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. |
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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
Optical Filters for Laser-based Fluorescence Microscopes
Additional Resources
Nikon's Microscopy U - TIRF Overview
JoVE - Measuring Near Plasma Membrane and Global Intracellular Calcium Dynamics in Astrocytes