Hi Everyone,
I have used Phalloidin to label F-actin in ethanol fixed cells ( I know formaldehyde might have been a better choice for fixation). I am new to new to this and I am experiencing that one part of image is bright while other portion is dark (please see attached image). I have already checked that its not due to the lever which we use to shift from bino mode to image mode (eye to camera mode). i have captured images previously and they looked perfect.I dont know what's wrong? Does anyone know what might be the problem?
As I said, I am new to this so it might be a very stupid question but I preferred to ask.
Thanks in advance!
Best,
Ankur
Hi Ankur,
From the image you show it is difficult to tell whether this is a problem with the fixation or with the microscope itself. The first question to ask is whether the difference in illumination is always the same or in other words when you move the sample does the contrast difference stay in the same place or does it move?
If it moves then it is probably something to do with the fixation process being patchy. If that is the case repeating the experiment should fix the problem.
If it does not move then it is more likely a problem with the illumination of the sample from the microscope. You should check that your microscope is achieving Kohler illumination. Here is a link to a tutorial on how to ensure that your microscope is set up in this way
https://www.zeiss.com/microscopy/int/solutions/reference/basic-microscopy/koehler-illumination.html
To be honest it is always good to check this is the case anyway as this will ensure that your images are the best quality that you can achieve from the microscope.
Hope this helps.
Alex
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