How do I calculate an optimal number of z-sections to be imaged for a given objective (or what interval size should I use when collecting a confocal z-stack)?
To capture all possible axial detail, the z-step spacing should be at least half the z-resolution of the system. For instance, when a 100x 1.4 NA objective is used, the z-resolution (PSF) is 0.5 µm. Therefore sampling should be done at 0.25 µm steps at minimum. For 60x 1.4 NA, the z-resolution is 0.8 µm; therefore, sampling should be done at 0.4 µm steps at minimum. During 3D rendering, an aspect ratio of the imaged sample is required. The aspect ratio is calculated by dividing the z-step size by the XY resolution. The XY resolution is usually measured; however, it can be calculated by dividing the pixel size by the magnification. For example, an ORCA ER camera has a pixel size of 6.4×6.4 µm. When a 100x objective is used, the XY resolution is 6.4/100= 0.064 µm. Therefore, the aspect ratio for a 100x objective at 0.25- µm steps would be 0.25/0.064= 3.9. When a 60x objective is used, the XY resolution is 6.4/60= 0.106 µm. The aspect ratio at 0.4 µm steps would be 0.4/0.106= 3.7. If the
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