Fluorescence Microscopy

Some chemicals absorb light of one wavelength and emit the visible rays of light of the greater wavelength. These substances are known as fluorescent substances and, the phenomenon is termed fluorescence.
It is an optical microscope that uses fluorescence instead of scattering, reflecting, attenuation, and absorption to study the property of the specimen.

By the phenomena of fluorescence, various types of bacteria and antigen-antibody reactions can be detected with accuracy.

Fluorescence Microscopy

Certain substances absorb light of a shorter wavelength and emit a light of a longer wavelength that is visible light such substances are known as fluorescent substances and, this phenomenon is called Fluorescence.

The proton absorbed energy at one wavelength and released it at a different wavelength in a higher wavelength of energy.

History

This phenomenon was first described by the British scientist Sir George strokes. He also coined the term Fluorescence in the year 1852. He observed that fluorescence emission always occurred at a longer wavelength than the excitation light.
This shift towards a longer wavelength is known as the stoke shift.

Principle

It is an ordinary compound microscope but it has a UV light source like a Mercury lamp or a xenon lamp as well as UV filters which screens the unwanted light and allow only the required wavelength of light to enter to be observed by the substance and the light which is emitted is off longer wavelength.
There are two working principles involved in a Fluorescence microscope;

  1. Only those wavelength of light that stimulates the phenomena of Fluorescence in the specimen is extracted from excitation of light and directed on to the specimen.
  2. Only those long wavelength of light which have been created by the fluorescent effect and the extracted light is utilized for the final image formation of the object which shows properties of the fluorescence.

Photo Luminance

It is the phenomenon that describes the process that produces light using light energy.

Phosphorescence

It is a long-lasting phenomenon than the fluorescence of the structure of the molecules at the quantum level. The energy transitions from excited higher energy level to the stable ground level are forbidden by quantum levels.

Working Principle

The specimen is to examine our first stained with fluorescent substances called fluorophores and, then they are viewed under the fluorescent microscope.
The UV light originates from a high-intensity light source like a Mercury or a xenon lamp.
The selective UV light is chosen with the help of an exciter filter.
The exciter filter allows only a narrow band of a specific wavelength to pass through it. The excitation light is redirected by a mirror so that it reaches the sample and as a result, the light of a long wavelength is emitted. This light is collected by the objective lens and re-emitted light passes through the barrier. The barrier filter further allows that light to pass through which is in the visible range of the spectrum. The real image is formed by the objective lens which is converted to a virtual image by the eyepiece.

Filters that are used in fluorescent microscopy

Fluorescence microscopy requires optical filters that have demanding spectral characteristics. There are three categories of filters used in a fluorescence microscope.

  1. Excitation filter [exciter]
  2. Emission filter [barrier filter]
  3. Dichroic beam splitter [dichromatic mirror]
    These are combined to form a block known as a filter cube.

Working

The exciter filter permits the selected wavelength of light to pass through the way towards the specimen. The barrier filters are designed to block or absorb the excitation wavelength and permits only. The related emission wavelength to pass towards the eye. The dichromatic mirrors are specialized filters that are designed efficiently to reflect the excitation wavelength and pass the emission wavelength. They are positioned in the light path after the exciter filter but before the barrier filter and these are oriented at an angle of 45 degrees to the light passing through the exciter filter as well as to the barrier filter. These fluorescent filters are name up of color glasses that are sandwiched between the glass pieces.

Applications

1.To identify drug abuse.

2. To locate certain substances present in the body fluids or tissues.

3. Detection of metallic poisons.

4. For the examination of bar body and y-body to find out the sex of the individual.

5. It is used in fluorescent immunoassay that is to study the antigen-antibody reaction by labeling with fluorescent markers.

6. Main fluorescent stains have been designed with a range of biological molecules.

7. It is used in immunology, cell and molecular biology, trace elements, and serology.

There are two main options for using fluorescent microscopy depending upon what is investigating;

1.Either the specimen itself contains the molecules that show fluorescence.

2. Specific fluorophores dyes have to be added to make the specimen fluorescent in nature.

It is the fluorescent chemical compound that can re-emit light upon; light excitation. For example DAPI and Hoechst.

It is a fluorescent stain that binds strongly to the adenine thymine-rich region in the DNA. It has an inherent binding capacity and is used for the staining of cells.

Green fluorescent protein [GFP] is inherently present itself and thus produce by the organisms themselves.

De-merits

1. Photobleaching or quenching

2. It is the process of fading of fluorescence with time and on repeated exposure to UV radiations.