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Focus stacking – also called focal plane merging, z-stacking, [1] or focus blending – is a digital image processing technique which combines multiple images taken at different focus distances to give a resulting image with a greater depth of field (DOF) than any of the individual source images.
Fluorescence and confocal microscopes operating principle. Confocal microscopy, most frequently confocal laser scanning microscopy (CLSM) or laser scanning confocal microscopy (LSCM), is an optical imaging technique for increasing optical resolution and contrast of a micrograph by means of using a spatial pinhole to block out-of-focus light in image formation. [1]
(a) Optically sectioned fluorescence images of a pollen grain. (b) Combined image. (c) Combined image of a group of pollen grains. [1]Optical sectioning is the process by which a suitably designed microscope can produce clear images of focal planes deep within a thick sample.
PSF Lab is a software program that allows the calculation of the illumination point spread function (PSF) of a confocal microscope under various imaging conditions. The calculation of the electric field vectors is based on a rigorous, vectorial model that takes polarization effects in the near-focus region and high numerical aperture microscope objectives into account.
Simultaneous two-color label-free stimulated Raman scattering z-stack imaging of mouse ear (red: protein, green: lipid, image is 220 by 220 microns the total depth is 60 microns, the pixel dwell time is 2 microsecond).
CombineZ processes a stack of images (or frames) and is most frequently used to blend the focused areas of several partially focused digital photographs, usually close-ups, in order to create a composite image with an extended depth of field, created from the in-focus areas of each image.
Most light sheet fluorescence microscopes are used to produce 3D images of the sample by moving the sample through the image plane. If the sample is larger than the field of view of the image sensor, the sample also has to be shifted laterally. An alternative approach is to move the image plane through the sample to create the image stack. [32]
The two-dimensional optical transfer function at the focal plane can be calculated by integration of the 3D optical transfer function along the z-axis. Although the 3D transfer function of the wide-field microscope (b) is zero on the z-axis for z ≠ 0; its integral, the 2D optical transfer, reaching a maximum at x = y = 0.