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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]
For a pinhole-to-film distance of 1 inch (25.4 mm), this works out to a pinhole of 0.185 mm in diameter. For f = 50 mm the optimal diameter is 0.259 mm. The equivalent f-stop value is f /193. The depth of field is basically infinite, but this does not mean that no optical blurring occurs.
Depth of field is a significant concern in macro photography, however, and there one sees smaller apertures. For example, the Canon MP-E 65mm can have effective aperture (due to magnification) as small as f /96. The pinhole optic for Lensbaby creative lenses has an aperture of just f /177. [13]
The hyperfocal distance has a property called "consecutive depths of field", where a lens focused at an object whose distance from the lens is at the hyperfocal distance H will hold a depth of field from H/2 to infinity, if the lens is focused to H/2, the depth of field will be from H/3 to H; if the lens is then focused to H/3, the depth of ...
Pinhole cameras have a couple of advantages over lenses - they have infinite depth of field, and they don't suffer from chromatic aberration, which can be cured in a refractive system only by using a multiple element lens. The smallest feature which can be resolved by a pinhole is approximately the same size as the pinhole itself.
A 4Pi microscope is a laser scanning fluorescence microscope with an improved axial resolution. With it the typical range of the axial resolution of 500–700 nm can be improved to 100–150 nm, which corresponds to an almost spherical focal spot with 5–7 times less volume than that of standard confocal microscopy .
With no modification to the microscope, i.e. with a simple wide field light microscope, the quality of optical sectioning is governed by the same physics as the depth of field effect in photography. For a high numerical aperture lens, equivalent to a wide aperture , the depth of field is small ( shallow focus ) and gives good optical sectioning.
As an example, the figure on the right shows the 3D point-spread function in object space of a wide-field microscope (a) alongside that of a confocal microscope (c). Although the same microscope objective with a numerical aperture of 1.49 is used, it is clear that the confocal point spread function is more compact both in the lateral dimensions ...