epifluorescence

Epifluorescence microscopy is a fluorescence microscopy technique in which illumination and detection use the same objective lens, achieving epi-illumination. The specimen is illuminated from above through the objective, and the emitted fluorescence is collected by the same objective and directed to a detector after spectral filtering.

In a typical setup, light from a source such as a mercury or metal halide lamp or LEDs is directed through an excitation filter. A dichroic mirror reflects the excitation light toward the specimen while transmitting the emitted fluorescence, which then passes through an emission filter before reaching a camera or photodetector. The system relies on objective lenses with high numerical aperture to maximize light collection, and detectors such as cooled CCD or sCMOS cameras are commonly used. Fluorophores frequently employed include DAPI, FITC, rhodamine, and fluorescent proteins like GFP.

Applications span a wide range of biological and medical fields. Epifluorescence is widely used for immunofluorescence, localization of cellular structures, and live-cell imaging, often with appropriate environmental control and time-lapse capabilities. It is a workhorse technique in research laboratories for qualitative imaging and, with careful calibration, semi-quantitative analyses of fluorescence intensity.

Advantages include straightforward instrument design, relatively high light efficiency, and broad compatibility with many fluorophores. Limitations include substantial out-of-focus light in thicker samples, leading to lower contrast compared with optical-sectioning methods; photobleaching and phototoxicity from light exposure; and potential autofluorescence from the specimen.