Fluorescence Microscopy Basics

What is the primary purpose of fluorescence microscopy?

What type of light is typically used to irradiate tissue sections in fluorescence microscopy?

What is the phenomenon that allows fluorescence microscopy to work?

Which compound is used as a fluorescent stain that binds to both DNA and RNA?

What do compounds like DAPI and Hoechst specifically stain?

What is the characteristic fluorescence color emitted by DAPI and Hoechst when bound to DNA?

What type of molecules are coupled with fluorescent compounds like fluorescein to identify specific cellular structures?

What is the purpose of using filters in a fluorescence microscope?

What is the term used to describe the binding of fluorescent compounds to specific cell macromolecules?

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.

Learn about the fundamentals of fluorescence microscopy, including the phenomenon of emission of light with a longer wavelength when irradiated, the use of UV light for tissue sections, and the appearance of fluorescent substances under microscopy.