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.

Most fluorescence microscopes are designed using epi-illumination. In epi-illumination excitation, light from the fluorescence microscope goes through the objective lens and illuminates the object. Light emitted from the specimen into the fluorescence microscope is collected the same. Sometimes the fluorescent molecule in the fluorescence microscope itself is a direct stain or probe for specific structures. In other situations the fluorescent dye is bound to another non-fluorescent probe that recognizes specific structures. For example, the fluorescence molecule in fluorescence microscope, rhodamine may be conjugated to phalloidin, which binds the filamentous actin. One important method to identify specific proteins is to couple fluorescent dyes to antibodies that bind very specifically to macromolecules in the cell in order to view them under a fluorescence microscope. Sometimes the fluorescent molecule itself is a direct stain or probe for specific structures. In other situations the fluorescent dye is bound to another non-fluorescent probe that recognizes specific structures.

Among the common fluorescence dyes used in a fluorescence microscope are fluorescein, which emits green light when exited with blue light and rhodamine, which emits a deep red fluorescence when excited by green-yellow light. Fluorescence microscopes are equipped with three fluorescent filter cubes, each containing specific barrier filters and a beam-splitting mirror. If you cannot locate using the fluorescence microscope you must be able to change the filter cube. The filter cubes for a fluorescence microscope provide blue light excitation fluorescein, green light excitation like rhodamine, and UV excitation for UV absorbing dyes.

Two improved fluorescence microscopes were reported to allow researchers to see individual protein molecules on the surface of a living cell. Both teams of researchers obtained fluorescence images by dipping a needle-like “tip” into the focus of the laser used to create the fluorescence. One team improved the positioning of the tip of fluorescence microscope, while the other channeled the laser light through a narrow aperture before letting it hit the tip. Optical fluorescence microscopy, in which a laser at one wavelength stimulates a sample to fluoresce at a different wavelength, should be a good way to view proteins without disrupting them. But the glaring drawback of normal fluorescence microscope is the resolution, one-half the wavelength of the laser light. That means 250 to 300 nanometers, whereas the average protein is 1 to 10 nanometers across. To break the resolution limit, researchers use the extremely sharp point or tip from an atomic force microscope to enhance the fluorescence signal–one form of a so-called scanning near-field optical microscope. The tip is placed at the focal point of a laser beam–just above the sample surface and the beam and tip move across the sample in tandem. This technique increases the electric field and therefore the fluorescence at the fluorescence microscope tip because charges tend to concentrate at sharp corners, such as the end of a lightning rod. Although this method resolves images at close to 10 nanometers, it is plagued by poor signal quality because metal tips can quench, or siphon excited electrons away from fluorescent dyes, preventing photon emission. The precise fluorescence measurements of the fluorescence microscope showed that the tip would have to touch the sample to get the best fluorescence and maximum resolution, whereas others had let the tip hover a few nanometers above the sample for convenience. Touching the sample increased its fluorescence up to 20-fold beyond the background signal from the laser alone, giving them four times the contrast of previous efforts. They used a silicon tip to avoid quenching, even though metal can theoretically create more fluorescence. By scanning it over the sample a 5 nanometer wide semiconductor nanocrystal sitting on a glass surface was able to observe high quality fluorescence with at least 10-nanometer resolution. An alternative to fluorescence microscope type device is to shine a laser through a narrow glass fiber onto a sample, which reduces background fluorescence. Such fluorescence microscopes usually have higher sensitivity to faint fluorescence but lower spatial resolution than tip-based fluorescence microscopes. The combined sensitivity and resolution by putting a metal-coated tip on the rim of a 100-nanometer-wide hole in a tapered glass fiber. The laser shined down the fiber to illuminate the tip and the sample, a mica surface covered with DNA molecules whose ends were labeled with fluorescent dye.
Although the team did have to contend with quenching, the low background signal allowed to resolve individual dye molecules separated by as little as 10 nanometers. Both groups say their methods should be able to achieve even finer resolution. The new results stand out because of the quality of fluorescence microscope. It clearly demonstrate that tip-enhanced microscopy is possible on the single molecule level” without significant quenching. That could point the way to resolving, mapping, or even manipulating single proteins on cell surfaces with the use of fluorescence microscopes.