filtered_imgs = deepcopy(imgs)
for selector in imgs._iter_axes():
data = filtered_imgs.get_slice(selector)[0]
scaled = data / scale_factors[selector[Axes.ROUND.value],
selector[Axes.CH.value]]
filtered_imgs.set_slice(selector, scaled, [Axes.ZPLANE])
# Decode with PixelSpotDecoder
psd = DetectPixels.PixelSpotDecoder(
codebook=experiment.codebook,
metric=
'euclidean', # distance metric to use for computing distance between a pixel vector and a codeword
norm_order=
2, # the L_n norm is taken of each pixel vector and codeword before computing the distance. this is n
distance_threshold=
0.5176, # minimum distance between a pixel vector and a codeword for it to be called as a gene
magnitude_threshold=
1.77e-5, # discard any pixel vectors below this magnitude
min_area=
2, # do not call a 'spot' if it's area is below this threshold (measured in pixels)
max_area=np.
inf, # do not call a 'spot' if it's area is above this threshold (measured in pixels)
)
initial_spot_intensities, prop_results = psd.run(filtered_imgs)
# filter spots that do not pass thresholds
spot_intensities = initial_spot_intensities.loc[initial_spot_intensities[
Features.PASSES_THRESHOLDS]]
# Example of how to access the spot attributes
print(
from starfish.spots import DetectPixels
from starfish.types import Features
# how much magnitude should a barcode have for it to be considered by decoding? this was set by looking at
# the plot above
magnitude_threshold = 0.5
# how big do we expect our spots to me, min/max size. this was set to be equivalent to the parameters
# determined by the Zhang lab.
area_threshold = (5, 30)
# how close, in euclidean space, should the pixel barcode be to the nearest barcode it was called to?
# here, I set this to be a large number, so I can inspect the distribution of decoded distances below
distance_threshold = 3
psd = DetectPixels.PixelSpotDecoder(codebook=experiment.codebook,
metric='euclidean',
distance_threshold=distance_threshold,
magnitude_threshold=magnitude_threshold,
min_area=area_threshold[0],
max_area=area_threshold[1])
initial_spot_intensities, results = psd.run(filtered_imgs)
spots_df = initial_spot_intensities.to_features_dataframe()
spots_df['area'] = np.pi * spots_df['radius']**2
spots_df = spots_df.loc[spots_df[Features.PASSES_THRESHOLDS]]
spots_df.head()
###################################################################################################
# Compare to benchmark results
# ----------------------------
# The below plot aggregates gene copy number across cells in the field of view and compares the
# results to the same copy numbers from the authors' pipeline. This can likely be improved by
sbp = Filter.Clip(p_max=99.8, level_method=Levels.SCALE_BY_CHUNK)
scaled = sbp.run(background_corrected, n_processes=1, in_place=False)
f = plot_scaling_result(background_corrected, scaled)
###################################################################################################
# Detect Spots
# ------------
# We use a pixel spot decoder to identify the gene target for each spot.
from starfish.spots import DetectPixels
psd = DetectPixels.PixelSpotDecoder(codebook=exp.codebook,
metric='euclidean',
distance_threshold=0.5,
magnitude_threshold=0.1,
min_area=7,
max_area=50)
pixel_decoded, ccdr = psd.run(scaled)
###################################################################################################
# plot a mask that shows where pixels have decoded to genes.
f, ax = plt.subplots()
ax.imshow(np.squeeze(ccdr.decoded_image), cmap=plt.cm.nipy_spectral)
ax.axis("off")
ax.set_title("Pixel Decoding Results")
###################################################################################################
# Get the total counts for each gene from each spot detector. Do the below values make sense for
# this tissue and this probeset?
scale_factors = {
(t[Axes.ROUND], t[Axes.CH]): t['scale_factor']
for t in experiment.extras['scale_factors']
}
filtered_imgs = deepcopy(imgs)
for selector in imgs._iter_axes():
data = filtered_imgs.get_slice(selector)[0]
scaled = data / scale_factors[selector[Axes.ROUND.value], selector[Axes.CH.value]]
filtered_imgs.set_slice(selector, scaled, [Axes.ZPLANE])
# Decode with PixelSpotDecoder
psd = DetectPixels.PixelSpotDecoder(
codebook=experiment.codebook,
metric='euclidean',
norm_order=2,
distance_threshold=0.5176,
magnitude_threshold=1.77e-5,
min_area=2,
max_area=np.inf,
)
initial_spot_intensities, prop_results = psd.run(filtered_imgs)
# Select only decoded spots that pass thresholds and map to genes in codebook
decoded = initial_spot_intensities.loc[initial_spot_intensities[Features.PASSES_THRESHOLDS]]
decoded_filtered = decoded[decoded.target != 'nan']
# Load cell mask
roi_path = os.path.join(os.path.dirname("__file__"), 'RoiSet.zip')
masks = BinaryMaskCollection.from_fiji_roi_set(path_to_roi_set_zip=roi_path, original_image=dapi)
# Assign spots to cells by labeling each spot with cell_id
al = AssignTargets.Label()
def process_fov(field_num: int, experiment_str: str):
"""Process a single field of view of MERFISH data
Parameters
----------
field_num : int
the field of view to process
experiment_str : int
path of experiment json file
Returns
-------
DecodedSpots :
tabular object containing the locations of detected spots.
"""
fov_str: str = f"fov_{int(field_num):03d}"
# load experiment
experiment = starfish.Experiment.from_json(experiment_str)
print(f"Loading fov: {fov_str}")
fov = experiment[fov_str]
imgs = fov.get_image(FieldOfView.PRIMARY_IMAGES)
print("Gaussian High Pass")
ghp = Filter.GaussianHighPass(sigma=3)
high_passed = ghp.run(imgs, verbose=True, in_place=False)
print("Deconvolve")
dpsf = Filter.DeconvolvePSF(num_iter=15,
sigma=2,
level_method=Levels.SCALE_SATURATED_BY_CHUNK)
deconvolved = dpsf.run(high_passed, verbose=True, in_place=False)
print("Guassian Low Pass")
glp = Filter.GaussianLowPass(sigma=1)
low_passed = glp.run(deconvolved, in_place=False, verbose=True)
scale_factors = {(t[Axes.ROUND], t[Axes.CH]): t['scale_factor']
for t in experiment.extras['scale_factors']}
filtered_imgs = deepcopy(low_passed)
for selector in imgs._iter_axes():
data = filtered_imgs.get_slice(selector)[0]
scaled = data / scale_factors[selector[Axes.ROUND.value],
selector[Axes.CH.value]]
filtered_imgs.set_slice(selector, scaled, [Axes.ZPLANE])
print("Decode")
psd = DetectPixels.PixelSpotDecoder(
codebook=experiment.codebook,
metric=
'euclidean', # distance metric to use for computing distance between a pixel vector and a codeword
norm_order=
2, # the L_n norm is taken of each pixel vector and codeword before computing the distance. this is n
distance_threshold=
0.5176, # minimum distance between a pixel vector and a codeword for it to be called as a gene
magnitude_threshold=
1.77e-5, # discard any pixel vectors below this magnitude
min_area=
2, # do not call a 'spot' if it's area is below this threshold (measured in pixels)
max_area=np.
inf, # do not call a 'spot' if it's area is above this threshold (measured in pixels)
)
initial_spot_intensities, prop_results = psd.run(filtered_imgs)
spot_intensities = initial_spot_intensities.loc[initial_spot_intensities[
Features.PASSES_THRESHOLDS]]
df = spot_intensities.to_decoded_spots()
return df