Cytology, General Histology and Embryology - V. K. Napkhanyuk 2002

Microscopes. Microscopy Technique
Microscopy Technique
Fluorescence Microscopy

The main advantage of fluorescence (luminescence) Cell/15.html">Microscopy is The ability to perform vital observations of cytological specimens, including conducting certain cyto- and histochemical reactions on living, unfixed material, with the method demonstrating exceptional sensitivity and Specificity in such cases.

The core principle of fluorescence is that atoms and molecules of certain substances absorb short-wave radiation and enter an excited state. The return transition from the excited state back to the ground state is accompanied by the emission of light, but with a longer wavelength. The general layout of a fluorescence Microscope differs very little from that of a standard Light Microscope (Fig. 2). To excite fluorescence, fluorescence microscopes utilize ultra-high-pressure mercury and xenon lamps as light sources, featuring high brightness within the 0.25-0.4 µm (near ultraviolet) and 0.4-0.5 µm (blue-violet) spectral ranges, operating in an over-voltaged mode.

The rays from the light source are directed by a collector lens and mirror onto the microscope condenser, which focuses them onto the specimen. The specimen is then examined or photographed in its visible fluorescence light using the standard Optical System of the microscope. Two optical filters are incorporated into the light path: the first isolates rays within a narrow spectral band (blue or long-wave ultraviolet) from the overall light source emission to excite fluorescence in the specimen, while the second absorbs these excitation rays but transmits the actual fluorescence emitted by the specimen itself.