Applications of Superresolution Imaging Using Single-Molecule Active-Control Microscopy (SMACM) and STED in Bacterial and Mammalian Cells
Current Subgroup Members: Dr. Andreas Gahlmann, Alex Diezmann, Lana Lau, Marissa Lee, Lucien Weiss
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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 or other active control schemes 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.
An alternative method for obtaining superresolution is to use STED (Stimulated Emission Depletion Microscopy) invented by Stephan Hell and its variants2, where a donut-shaped beam with a dark hole in the middle is used to quench or turn off the emission of molecules outside the dark region. Implemented in a confocal scanning microscope, this method also produces resolution beyond the optical diffraction limit. Using our home-built STED microscope, we are exploring what new structures can be observed in various cellular systems.
We are pursuing several thrusts in collaboration with our colleagues:
- Direct superresolution imaging of protein localization patterns in bacterial cells in collaboration with the group of Prof. Lucy Shapiro, Stanford, with emphasis on polar protein superstructure and organization.
- Imaging of nanoscale protein superstructures in cells.
- Single-molecule tracking as a probe of cellular signaling with Prof. Matt Scott, Stanford.
Recent Review: W. E. Moerner, “Microscopy beyond the diffraction limit using actively controlled single molecules,” J. Microsc. 246, 213-220 (2012), published online 12 April 2012. DOI
Quantitative multicolor subdiffraction imaging of bacterial protein ultrastructures in 3D
We demonstrate quantitative multicolor 3D subdiffraction imaging of the structural arrangement of fluorescent protein fusions in living Caulobacter crescentus bacteria. Given single-molecule localization precisions of 20-40 nm, a flexible locally-weighted image registration algorithm is critical to accurately combine the super-resolution data with <10 nm error. Simple surface-relief dielectric phase masks implement a double-helix response at two wavelengths to distinguish two different fluorescent labels and to quantitatively and precisely localize them relative to each other in 3D. This work demonstrates that the DH-PSF approach to 3D localization can easily be implemented with minimal modification to a conventional fluorescence microscope.
Andreas Gahlmann, Jerod L. Ptacin, Ginni Grover, Sean Quirin, Alexander R. S. von Diezmann, Marissa K. Lee, Mikael P. Backlund, Lucy Shapiro, Rafael Piestun, and W. E. Moerner, “Quantitative Multicolor Subdiffraction Imaging of Bacterial Protein Ultrastructures in Three Dimensions,” Nano Lett. (published online February 15, 2013). DOI [Slide]
Enzymatic Activation of Nitro-Aryl Fluorogens in Live Bacteria for Enzymatic Turnover-Activated Localization Microscopy Beyond the Diffraction Limit
Many modern super-resolution imaging methods based on single-molecule fluorescence require the conversion of a dark fluorogen into a bright emitter to control emitter concentration. We have synthesized and characterized a nitro-aryl fluorogen which can be converted by a nitroreductase enzyme into a bright push-pull red-emitting fluorophore. Synthesis of model compounds and optical spectroscopy identify a hydroxyl-amino derivative as the product fluorophore, which is bright and detectable on the single-molecule level for fluorogens attached to a surface. Solution kinetic analysis shows Michaelis-Menten rate dependence upon both NADH and the fluorogen concentrations as expected. The generation of low concentrations of single-molecule emitters by enzymatic turnovers is used to extract subdiffraction information about localizations of both fluorophores and nitroreductase enzymes in cells. Enzymatic Turnover Activated Localization Microscopy (ETALM) is a complementary mechanism to photoactivation and blinking for controlling the emission of single molecules to image beyond the diffraction limit.
Marissa K. Lee, Jarrod Williams, Robert J. Twieg, Jianghong Rao, and W. E. Moerner, “Enzymatic Activation of Nitro-Aryl Fluorogens in Live Bacterial Cells for Enzymatic Turnover-Activated Localization Microscopy,” Chemical Science4 (1), 220-225 (2013), published online 5 October 2012. DOI [Slide]
Fluorescent Saxitoxins for Live Cell Imaging of Single Voltage-Gated Sodium Ion Channels Beyond the Optical Diffraction Limit
A desire to better understand the role of voltage-gated sodium channels (NaVs) in signal conduction and their dysregulation in specific disease states motivates the development of high precision tools for their study. Nature has evolved a collection of small molecule agents, including the shellfish poison (+)-saxitoxin, that bind to the extracellular pore of select NaV isoforms. As described in this report, de novo chemical synthesis has enabled the preparation of fluorescently labeled derivatives of (+)-saxitoxin, STX-Cy5 and STX-DCDHF, which display reversible binding to NaVs in live cells. Electrophysiology and confocal fluorescence microscopy studies confirm that these STX-based dyes function as potent and selective NaV labels. The utility of these probes is underscored in single-molecule and super-resolution imaging experiments, which reveal NaV distributions well beyond the optical diffraction limit in subcellular features such as neuritic spines and filopodia.
