Total Internal Reflection Fluorescence Microscopy
Trapezoidal Prism Microscope Configuration
The simplest approach to achieve total internal reflection from a culture chamber on an inverted microscope is direct laser illumination through a glass cube, prism, or trapezoidal block positioned on top of the chamber. This tutorial explores the effects of variations in refractive index and prism side angles on the critical angle and resulting incident laser angles.
The tutorial initializes with the trapezoidal prism side angle set to 48 degrees, which corresponds to an incident angle of 68.4 degrees and a critical angle of 57.6 degrees when the prism refractive index is 1.575. To operate the tutorial, use the Prism Shape slider to adjust the side angles between a value of 45 to 77 degrees. As the slider is translated, new incident angles are calculated and presented in the tutorial window beneath the objective drawing. The Glass Refractive Index slider will modify the critical angle, but will have no effect on the incident angle or prism angle. Use the Beam Width slider to adjust the size of the laser beam entering the focusing lens. Laser emission wavelengths can be adjusted with the Laser Wavelength slider between a range of 395 to 700 nanometers.
When mounted on the condenser unit of an inverted tissue culture microscope, a 60-degree trapezoidal prism is the most convenient and reproducible configuration yet developed for TIRFM above the stage. The incoming laser beam is vertical, so the total internal reflection area shifts laterally to a very small degree when the prism is raised and relowered during specimen changes. In addition, conventional transmitted light techniques (phase contrast, brightfield, etc.) are compatible with this experimental design. Because the incident angle is fixed at 60 degrees, ordinary optical glass (refractive index of 1.52) is not able to support total internal reflection, and a prism having a high refractive index is required. Prisms fabricated with flint glass (refractive index of 1.64) will meet these specifications, and are commercially available. The beam will then refract away from the normal at an angle of 69 degrees in passing from the prism into the coverslip, thereby exceeding the critical angle at the coverslip/buffer or coverslip/cell interface. A trapezoid with walls ranging between 45 and 60 degrees is ideal, but these units are not readily available and must be manufactured to custom specifications. Unfortunately, 45 or 60-degree trapezoids are also not commercially available, but they can be cheaply produced by truncating and polishing the apex of a commercially available triangular prism.
Daniel Axelrod - Department of Biophysics, University of Michigan, 930 North University Ave., Ann Arbor, Michigan 48109.
Matthew J. Parry-Hill and Michael W. Davidson - National High Magnetic Field Laboratory, 1800 East Paul Dirac Dr., The Florida State University, Tallahassee, Florida, 32310.
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