Fluorescence Microscope: Introduction, Principle, Components, Application

Fluorescence Microscope: Introduction, Principle, Components, Applications and Keynote

Introduction of Fluorescence Microscope

The fluorescence microscope is showing its special parts as shown above picture. It is an 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 is the emission of light by a substance that has absorbed light or other electromagnetic radiation. Phosphorescence is a specific type of photoluminescence related to fluorescence. Unlike fluorescence, a phosphorescent material does not immediately re-emit the radiation it absorbs. Sir George G. Stokes, a British scientist first described fluorescence in 1852 and observed that the mineral fluorspar emitted red light when it was illuminated by ultraviolet excitation. He noted that fluorescence emission always occurred at a longer wavelength than the excitation light. This shift towards a longer wavelength is known as Stokes Shift. The “fluorescence microscope” refers to any microscope that uses fluorescence to generate an image, whether it is a more simple setup like an epifluorescence microscope, or A more complicated design such as a confocal microscope, which uses optical sectioning to get better resolution of the fluorescent image. The development of super-resolved fluorescence microscopy that brings optical microscopy into the nano dimension made Nobel laureates in Chemistry to Eric Betzig, William Moerner, and Stefan Hell On 8 October 2014,

Principle of Fluorescence Microscope

The specimen is illuminated with light of a specific wavelength. and it is absorbed by the fluorophores, causing them to emit light of longer wavelengths. The illumination light is separated from the much weaker emitted fluorescence through the use of a spectral emission filter. The most common filter is the dichroic filter. A dichroic filter or thin-film filter is a very accurate color filter used to selectively pass light of a small range of colors while reflecting other colors. A fluorophore is a fluorescent chemical compound that can re-emit light upon light excitation and it typically contains several combined aromatic groups, or plane or cyclic molecules with several π bonds.

Components of Fluorescence Microscope

The typical components are-

Fluorescent dyes (Fluorophore): There are many fluorescent molecules called fluorophores or fluorochromes such as fluorescein, Alexa Fluors, or DyLight 488, which can be chemically linked to a different molecule that binds the target of interest within the sample.
A light source (xenon arc lamp or mercury-vapor lamp or high-power LEDs and lasers): Four main types of light sources are used, including xenon arc lamps or mercury-vapor lamps with an excitation filter, lasers, and high-power LEDs.
The excitation filters
The dichroic mirror
The emission filters.

Epifluorescence microscope is the most fluorescence microscope in use where excitation of the fluorophore and detection of the fluorescence are done through the same light path (i.e. through the objective). This microscope is widely used in biology and is the basis for more advanced microscope designs.

Epifluorescence Microscope

The majority of fluorescence microscopes, especially those used in the life sciences, are of the epifluorescence design shown in the above picture. Light of the excitation wavelength is focused on the specimen through the objective lens. The fluorescence emitted by the specimen is focused on the detector by the objective. Since most of the excitation light is transmitted through the specimen, only reflected excitatory light reaches the objective together with the emitted light. As a light source, any of four is used as mentioned in components. LASER (light amplification by stimulated emission of radiation) is mostly used for complex fluorescence microscopy techniques, while xenon lamps, and mercury lamps, and LED ( light-emitting diode) with a dichroic excitation filter are commonly used for wide-field epifluorescence microscopes. A xenon arc lamp is a specialized type of gas discharge lamp, an electric light that produces light by passing electricity through ionized xenon gas at high pressure. It produces a bright white light that closely mimics natural sunlight. Many fluorescent stains have been designed for a range of biological molecules. Some of these are small molecules that are intrinsically fluorescent and bind a biological molecule of interest. Major examples of these are nucleic acid stains like DAPI(4′,6-diamidino-2-phenylindole) and Hoechst. DAPI is a fluorescent stain that binds strongly to A- T rich regions in DNA and Hoechst. Hoechst stains are part of a family of blue fluorescent dyes used to stain DNA. A major example of fluorescent stain is phalloidin that is used to stain actin fibers in mammalian cells.

Applications of Fluorescence Microscope

To recognize structures in fixed and live biological specimens
Because it allows for multicolor staining, labeling of structures within cells, and evaluation of a cell’s physiological status, it is a common technique in today’s life science.
It is also a common application in diagnostic laboratories. e.g. Staining Mycobacterium tuberculosis for Auramine-rhodamine staining, Immunohistopathology, etc.

Keynote

Technique based classification of Fluorescence microscopy-

Fluorescent Widefield Microscopy
Point Scanning Confocal Microscopy
Parallelized Confocal Microscopy (Spinning Disk)
2-Photon Microscopy
Light Sheet Microscopy
Total Internal Reflection Microscopy (TIRF)
Super-resolution microscopy

Mycobacterium under Fluorescence Microscope

Nocardia asteroides, auramine O Stained Slide Under Fluorescence Microscope

Acridine Orange Stained Smear Showing Structures of Staphylococcus aureus Under Fluorescence Microscope

universe84a