Fluorescence Microscope

WHAT IS A FLUORESCENCE MICROSCOPE?

admin — Wed, 20 Jun 2007 09:26:29 +0000

A fluorescence microscope is basically a conventional light microscope with added features and components that extend its capabilities. A conventional microscope uses light to illuminate the sample and produce a magnified image of the sample. A fluorescence microscope uses a much higher intensity light to illuminate the sample. This light excites fluorescence species in the sample, which then emit light of a longer wavelength.

A fluorescent microscope also produces a magnified image of the sample, but the image is based on the second light source — the light emanating from the fluorescent species — rather than from the light originally used to illuminate, and excite, the sample.

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Background of the Fluorescence Microscope

admin — Wed, 20 Jun 2007 09:23:19 +0000

The fluorescence microscope is used to detect structures, molecules or proteins within the cell. Fluorescent molecules in the fluorescence microscope absorb light at one wavelength and emit light at another, longer wavelength. When fluorescent molecules absorb a specific absorption wavelength for an electron in a given orbital, the electron rises to a higher energy level (the excited) state. Electrons in this state are unstable and will return to the ground state, releasing energy in the form of light and heat. This emission of energy is fluorescence. Because some energy is lost as heat, the emitted light contains less energy and therefore is a longer wavelength than the absorbed or excitation light. In fluorescence microscope, a cell is stained with a dye and the dye is illuminated with filtered light at the absorbing wavelength; the light emitted from the dye is viewed through a filter that allows only the emitted wavelength to be seen. The dye glows brightly against a dark background because only the emitted wavelength is allowed to reach the eyepieces or camera port of the microscope.

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Specimen Preparation under a Fluorescence Microscope

admin — Wed, 20 Jun 2007 09:21:19 +0000

Studying the dynamics in the cell is essential for understanding cell function. Fluorescence microscopy is one of the most used approaches in studying the location and movement of molecules and subcellular components in the cell using a fluorescence microscope. Usually, cellular components do not fluoresce themselves. Fluorescent markers are therefore introduced in the specimen to be viewed under a fluorescence microscope. Fluorescent dyes are directly taken up by the cells. They are incorporated and concentrated in specific subcellular compartments. The living cells are then mounted on a microscope slide and examined in a fluorescence microscope.

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Fundamentals of Excitation and Emission of the Fluorescence Microscope

admin — Wed, 20 Jun 2007 09:18:05 +0000

The basic function of a fluorescence microscope is to irradiate the specimen with a desired and specific band of wavelengths, and then to separate the much weaker emitted fluorescence from the excitation light.

In a properly configured microscope, only the emission light should reach the eye or detector so that the resulting fluorescent structures are superimposed with high contrast against a very dark or black background when viewed in the microscope. The limits of detection in a fluorescence microscope are generally governed by the darkness of the background, and the excitation light is typically several hundred thousand to a million times brighter than the emitted fluorescence.

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Fluorescence Microscope Introduction

admin — Wed, 20 Jun 2007 09:15:17 +0000

The absorption and subsequent re-radiation of light by organic and inorganic specimens is typically the result of well-established physical phenomena described as being either fluorescence or phosphorescence. The emission of light through the fluorescence process is nearly simultaneous with the absorption of the excitation light due to a relatively short time delay between photon absorption and emission, ranging usually less than a microsecond in duration. When emission persists longer after the excitation light has been extinguished, the phenomenon is referred to as phosphorescence.

British scientist Sir George G. Stokes first described fluorescence in 1852 and was responsible for coining the term when he observed that the mineral fluorspar emitted red light when it was illuminated by ultraviolet excitation. Stokes noted that fluorescence emission always occurred at a longer wavelength than that of the excitation light.

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