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, Ankurenter image description here

  • $\begingroup$ My fluorescence microscopy is rather rusty, but it looks like you have a light gradient from left to right (as opposed to a fixed line). Is it possible that you have a light focusing adjustment that's out of wack? $\endgroup$ – rotaredom Nov 22 '17 at 14:31
  • $\begingroup$ Also, the background (at least on my screen) appears to follow that gradient, suggesting to me that it's not an issue with the staining, but rather with a light source direction. $\endgroup$ – rotaredom Nov 22 '17 at 14:33
  • $\begingroup$ @rotaredom explanation is the most likely, i.e. that the light source is not properly aligned. You can test that by, for example, imaging another specimen that you know has worked before. Alternatively, you may want to double-check that the slide is truly parallel to the objective, although everything looks reasonably in focus to me. $\endgroup$ – vkehayas Nov 23 '17 at 8:51

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