Research interests

 

RESEARCH INTERESTS

• Experimental and Theoretical Biophysics,
• Fluorescence Microscopy and Spectroscopy,
• Function and Lateral Organization of Biomembranes,
• Single Molecule Fluorescence Photophysics,
• Green Fluorescent Proteins.

 

Overcoming "The Diffraction Limit"

Resolution in light microscopes is limited by diffraction to approximately 200-250 nanometers, but much of biology occurs on much shorter (molecular) length scales. Scientists have been struggling to circumvent the limits of diffraction for more than one hundred years. Electron microscopy can image with nanometer resolution, but so far cannot be used to image living specimens. Super-resolution imaging techniques, which can break the diffraction barrier, are in great demand.

 

Breakthrough in Light Microscopy

We have developed a technique called fluorescence photoactivation localization microscopy (FPALM) which can image below the diffraction limit. The diffraction limit normally obscures features in the sample which are smaller than roughly half of a wavelength in size, or 200-250 nm for visible light. The FPALM method gets around the diffraction limit by using optical control of the molecules, such that only a small number are visible at any given time, allowing them to be visualized as individuals. Initially, the molecules are all in a non-fluorescent (inactive state). A light source (typically a 405 nm laser) called the activation beam is applied to the sample in a brief pulse at low intensity to activate a small number of molecules. A second light source (typically a 496 nm laser) called the activation beam then illuminates the sample, causing only the active molecules to light up and fluoresce, while all other (inactive) molecules are essentially non-fluorescent and remain invisible. The small number of active molecules are imaged using a high-sensitivity camera and their positions are measured (i.e. the molecules are localized) from the recorded images. After illumination for some short period (a fraction of a second, or roughly a few camera frames), the active molecules are bleached by the high-intensity illumination beam and become non-fluorescent once again. Then, another activation pulse is used, which activates another small subset of the total molecules, which are then imaged under readout beam illumination until they bleach. This iterative process of activation, readout, and bleaching, is repeated until as many molecules as possible are imaged and localized. The plotted positions of all molecules (typically 10,000 to 1,000,000) provides an image of the distribution of the molecules in the sample.

The initial proof-of-principle paper on this technique was published in the Biophysical Journal in late 2006 (1), essentially coincident with the work of two other groups (2; 3). These developments were ranked by Science magazine as one of the top ten breakthroughs of 2006, and awarded “Method of the Year” of 2008 by Nature Methods.

Because of the interest in imaging living cells, we pushed with particular effort to prove that FPALM works in living cells, and succeeded in imaging individual molecules of fluorescently-tagged hemagglutinin (HA. the fusion protein from influenza virus) inside living fibroblast cell membranes. This work revealed that molecules of HA could move within the membrane clusters they form, eliminating solid phase clusters as a possibility. Furthermore, the boundaries of the clusters are jagged, rather than rounded as would be predicted by energy minimization of the boundary perimeter due to line tension. Instead, some cellular factors must be preventing the clusters from becoming rounded, perhaps interactions between the cytoskeleton and the membrane which are postulated in the literature. Thus, several existing models of biological membrane organization were inconsistent with our observations, and our results strengthened the standing of at least one other model. These results were published in the Proceedings of the National Academy of Sciences in 2007.(4)

 

 

 

Figure 1. Concept of FPALM. (A) In normal fluorescence microscopy, large numbers of fluorescent molecules are visible at once, and diffraction blurs objects smaller than 200-250 nm, obscuring fine details. In FPALM, light is used to limit the number of visible (fluorescent) molecules. In contrast to normal fluorescent molecules, FPALM uses photoactivatable fluorescent probes, which are initially non-fluorescent (inactive). (B) Even under normal illumination, inactive molecules are invisible. (C) The low-intensity 405 nm activation laser converts a small subset of inactive molecules into active ones (large spots). (D) Active molecules are imaged and localized to precisely determine their positions (small spots). (E) Photobleaching turns active molecules permanently off. (F-I) Starting with the remaining inactive molecules, the process of activation, imaging, localization, and photobleaching is repeated, this time yielding the coordinates of a new subset of molecules. The process is repeated until enough molecules have been localized to reveal the structure of the sample. The plotted positions of the localized molecules is the FPALM image. (J-M) Simulated FPALM images of a sample with increasing numbers of molecules illustrate how the data is built up iteratively. FPALM can be used to image a variety of samples in two or three dimensions.

