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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]
In both cases the numerical aperture of the objective is 1.49 and the refractive index of the medium 1.52. The wavelength of the emitted light is assumed to be 600 nm and, in case of the confocal microscope, that of the excitation light 500 nm with circular polarization. A section is cut to visualize the internal intensity distribution.
In epifluorescence microscopy, the objective lens acts not only as a magnifier for the light emitted by the fluorescing object, but also as a condenser for the incident light. The Arlow-Abbe condenser is a modified Abbe condenser that replaces the iris diaphragm, filter holder, lamp and lamp optics with a small OLED or LCD digital display unit.
The optical microscope, also referred to as a light microscope, is a type of microscope that commonly uses visible light and a system of lenses to generate magnified images of small objects. Optical microscopes are the oldest design of microscope and were possibly invented in their present compound form in the 17th century.
These lenses are often color coded for easier use. The least powerful lens is called the scanning objective lens, and is typically a 4× objective. The second lens is referred to as the small objective lens and is typically a 10× lens. The most powerful lens out of the three is referred to as the large objective lens and is typically 40–100×.
By virtue of the linearity property of optical non-coherent imaging systems, i.e., . Image(Object 1 + Object 2) = Image(Object 1) + Image(Object 2). the image of an object in a microscope or telescope as a non-coherent imaging system can be computed by expressing the object-plane field as a weighted sum of 2D impulse functions, and then expressing the image plane field as a weighted sum of the ...
Raman microscopy, and in particular confocal microscopy, can reach down to sub-micrometer lateral spatial resolution. [7] Because a Raman microscope is a diffraction-limited system, its spatial resolution depends on the wavelength of light and the numerical aperture of the focusing element. In confocal Raman microscopy, the diameter of the ...
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.
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