Hello all. Can someone shed light on the relationship between canvas size, Nyquist sampling and FOV in confocal microscope?

I can calculate the needed pixel size for Nyquist sampling if I know the lens NA, magnification and excitation wavelength. In the commercial confocal acquisition software, it doesn’t let you enter the pixel size. Instead, one can press the “optimal” or “Nyquist” button to set the canvas size to match the needed pixel size. What is the math behind it? Do I have an image plane that has a max diameter I can use to calculate? I imagine this image plane must be smaller than the objective field number. And FOV is limited by this image plane diameter, not the objective FN.

Field of view depends on the objective magnification and the relative scan area (this is manufacturer specific). You can generally find the approximate field of view (in microns) within the metadata of the image file, however the most accurate method is to use a stage micrometer to determine absolute values for pixel size then multiply by the image array size.

Nyquist sampling can be thought of as the proper method for digitally representing the optical resolution of a microscope. Without getting into the details (which you can read about elsewhere), the commonly held theory is that your pixel size should be 2-3 times your theoretical optical resolution to be sampled according to Nyquist. There are a variety of places to help understand this better - my favorite is here:

But the basics are (0.61*emission center wavelength) / Objective NA gives you lateral resolution. Divide this by your sampling value (2-3) and you have your ideal pixel size.

Most vendors use some value between 2-3x for their ‘Optimal’ multipliers – but the precise value does vary between them so to know what your specific scalar is you’d have to tell us your confocal model. But generally speaking, the pixel size is reported either in the software interface (usually right next to the frame size or scan area) or in the metadata of the image file, so it is quite easy to find.

just to add to @jasonkirk’s answer. Most confocal acquisition software offers you one (or both) of two modes when you click the nyquist button (sounds like you have the first one).

keep the total number of pixels fixed, and adjust the field of view

keep the field of view fixed, and adjust the number of pixels

beyond the nyquist math, the fov math is super basic: fov = num_pixels * pixel_size

likely, your software also has some maximum number of pixels? and yes… the final fov will also be no larger than what the field number of your system can accomodate, and likely smaller to much smaller depending on additional parameters

Thanks for sharing. If I know max # of pixels my system allows (is this limited by the scan head? sorry, this is another question), can I increase the pixel size (more than Nyquist) to achieve larger FOV at the cost of lateral resolution? I guess my question becomes what sets the limit of FOV in confocal assuming the same objective.