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Epi-Fluorescence

In Epi-Fluorescence microscopy, the light source is positioned between the eyepiece and the objective. The illuminator is designed to direct light on the sample before making travel through the objective of the microscope, which in this configuration acts as a condenser, and then using the same objective to capture the emitted fluorescence. The advantage of this configuration is that the component objective/condenser will always be in perfect alignment. Most of the excitation light that reaches the sample passes without any kind of influence and the area illuminated corresponds to the observed area.

Scheme of a microscope for Epi-Fluorescence microscopy

The illuminator uses a short arc discharge lamp, usually a mercury or xenon lamp, these lamps are used because they provide a high intensity of light in a very small area, ideal for this type of use. The excitation light travels along the optical axis perpendicular to the illuminator of the microscope, passes through any lenses (heat filter, infrared ...) and finally in the diaphragm. The light passes through the excitation filter that lets only the desired frequency band and blocks unwanted frequencies. The wavelengths you want, reach, therefore, the dichroic mirror, which is a specialized filter to reflect the short wavelengths (excitation) and passing the longer wavelengths. This filter is positioned at 45 ° to the excitation light and reflects this light 90 ° through the optical system up to sample. The emission produced by the illuminated sample is collimated by the lens. Since the emitted light is of longer wavelength light excitation, it is capable of passing through the dichroic mirror, and reach the observer.
Part of the excitation light that reaches the dichromatic mirror is reflected towards the light source, a small part is, instead, absorbed from the lining of the mirror. Before light reaches the eye of the emitted fluorescence, it must first pass emission filter. The filter blocks the excitation light and passes the desired wavelength. In modern microscopes the excitation filter, dichroic mirror, and the transmit filter is incorporated into a single block called block filters.