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When a bright object is encontered, the image goes out of focus without adjusting anything (e.g., stage position, gain, exposure), so it seems as if something is happening within the camera in response to the sudden change in brightness. I typically have to move the stage up to get the image back in focus. I’m not aware of any lens/apreture or automatic feature on the camera itself that could be causing this (although I’ll admit I know next-to-nothing about microscopy cameras).

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If it is not adjusting focus, could it be that it auto-adjusts the gain, and that you are seeing particles that are way to bright (or dim) because auto-adjustment did not finish its job yet?

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I typically have to adjust the focus by +/- 10 um every few frames since the depth of field changes sligtly depending on the morphology of the particles I’m imaging. However, even if I keep the focus level consistant, the camera itself seems to be adjusting when moving form a dark area to an area with fluoroescent particles.

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Any explanation or advice would be great! Is there a way to stop the camera form adjusting when there are bright particles?

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Is the Autostretch feature of the histogram turned on? Have you tried turning it off and seeing if that explains what you are seeing?

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I’m using a Basler Ace A2440-35 um to take a mosaic image of a filter paper containing various fluorescent particles ranging from 1-125 um. I’m using micromanager MDA with autofocus and a prior Proscan III stage with a motorized Z stage attachment.

You might like to add the tag “micromanager” to your post to ensure that it would be seen by forum members who are interested/experienced in that software.

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You can go to Device > Property Browser and look for the cameras properties. Usually, you want anything that is auto-adjusting to be switched off.

I can not see any physical way how a bright object would change the focus without any components (stages, lenses) changing their position. Is it possible that the bright object is at a different focal plane than the dim objects?

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During acquisition, when the field of view moves from an area with no particles (i.e., dark) to an area with particles, the camera seems to go out of focus, and the following mosaic positions are out of focus until the stage autofocus catches up with the change of focus from the camera.

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What does “the camera itself seems to be adjusting” mean? Not to be pedantic, but normally cameras attached to microscopes have no ability to adjust focus. If your camera has some kind of lens that it can manipulate, then yes, that could be the case.

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Is the depth of field such that if you were to (manually) set the focus once, your images would all be in focus? (sorry if this is a daft question - not a microscopy expert!)

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Photography in the near-UV range requires the use of a light source of these wavelengths. UV-A (320-400 nm) is the band closest to visible light (400-700 nm).

I have been making sure to turn off auto gain and exposure adjustment, and it doesn’t seem like ther are any other atomatic parameters for the camera.

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I have tried lowering the exposure and incident light intensity and this helped a bit, but I lost visibility of some of my fluorophores.

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