Fluorescence Microscopy: Working, Principal, Advantages, Limitation And Application.

 Fluorescence Microscope

⦁ A fluorescence microscope is a type of light microscope that uses a higher intensity (lower wavelength) light source that exists a fluorescent molecule called fluorophore (also known as fluorochrome).

⦁ Fluorescence is a phenomenon that takes place when the substance (fluorophore) absorbs light at a given wavelength and emits light at a higher wavelength.

⦁ Thus, fluorescence microscopy combines that magnify properties of the light microscope with fluorescence technology.

⦁ The fluorophore absorbs photons leading to electrons moving to a higher energy state (excited state). When the electrons return to the ground state by losing energy, the fluorophore emits light of a longer wavelength.

Working Mechanism:

⦁The specimen to be observed are stained or labeled with a fluorescent dye and then illuminated with high-intensity ultraviolet light from a mercury arc lamp.

⦁ The light passes through the exciter filter that allows only blue light to pass through.

⦁ Then the blue light reaches dichronic mirror and reflected downward the specimen.

⦁ The specimen labeled with fluorescent dye absorbs blue light (shorter wavelength) and emits green light.

⦁ The emitted green light goes upward and passes through dichronic mirror, reflects back blue light and allows only green light to pass the objective lens, then it reaches the barrier filter which allows only green light.

⦁ The filtered fluorescent emmision is sent to the detector where the image can be digitized.

Principal:

                               

⦁ Light source such as Xenon or Mercury Arc Lamp which provides light in a wide range of wavelength, from ultraviolet to the infrared is directed through an exciter filter (selects the excitation wavelength).

⦁ This light is reflected toward the sample by a special mirror called a dichronic mirror, which is designed to reflect light only at the excitation wavelength.

⦁ The reflected light passes through the objective where it is focused onto the fluorescent specimen.

⦁ The emission from the specimen are in turn, passed back up through the objective where the magnification of the image occurs and through the dichronic mirror.

⦁ This light is filtered by the barrier filter, which selects for the emission wavelength and filters out contaminating light from the arc lamp or other sources that are reflected off from the microscope components.

⦁ Finally, the filters fluorescent emission is sent to the detector where the image can be digitized.

Application:

                               

⦁ To identify structure in fixed and live biological samples.

⦁ Fluorescence microscopy is a common tool for today's life science research because it allows the use of multicolor staining, and labeling of structures within cells.

⦁ Study of microorganism that causes the disease and how they caused diseases.

⦁ Study of the position of protein within a cell.

⦁ Study of the pathway of protein.

⦁ Chromosomal study in fishes.