Hello all. I was trying to calculate the illumination area in fluorescence microscopy to estimate the irradiance. Can you help me confirm if the following is correct?
In point scanning confocal, we overfill the objective BFP to create a diffraction-limited illumination spot on the sample. The illumination area = pi * square (0.61*lamda / NA)/(4 * ln(2)). The lamda is the Ex wavelength, and NA is the objective numerical aperture, and 4 * ln(2) is the gaussian beam estimation.
In the case of Yokogawa spinning disk confocal, can I still use the above equation to calculate the single illumination area, and then multiply by the total # of illumination spots (~1000)? In this case, the Ex laser beam didn’t overfill the objective BFP, can each individual beam still create a diffraction-limited spot on the sample plane?
Thank you for your help in advance.