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TIRF (Total Internal Reflection Fluorescence)

TIRF microscopy exploits the unique properties of an induced evanescent wave or field in a limited specimen region immediately adjacent to the interface between two media having different refractive indices.  With adjustment of the laser excitation incidence angle to a value greater than the critical angle, the illuminating beam is entirely reflected back into the microscope slide upon encountering the interface, and an evanescent field is generated in the specimen medium immediately adjacent to the interface where the fluorophores nearest the glass surface are selectively excited.  


Adjusting the angle of the laser controls the depth of the evanescent wave.

TIRF microscopy

Learn more about TIRF microscopy:

Technical Notes

Working with Optical Density

Laser Damage Threshold

Optical Filters for Laser-based Fluorescence Microscopes

Related Publications

Read the feature article Perfecting TIRF Optics in BioOptics World (Jan/Feb 2009)

Semrock White Paper Series

Maximizing the Performance of Advanced Microscopes by Controlling Wavefront Error Using Optical Filters

Additional Resources

Nikon's Microscopy U - TIRF Overview

JoVE - Measuring Near Plasma Membrane and Global Intracellular Calcium Dynamics in Astrocytes

TIRF Total-Internal-Reflection-Fluorescence