Illumination Correction

The goal of illumination correction is to remove uneven illumination of the image caused by non uniform illumination of the field of view, characteristics of the sensor, (like vignetting), or orientation of the tissue’s surface with respect to the light source.

Prospective Correction

The simplest forms of illumination correction are called “prospective correction” and are based on background subtraction. This involves taking additional images using the microscopy apparatus to help calibrate. These can either be acquired by averaging a series of images captured with no sample and no light (dark image), or with no sample and light (bright image).

Starfish can apply this type of background correction by exposing the ElementWiseMultiply Filter. The user is responsible for transforming their calibration images into the correct matrix to correct for background, and then ElementWiseMultiply can apply a transformation to correct any uneven illumination.

The below plot shows how to use ElementWiseMultiply on a single plane of an in-situ sequencing experiment.

import matplotlib.pyplot as plt
import numpy as np
import xarray as xr

import starfish
import starfish.data
from starfish.types import Axes

experiment = starfish.data.ISS(use_test_data=True)
image: starfish.ImageStack = experiment['fov_001'].get_image('primary')

image_2d = image.sel({Axes.CH: 0, Axes.ROUND: 0, Axes.ZPLANE: 0})

plt.imshow(np.squeeze(image_2d.xarray.values))
plt.show()
../../_images/sphx_glr_plot_illumination_correction_001.png

Out:

  0%|          | 0/16 [00:00<?, ?it/s]
100%|##########| 16/16 [00:00<00:00, 269.21it/s]

This image was corrected before it was sent to us, but we can introduce an uneven illumination profile. Below we mock an extremely severe 200% decrease in illumination from left to right.

lightness = np.linspace(4, 1, image_2d.xarray.sizes[Axes.X])
gradient_data = np.tile(lightness, reps=(image_2d.xarray.sizes[Axes.Y], 1))
gradient = xr.DataArray(
    data=gradient_data[np.newaxis, np.newaxis, np.newaxis, :, :],
    dims=(Axes.ROUND.value, Axes.CH.value, Axes.ZPLANE.value, Axes.Y.value, Axes.X.value)
)

# introduce the gradient, overwriting the ImageStack
data = image_2d.xarray.values / gradient.values
image_2d = starfish.ImageStack.from_numpy(data)

# display the resulting image
plt.imshow(np.squeeze(image_2d.xarray.values))
plt.show()
../../_images/sphx_glr_plot_illumination_correction_002.png

Out:

/home/docs/checkouts/readthedocs.org/user_builds/spacetx-starfish/checkouts/latest/starfish/core/imagestack/imagestack.py:413: UserWarning: ImageStack detected as float64. Converting to float32...
  warnings.warn(f"ImageStack detected as {array.dtype}. Converting to float32...")
/home/docs/checkouts/readthedocs.org/user_builds/spacetx-starfish/envs/latest/lib/python3.6/site-packages/skimage/util/dtype.py:135: UserWarning: Possible precision loss when converting from float64 to float32
  .format(dtypeobj_in, dtypeobj_out))

  0%|          | 0/1 [00:00<?, ?it/s]
100%|##########| 1/1 [00:00<00:00, 346.15it/s]

The illumination profile has increased the intensity of the background in the right side of the image. This is problematic for many spot finding methods that set thresholds for peak intensities globally across the image; spots can be incorrectly excluded in low-illumination areas, and this spatial phenomenon can lead to incorrect spatial hypotheses.

We use starfish’s ElementWiseMultiply to multiply the image with a gradient. Here, it’s just the same gradient we divided the image by. However, in typical microscopy experiments this should be derived from the additional black or bright images taken to calibrate the microscope, and the correction is likely to be more more complex than a simple gradient.

ewm = starfish.image.Filter.ElementWiseMultiply(mult_array=gradient)
corrected_image_2d = ewm.run(image_2d, in_place=False)

the image should now be returned to normal

plt.imshow(np.squeeze(corrected_image_2d.xarray))
plt.show()
../../_images/sphx_glr_plot_illumination_correction_003.png

Retrospective Correction

When additional images were not acquired, or cannot be used calibrate the microscope, then uneven background illumination can be subtracted by estimating the background. This is called “retrospective correction”. Low pass filters like GaussianLowPass and morphological filters are common ways to compute an approximate background image.

A simple one-step process is to use the WhiteTophat, which will perform the background estimation and subtraction. See the White Top-Hat Filtering example in the Removing Autofluorescence tutorial.

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