Single Molecule Projects:

Superresolution Imaging using Single-Molecule Active-Control Microscopy (SMACM)

Optical fluorescence microscopy is an important tool for cell biology because light can be used to non-invasively probe a sample with relatively small perturbation of the specimen, enabling dynamical observation of the motions of internal structures in living cells, but with resolution usually limited to ~250 nm by optical diffraction. Single-molecule epifluorescence microscopy achieves nanometer-scale resolution by taking advantage of the fact that the point spread function of an isolated nanoscale emitter can be fit to a precision far greater than the standard diffraction limit. Over the past few years, the utility of this technique has been extended to the regime of biologically relevant, room-temperature experiments with clever use of photoactivation to control the emitting concentration of single nanoscale fluorescent labels (PALM, F-PALM, STORM).1

All such superresolution techniques are based on the critical requirement of imaging nanometer-sized single-molecule emitters and on the use of an active control mechanism to produce sparse sub-ensembles. For convenience, we refer to these techniques as a group using the inclusive, general term, Single-Molecule Active-Control Microscopy (SMACM). In SMACM experiments, structures labeled by an ensemble of photoactivatable fluorophores too dense to be imaged simultaneously are resolved over repeated cycles in each of which only a sparse subset of the fluorophores is activated. The final superresolution image is reconstituted from a superposition of single-molecule (low-concentration) images.

We are pursuing several thrusts in collaboration with our colleagues:

  • Development and evaluation of new photoswitchable single-molecule fluorophores in collaboration with the group of Prof. Robert J. Twieg, Kent State University
  • Implementation of new targeting protocols in cells for small-molecule photoswitchable emitters in collaboration with the group of Prof. Jianghong Rao, Stanford
  • Direct superresolution imaging of protein localization patterns in cells in collaboration with the group of Prof. Lucy Shapiro, Stanford

Superresolution Imaging in Live C. Crescentus Cells Using Photoswitchable EYFP

Most SMACM experiments rely on specialized photoactivatable or photoswitchable fluorescent protein labels. In our lab, however, we have shown that this is not an imperative. Rather, we have used fusions to the common fluorescent protein EYFP to perform in vivo superresolution imaging in live bacteria. We also take advantage of the fact that, rather than being photoactivated, EYFP can be reactivated with violet light after apparent photobleaching.2 To address limitations arising from physiologically imposed upper boundaries on the concentration of fluorophores, we employ dark time-lapse periods to allow single-molecule motions to fill in filamentous structures, increasing the effective labeling concentration while localizing each emitter at most once per resolution-limited spot. We image cell-cycle-dependent superstructures of the bacterial actin protein MreB in live C. crescentus cells3 with sub-40-nm resolution for the first time, showing that EYFP is a useful emitter for in vivo superresolution imaging of intracellular structures in bacterial cells.

J.S. Biteen, M.A. Thompson, N.K. Tselentis, G.R. Bowman, L. Shapiro, and W.E. Moerner, Nature Methods 5, 947 (15 Sept 2008) [Slide] [journal link: Nature Meth.]

 

New Photoactivatable Single-Molecule Fluorophores

For several years, we have been exploring the properties of push-pull fluorophores containing an amine donor covalently linked to a dicyanomethylenedihydrofuran (DCDHF) acceptor, described elsewhere on these pages. In this new effort, we created a new class of photoactivatable single-molecule fluorophores by replacing the amine with an azide (A). With long-wavelength pumping at 594 nm, the azido-DCDHF fluorogenic molecules are dark, but applying low-intensity activating light at 407nm converts the azide to an amine (B), restoring the donor-acceptor character, the redshifted absorption, and the bright fluorescent emission. Because the emitters are not specifically targeted in this preliminary study, the fluorophores either diffuse in the cytosol, or attach to relatively immobile proteins via insertion into C-C bonds.

S. J. Lord, N. R. Conley, H.-l. D. Lee, R. Samuel, N. Liu, R. J. Twieg, W. E. Moerner, JACS 130, 9204 (2008) [Slide] [journal link: JACS]

 

Cy3-Cy5 Covalent Heterodimers for Superresolution Imaging

Covalent heterodimers of the Cy3 and Cy5 fluorophores have been prepared from commercially available starting materials and characterized at the single-molecule level. This system behaves as a discrete molecular photoswitch, in which photoexcitation of the Cy5 results in fluorescence emission or, with a much lower probability, causes the Cy5 to enter into a long-lived, but metastable, dark state. Photoinduced recovery of the emissive Cy5 is achieved by very low intensity excitation (5 W/cm2) of the Cy3 fluorophore at a shorter wavelength. A similar system consisting of proximal, but not covalently linked, Cy3 and Cy5 has found application in stochastic optical reconstruction microscopy (STORM), a single-molecule localization-based technique for superresolution imaging that requires photoswitching. The covalent Cy3-Cy5 heterodimers described herein eliminate the need for probabilistic methods of situating the Cy3 and Cy5 in close proximity to enable photoswitching. As proof of principle, these heterodimers have been applied to superresolution imaging of the tubular stalk structures of live Caulobacter crescentus bacterial cells (yellow in the figure).

N. R. Conley, J. S. Biteen, and W. E. Moerner, J. Phys. Chem. B Letters 112, 11878 (2008) [Slide] [journal link: JPC B Letter]

 

Single Molecules of Bacterial Actin MreB Undergo Directed Treadmilling Motion in Caulobacter Cells - Where the Single Molecule Moves Through a Filament to Yield Superresolution Information
S. Y. Kim, Z. Gitai, A. Kinkhabwala, L. Shapiro, and W. E. Moerner, PNAS 103, 10929 (2006) [ Full Text] [Journal link] [Supporting material] [movie1] [movie2] [movie3] [movie4]

 

 

  1. E. Betzig, G.H. Patterson, R. Sougrat, O.W. Lindwasser, S. Olenych, J.S. Bonifacino, M.W. Davidson, J. Lippincott-Schwartz, and H.E. Hess, "Imaging intracellular fluorescent proteins at nanometer resolution," Science, 2006, 313, 1642-1645; M.J. Rust, M. Bates, and X. Zhuang, "Sub-diffraction-limit imaging by stochastic optical reconstruction microscopy (STORM)," Nature Meth., 2006, 3, 793-795; S.T. Hess, T.P. Girirajan, and M.D. Mason, "Ultra-High Resolution Imaging by Fluorescence Photoactivation Localization Microscopy," Biophys. J., 2006, 91, 4258-4272.
  2. R.M. Dickson, A.B. Cubitt, R.Y. Tsien, and W.E. Moerner, "On/off blinking and switching behaviour of single molecules of green fluorescent protein," Nature, 1997, 388, 355-358.
  3. S.Y. Kim, Z. Gitai, A. Kinkhabwala, L. Shapiro, and W.E. Moerner, "Single molecules of the bacterial actin MreB undergo directed treadmilling motion in Caulobacter crescentus," PNAS, 2006, 103, 10929-10934.
This work was supported in part by The National Institutes of Health, NIGMS Grant R01 GM086196