Session H7: Cellular Imaging at the Nanometer Scale

Irving Langmuir Prize Talk: Single-Molecule Fluorescence Imaging: Nanoscale Emitters with Photoinduced Switching Enable Superresolution.

In the two decades since the first optical detection and spectroscopy of a single molecule in a solid (Phys. Rev. Lett. \textbf{62}, 2535 (1989)), much has been learned about the ability of single molecules to probe local nanoenvironments and individual behavior in biological and nonbiological materials in the absence of ensemble averaging that can obscure heterogeneity. The early years concentrated on high-resolution spectroscopy in solids, which provided observations of lifetime-limited spectra, optical saturation, spectral diffusion, optical switching, vibrational spectra, and magnetic resonance of a single molecular spin. In the mid-1990's, much of the field moved to room temperature, where a wide variety of biophysical effects were subsequently explored, but it is worth noting that several features from the low-temperature studies have analogs at high temperature. For example, in our first studies of yellow-emitting variants of green fluorescent protein (EYFP) in the water-filled pores of a gel (Nature \textbf{388}, 355 (1997)), optically induced switching of the emission was observed, a room-temperature analog of the earlier low-temperature behavior. Because each single fluorophore acts a light source roughly 1 nm in size, microscopic imaging of individual fluorophores leads naturally to superlocalization, or determination of the position of the molecule with precision beyond the optical diffraction limit, simply by digitization of the point-spread function from the single emitter. Recent work has allowed measurement of the shape of single filaments in a living cell simply by allowing a single molecule to move through the filament (PNAS \textbf{103}, 10929 (2006)). The additional use of photoinduced control of single-molecule emission allows imaging beyond the diffraction limit (superresolution) by several novel approaches proposed by different researchers. For example, using photoswitchable EYFP, a novel protein superstructure can now be directly imaged in a living bacterial cell at sub-40nm resolution (Nat. Meth. \textbf{5}, 947 (2008)). These important advances provide the impetus for the further development of both new imaging schemes with 3-D capability as well as invention of new photoswitchable single-molecule emitters for use in polymers and in biological systems (JACS \textbf{130}, 9204 (2008); J. Phys. Chem. B \textbf{112}, 11878 (2008)).   

Far-Field Fluorescence Nanoscopy

The resolution of a far-field optical microscopy is usually limited to $d=\lambda \mathord{\left/ {\vphantom {\lambda {\left( {2\,n\sin \alpha } \right)}}} \right. \kern-\nulldelimiterspace} {\left( {2\,n\sin \alpha } \right)} \quad >$ 200 nm, with $n\sin \alpha $ denoting the numerical aperture of the lens and $\lambda $ the wavelength of light. While the diffraction barrier has prompted the invention of electron, scanning probe, and x-ray microscopy, the 3D-imaging of the interior of (live) cells requires the use of focused visible light. I will discuss new developments of optical microscopy that I anticipate to have a lasting impact on our understanding of living matter. Emphasis will be placed on physical concepts that have overcome the diffraction barrier in far-field fluorescence microscopy. To set the scene for future directions, I will show that all these concepts share a common strategy: exploiting selected states and transitions of the fluorescent marker to neutralize the limiting role of diffraction. The first viable concept of this kind was Stimulated Emission Depletion (STED) microscopy where the spot diameter follows$d\approx \lambda \mathord{\left/ {\vphantom {\lambda {\left( {2\,n\sin \alpha \sqrt {1+I \mathord{\left/ {\vphantom {I {I_s }}} \right. \kern-\nulldelimiterspace} {I_s }} } \right)}}} \right. \kern-\nulldelimiterspace} {\left( {2\,n\sin \alpha \sqrt {1+I \mathord{\left/ {\vphantom {I {I_s }}} \right. \kern-\nulldelimiterspace} {I_s }} } \right)}$; $I \mathord{\left/ {\vphantom {I {I_s }}} \right. \kern-\nulldelimiterspace} {I_s }$is a measure of the strength with which the molecule is send from the fluorescent state to the dark ground state. For $I \mathord{\left/ {\vphantom {I {I_s }}} \right. \kern-\nulldelimiterspace} {I_s }\to \infty $ it follows that $d\to 0$, meaning that the resolution that can, in principle, be molecular. The concept underlying STED microscopy can be expanded by employing other transitions that shuffle the molecule between a dark and a bright state, such as (i) shelving the fluorophore in a dark triplet state, and (ii) photoswitching between a `fluorescence activated' and a `fluorescence deactivated' conformational state. Examples for the latter include photochromic organic compounds, and fluorescent proteins which undergo a cis-trans photoisomerizations. Photoswitching provides ultrahigh resolution at ultralow light levels. Switching can be performed in an ensemble or individually in which case the image is assembled molecule by molecule at high resolution. By providing molecular markers with the appropriate transitions, synthetic organic chemistry and protein biotechnology plays a key role in this endeavor. Besides being a fascinating development in physics, far-field optical ``nanoscopy'' is highly relevant to the life sciences. In fact, it has already been a key to answering important questions in biology [1, 2]. Due to its simplicity and improving performance, I expect far-field optical nanoscopy to enter virtually every cell biology laboratory in the near future. \\[4pt] [1] S. W. Hell, Far-field optical nanoscopy, \textit{Science} 316 (2007) 1153. \\[0pt] [2] Westphal, V., S. O. Rizzoli, M. A. Lauterbach, D. Kamin, R. Jahn, S. W. Hell, Video-Rate Far-Field Optical Nanoscopy Dissects Synaptic Vesicle Movement, Science (2008) DOI: 10.1126/science.1154228.   

