The following guidelines offer an outline of the basic requirements for TIRFM microscope configuration using a high numerical aperture objective. 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.

• Inverted 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.

• High Numerical Aperture Objective - Objectives having a numerical aperture ranging between 1.4 and 1.65 are suitable for TIRFM.

• Optical Mounts - A small assortment will be required including a lens mount, optical fiber coupler, and possibly a beamsplitter holder.

• Optical Elements - Fiber optics or lenses as required to couple the laser beam expander to the microscope port, a plano-convex lens of short radius of curvature or alternatively, a hemispherical or triangular prism. In addition, a converging lens having a focal length of several centimeters will be necessary.

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

Procedure

1 - Place a bare coverslip on the microscope stage. Add immersion oil between the objective and the coverslip and focus on the upper surface of the coverslip.

2 - Remove all obstructions between the coverslip sample and the room ceiling. Allow a collimated laser beam, the "raw" beam, to enter the standard epi-illumination port and field diaphragm along the optical axis. A large area of laser illumination will be seen on the ceiling, roughly straight up.

3 - Next, place the triangular or hemispherical prism or plano-convex lens (flat side down) on the coverslip over the objective, making optical contact through a layer of immersion oil. This prism or lens is not going to be used in actual experiments. It is used here to avert total internal reflection and thereby couple light out of the coverslip and on to the wall or ceiling of the room. During this step, safety goggles are advisable.

4 - Reposition the laser beam with the mirrors placed upbeam from the microscope so that the beam still enters the center of the field diaphragm, but now at a small angle to the optical axis. This angle should be continuously adjustable. Slowly increase the angle. The ceiling laser illumination will "set" to an ever-lower position on the wall until it just disappears. At this angle, it is just blocked by the internal aperture of the objective. Back off the entrance angle so that half the illuminated area is visible.

5 - Place the converging lens about 200 millimeters "upbeam" from the field diaphragm and concentric with the incoming beam. The illuminated region on the wall will now be a different size, probably smaller.

6 - Move the converging lens longitudinally (along the axis of the laser beam) to minimize the illuminated region on the wall. This will occur where the converging lens focal point falls exactly at a plane outside the microscope equivalent to the objective's rear focal plane. At this position, the beam is also focused at the objective's actual rear focal plane and emerges from the objective front lens in a roughly collimated form.

7 - Following the focusing step, fine-tune the lateral position of the converging lens and the raw beam mirrors so that the beam on the wall is just barely above the point at which it disappears. This ensures that the beam is propagating upward along the inside periphery of the objective.

8 - Now, verify that total internal reflection is achievable by moving to the sample to a clean, new spot that is not under the prism. Almost no light should emerge, except for some scattering, because of total internal reflection at the glass coverslip-air interface.

9 - Replace the bare coverslip with an identical one coated with the fluorescent dye DiI. Place a droplet of water on the DiI coverslip directly above the objective, and again verify that no light emerges even at this glass-water interface.

10 - Next, view the DiI fluorescence through the microscope with the illuminated region circular or elliptical and roughly centered. Fine-tune the mirrors and converging lens to center the total internal reflection fluorescence in the field of view.

11 - The size of the total internal reflection fluorescence area on the sample is directly proportional to the laser beam size at the field diaphragm. To change this size, replace the converging lens with another one of different focal length, but always keep its focal point at the objective's equivalent rear focal plane, which is at a fixed position upbeam from the microscope.

12 - Replace the DiI coverslip with the actual cell sample. When the cells are in-focus, the total internal reflection optics should be perfectly aligned without need of further adjustment.

13 - If switching back and forth between total internal reflection and epi-illumination is desirable, design the optics with movable mirrors that are easily reachable and can select angular or straight-on laser paths.

14 - If interference fringe total internal reflection is chosen, use a beamsplitter and arrange it so that the second beam enters the center of the field diaphragm from an approach at the same angle, but at a different azimuthal position around the optical axis. A relative azimuthal angle of 180 degrees will give the closest spaced fringes. It may be most convenient to use the same converging lens for both beams. In this case, the converging lens should be positioned on-axis, but each beam should enter off-axis so they still arrive at the center of the field diaphragm. Be sure that any difference in path length of the two beams from the beamsplitter to their intersection point is less than the coherence length of the laser (a few millimeters). Otherwise, interference will not occur.

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