Excitation by an Evanescent Wave
Total internal reflection fluorescence microscopy (TIRFM) is employed to investigate the interaction of molecules with surfaces, an area that is of fundamental importance to a wide spectrum of disciplines in cell and molecular biology. This interactive tutorial explores TIRFM excitation of fluorophores residing in the membranes of living cells in tissue culture.
The concept of TIRFM is schematically presented in the tutorial, which depicts selective excitation of fluorophores in a tissue culture cell (refractive index, n = 1.33 to 1.37) resting on the surface of a glass slide (refractive index, n = 1.518). Wavefronts from a virtual tunable laser excitation source pass through the glass and are reflected from the glass-buffer boundary at angles restricted by the applet to reside just above the critical angle, θ(c). A small portion of the reflected light passes through the interface to establish an evanescent wave that travels several hundred nanometers into the cell membrane. Fluorophores residing in the membrane near the glass interface (small dark yellow spheres) are excited by the evanescent wave and subsequently emit secondary fluorescence (blue to red, depending upon the excitation wavelength), while those farther away from the interface are not excited.
To operate the tutorial, first adjust the wavelength of the virtual laser source. As the wavelengths are toggled between 400 nanometers (purple) and 550 nanometers (green), fluorophores residing in the cell membrane are excited and subsequently emit fluorescent wavelengths varying in size from 450 to 700 nanometers (blue to red). The Reflection Angle slider can be employed to adjust this angle, but it is constrained by the applet never to exceed the critical angle, which is defined by the refractive index difference between the glass and water (buffer) layers. To change the refractive index of the glass phase, use the High Refractive Index slider. Equations presented below the slider calculate the critical angle as the refractive index is varied.