Edges of field of view out of focus

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I got the scope running well for the most part but the edges of the field of view are consistently blurry/a little out of focus compared to the center. Is there something physical with the scope I should be looking at that might help with this? Like the illumination being off center or the camera lens not sitting flat in the housing? Or is this an inherent limitation of using the basic quality camera module optics?

The opposite corners seem to be out of focus in the opposite direction (for example if I scroll from below the cells to above the cells the upper left will come into focus, then the center, then the lower right) which also makes me suspect the sample is just not level in the stage. But I’ve also tried leveling different sides of the stage by placing layers of tape under the sample to raise one side and that hasn’t seemed to change anything.

If anyone has ideas for other simple solutions I may have missed I’d love to hear them. Or if it would just be easier to image a grid with overlap and stitch the images together for an in focus composite?

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That does sound like a slope on the sample. From your image and description it looks more as though most of the left is at the same focus, and is the opposite defocus from the right side. The cells look ‘embossed’ on one side, and fuzzy sticking out on the other.

It is not just a slope, there is not a stripe of perfect focus right across the middle. That will be the quality of the lens, it is hard to make a lens with a flat field. With the tilt removed it looks as though you will have pretty good focus across the sample, although a little off around the edges. The only solution to that is tiling the good bits.

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Welcome to the forum @quigly. It looks like there is some field curvature. What lens are you using?

If you have an objective and tube lens set up it is worth checking whether it is a “plan” or “plano” objective.

If you have the pi lens there is not much you can do about this short of buying an plan objective. The microscope plan objective flattens the field by using a series of different lenses.