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In epi-illumination scanning confocal microscopy, the laser light source and photomultiplier detectors are separated from the specimen by the objective lens, which acts as a condenser and objective combination. The light path includes internal filters and neutral density filters that can be inserted into the light path by the operator. The excitation laser beam is expanded to fill the objective rear aperture, and the expanded light is scanned across the specimen using galvanometer mirrors. The pinhole aperture is a critical component that acts as a spatial filter at the conjugate image plane, excluding out-of-focus fluorescence signals and eliminating stray light. The pinhole aperture is adjustable, allowing the operator to control the optical section thickness and exclude fluorescence signals from planes away from the focal point. When comparing widefield and confocal microscopes, the type of illumination used is a key difference. Widefield microscopes use a wide cone of illumination that simultaneously illuminates a large volume of the specimen, resulting in a significant amount of background light and autofluorescence. Confocal microscopes, on the other hand, use a focused spot of illumination that is centered in the focal plane, allowing for a much smaller amount of background light and higher image contrast. The size of the illumination spot in confocal microscopes can range from 0.25 to 0.8 micrometers in diameter, depending on the objective numerical aperture and other factors. Overall, confocal microscopes provide higher resolution and image contrast than widefield microscopes due to their more targeted and controlled illumination. In laser scanning confocal microscopy, a focused laser beam is scanned across a specimen in a raster pattern to generate an image. The beam is moved along the x and y axes using galvanometer mirrors, and the fluorescence emission is collected by the objective and passed through the confocal optical system. The fluorescence emission is then focused at a pinhole aperture and converted into an analog electrical signal by a photomultiplier. The analog signal is periodically sampled and converted into pixels by an analog-to-digital converter, and the image information is temporarily stored in an image frame buffer card before being displayed on a monitor. The confocal image is reconstructed point by point from emission photon signals, but it cannot be observed through the microscope eyepieces. Write your response using less than 100 words.
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