Fluorescence filter size advantages

Hi There, I need your advices. We are about to purchase fluorescence filter (cubes) for our new microscope (IX83). We could opt for “standard” 25mm diameter filters or larger 32mm ones (obviously more expensive). Which are the main advantages of the larger ones?
I assume it is a matter of field of view, vignetting and size of the camera sensor, but, practically, which are the situations where the 32mm filters have an edge over the others?
I cannot find any information online and I would prefer to have an unbiased review from your side rather than from the reseller.

The advantage is not clear to me as there are lots of apertures in a microscope that might be limiting, not just the filter cube.


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Vignetting is the concern. If you know details about optic apertures and spacings then you can work out whether or not you need the larger filter apertures. Generally speaking the required aperture is largest for low-mag objectives because they tend to have the largest back aperture sizes. Also if you are using a large-format camera sensor all the way to the edge then you will notice vignetting sooner than if you are cropping or using a smaller sensor.

Here is the equation for the required aperture diameter ⌀aperture placed at distance Laperture from the objective back aperture (commonly located inside the objective body), for utilized sensor diagonal ⌀sensor, tube lens focal length EFLtube (180mm for standard Olympus stand), and objective lens numerical aperture NAobj and focal length EFLobj (EFL is 180mm / nameplate magnification for Olympus objectives). Note the final term is simply the diameter of the objective’s back aperture.

aperture >= Laperture * ⌀sensor / EFLtube + 2*NAobj*EFLobj


JonD gives a great answer. From a purchase and user perspective, the trend in systems (objectives, the scope itself and cameras) is accommodate larger and larger fields, so in a way you are future proofing so you can take advantage of larger fields without vignetting. I was recently demoing a camera for color brightfield imaging, on a system with 25mm filters and I couldn’t use the entire chip with the 4x as there was a huge amount of vignetting, so there is an advantage in 1) larger field possible, 2) ‘better’ cameras that have bigger sensors, and 3) possibly matches better with potential of objectives with large back apertures. Here, if you have the right light source that allows you to fill the back aperture with enough photons, you can better achieve the theoretical NA with brightness (I recently experienced this on an Olympus ‘Super 10’ 10X 0.6NA water and their 25X NA 1.05NA water (multiphoton application).

Thank you all for the answers!
So if I have a sensor with a diagonal of 18mm, a 180mm tube, a 10x lens (my worst case scenario for large FOV) with NA=0.3, the equation provided by @JonD says:
⌀aperture >= Laperture * 18 / 180 + 2 * 0.3 * 18

I just have some doubts on Laperture. Is this the distance from the objective back aperture to the sensor (thus summing all the “vertical” and “horizontal” sections in the light path) or do I have to consider only the distance between the camera port and the sensor or some other length?

Laperture is the distance from the objective back aperture to the a potentially-vignetting element with diameter ⌀aperture. So that distance is inside the microscope body for the filter, but you can probably decently estimate that distance.

Even though low-mag objectives commonly have the largest back apertures, the axial location of the back aperture or back focal plane is usually “less inside” than with high-mag objectives. Olympus is good about listing back aperture positions on their website (https://www.olympus-lifescience.com/en/objective-finder/); other manufacturers will sometimes tell you where the back focal plane is if you ask. (It is also possible to measure it yourself if you have a lens with known focal position and well-collimated light.)

That objective has back aperture diameter 2 * 0.3 * 18mm = 10.8mm, so that’s the second term. Assuming the 25mm filter has 23mm usable aperture, for distance between the objective back aperture and filter less than 122mm you should be fine with 25mm filters. The tube lens is another possible vignetting aperture, but microscope manufacturers will make sure that doesn’t limit you.

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