Fluorescence microscopy is a widely used phenomenon to study some special types of molecules. The principle of the fluorescence microscopy is based on the fact that some molecules when are illuminated with a light of specific wavelength, emit a different light with a longer wavelength which causes emission of light of a different color. Such substances are called fluorophore. This happens due to the molecular structure of those substances. A fluorescence microscope has components like a xenon arc or mercury vapor lamp for illumination. In more advanced microscopes, high power LED lamps or lasers are used. In addition to that, they have excitation filters, a dichroic beam splitter, and an emission filter.
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Most of the fluorescence microscopes used are epifluorescence microscopes. In this type of microscope, the excitation of the fluorophore and the detection of the fluorescence is performed in the same path of light. Such microscopes are mainly used in the field of biological research. In these microscopes, the excitation light is focused on the sample with the help of the objective lens, and the fluorescence that is emitted by the sample is focused by the same objective lens on the detector. It is important that the objective lens has a greater numeric aperture for a better resolution of the images.
Preparation of Sample for Fluorescence Microscopy
To perform the Fluorescence microscopy on a sample, it is essential that the sample is also fluorescent. There are several ways in which the fluorescent samples can be created such as labelling with fluorescent stains. For biological samples, the sample is bonded with fluorescent proteins. The fluorescent proteins can easily be created with the help of the modern DNA altering techniques and genetics.
Another technique that is used for the preparation of fluorescent specimen is the Immunofluorescence In which the sample is bonded with an antibody or antigen which labels the specific proteins in a cell. A fluorophore is directly attached to the primary antibody in this technique.
Limitation of Fluorescence Microscopy
The fluorophores are not affective when they are subjected to a phenomenon called photobleaching. When the fluorescent molecules are excited, then there are electrons released from the molecule which causes photobleaching. With photobleaching, there is very limited time for the users to observe the sample during the fluorescent microscopy.
Applications of Fluorescence Microscopy
The fluorescence microscopy can be used for many different applications such as Imaging of the structures of very small specimen such as cells. It is also widely used studying the cell populations and imaging of the genetic materials. This method of microscopy is also used for viewing a specific cell within a large group of cells.
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