Z-Maximum resolution

I’m using spinning disk confocal of YOKOGAWA to capture 3D live imaging of tissue. I want to calculate the optimal resolution of Z. What kind of equation should I use for that?

  1. My Objective magnification: 63x and Objective N.A: 1.3, it says pinhole FWHM(z) eq.5(um) is 1.45um from the table. Does it mean my maximum resolution is 1.45um?

FWHM axial = [(0.88 × λ Em / (n - (n 2 - NA2)^1/2))2+ ((2^1/2)× n × PH / NA)2]^1/2

5th equation of this site ZEISS Microscopy Online Campus

  1. Then, what should be the correct interval of z stack? Should the maximum resolution(1.45um) be divided by 2 or 3? why?

  2. Even if I measure the resolution of z-stack using beads experimentally, why should I divide the value with 2 or 3?

Your equation #1 purports to be the theoretical axial resolution measured by the FWHM criterion. I am not expert enough to judge its correctness but it seems within reason.

Questions #2 and #3 relate to the question of proper sampling. I won’t pretend to understand the math to derive exactly how much oversampling is “required” but I think it depends on your definition of “required” since the frequency information is rolling off according to the OTF. Practical limitations are also paramount, e.g. are you willing to accept more dose/bleaching to get a bit better axial reconstruction? (there is no universally-correct answer). Nyquist sampling just says that you cannot reconstruct information at higher frequencies than twice the sampling rate, and often people pick that to match the diffraction-limited optical resolution.

Maybe someone with more mathematical chops can chime in.