The following guidelines offer an outline of the basic requirements for TIRFM microscope configuration using a prism, laser light source, and a focusing lens. They are intended to serve as a starting point for those interested in exploring fluorescence excitation at an interface of dissimilar refractive indices.

Equipment

• Laser - Choose an air or water-cooled model (argon-ion or helium-neon) with blue or green continuous wave output and at least one watt of total power.

• Fluorescence Microscope - The microscope should be equipped with an epi-illumination dichroic mirror and barrier filter that is appropriate for the laser color, but with the excitation filter removed.

• Prism - Select the prism shape to match the desired configuration (cubic, rectangular, equilateral, trapezoidal, etc.).

• Optical Mounts - A small assortment will be required, including an adjustable beamsplitter support, mirror bracket, and focusing lens holder.

• Focusing Lens - Select a biconvex, positive meniscus, or planar-convex lens having approximately a 50 to 150-nanometer focal length.

• Safety Goggles - To ensure eye safety, purchase a pair of safety goggles certified for the laser source.

Procedure

1 - Mount the prism on the condenser mount carrier (if possible). This need not be done in a precise manner, but only accurate enough for a usable area at the sample-contacting surface of the prism to lay directly in the optical axis of the microscope objective. The mounting may require some custom machining of acrylic or brass plates and use of a glue (e.g., Duco Cement, or the equivalent) that can be easily cracked off and glued again if necessary. If a standard condenser will be used for simultaneous phase contrast or darkfield microscopy, and the condenser mount cannot hold two separate carriers, then the prism must be mounted on a separate holder with the capability of vertical motion. If the microscope focuses by moving the stage up and down, then this separate holder must be fixed to the microscope stage. Otherwise, the prism holder can be fixed directly to the optical table.

2 - Depending on the configuration, a system of mirrors with adjustable angle mounts fixed to the table must be used to direct the beam toward the prism. One of these mirrors (or a system of shutters) should be movable and placed near the microscope so that switching between standard episcopic illumination and total internal reflection is possible without interrupting viewing. Mounts for a focusing lens should also be prepared.

3 - Next, place a coverslip with a uniform dried spread of fluorescent dye in the same type of sample holder to be used for living cell experiments. A convenient, reproducible, and durable film is made from 3,3' dioctadecylindocarbocyanine (also known as "DiI"; available from Molecular Probes, Eugene, Oregon). If DiI is to be used, dissolve it at about 0.5 milligrams per milliliter in ethanol, and place a single droplet of the solution on a glass coverslip. Before the solution dries, rinse off the coverslip with distilled water. A monolayer of fluorophore, which is stable either in air or water, will remain adhered to the glass. Filter sets appropriate for either fluorescein or rhodamine will work with DiI.

4 - With the uniform sample on the stage, focus on the fluorescent surface using transmitted (usually tungsten-halogen) illumination. Often, dust and defects can be easily seen well enough to assay the focus. However, a deliberately scribed scratch in the fluorescent surface can be made to aid this focusing process. Fluorescent epi-illumination can also be used to find the focus, because only at the focal position are laser interference fringes seen sharply.

5 - Place a small droplet of immersion oil the non-DiI surface of the sample coverslip or directly on the prism (depending on which one faces upward in the chosen configuration) and carefully translate the prism vertically so it touches and spreads the oil, but does not squeeze it so tightly that lateral sliding motion is inhibited. Too much oil will bead up around the edges of the prism and possibly interfere with the illumination path.

6 - By naked eye (perhaps with safety goggles to attenuate errant reflections) and without any focusing lens in place, adjust the unfocused ("raw") collimated laser beam position with the mirrors so that total internal reflection occurs directly in line with the objective's optical axis. This can usually be seen by observing the scattering of the laser light as it traverses through the prism, oil, and the total internal reflection surface.

7 - Now, insert the focusing lens so that the focus is roughly at the total internal reflection interface under observation. Again by naked eye, adjust the lateral position with translators on the focusing lens mount (not with the mirrors controlling the raw laser beam) so that the total internal reflection region occurs directly in line with the objective. To guide this adjustment, look for three closely aligned spots of scattered light, corresponding to where the focused beam first crosses the immersion oil layer, where it totally reflects off the sample surface, and where it exits by crossing the oil again.

8 - The total internal reflection region should now be positioned well enough to appear in the view of the microscope when seen as fluorescence with the standard filters in place. In general, the total internal reflection region will appear as a yellow ellipse or streak. Make final adjustments with the focusing lens to center this area. The reflection area can be distinguished from two out-of-focus blurs (arising from autofluorescence of the immersion oil) past either end of the ellipse or streak because the total internal reflection spot contains sharply focused images of defects in the DiI coating. The focusing lens can be moved forward or backward along the laser optical path to achieve the desired size of the total internal reflection area.

9 - With the optics now correctly aligned for total internal reflection, translate the prism vertically to remove the DiI sample, and replace it with the actual cell sample. Lower the prism again to make optical contact. Although the total internal reflection region will not be exactly in the same spot because of irreproducibility in the prism height, it will be close enough to make final adjustments with the focusing lens while observing fluorescence from the cell sample.

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