Briefly, one of the chief problems with TIRFM is that, while it can be very useful for tracking objects and determining their positions, it only gives you a 2D projection of position, in the focal plane of the microscope objective. Getting information about the z-axis position of an object is very difficult. There are basically two standard ways to get at this information, and a large number of papers proposing different schemes for realizing them:
1) Some information about the z-axis position of objects in the field of view can be determined by the shape of the point spread function. As an object goes out of focus, the point spread function broadens, and by measuring the width of the PSF, you can learn about its z-axis position. See, for instance:
- Three-dimensional tracking of fluorescent nanoparticles with subnanometer precision by use of off-focus imaging
2) The other method is to exploit the fact that the TIRF field has exponentially decaying intensity, and to determine the axial position based on fluorescence intensity. This has a lot of potential problems due to fluctuations in intensity that are not related to axial position (for instance, due to dye fluctuations, blinking, or local environment). So, most of these approaches are differential, comparing intensities at different TIRF angles. Two good examples are:
- Observing secretory granules with a multiangle evanescent wave microscope.
- Variable-angle total internal reflection fluorescence microscopy (VA-TIRFM): realization and application of a compact illumination device.
While this isn't technically (or even mostly) single molecule work, novel approaches to fluorescence imaging is another one of my areas of interest, and I did a significant amount of work on 3D single molecule tracking in my doctoral work (as you may have already guessed.) My work also involved implementing a z-axis stabilizer for the microscope stage, since drift of the microscope stage is a serious issue for high resolution tracking, especially along the axis of the microscope focuser.
If you're really interested, you can get the relevant chapter of my thesis here, and a poster I presented on 3D single molecule particle tracking at BPS in 2006 here.