Microscopy And Study Of Fluorescence Microscope

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Fluorescence Microscopy and Study of Fluorescence Labelled Developing Heart of Chick Embryo

Introduction:
Fluorescence:
The fluorescence microscopy is a kind of optical microscopy in which the phenomena of fluorescence instead of, or in addition to, reflection and absorption are used to generate the image of the specimen under study. When organic or inorganic specimens absorb photon from a radiation of shorter wavelength (UV or visible spectrum), get excited and subsequently emit light of longer wavelength (in the form of visible light), the process is called fluorescence. The molecules that exhibit fluorescence are called fluorochromes or fluorophores.
When electrons go from the excited state to the ground state, there is a loss of vibrational …show more content…

Only the emission light should reach the eye or detector so that the resulting fluorescing areas can be contrasted against a dark background. Most fluorescence microscopes in use are epifluorescence microscopes. This is basically a reflected light microscopy mode where the illuminator and objective lens are positioned on the same side of the specimen, the light does not pass through the specimen and excitation of the fluorophore and detection of the fluorescence are done through the same light …show more content…

The selected excitation wavelength light reaches the dichroic mirror.
3. Dichroic Mirror/Dichromatic Mirror/Beam Splitting Mirror:
A dichroic mirror is a special type of interference filter that efficiently reflects shorter wavelength (excitation) light and efficiently passes longer wavelength (fluorescent) light. It is oriented at 45O angle to the incoming excitation light path and reflects the excitation light at a 90O angle directly through the objective and onto the specimen (Fig.1). It also reflects any scattered excitation light back in the direction of the illuminator.
4. Objective Lens:
The excitation light is first focused on the specimen through the objective lens and then using that same objective the emitted fluorescent light is gathered and sent towards dichroic mirror. The objective lens has a high numerical aperture, thus providing highest sensitivity.

5. Emission Filter/Barrier Filter/Second Barrier Filter:
Before the emitted light can reach the eyepiece or detector, it is incident upon and passes through the barrier or emission filter. This filter blocks any residual excitation light and passes the desired longer emission

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