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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.
(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.
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]
As the complete stack of image planes may be calculated from a single hologram, it is possible to use any passive autofocus method to digitally select the focal plane. [7] The digital auto focusing capabilities of digital holography opens up the possibility to scan and image surfaces extremely rapidly, without any vertical mechanical movement.
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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.
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).