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In fluorescence microscopy a point source of excitation light is focused on the specimen as a diffraction limited spot, which, in the case of a high NA oil objective (NA=1.4 or above) ,for example, will have a diameter of 200-300 nm, depending on the excitation wavelength. The blurred extended diffraction image is described by a point-spread function, and is a measure of image degradation. It can be determined theoretically using the microscope control software or commercial algorithms, or by taking image stacks through sub-resolution fluorescent beads mounted in the same medium and emitting at a similar wavelength as fluorophores as the specimen. Alternatively, it can be calculated theoretically using the microscope control software or commercial algorithms.
Working Distance (WD)
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