data for PSF z section " + str(i)
# Normalize by the number of sample per z section.
#for j in range(samples.size):
# psf[j,:,:] = psf[j,:,:]/samples[j]
# Keep track of total number of samples.
if total_samples is None:
total_samples = samples
else:
total_samples += samples
psfs.append(psf)
# Set the PSF to have zero average on the X/Y boundaries. We are
# matching the behavior of spliner.measure_psf here.
#
sum_psf = measurePSFUtils.sumPSF(psfs)
for i in range(sum_psf.shape[0]):
mean_edge = measurePSFUtils.meanEdge(sum_psf[i, :, :])
for j in range(len(psfs)):
psfs[j][i, :, :] -= mean_edge / float(len(psfs))
# Align the PSFs to each other. This should hopefully correct for
# any small errors in the input locations, and also for fields of
# view that are not completely flat.
#
if refine:
print("Refining PSF alignment.")
# Normalize each PSF by the number of z sections.
for psf in psfs:
for i in range(samples.size):
psf[i, :, :] = psf[i, :, :] / samples[i]
[average_psf, i_score] = measurePSFUtils.alignPSFs(psfs)
else:
average_psf = measurePSFUtils.averagePSF(psfs)
# Normalize PSF.
#
# This normalizes the PSF so that sum of the absolute values
# of each section is 1.0. This only makes sense if the AOI is
# large enough to capture all the photons, which might not be
# true. Not clear how important this is as Spliner will fit
# for the height anyway.
#
for i in range(average_psf.shape[0]):
print("z plane {0:0d} has {1:0d} samples".format(i, total_samples[i]))
section_sum = numpy.sum(numpy.abs(average_psf[i, :, :]))
# Do we need this test? We already check that we have at
# least one sample per section.
if (section_sum > 0.0):
average_psf[i, :, :] = average_psf[i, :, :] / section_sum
# Normalize to unity maximum height.
if (numpy.max(average_psf) > 0.0):
average_psf = average_psf / numpy.max(average_psf)
else:
print("Warning! Measured PSF maxima is zero or negative!")
# Save PSF (in image form).
if True:
tif_name = os.path.splitext(psf_name)[0]
with tifffile.TiffWriter(tif_name + "_beads.tif") as tf:
for i in range(average_psf.shape[0]):
tf.save(average_psf[i, :, :].astype(numpy.float32))
# Save PSF.
#
# For historical reasons all the PSF z values are in nanometers.
# At some point this should be fixed.
#
z_range = 1.0e+3 * z_range
z_step = 1.0e+3 * z_step
cur_z = -z_range
z_vals = []
for i in range(average_psf.shape[0]):
z_vals.append(cur_z)
cur_z += z_step
psf_dict = {
"psf": average_psf,
"pixel_size": 0.5 * pixel_size,
"type": "3D",
"version": 1.0,
"zmin": -z_range,
"zmax": z_range,
"zvals": z_vals
}
pickle.dump(psf_dict, open(psf_name, 'wb'))
def measurePSFBeads(movie_name, zfile_name, beads_file, psf_name, aoi_size = 12, pixel_size = 0.1, refine = False, z_range = 0.6, z_step = 0.05):
"""
movie_name - The name of the movie, presumably a z stack for PSF measurement.
zfile_name - The text file containing the z offsets (in microns) for each frame.
beads_file - The text file containing the locations of the beads.
psf_name - The name of the file to save the measured PSF in (as a pickled Python dictionary).
aoi_size - The AOI of interest in pixels. The final AOI is 2x this number.
pixel_size - The pixel size in microns.
refine - Align the measured PSF for each bead to the average PSF.
z_range - The range the PSF should cover in microns.
z_step - The z step size of the PSF.