 

Because of strong interest in imaging three-dimensional samples, and because FPALM as originally published images two-dimensional samples, we worked together with Dr. Joerg Bewersdorf, a scientist at the Jackson Laboratory, and an adjunct member of the Department of Physics and Astronomy at U. Maine, to develop a new version of FPALM called Biplane-FPALM, or BP-FPALM. BP-FPALM can image three-dimensional samples with a resolution of 30 nm x 30 nm x 75 nm, using methods similar to that of normal FPALM, but with simultaneous detection of fluorescence in two different focal planes, which was recently published in Nature Methods. (5)

Because much of biology at the molecular level relies on relative orientations of molecules (not just proximity), we developed a version of FPALM which can image molecular orientations. Using the information encoded in the polarization of each photon, we are able to calculate the two-dimensional anisotropy of each localized molecule, which can be used to determine the orientation of the molecule. This method was published separately in Nature Methods in 2008. (6)

The number of potential biomedical applications of localization microscopy is very large. These methods have been successfully used to image living and fixed fibroblasts, kidney cells, neurons, muscle fibers, nanopores in polymer membranes, crystalline surfaces, and nanostructures. The opportunities extend beyond biological systems to any three-dimensional sample which can be labeled with a photoactivatable fluorescent dye. A variety of photoactivatable dyes and photoactivatable fluorescent proteins such as the photoactivatable green fluorescent protein (PA-GFP),(7) EosFP,(8) and Dendra,(9) have been described in literature (10) and are available commercially.  Recent publications have demonstrated imaging multiple species(11; 12), living cells(13-15), three-dimensional samples(5; 16), and molecular orientations(6), presenting several powerful capabilities for studying biological systems.

 

 

REFERENCES

1. Hess, S.T., T.P. Girirajan, and M.D. Mason. 2006. Ultra-high resolution imaging by fluorescence photoactivation localization microscopy. Biophys J. 91(11): 4258-4272.

2. Betzig, E., G.H. Patterson, R. Sougrat, O.W. Lindwasser, S. Olenych, J.S. Bonifacino, M.W. Davidson, J. Lippincott-Schwartz, and H.F. Hess. 2006. Imaging Intracellular Fluorescent Proteins at Nanometer Resolution. Science. 313: 1642-1645.

3. Rust, M.J., M. Bates, and X. Zhuang. 2006. Sub-diffraction-limit imaging by stochastic optical reconstruction microscopy (STORM). Nat Methods. 3(10): 793-796.

4. Hess, S.T., T.J. Gould, M.V. Gudheti, S.A. Maas, K.D. Mills, and J. Zimmerberg. 2007. Dynamic Clustered Distribution of Hemagglutinin Resolved at 40nm in Living Cell Membranes Discriminates Between Raft Theories. Proc Natl Acad Sci U S A. 104(44): 17370-17375.

5. Juette, M.F., T.J. Gould, M.D. Lessard, M.J. Mlodzianoski, B.S. Nagpure, B.T. Bennett, S.T. Hess, and J. Bewersdorf. 2008. Three-dimensional sub-100 nm resolution fluorescence microscopy of thick samples. Nat Methods.

6. Gould, T.J., M.S. Gunewardene, M.V. Gudheti, V.V. Verkhusha, S.R. Yin, J.A. Gosse, and S.T. Hess. 2008. Nanoscale imaging of molecular positions and anisotropies. Nature Methods. 5(12): 1027-1030.

