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I'm looking for ways to set up a phase contrast microscope to reduce the brightness gradient that appears across the background of my images.

I use phase contrast to find the outlines of cells in my images. My problem is that the automated processing algorithms I use get confused by strong brightness gradients in some images. Post-processing of the images disrupts the continuous nature of the histogram, again throwing off the algorithms.

Does anyone know why these gradients appear and how to reduce their formation?

Thanks,

Oliver

Edited the question to clarify that I am interested in microscope usage, rather than image manipulation

Sample image:

   enter image description here

The image background is darker at the left of the image than at the right

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  • $\begingroup$ Are you using the images to count cells or to determine shape, volume etc? Also, the question is borderline off-topic since it is mostly about image manipulation, so it would probably be good if you added some more info on the biological background/problem you are studying. $\endgroup$ – fileunderwater Feb 6 '15 at 14:31
  • $\begingroup$ @fileunderwater I use the phase image to create ROIs for collecting data from epifluorescent images. How I use the images is not particularly important with respect to the question, though. I'm just interested to know if there are ways to configure my microscope such that the brightness gradient across the field is less pronounced. $\endgroup$ – Olly Feb 6 '15 at 14:47
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    $\begingroup$ Add this information directly to the question by editing. For the record, I think the question is fine since its an important lab techique (but I cannot answer it), but it might attract close-votes for being "only" about microscopy and not biology. $\endgroup$ – fileunderwater Feb 6 '15 at 14:50
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    $\begingroup$ I hope not - there isn't a microscopy stack exchange I can post in instead! The biology exchange does contain many other questions purely related to microscopy, though... Furthermore, I expect biologists to comprise the largest demographic of microscope users, so I'm more likely to find an answer here than anywhere else. Thanks for your comments, I've edited the question accordingly. $\endgroup$ – Olly Feb 6 '15 at 14:57
  • $\begingroup$ Is it possible to provide an image of the problem? $\endgroup$ – James Feb 6 '15 at 15:29
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If you're not already doing so, make sure you set up Kohler illumination every time you use the microscope -- see this tutorial by Steven Ruzin.

The lamp or the phase ring may be misaligned. There are usually small hexagonal set screws on the lamp housing and the condenser, respectively, to adjust these.

Imaging near the edge of a dish or well can lead to uneven illumination as the light is bent by the walls of the culture vessel. If this might be the cause, try re-doing the Kohler illumination before you take each image.

If you can't find the cause of the problem, there are several ways to correct for uneven background described here. Maybe one of them will work with your analysis.

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  • $\begingroup$ Excellent first post! Welcome to Biology S.E.! If you need any assistance, please visit The Help Center. $\endgroup$ – L.B. Mar 20 '15 at 18:03

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