Does STED really break the law?


Ernst Abbe, legendary German physicist of yore, defined the resolution of a microscope as a function of the ratio of the objective aperture radius to the working distance of the image object. Meaning that the closer the image object (the shorter the focal length) and the wider the objective lens is, the greater the resolution, all else being equal. It’s commonly referred to as “Abbe’s diffraction limit”-a fundamental, physical limit to our ability to form images with lenses. Wavelength plays into it too, and that’s why the longer wavelengths used in say, two-photon microscopy can only resolve different light sources over a longer distance.

I remember sitting in a neurology seminar last year, the topic was the imaging capabilities of STED (STimulated Emission Depletion) confocal microscopy-a superresolution technique-for living neurons. Immediately prior to the section where the method was described (a doughnut-shaped point-spread function photobleaches nearby fluorophores, preventing them from contributing to the intensity measurement at a given point) was a cartoon involving a police officer and some sort of criminal hijinks taking place. The title of the slide was something along the lines of “How to break the law.”

As one audience member was quick to point out, the fluorescence emission (from the middle of the doughnut-shaped stimulated emission PSF) still must travel back through the objective and all the other optics, convolving with the various transfer functions of the lens elements at every step of the way. How can this be said to be breaking the diffraction limit? I agree.

Because a confocal microscope is a scanning apparatus, it only illuminates and records a single point at a time. The illumination path has a point spread function just as in the imaging path, so normally a fairly large volume, capable of containing many fluorophores, is illuminated and contributes to the light brought to focus by the imaging path. By bleaching a large volume around surrounding the region of interest, the only fluorophores capable of excitation by the non-toroidal excitation PSF reside in this central volume. Although the PSF incident on the photodetector is still diffraction limited, the only fluorophores contributing to the signal are in that small, central, un-depleted volume.

I’m not convinced that this corresponds to superresolution, at least not without qualification. It certainly allows you to superlocalize the fluorophores, but resolution in microscopy is differentiating between two proximal light sources in space and, I think this is crucial, in time. So there is a time scale involved in STED, and all scanning methods, for which two objects can be resolved, as long as their movements are much slower than the scan. A highly dynamic process like cell membrane fluctuations would probably fall outside of the processes that could be imaged by STED. I think a more suitable term would be appropriate for this and similarly limited techniques.

I don’t make this claim simply because I’m catching my grumps early this year. I think the terminology is genuinely misleading. Consider the title of this paper: “Three-dimensional, single-molecule fluorescence imaging beyond the diffraction limit by using a double-helix point spread function” [1].
They demonstrate an interesting method for recovering depth in a widefield microscope, and they do pinpoint a dense population of fluorophores, some of which are only a few nanometres apart. But the fluorophores are effectively immobilized, and the entire imaging processing takes 450 seconds.
“Resolution” and “sensitivity” are two very different things, and under certain constraints you can use one to inform the other. The title in this case misleads the reader to assume that physical laws are being broken, which is not the case. In particular this mild misdirection will lead undergraduates and laypeople to misinterpret the claims of science. We should take efforts to avoid it, even if your rival is pulling in grant money with these sorts of “impossible” claims. After all, I’m sure that Ernst Abbe wouldn’t buy it.

[1]S.R.P. Pavani, M.A. Thompson, J.S. Biteen, S.J. Lord, N. Liu, R.J.Twieg, R. Piestun, W.E. Moerner. Three-dimensional, single-molecule fluorescence imaging beyond the diffraction limit by using a double-helix point spread function. Proc. Nat. Ac. Sci. 106:9. (2009)

Note: The 3D localization of dense fluorophores they report, again by multiple rounds of partial photoactivation and photobleaching, was generated over 30 cycles of 30 exposures, each frame consisting of a 500 ms exposure. That’s over five minutes of acquisition time.