Total Internal Reflection Fluorescence Microscopy
Variable Prism Configurations
A majority of the currently utilized TIRFM configurations rely on an added prism to direct laser illumination toward the interface where total internal reflection occurs, which is in the specimen conjugate plane of the microscope. This interactive tutorial explores TIRFM with a variable prism that morphs between a trapezoidal and cubic geometry with adjustable side angles and refractive index.
The tutorial initializes with the trapezoidal prism side angle set to 57 degrees, which corresponds to an incident angle of 65 degrees and a critical angle of 61 degrees when the prism refractive index is 1.52. To operate the tutorial, use the Prism Shape slider to adjust the side angles between a value of 50 and 90 degrees. As the slider is translated, new incident angles are calculated and presented in the tutorial window beneath the objective drawing. The Refractive Index slider will modify both the incident and critical angles. Use the Focus Lens Position slider to adjust the angle of the laser beam entering the prism. Laser emission wavelengths can be adjusted with the Laser Wavelength slider between a range of 395 to 700 nanometers.
As illustrated in the tutorial window, the laser beam first enters a focusing lens positioned obliquely above the microscope stage. The purpose of the lens is to concentrate laser illumination in much the same manner as an objective during epi-illumination, but the lens also serves to narrow the beam width for easier alignment. The focal length of the lens is not critical and can range between 50 and 100 millimeters, but should allow the investigator to easily change the size of the illuminated area over a narrow range. As with other components in the illumination system, the focusing lens should be mounted on an x-y-z translator having one of the axes aligned with the incoming laser beam direction. The translator can be mounted either on the laboratory bench or the microscope stage, but will be easier to align when it is mounted on the stage.
When properly aligned, the collimated and focused laser beam enters the glass cube, and is directed by refraction to strike the total internal reflection interface (beneath the cube's lower surface) with an incident angle exceeding the critical minimum. Neither the size nor shape of the cube is vital, and a prism or rectangular glass block can easily be substituted. Equilateral and 45-45-90-degree prisms are standard commercial items and can be purchased from a variety of sources. A prism with a flat top, such as the cube described above or a truncated triangle, allows placement of a tungsten halogen lamp and condenser system above the prism. In this configuration, conventional illumination techniques such as brightfield, darkfield, phase contrast, and differential interference contrast can be coupled to TIRFM investigations to assist in determining the spatial location of fluorescence originating at the interface.
In cases where a prism is employed (as opposed to a glass block) to achieve total internal reflection, the maximum incidence angle is obtained by introducing the laser beam from the horizontal direction. For standard glass (having a refractive index of 1.52), the maximum incidence angle is 73 degrees for a right angle prism and 79 degrees for an equilateral prism. Phase contrast and other transmitted light techniques are not compatible with this configuration because the upper surface of the triangular prism is not flat. However, custom truncation and polishing of the prism top produces a surface that can easily pass incident light from above by an inverted microscope condenser system.
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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