4. Kymographs

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To load an HDF5 file and lists all of the kymographs inside of it, run:

import lumicks.pylake as lk

file = lk.File("example.h5")
list(file.kymos)  # e.g. shows: "['cas9', 'reference']"

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

# Plot all kymos in a file
>>> for name, kymo in file.kymos.items():
        print(f"{name}, starts at {kymo.start} ns")
cas9, starts at 1586776312560250200 ns
reference, starts at 1586777007674285402 ns

Or just pick a single one:

kymo = file.kymos["cas9"]
kymo.plot(channel="green", aspect="auto", vmax=5)

Here we see the plot() convenience function. The channel argument accepts the strings “red”, “green”, “blue”, or “rgb”. This function accepts keyword arguments that are passed to plt.imshow() internally. Note also, the axes are labeled with the appropriate time and position units.

The kymograph can also be exported to TIFF format:

kymo.save_tiff("image.tiff")

4.1. Kymo data and details

We can access the raw image data as numpy arrays:

rgb = kymo.rgb_image  # matrix with `shape == (h, w, 3)`
blue = kymo.blue_image  # single color so `shape == (h, w)`

# Plot manually
plt.imshow(kymo.green_image, aspect="auto", vmax=5)

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

• kymo.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
• kymo.size_um provides a list of scan sizes in micrometers along the axes of the scan
• kymo.pixelsize_um provides the pixel size in micrometers
• kymo.pixels_per_line provides the number of pixels in each line of the kymograph
• kymo.fast_axis provides the axis that was scanned (x or y)
• kymo.line_time_seconds provides the time between successive lines

4.2. Cropping and slicing

It is possible to crop a kymograph to a specific coordinate range, by using the function Kymo.crop_by_distance For example, we can crop the region from 6 micron to 24 micron using the following command:

kymo.crop_by_distance(6, 24).plot("green)

Kymographs can also be sliced in order to obtain a specific time range. For example, one can plot the region of the kymograph between 114.2 and 164.6 seconds using:

kymo["114.2s":"164.6s"].plot("green")

Note, slicing in time is currently only supported for unprocessed kymographs. If you want to both crop and slice a kymo, the order of operations is important:

kymo_sliced = kymo["114.2s":"164.6s"]
kymo_cropped = kymo_sliced.crop_by_distance(6, 24)

kymo_cropped.plot("green")

4.3. Calibrating to base pairs

By default, kymographs are constructed with units of microns for the position axis. If, however, the kymograph spans a known length of DNA (for example, lambda DNA) we can calibrate the position axis to kilobase pairs:

kymo_kbp = kymo_cropped.calibrate_to_kbp(48.502)

Now if we plot the image, the y-axis will be labeled in kbp:

kymo_kbp.plot("green")

These units are also carried forward to any downstream operations such as kymotracking algorithms and MSD analysis, . Note: currently this is a static calibration, meaning it is only valid if the traps do not change position during the time of the kymograph.

We can also interactively slice, crop, and calibrate kymographs using:

widget = kymo.crop_and_calibrate(channel="green", tether_length_kbp=48.502)
plt.show()

Simply click and drag the rectangle selector to the desired ROI. After closing the widget, we can access the edited kymograph with:

new_kymo = widget.kymo
new_kymo.plot("green")

If the optional tether_length_kbp argument is supplied, the kymograph is automatically calibrated to the desired length in kilobase pairs. If this argument is missing (the default value None) the edited kymograph is only sliced and cropped.

4.4. Downsampling

We can downsample a kymograph in time by invoking:

kymo_ds = kymo_cropped.downsampled_by(time_factor=2)

Or in space by invoking:

kymo_ds = kymo_cropped.downsampled_by(position_factor=2)

Or both:

kymo_ds = kymo_cropped.downsampled_by(time_factor=2, position_factor=2)

Note however, that not all functionalities are present anymore when downsampling a kymograph. For example, if we downsample a kymograph by time, we can no longer access the per pixel timestamps:

>>> kymo_ds.timestamps
AttributeError: Per pixel timestamps are no longer available after downsampling a kymograph in time since they
are not well defined (the downsampling occurs over a non contiguous time window). Line timestamps are still
available however. See: `Kymo.line_time_seconds`.

4.5. Plotting and exporting

There are also convenience functions to plot individual color channels and the full RGB image:

plt.subplot(2, 1, 1)
kymo.plot("rgb")
plt.subplot(2, 1, 2)
kymo.plot("blue")

The images can also be exported in the TIFF format:

kymo.save_tiff("image.tiff")

4.6. Correlating with force

We can plot a kymograph along its force trace using:

kymo.plot_with_force("1x", "green")

This will average the forces over each Kymograph line and plot them in a correlated fashion. The function can also take a dictionary of extra arguments to customize the kymograph plot. These parameter values get forwarded to matplotlib.pyplot.imshow(). For instance, if a few pixels dominate the image, it might be preferable to set the scale by hand. This can be accomplished by setting vmax:

kymo.plot_with_force("1x", "green", kymo_args={"vmax": 3})