Alison E. Ondrus*, Hsiao-lu D. Lee*, Shigeki Iwanaga, William H. Parsons, Brian M. Andresen, W. E. Moerner, and J. Du Bois (equal contributions), “Fluorescent Saxitoxins for Live Cell Imaging of Single Voltage-Gated Sodium Ion Channels Beyond the Optical Diffraction Limit,” Chemistry and Biology 19, 902-912 (2012), published online 26 July 2012. (*equal contributions) DOI [Slide]
STED Microscopy with Optimized Labeling Density Reveals 9-Fold Arrangement of a Centriole Protein
Super-resolution fluorescence microscopy can achieve resolution beyond the optical diffraction limit, partially closing the gap between conventional optical imaging and electron microscopy for elucidation of subcellular architecture. The centriole, a key component of the cellular control and division machinery, is 250 nm in diameter, a spatial scale where super-resolution methods such as stimulated emission depletion (STED) microscopy can provide previously unobtainable detail. We use STED with resolution of 60 nm to demonstrate that the centriole distal appendage protein Cep164 localizes in 9 clusters spaced around a ring of ~300 nm in diameter, and quantify the influence of the labeling density in STED immunofluorescence microscopy. We find that the labeling density dramatically influences the observed number, size and brightness of labeled Cep164 clusters, and estimate the average number of secondary antibody labels per cluster. The arrangements are morphologically similar in centrioles of both proliferating cells and differentiated multiciliated cells, suggesting a relationship of this structure to function. Our STED measurements in single centrioles are consistent with results obtained by electron microscopy, which involve ensemble averaging or very different sample preparation conditions, suggesting that we have arrived at a direct measurement of a centriole protein by careful optimization of the labeling density.
Lana Lau, Yin Loon Lee, Steffen J. Sahl, Tim Stearns, and W. E. Moerner, “STED Microscopy with Optimized Labeling Density Reveals 9-fold Arrangement of a Centriole Protein,” Biophys. J. 102, 2926-2935 (2012) published online 19 June 2012. DOI [Slide]
Super-Resolution Imaging of the Nucleoid-Associated Protein HU in Caulobacter crescentus
Little is known about the structure and function of most nucleoid-associated proteins (NAP) in bacteria. One reason for this is that the distribution and structure of the proteins is obfuscated by the diffraction limit in standard widefield and confocal fluorescence imaging. In particular, the distribution of HU, which is the most abundant NAP, has received little attention. In this study we investigate the distribution of HU in Caulobacter crescentus using a combination of super-resolution (SR) fluorescence imaging and spatial point statistics. By simply increasing the laser power, single molecules of the fluorescent protein fusion HU2-eYFP can be made to blink on and off to achieve SR imaging with a single excitation source. Through quantification by Ripley’s K-test and comparison with Monte Carlo simulations, we find the protein is slightly clustered within a mostly uniform distribution throughout the swarmer and stalked stages of the cell cycle but more highly clustered in pre-divisional cells. The methods presented in this paper should be of broad applicability in the future study of prokaryotic NAPs.
Steven F. Lee*, Michael A. Thompson*, Monica Schwartz, Lucy Shapiro, and W. E. Moerner, “Super-Resolution Imaging of the Nucleoid-Associated Protein HU in Caulobacter crescentus," Biophys. J. Lett. 100, L31-L33 (2011). SIandMovies, DOI [Slide]
A New Photoactivatable Organic Fluorophore Enables Superresolution Imaging of Targeted Proteins in Fixed and Living Cells
Superresolution imaging techniques based on sequential imaging of sparse subsets of single molecules require fluorophores whose emission can be photoactivated or photoswitched. Because typical organic fluorophores can emit significantly more photons than average fluorescent proteins, organic fluorophores have a potential advantage in super-resolution imaging schemes, but targeting to specific cellular proteins must be provided. We report the design and application of HaloTag-based target-specific azido DCDHFs, a class of photoactivatable push–pull fluorogens which produce bright fluorescent labels suitable for single-molecule superresolution imaging in live bacterial and fixed mammalian cells.
