epifluorescenza

Epifluorescenza is a fluorescence microscopy technique where the excitation light illuminates the specimen from above, through the objective lens, and the emitted fluorescence is observed through the same objective lens. This configuration is in contrast to transmitted light microscopy where the light source is below the specimen. In epifluorescence, excitation light, typically from a high-intensity lamp or a laser, passes through an excitation filter that selects a specific wavelength. This filtered light then travels to a dichroic mirror, which reflects it down through the objective lens and onto the sample.

When the excitation light strikes fluorophores within the sample, these molecules absorb the energy and then re-emit light at a longer wavelength. This emitted fluorescence is then collected by the objective lens and travels back towards the dichroic mirror. The dichroic mirror, designed to transmit longer wavelengths, allows the emitted fluorescence to pass through to an emission filter. This emission filter blocks any remaining excitation light, allowing only the fluorescence signal to reach the detector, which is usually the observer's eye or a camera.

The primary advantage of epifluorescence microscopy is its efficiency in detecting weak fluorescence signals, as the excitation and emission paths are optimized. It is widely used in biological research for visualizing specific cellular structures, proteins, or molecules that have been labeled with fluorescent dyes or probes. The technique allows for high contrast imaging and can be used for both fixed and live specimens, depending on the fluorophores and experimental conditions.