Total Internal Reflection Fluorescence (TIRF) Microscopy

Abstract

Total internal reflection fluorescence (TIRF) microscopy (TIRFM) is an elegant optical technique that provides for the excitation of fluorophores in an extremely thin axial region ("optical section"). The method is based on the principle that when excitation light is completely internally reflected in a transparent solid (e.g., coverglass) at its interface with liquid, an electromagnetic field, called the evanescent wave, is generated in the liquid at the solid-liquid interface and is the same frequency as the excitation light. Since the intensity of the evanescent wave exponentially decays with distance from the surface of the solid, only fluorescent molecules within a few hundred nanometers of the solid are efficiently excited. This overview will review the history, optical theory, and hardware configurations used in TIRFM. In addition, it will provide experimental details and methodological considerations for studying receptors at the plasma membrane in neurons. © 2022 Wiley Periodicals LLC.

Keywords: axial resolution; fluorescence microscopy; live cell imaging; neurons; receptor trafficking.

Figure 1

Information revealed by standard epifluoresence (A) and TIRF microscopy (B). HeLa cells were cultured on 18-mm round 1.78 RI coverslips. Post fixation with 4% paraformaldehyde, rhodamine-conjugated phalloidin was used to visualize F-actin. The cell was imaged in HBSS using a 100× 1.65 NA objective and 1.78 RI immersion liquid. Both images were taken at the same plane with the same exposure settings.

Figure 2

The evanescent wave. (A) The incident angle can be described using a coordinate system arranged to display all three orthogonal directions. The x-y plane represents the interface between the coverglass and the cell cytosol. The plane of incidence is the x-z plane, which is parallel to the excitation light beam. (B) The evanescent field intensity decays exponentially with increasing distance from the interface according to Equation 3. The penetration depth, which is usually between 50 and 300 nm, decreases as the reflection angle grows larger and is dependent on the refractive indices at the interface and the illumination wavelength. Illustrations were adapted from ones provided by Michael W. Davidson, Florida State University.

Figure 3

TIRFM Calculator. The calculation of max and critical angles, penetration depth, and evanescent field intensity for performing TIRFM has been simplified by an ImageJ plugin called Calc TIRF (https://imagej.nih.gov/ij/plugins/tirf/index.html ) written by Sebastian Rhode. Using this calculator, the max and crtical angles were calculated for the popular 60x 1.49 N.A. objective (inset text A). (A) Penetration depth curves for multiple laser lines and (B) the evanescent field intensity curve is shown.

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.

Figure 5

Visualizing the arrival of VSVG-GFP to the PM. VSVG-GFP plasmid DNA was transported into neurons by electroporation prior to plating. At 7 days in vitro, the trafficking of VSVG-GFP vesicles to the PM was imaged by TIRFM using a 60× 1.45 NA objective at 11.32 frames per sec. The panels labeled 1 through 10 are sequential frames. As a VSVG-GFP vesicle nears the PM, it becomes brighter. Upon fusing with the PM, the signal rapidly diffuses. Bar = 2 μm.

Figure 6

Visualization of Y1r arrival at the PM by TIRFM. (A) Taken shortly after NPY-488 was added to the culture medium. The signal-to-background ratio is not affected by the presence of free fluorescently labeled agonist in the medium. (B) Captured 10 min after the image in A. Note the loss of 488 fluorescence along the dendritic branches and in some cases (open arrows) the detection of new receptors at the PM. Bar = 2 μm.