Fluorescence Microscopy

Certain molecules, by virtue of their chemical structure, have the ability to emit light of a specific wavelength following absorption of light of a shorter, higher energy wavelength. This process of light absorption and re-emission is termed 'fluorescence' and the molecules which exhibit this behavior are termed 'fluorochromes'. All fluorochromes have characteristic light absorption and emission spectra. Upon absorption of photons of the excitation wavelength, fluorochromes become excited into a higher, unstable energy state. This instability is then relieved by the subsequent production of photons of a lower energy emission wavelength. For example, fluorescein - a commonly used fluorescent dye - absorbs blue light and emits green light. The difference in wavelength between a fluorochrome's excitation and emission is termed its Stokes shift after its discoverer.

If a fluorescent dye can be made to interact with specific cellular components - attached to an antibody that binds to a cellular protein, for example - then it can be used as a probe for microscopy. A specimen stained with this probe may be illuminated with pure, filtered light corresponding to its excitation wavelength and then viewed through an emission filter which is opaque to all other light except for its emission wavelength. The structures tagged with the fluorescent probe will appear to light up against a black background in a (hopefully) high contrast image.

The light path of a fluorescence microscope is designed to perform the illumination and the detection described above. All commonly used fluorescence microscopes utilize 'epi-illumination', which means they illuminate the samples by shining light through the objective lens. This design essentially uses the objective lens as a 'condenser' - providing efficient illumination and detection at high numerical apertures. Light is produced by a bright lamp that produces light with spectral peaks corresponding to the fluorescent dyes that to be viewed. Typically a mercury lamp is used for this as these light sources closely match the red, green, and blue dyes most commonly used by biologists. The light is first passed through a heat filter to prevent damage to the specimens, as well as to the microscope itself. It then passes through an excitation filter which blocks all incoming light except for the wavelength of the specific dye being examined. This filtered light then hits a special mirror sitting directly above (or below the objective in the case of an inverted microscope) the objective lens called a dichroic filter. Dichroics are mirrors that have special coatings which make them reflective to certain wavelengths and transparent to other, longer wavelength light. The excitation light is reflected off the dichroic, through the lens, and onto the specimen. If any fluorescent dye in the field becomes excited, its emitted light is collected by the objective and passes through the dichroic. Emitted light then passes through a third emission, or barrier, filter which absorbs all light except for the emitted wavelength. This last filter ensures that any stray excitation light does not contribute to the final image. Once passed through the barrier filter, the distribution of the dye within the sample is registered on your eye or on a camera, producing an image of its distribution. Typically each class of dye (blue, green, or red) has its own set of excitation, dichroic, and emission filters which work together and are associated as a 'cube'. Different cubes are selected by moving a slider or rotating a wheel within the body of the microscope.

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