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. Author manuscript; available in PMC: 2023 Aug 1.
Published in final edited form as: Curr Protoc. 2022 Aug;2(8):e517. doi: 10.1002/cpz1.517

Figure 4.

Figure 4

TIRFM System calibration. (A) Schematic drawing of a TIRF microscope that uses a micrometer to position a fiber that delivers laser light to the microscope. (B) Schematic drawing of how to use a hemicylindrical glass prism to determine the angle of incidence. (C) Schematic drawing of how to empirically determine the penetration depth of the evanescent wave using 1-μm diameter fluorescent microspheres. Note that in the example 370 equals the measured radius of the microsphere and that a is calculated using the Pythagorean theorem and 500-a = the empirically determined penetration depth. (D) Demonstrates how the objectives numerical aperture and the illumination wavelength affect the penetration depth of the evanescent field. Panel A was provided by Michael W. Davidson, Florida State University.