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09 Nov

Fluorescence Microscopy

Posted by Paul

Fluorescence microscopy is a technique where a microscope uses fluorescence to create an image. A sample is illuminated by light of an excitation wavelength and it emits light of a longer wavelength which is collected by the objective. A filter cube is placed in the light path and is located above the objective. The filter cube consists of an excitation filter, dichoric mirror and an emission filter. Xenon arc lamps and mercury-vapor lamps are used as light sources. Light passes through the excitation filter and reaches the sample. The sample is tagged with a fluorophore which releases light in a specific wavelength range. This light then passes through the emission filter and reaches the eyepiece. The dichroic mirror reflects the excitation light while allowing the emission light to pass through. Prior to being viewed with a fluorescent microscope samples must be prepared properly. This may involve the immunostaining of cells. This is a process in which an antibody binds to a specific protein within the cell. A secondary antibody conjugated to a fluorophore may be used to bind to the primary antibody. It is the fluorophore conjugated to the secondary antibody that emits the light that eventually passes through the emission filter.


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