A TIRFM instrument configuration that is compatible with simultaneous microinjection or patch clamp experiments utilizes a substage prism as illustrated in the tutorial window. In order to provide easy and continuous access from above to a tissue culture bathed in buffer, the prism is deployed below stage level to contact the substrate glass. This tutorial explores multiple total internal reflection by the laser illumination source in designs of this type.

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The tutorial initializes with the laser illumination beam incident on a focusing lens that directs light into a small prism attached to the specimen slide. Light passing through the prism undergoes multiple total internal reflection in the microscope slide or coverslip while simultaneously creating an evanescent field that excites fluorophores in the specimen. To operate the tutorial, use the Lens Position slider to adjust the incidence angle of the laser source. Moving the slider to the right changes the incidence angle only slightly, but this action does affect the position of the specimen area under illumination by the laser. When the slider is moved to the left, the total internal reflection geometry changes until the incident angle forces the light beam to refract through the specimen and above the stage.

The TIRFM configuration presented in this tutorial cannot be employed for all inverted microscopes, and suffers from tight geometry because the objective also resides in the immediate area. In a manner similar to other designs, the substage prism initiates multiple total internal reflection in the coverslip, which transfers excitation light as a waveguide from far off-axis to the center of the viewfield.

In the simplest form, a small triangular prism (commercially available) is placed, via immersion oil or glycerol, in optical contact with the bottom of the coverslip containing the cells and buffer solution. The specimen can be translated while the prism remains laterally fixed, although a smear of oil may be left on the lower side of the coverslip that can destroy the first internal reflection (and the experiment). A feasible alternative is to employ an additional intervening coverslip fixed to the prism with optically transparent glue. The sliding motion occurs between the intervening coverslip and the cell coverslip, which are in optical contact and lubricated by a thin layer of immersion oil or glycerol. A major disadvantage of this configuration is that oil or glycerol immersion objectives cannot be used because the immersion medium could destroy the internal reflections prior to formation of the illumination spot. However, air (dry) or water immersion objectives work very well under these circumstances.