"""
# Load the z-offset information for the dax file.
#
# This is a text file with one line per frame that contains the
# z-offset (in microns) for that frame. Each line is a space separated
# valid, z_pos pair. If valid if 0 the frame will be ignored,
# otherwise it will be used.
#
z_offsets = numpy.loadtxt(zfile_name)
# Create array specifying what frame corresponds to what
# Z slice in the PSF.
#
z_index = measurePSFUtils.makeZIndexArray(z_offsets, z_range, z_step)
# Load the locations of the beads.
#
# This is a text file the contains the locations of the beads that
# will be used to construct the PSF. Each line is a space separated
# x, y pair of bead locations (in pixels).
#
# One way to create this file is to look at the bead movie with
# visualizer.py and record the center positions of several beads.
#
data = numpy.loadtxt(beads_file, ndmin = 2)
bead_x = data[:,1] + 1
bead_y = data[:,0] + 1
# Create a reader of the movie.
#
# We assume that the bead stack was measured with a camera
# that does not have a large pixel to pixel variation in
# gain and offset. The offset and magnitude are not that
# important at we will estimate and subtract the offset
# and normalize 1.
#
# Movie (frame) reader.
frame_reader = analysisIO.FrameReaderStd(movie_file = movie_name,
camera_gain = 1.0,
camera_offset = 0.0)
# Measure PSFs for each bead.
#
total_samples = None
psfs = []
for i in range(bead_x.size):
[psf, samples] = measurePSFUtils.measureSinglePSFBeads(frame_reader,
z_index,
aoi_size,
bead_x[i],
bead_y[i],
zoom = 1)
# Verify that we have at least one sample per section, because if
# we don't this almost surely means something is wrong.
if (i == 0):
for j in range(samples.size):
assert(samples[j] > 0), "No data for PSF z section " + str(j)
# Normalize by the number of sample per z section.
#for j in range(samples.size):
# psf[j,:,:] = psf[j,:,:]/samples[j]
# Keep track of total number of samples.
if total_samples is None:
total_samples = samples
else:
total_samples += samples
psfs.append(psf)
# Set the PSF to have zero average on the X/Y boundaries. We are
# matching the behavior of spliner.measure_psf here.
#
sum_psf = measurePSFUtils.sumPSF(psfs)
for i in range(sum_psf.shape[0]):
mean_edge = measurePSFUtils.meanEdge(sum_psf[i,:,:])
for j in range(len(psfs)):
psfs[j][i,:,:] -= mean_edge/float(len(psfs))
# Align the PSFs to each other. This should hopefully correct for
# any small errors in the input locations, and also for fields of
# view that are not completely flat.
#
if refine:
print("Refining PSF alignment.")
# Normalize each PSF by the number of z sections.
for psf in psfs:
for i in range(samples.size):
psf[i,:,:] = psf[i,:,:]/samples[i]
[average_psf, i_score] = measurePSFUtils.alignPSFs(psfs)
else:
average_psf = measurePSFUtils.averagePSF(psfs)
# Normalize PSF.
#
# This normalizes the PSF so that sum of the absolute values
# of each section is 1.0. This only makes sense if the AOI is
# large enough to capture all the photons, which might not be
# true. Not clear how important this is as Spliner will fit
# for the height anyway.
#
for i in range(average_psf.shape[0]):
print("z plane {0:0d} has {1:0d} samples".format(i, total_samples[i]))
section_sum = numpy.sum(numpy.abs(average_psf[i,:,:]))
# Do we need this test? We already check that we have at
# least one sample per section.
if (section_sum > 0.0):
average_psf[i,:,:] = average_psf[i,:,:]/section_sum
# Normalize to unity maximum height.
if (numpy.max(average_psf) > 0.0):
average_psf = average_psf/numpy.max(average_psf)
else:
print("Warning! Measured PSF maxima is zero or negative!")
# Save PSF (in image form).
if True:
tif_name = os.path.splitext(psf_name)[0]
with tifffile.TiffWriter(tif_name + "_beads.tif") as tf:
for i in range(average_psf.shape[0]):
tf.save(average_psf[i,:,:].astype(numpy.float32))
# Save PSF.
#
# For historical reasons all the PSF z values are in nanometers.
# At some point this should be fixed.
#
z_range = 1.0e+3 * z_range
z_step = 1.0e+3 * z_step
cur_z = -z_range
z_vals = []
for i in range(average_psf.shape[0]):
z_vals.append(cur_z)
cur_z += z_step
psf_dict = {"psf" : average_psf,
"pixel_size" : pixel_size,
"type" : "3D",
"version" : 2.0,
"zmin" : -z_range,
"zmax" : z_range,
"zvals" : z_vals}
pickle.dump(psf_dict, open(psf_name, 'wb'))