3. Confocal images

Download this page as a Jupyter notebook

The following code uses scans as an example. Kymographs work the same way – just substitute file.scans with file.kymos. To load an HDF5 file and lists all of the scans inside of it, run:

import lumicks.pylake as lk

file = lk.File("example.h5")
list(file.scans)  # e.g. shows: "['reference', 'bleach', 'imaging']"

Once again, .scans is a regular Python dictionary so we can easily iterate over it:

# Plot all scans in a file
for name, scan in file.scans.items():
    scan.plot(channel="rgb")
    plt.savefig(name)

Or just pick a single one:

scan = file.scans["name"]
scan.plot("red")

3.1. Scan data and details

You can access the raw image data directly. For a Scan with only a single frame:

rgb = scan.get_image("rgb")  # matrix with `shape == (h, w, 3)`
blue = scan.get_image("blue")  # single color so `shape == (h, w)`

# Plot manually
plt.imshow(rgb)

For scans with multiple frames:

# returned data has `shape == (n_frames, h, w, 3)`
rgb = multiframe_scan.get_image("rgb")
# returned data has `shape == (n_frames, h, w)`
blue = multiframe_scan.get_image("blue")

# Manually plot the RGB image of the first frame.
plt.imshow(rgb[0, :, :, :])

We can also slice out a subset of frames from an image stack:

sliced_scan = multiframe_scan[5:10]

This will return a new Scan containing data equivalent to:

multiframe_scan.get_image("rgb")[5:10, :, :, :]

The images contain pixel data where each pixel represents summed photon counts. For an even lower-level look at data, the raw photon count samples can be accessed:

photons = scan.red_photons
plt.plot(photons.timestamps, photons.data)

There are also several properties available for convenient access to the scan metadata:

  • scan.center_point_um provides a dictionary of the central x, y, and z coordinates of the scan in micrometers relative to the brightfield field of view

  • scan.size_um provides a list of scan sizes in micrometers along the axes of the scan

  • scan.pixelsize_um provides the pixel size in micrometers

  • scan.lines_per_frame provides the number scanned lines in each frame (number of rows in the raw data array)

  • scan.pixels_per_line provides the number of pixels in each line of the scan (number of columns in the raw data array)

  • scan.fast_axis provides the fastest axis that was scanned (x or y)

  • scan.num_frames provides the number of frames available

3.2. Plotting and Exporting

As shown above, there are convenience functions for plotting either the full RGB image or a single color channel.

scan.plot(channel=”red”)

Multi-frame scans are also supported:

print(scan.num_frames)
print(scan.get_image("blue").shape)  # (self.num_frames, h, w) -> single color channel
print(scan.get_image("rgb").shape)  # (self.num_frames, h, w, 3) -> three color channels

# plot frame at index 3 (first frame is index 0)
# defaults to the first frame if no argument is given
scan.plot("green", frame=3)

Sometimes a few bright pixels can dominate the colormap of a scan. When this is the case, it may be beneficial to manually set the color limits for each of the channels. This can be accomplished by providing a ColorAdjustment to plotting or export functions:

scan.plot(channel="red", adjustment=lk.ColorAdjustment([50, 50, 50], [100, 250, 196]))

Gamma adjustments can be applied in addition to the bounds by supplying an extra argument named gamma. For example, a gamma adjustment of 2 to the red channel can be applied as follows:

scan.plot(channel="red", adjustment=lk.ColorAdjustment([50, 50, 50], [100, 250, 196], gamma=[2, 1, 1]))

The limits can also be specified in percentiles when this is more practical:

scan.plot(channel="red", adjustment=lk.ColorAdjustment([5, 5, 5], [95, 95, 95], mode="percentile"))

The images can also be exported in the TIFF format:

scan.save_tiff("image.tiff")

Scans can also be exported to video formats. Exporting the red channel of a multi-scan GIF can be done as follows for example:

scan.export_video_red("test_red.gif")

Or if we want to export a subset of frames (the first frame being 10, and the last frame being 40) of all three channels at a frame rate of 40 frames per second, we can do this:

scan.export_video_rgb("test_rgb.gif", start_frame=10, end_frame=40, fps=40)

For other video formats such as .mp4 or .avi, ffmpeg must be installed. See installation instructions for more information on this.

3.3. Correlating scans

We can downsample a scan according to the frames in a scan. We can use frame_timestamp_ranges() for this:

frame_timestamp_ranges = scan.frame_timestamp_ranges()

This returns a list of start and stop timestamps that can be passed directly to downsampled_to(), which will then return a Slice with a datapoint per frame:

downsampled = f.force1x.downsampled_over(frame_timestamp_ranges)

We can also correlate multi-frame confocal scans with a channel Slice using a small interactive plot:

scan.plot_correlated(f.force1x)