Microscope users: Horses wear blinders to restrict their field-of-view (FOV). You might not be getting your full field of view either. Learn why in this thread. (1/ 8) #FluorescenceFriday
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What is your field-of-view in practice? You can get a rough idea by measuring with stage movement, or a calibrated slide. There’s a really nice procedure written up here: https://doi.org/10.1101/2024.01.23.576417 (2/8) #FluorescenceFriday
If you have the field number (FN), that’s the diagonal measurement in mm across the FOV in an intermediate image plane. You can divide by the objective mag. to get the FOV. E.g., a FN 28 with a 16x obj. should give you a FOV with a diagonal of 1.75 mm (28/16), or 1.2 mm x 1.2 mm (1.75/sqrt(2)).(3/8)
However, you might not be getting all of that. Maybe the resonant scanner has a smaller maximum scan angle (e.g., many 12 kHz resonant scanners max out at 10 degrees). (4/8)
Or maybe there are optical aberrations or clipping in your system that make the edges dim and/or lower resolution (i.e., vignetting). It might look like a big FOV, but it’s actually only the center that is nice and the image at the edges isn’t usable. (5/8)
It is also possible that your system isn’t providing a large enough beam for the objective. If the back aperture is 20 mm in diameter, then your beam should be that wide too. If it’s smaller, you can still images, but the resolution can be lower. (6/8)
So just because the FN is high, or the FOV looks large, the actual resolution and brightness can be lower outside of the central region. Your scope might still have blinders on. (7/8)
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https://doi.org/10.1101/2024.01.23.576417 (2/8) #FluorescenceFriday