Raw signals do not match suite2p raw signals for the same rois

[just-meng]

Hi there,

I was running fissa on suite2p motion-corrected reg_tif files following this instruction. When comparing the result with the subtraction method (F = 0.7*Fneu) I noticed that they were nowhere similar to each other. Having a look at the extracted raw signals by fissa (experiment.raw) and by suite2p (F.npy) of the same roi I noticed that also the raw traces were not similar.

I triple-checked the roi identity and plotted experiment.roi_polys[i_roi, trial][0]to compare with suite2p GUI). It definitely is the same roi. Any idea why the raw traces do no agree?

• python: 3.9.12
• suite2p: 0.11.0
• fissa: 1.0.0
• operating system: Windows 11

Just to be sure, the code is attached here:

images = "./reg_tif"

# Get image size
Lx = ops["Lx"]
Ly = ops["Ly"]

# Generate ROI masks in a format usable by FISSA (in this case, a list of masks)
rois = [np.zeros((Ly, Lx), dtype=bool) for n in range(n_cells)]

for i, n in enumerate(cell_ids):
    # i is the position in cell_ids, and n is the actual cell number
    ypix = stat[n]["ypix"][~stat[n]["overlap"]] # get only the non-overlapping pixels
    xpix = stat[n]["xpix"][~stat[n]["overlap"]]
    rois[i][ypix, xpix] = 1

fissa_folder = "../../fissa"
fissa_experiment = fissa.Experiment(images, [rois], fissa_folder)
fissa_experiment.separate()

n_cells and cell_ids are previously obtained from F.npy and iscell.npy.

[just-meng]

I found the bug: if the folder of the suite2p registered images is passed to fissa.Experiment(images, rois, folder) as argument images, the tif files will be sorted according to self.images = sorted(glob.glob(os.path.join(images, "*.tif*"))) This leads to incorrectly sorted images simply due to the naming of the tif files: file000_chan0, file500_chan0, file1000_chan0 After sorting it lists: file000_chan0, file1000_chan0, file500_chan0 The result is temporally mis-appended signals.

A quick fix to my code (not elegant but works):

images_folder = "./reg_tif"
images_path = glob.glob(os.path.join(images_folder, "*.tif*"))
# extract the interger that indicates the number of the starting frame of the tif and add to the path as a tuple
images_path = [(int(path.split('_')[-2].split('file')[-1]), path) for path in images_path]
# sort the path list according to the frame numbers
sorted_path = sorted(images_path)
# get rid of the integer and make a list of the now sorted paths
sorted_path = list(np.array(sorted_path)[:,1])

Now one can run fissa.Experiment(sorted_path, rois, folder).