Fluorescence Microscopy

Many dyes exhibit the phenomenon of absorption when they are exposed to light. There are also compounds that emit part of the absorbed energy as light rays. This emission is known as fluorescence if the emission takes place very shortly after the absorption of light.

There are at least three components necessary for the generation and observation of fluorescence. A fluorochrome bound to the object of interest, a high intensity light source, and a detection system (i.e. eye or light sensitive detector). An excitation filter is placed in front of the light source that selectively chooses the wavelengths of light necessary for proper excitation of the fluorochrome. Thus, the choice of the filter varies according to the interested stain that is used. Following excitation, the fluorescing object emits light of wavelengths longer than the excitation wavelength. This light is then selectively passed through a dichroic mirror and collected for visualization.

Images were obtained using a Zeiss Axiovert 100 inverted microscope equipped for both light and fluorescence microscopy. Conventional brightfield imaging, phase contrast, and differential-interference (DIC - Normarki) microscopy were also utilized. These three techniques are especially useful for viewing specimens that absorb visible light, such as histological stains, or for unstained specimens. The fluorescence filter set is equipped for dyes such as fluorescein, DAPI, and rhodamine. An AttoArc HBO 100W mercury lamp was used as the light source. Images were acquired through a Hammamatsu CCD camera that is remotely controlled via Adobe Photoshop through an attached Macintosh PowerPC. Images were obtained using a 63X oil immersion lens.

Cultured epithelial cells triple stained for the nucleus (blue), microtubules (green), and actin (red). Images were acquired with a 20X objective (left) and a 100X objective (right).

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