Advances in super-resolution imaging technologies

Superresolution techniques such as photoactivated localization microscopy (PALM) enable the imaging of fluorescent protein chimeras to reveal the organization of genetically-expressed proteins on the nanoscale with a density of molecules high enough to provide structural context. Various applications of this new technology are now possible. One application is for in cellula pulse-chase analysis to follow protein turnover and diffusion of photoactivated fluorescent proteins. Another approach combines the techniques of PALM and single particle tracking to resolve the dynamics of individual molecules by tracking them in live cells. Called single particle tracking PALM (sptPALM), the technique involves activating, localizing and bleaching many subsets of photoactivatated fluorescent protein chimeras in live cells. Spatially-resolved maps of single molecule motions can be obtained by imaging membrane proteins with this technique, providing several orders of magnitude more trajectories per cell than by traditional single particle tracking. By probing distinct subsets of molecules, including Gag and VSVG, sptPALM can provide a powerful means for exploring the origin of spatial and temporal heterogeneities in membranes. Examples such as these will be presented to illustrate the value of super-resolution imaging in providing quantitative insights into protein organization and dynamics at the nanoscale.   

Bacterial cryotomography

Electron cryotomography (ECT) is an emerging technique that allows thin samples such as small cells, viruses, or tissue sections to be imaged in 3-D in a near-native, ``frozen-hydrated'' state to molecular ($\sim $4 nm) resolution. Thus ECT fills a critical gap between light microscopy and higher resolution structural techniques like X-ray crystallography and NMR. In a combination of technology development and biological application, during the past few years our lab has been studying bacterial ultrastructure through ECT of intact, plunge-frozen cells. We have now collected over a thousand tomograms of more than ten different species. This work has revealed the surprising complexity of the bacterial cytoskeleton as well as the architectures of several important ``supramolecular'' complexes including the chemoreceptor array, the flagellar motor, and the cell wall peptidoglycan. Example results highlighting both the potential and limitations of this technology will be shown.   

The Role of MreB in Escherichia Coli's Cellular Rigidity

Bacteria possess homologs of all three classes of eukaryotic cytoskeletal proteins. These filamentous proteins have been shown to localize proteins essential for a number of cell-biological processes in prokaryotes such as cell growth and division. However, to date, there has been no direct evidence that the cytoskeleton in bacteria bears mechanical loads or can generate physical forces than are used by the cell. I will present evidence from combined fluorescence and force microscopy measurements that MreB, an actin homolog, is responsible for half of Escherichia coli's cellular rigidity. These data support an interpretation in which the cytoskeleton, the peptidoglycan cell wall and a large turgor pressure work together to give gram-negative cells their mechanical properties.