However, this calculator is designed to help with the optimization process. For most light microscopes, this usually ranges from 4x to 100x. We will usually not be able to find a setting on spinning disk microscope that allows us to image with both "optimal" confocality of 1 AU and a sampling rate of 2 pixels per airy radius.
This means that when using a 6.5 um pixel camera with a 60x objective, the pixel size at the sample is in fact 6.5/60 0.108 um or 108 nm, resulting in a very high resolution. Thus, we must take advantage of differing combinations of objectives and optical relays to optimize the sampling rate in our digital image. The microscope objective decreases the pixel in size, relative to the magnification. Similarly, though CCD and sCMOS chips have significantly better quantum efficiency than the PMTs used in laser scanning confocals, they do not grant the user the same sort of flexibility with regards to Nyquist sampling that the "arbitrary" pixel size of the PMT does. The formula for the total magnification of a microscope is the objective lens magnification times the ocular lens magnification. where is the wavelength of light, represents the refractive index of the imaging medium as described above, and the combined term sin() is known as the. While newer systems such as the Yokogawa CSU-W1 allow one to change the physical disk and the Spectral Borealis modification to the CSU-X1 grants some flexibility with the size of the "effective" (backprojected) pinhole, we are still largely limited when it comes to the size of the pinhole in spinning disk microscopy. When attempting to match microscope optical resolution to a specific digital camera and video coupler combination, use this calculator for determining the. Decreasing the distance between the line pairs to a spatial period of 0.5 microns (spatial frequency equal to 2000 lines per millimeter) would further reduce contrast in the final image, but increasing the spatial period to 2 microns (spatial frequency equal to 500 lines per millimeter) would produce a corresponding increase in image contrast. Spinning Disk vs Laser Scanning ConfocalsĪlthough it has many benefits with regards to photon efficiency and speed, one of the major disadvantages of a spinning disk confocal microscope is the inability to change the size of the pinhole. The lateral (X-Y) resolution of fluorescence and Raman microscopes is frequently calculated using the famous Rayleigh Criterion for resolution, 0. The calculation of a volume is subject to error propagation, namely the magnification of an error when deriving a figure from one or more measured variables.
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