Fluorescence Microscopy

Principle

Many biological objects are large enough to be seen through a microscope but their optical properties are so similar to the surrounding environment that the optical microscope cannot detect them. When such objects are made fluorescent, they can be readily detected by a fluorescence microscope. For instance, endosomes in a microphage cannot be distinguished from other small objects, like granules, fat droplets, etc. They can be visualised, however, when loaded with a red fluorescing dye:

Macrophages in culture, seen through a standard optical microscope (left) and the same cell seen in a fluorescence microscope (right) . In a standard mode of observation cell edges, nucleus and some granules can be seen but endosomes cannot be identified. Fluorescence image reveals the endosomes loaded with a red fluorescent dye, while cell shape, nucleus and various granules are not visible; the blue light, which excites red fluorescence, has been filtered out. The localisation of endosomes within cells can be revealed by superimposing the fluorescence image over the standard (transmitted light) image:

 

Fluorescent molecules absorb light of a given wavelength and subsequently emit light of a longer wavelength. A good example is a popular fluorescent dye, fluorescein. It absorbs blue light and emits green fluorescence (with some yellow and red emission). It is possible to attach molecules of fluorescein to an antibody which binds to microtubuls in a fibroblast, and subsequently illuminate this cell with blue light. In a cell treated that way only microtubules emit green light. If the blue excitation light which excites fluorescence is removed from the final image, microtubules appear as green threads against black background. This method of fluorescently labelling subcellular structures is called immunofluorescence:

An image of a part of fibroblast in culture seen through a fluorescence microscope; green threads are microtubules stained with fluorescein-conjugated antibody.

 

Schematics

Fluorescence microscope is a modification of a standard optical microscope. The diagram below shows main components of a fluorescence microscope. The source of excitation light (Dl1) is usually a mercury arc lamp (LR). Excitation light is separated from fluorescence (Dl2) of a preparation at a dichroic filter (ZD). An image of fluorescing cell components in a specimen (S) is viewed through binoculars (OK) or can be registered by a CCD camera.

 
Basic components of a fluorescence microscope.