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Fluorescence Principles

When irradiated with short wavelength light, fluorescent substances emit light of a longer wavelength; non-fluorescent objects, such as the background, remain dark.

This property, possessed by many different materials, is known as primary or auto-fluorescence. The majority of microscopically interesting specimens, however, do not have this property.

In order to achieve the aim in fluorescence microscopy of rendering certain structures visible or of highlighting details for specific analysis, such specimens must first be stained or, in an immunochemical reaction, labelled with a fluorescent dye, known as a fluorochrome.

The light emitted from a substance stained with a fluorochrome is called secondary fluorescence.

Important biomedical applications of fluorescence microscopy include the classic immuno-fluorescence technique for detecting infectious diseases or, as examples of recent molecular-genetic developments, fluorescence in situ hybridisation (FISH) or comparative genomic hybridisation (CGH).

The FISH method is used for direct localisation of genes and other DNA/RNA sequences in chromosomes or tissue (e.g. antenatal karyotyping), while CGH involves examining complete genomes for genetic changes, a method of screening which provides valuable information, particularly for tumour pathology, on all un-balanced genetic changes of the examined DNA.

Picture Leica DMI6000 B

Leica DMI6000 B Light Path