Controlling the TIRF Penetration Depth is Mandatory for Reproducible Results

Employing a highly flexible TIRF scanner has additional advantages. As mentioned, the system exactly recognizes at which laser positions (or scanner positions) total internal reflection occurs and, vice versa, where this is not the case. The knowledge of exactly where total internal reflection occurs and where not can be used to illuminate the sample in a special so-called HILO mode. If the laser encounters the glass/water interface at an angle lower than the critical angle necessary for total internal reflection, the laser beam will pass through the specimen obliquely. Consequently, only parts of the specimen are encountered by the laser beam and only the fluorophores in this volume are excited. This results in images with low background and a very good signal-to-noise ratio compared to conventional epifluorescence images. An additional benefit of this method is that bleaching and phototoxicity are greatly reduced, which prolongs cell viability, especially in long-term experiments. The HILO mode is particularly suitable for quantitative studies of dynamic movement, the distribution or interaction of molecules and single molecule tracking. A standard application is vesicle tracking.

Another advantage of a TIRF scanner with freely definable in coupling positions is that the direction (the so-called azimuth) from which the laser light encounters the glass/water interface can be chosen. This means that the direction of the wavefront of the evanescent wave can be selected. This, in turn, has an impact on the direction of the propagation of the evanescent wave (horizontal or vertical in respect to the glass/water interface). The propagation direction in turn can influence the efficiency of fluorophore excitation, as they are usually dipoles which have a certain orientation. In the Leica TIRF system it is possible to select between four azimuth positions to ensure optimal excitation of the fluorophore.

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