Fluorescence Microscopy

Hello everyone,

Today I will be talking about some important point about Fluorescence Microscopy
 Before going to the main topic i would like to discuss some important points

After irradiation with Ultraviolet, certain substance have property of emitting radiation of their own at a wavelength longer than that of the exciting source.

If the luminescence has capability of persisting for an appreciable period of time, the luminescence is called phosphorescence, But if the luminescence has capability of persisting only while the exiting source acts a on the substance  the luminescence is called Fluorescence. 

Also fluorescence is observed in specimen that have not been treated with fluorochrome , the emission is called Autoflourescence. The fluorescence acquired after treatment of a specimen with a fluorochrome is called Secondary fluorescence 

In fluorescence microscopy, the sample you want to study is itself the light source. The technique is used to study specimens, which can be made to fluoresce..The "fluorescence microscope" refers to any microscope that uses fluorescence to generate an image, whether it is a more simple set up 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.

Principle

The fluorescence microscope is based on the phenomenon that certain material emits energy detectable as visible light when irradiated with the light of a specific wavelength. 

A suitable exiting light source is employed which passes through exiting filters. Four main types of light source are used, including xenon arc lamps or mercury-vapor lamps with an excitation filter,lasers, super continuum sources, and high-power LED's. 

The basic task of the fluorescence microscope is to let excitation light radiate the specimen and then sort out the much weaker emitted light to make up the image. First, the microscope has a filter that only lets through radiation with the desired wavelength that matches your fluorescing material. The radiation collides with the atoms in your specimen and electrons are excited to a higher energy level. When they relax to a lower level, they emit light.

To become visible, the emitted light is separated from the much brighter excitation light in a second filter. Here, the fact that the emitted light is of lower energy and has a longer wavelength is used. The fluorescing areas can be observed in the microscope and shine out against a dark background with high contrast.

How specific areas detected

If a fluorescent dye can be made to interact with specific cellular components - attached to an antibody that binds to a cellular protein, for example - then it can be used as a probe for microscopy. A specimen stained with this probe may be illuminated with pure, filtered light corresponding to its excitation wavelength and then viewed through an emission filter which is opaque to all other light except for its emission wavelength. The structures tagged with the fluorescent probe will appear to light up against a black background in a (hopefully) high contrast image.

Mounting Mediums

• Xylene or toluene solution of isopbutyl methcrylate is satisfactory mounting medium for temporary mounts.
• Liquid petroleum or glycerol
• Auramine stained with dry lenses

Fixatives

Formalin,alcohol, acetic acid Formalin, Regaud's fluid. the most common in use is Picric acid

What can you do with a fluorescence microscope?

Determine the localisation of specific (multiple) proteins

Determine the shape of organs, cells, intracellular structures  

Examine the dynamics of proteins

Study protein interactions or protein conformation

Examine the ion concentration etc.

 with love 
-Dixy