Differential Evanescence Nanometry

This is an extraordinarily confusing paper titled Differential Evanescence Nanometry: Live-Cell Fluorescence Measurements with 10-nm Axial Resolution on the Plasma Membrane in Biophys J. After looking over it a few times, I think I get the gist of it, but I'm hampered by the fact that I know almost nothing about "clathrin coated pits". But, I find even the descriptions of the theory and control experiments to be incredibly confusingly written. But, basically, it appears to be basically just another realization of VA-TIRFM.

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

There's also a more clever version of this realized recently by Watanabe, et al, which uses a dual-view type splitting to image two focal planes at once, and thereby get differential information:

• Three-dimensional nanometry of vesicle transport in living cells using dual-focus imaging optics

Yet another twist on this approach is to utilize a long focal-length cylindrical lens in the imaging path to create elliptical PSFs. One axis of the PSF will focus to a different plane than the other, and the eccentricity of the PSF can be used to determine the axial position. This approach was utilized in one of the Zhuang lab's recent papers which I blogged about briefly:

• Three-Dimensional Super-Resolution Imaging by Stochastic Optical Reconstruction Microscopy
  

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.

The recent paper referenced at the beginning is basically an example of this, except that instead of varying the TIR angle, the switched between TIR and widefield, using widefield imaging as their "reference". They were also able to do imaging in two colors, in order to compare differential aggregation of two different proteins in to cytosolic pits.

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.