Definition of Fluorescence Microscope
- A fluorescence microscope is a type of optical microscope that uses fluorescence and phosphorescence instead of, or in addition to, reflection and absorption to study properties of organic or inorganic substances.
- Fluorescence refers to the emission of light rays from an exciting substance, which is excited by UV, blue, etc.
- Phosphorescence refers to as photoluminescence related to fluorescence.
- When some molecules absorb Radiant energy, they become excited and later release much of their trapped energy as light. This light is called fluorescence light, which emitted very quickly by the excited molecule. using this technology fluorescence microscope is Discover.
- In 1852, British scientist Sir George G. Stokes first described fluorescence.
- Eric Betzig, William Moerner, and Stefan Hell developed super-resolved fluorescence microscopy and they got Nobel Prize On 8 October 2014.
Principle of Fluorescence Microscope
The fluorescence microscope exposes a specimen to excited light (UV, blue, etc.) and forms an image of the object with the resulting fluorescent and light. A Mercuric vapour arc lamp produces an intense beam and heat transfer is limited by a special filter.
The light passes through an exciter filter and transmits only the desired wavelength. A darkfield condenser provides a background against which the fluorescent objects glow. Usually, the specimen has been stained with dye, called fluorochromes.
Sample Preparation for Fluorescence Microscope
There are present different types of fluorescent staining techniques such as;
- Biological fluorescent stains
- DAPI, Hoechst, DRAQ5, and DRAQ7 stains are used for the staining of nucleic acids.
- Phalloidin used to stain actin fibers in mammalian cells.
- This method uses the specificity of antibodies to their antigen to target fluorescent dyes to specific biomolecule targets within a cell, and therefore allows visualization of the distribution of the target molecule through the sample.
- Fluorescent proteins
- In this method, different Fluorescent proteins are used such as, Green fluorescent protein (GFP), Yellow fluorescent protein (YFP), Red fluorescent protein (RFP)
Light Path In Fluorescence Microscopy
- Fluorescence microscope uses a high-intensity Mercury lamp as a Source of light. This lamp emits white light..
- The exciter filter transmits only blue lights to the specimen and blocks out all other colors.
- The blue light is reflected downward to the specimen by a dichroic mirror, which reflects the lights of certain colors but transmits light of other colors.
- The specimen is stained with a fluorescent dye certain portions of the specimen retains the dye others do not.
- The stained portion absorbs blue light and emits green light, which passes upward penetrates the dichroic mirror and reaches the barrier filter.
- This filter allows the green light to pass to the eye; however, it blocks out any residual blue lights from the specimen which may not have been completely deflected by the dichroic mirror.
- Thus the eye perceives the stained portion of the specimen as glowing green against a jet black background whereas the unstained portion of the specimen is invisible.
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Parts of Fluorescence Microscope
- Light Source
- Most of the Fluorescence Microscope Uses Mercury light lam as a primary source of light. This lam emits white lights.
- Fluorescent dyes or Fluorochromes
- Fluorescent dyes are a type of chemical compound that emits light rays when they are excited with UV, blue rays.
- It consists of aromatic groups, or plane or cyclic molecules which contain several π bonds.
- Excitation filter
- It is a type of bandpass filter, which passes the wavelengths absorbed by the fluorophore.
- It minimizes the excitation of other sources of fluorescence.
- Block the excitation light in the fluorescence emission band.
- Emission filter
- It is also a type of bandpass filter.
- It only passes only those wavelengths are emitted from a fluorophore.
- It blocks all unwanted wavelengths outside this band – especially the excitation light. As a result of this action, it creates a dark background.
- Dichroic mirror
- It is a type of accurate color filter.
- It only passes small range of colors and reflects other colors.
Application of Fluorescence Microscope
- The Fluorescence microscope has become an essential tool in medical microbiology and microbial ecology.
- Bacterial pathogens can be identified after staining them with fluorescent or specifically labeling them with fluorescent antibodies using immunofluorescence produce.
- In ecological studies, the Fluorescence microscope is used to observe microorganisms stained with Fluorochrome-label probes or Fluorochromes that bind specific call constitutes.
- Another Important use of the fluorescence microscope is the localization of specific proteins within the cell.
Advantages of Fluorescence Microscope
- It is used to study the dynamic behavior exhibited in live-cell imaging.
- It can trace the location of a specific protein in the cell.
- Due to the presence of higher sensitivity, it can detect the 50 molecules per cubic micrometer.
- It allows multicolor staining of the specimen.
Disadvantages of Fluorescence Microscope
- During the process of photobleaching, the Fluorophores lose their ability to fluoresce.
- Fluorescent molecules can generate reactive chemical species during the illumination process which enhances the phototoxic effect.
- The specimen must be stained with the fluorescent dyes.
- Specimen preparation is a costly process.
Images of Fluorescence Microscopy