Fluorescence Microscopy Basics

Fluorescence Microscopy

• Fluorescence occurs when certain cellular substances emit light with a longer wavelength after being irradiated by light of a proper wavelength.
• In fluorescence microscopy, tissue sections are typically irradiated with ultra-violet (UV) light, and the emission is in the visible portion of the spectrum.
• Fluorescent substances appear bright on a dark background in fluorescence microscopy.

Instrumentation

• The instrument used in fluorescence microscopy has a source of UV or other light.
• The instrument also has filters that select rays of different wavelengths emitted by the substances to be visualized.

Fluorescent Stains

• Fluorescent compounds with affinity for specific cell macromolecules can be used as fluorescent stains.
• Acridine orange binds to both DNA and RNA, emitting slightly different fluorescence, allowing them to be localized separately in cells.
• DAPI and Hoechst stains specifically bind to DNA, emitting a characteristic blue fluorescence under UV, and are used to stain cell nuclei.

Applications

• Fluorescence microscopy can be used to identify specific cellular components by coupling compounds like fluorescein to molecules that will bind to those components.
• Antibodies labeled with fluorescent compounds are important in immunohistologic staining, allowing for the visualization of specific molecules.