Hsiao-lu D. Lee, Samuel J. Lord, Shigeki Iwanaga, Ke Zhan, Hexin Xie, Jarrod C. Williams, Hui Wang, Grant R. Bowman, Erin D. Goley, Lucy Shapiro, Robert J. Twieg, Jianghong Rao, and W. E. Moerner, “Superresolution Imaging of Targeted Proteins in Fixed and Living Cells Using Photoactivatable Organic Fluorophores," J. Amer. Chem. Soc. 132, 15099-15101 (2010), published online October 11, 2010. [Slide] [journal link]
A Spindle-like Apparatus Guides Bacterial Chromosome Separation
Until recently, a dedicated mitotic apparatus that segregates newly replicated chromosomes into daughter cells was believed to be unique to eukaryotic cells. Here we demonstrate that the bacterium Caulobacter crescentus segregates its chromosome using a partitioning (Par) apparatus that has surprising similarities to eukaryotic spindles. We show that the C. crescentus ATPase ParA forms linear polymers in vitro and assembles into a narrow linear structure in vivo detected by superresolution imaging. The centromere-binding protein ParB binds to and destabilizes ParA structures in vitro. We propose that this ParB-stimulated ParA depolymerization activity moves the centromere to the opposite cell pole through a burnt bridge Brownian ratchet mechanism. Finally, we identify the pole-specific TipN protein1, 2 as a new component of the Par system that is required to maintain the directionality of DNA transfer towards the new cell pole. Our results elucidate a bacterial chromosome segregation mechanism that features basic operating principles similar to eukaryotic mitotic machines, including a multivalent protein complex at the centromere that stimulates the dynamic disassembly of polymers to move chromosomes into daughter compartments.
Jerod L. Ptacin, Steven F. Lee, Ethan C. Garner, Esteban Toro, Michael Eckart, Luis R. Comolli, W.E. Moerner, and Lucy Shapiro, “A spindle-like apparatus guides bacterial chromosome segregation,” Nature Cell Biology 12, 791-798 (2010), published online July 25, 2010. [Slide] [journal link]
Photoactivatable Push-Pull Fluorophores Form a General Class and Enable Fluorogenic Photoaffinity Labeling
Dark azido push−pull chromophores have the ability to be photoactivated to produce bright fluorescent labels suitable for single-molecule imaging. Upon illumination, the aryl azide functionality in the fluorogens participates in a photochemical conversion to an aryl amine, thus restoring charge-transfer absorption and fluorescence. Previously, we reported that one compound, DCDHF-V-P-azide, was photoactivatable. Here, we demonstrate that the azide-to-amine photoactivation process is generally applicable to a variety of push−pull chromophores, and we characterize the photophysical parameters including photoconversion quantum yield, photostability, and turn-on ratio. Azido push−pull fluorogens provide a new class of photoactivatable single-molecule probes for fluorescent labeling and super-resolution microscopy. Lastly, we demonstrate that photoactivated push−pull dyes can insert into bonds of nearby biomolecules, simultaneously forming a covalent bond and becoming fluorescent (fluorogenic photoaffinity labeling).
S. J. Lord, H-L. D. Lee, R. Samuel, R. Weber, N. Liu, N. R. Conley, M. A. Thompson, R. J. Twieg, and W. E. Moerner, “Azido Push–Pull Fluorogens Photoactivate to Produce Bright Fluorescent Labels,” appearing in J. Phys. Chem. B (Michael R. Wasielewski Festschrift), published online October 27, 2009, doi: 10.1021/jp907080r. [journal link]
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.3 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.
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.
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).
Molecules of Bacterial Actin MreB Undergo Directed
Motion in Caulobacter Crescentus Cells4
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]
- 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. W. E. Moerner, “New Directions in Single-Molecule Imaging and Analysis,” Invited Perspective, Proc. Nat. Acad. Sci. (USA) 104, 12596-12602 (2007).
- S. W. Hell, "Far-Field Optical Nanoscopy," Science 2007, 316, 1153-1158.
- 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.
- 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.