7. Patterson, G.H., and J. Lippincott-Schwartz. 2002. A photoactivatable GFP for selective photolabeling of proteins and cells. Science. 297(5588): 1873-1877.

8. Wiedenmann, J., S. Ivanchenko, F. Oswald, F. Schmitt, C. Rocker, A. Salih, K.D. Spindler, and G.U. Nienhaus. 2004. EosFP, a fluorescent marker protein with UV-inducible green-to-red fluorescence conversion. Proc Natl Acad Sci U S A. 101(45): 15905-15910.

9. Gurskaya, N.G., V.V. Verkhusha, A.S. Shcheglov, D.B. Staroverov, T.V. Chepurnykh, A.F. Fradkov, S. Lukyanov, and K.A. Lukyanov. 2006. Engineering of a monomeric green-to-red photoactivatable fluorescent protein induced by blue light. Nature Biotechnology. 24(4): 461-465.

10. Lukyanov, K.A., D.M. Chudakov, S. Lukyanov, and V.V. Verkhusha. 2005. Photoactivatable fluorescent proteins. Nat Rev Mol Cell Bio. 6(11): 885-891.

11. Bates, M., B. Huang, G.T. Dempsey, and X. Zhuang. 2007. Multicolor super-resolution imaging with photo-switchable fluorescent probes. Science. 317(5845): 1749-1753.

12. Shroff, H., C.G. Galbraith, J.A. Galbraith, H. White, J. Gillette, S. Olenych, M.W. Davidson, and E. Betzig. 2007. Dual-color superresolution imaging of genetically expressed probes within individual adhesion complexes. Proc Natl Acad Sci U S A. 104(51): 20308-20313.

13. Shroff, H., C.G. Galbraith, J.A. Galbraith, and E. Betzig. 2008. Live-cell photoactivated localization microscopy of nanoscale adhesion dynamics. Nature Methods. 5(5): 417-423.

14. Hess, S.T., T.J. Gould, M.V. Gudheti, S.A. Maas, K.D. Mills, and J. Zimmerberg. 2007. Dynamic clustered distribution of hemagglutinin resolved at 40 nm in living cell membranes discriminates between raft theories. Proc. Natl. Acad. Sci. U. S. A. 104(44): 17370-17375.

15. Manley, S., J.M. Gillette, G.H. Patterson, H. Shroff, H.F. Hess, E. Betzig, and J. Lippincott-Schwartz. 2008. High-density mapping of single-molecule trajectories with photoactivated localization microscopy. Nat Methods. 5(2): 155-157.

16. Huang, B., W. Wang, M. Bates, and X. Zhuang. 2008. Three-dimensional super-resolution imaging by stochastic optical reconstruction microscopy. Science. 319(5864): 810-813.

 

 

 

Publications

• Hess ST. 2009. Red Lights, Camera, Photoactivation! Nature Methods 6: 124-125.

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• Juette MF, Gould TJ, Lessard MD, Mlodzianoski MJ, Nagpure BS, Bennett BT, Hess ST, Bewersdorf J. 2008. Three-Dimensional sub-100 nm Resolution Fluorescence Microscopy of Thick Samples. Nature Methods, 5: 527-9.

  For more information: Download PDF

• Gould TJ, Gunewardene MS, Gudheti MS, Verkhusha VV, Yin SR, Gosse JA, Hess ST. 2008. Imaging Molecular Positions and Anisotropies. Nature Methods 5: 1027-30.

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• Gould TJ, Hess ST. 2008. Nanoscale Imaging of Intracellular Fluorescent Proteins: Breaking the Diffraction Barrier. Biophysical Tools for Biologists, Vol. 2. Ed. Detrich HW, Methods in Cell Biology 89: 329-358.
• Rochira J, Gudheti M, Gould T, Laughlin R, Nadeau J, Hess ST. Fluorescence Intermittency Limits Brightness in CdSe/ZnS Nanoparticles Quantified by Fluorescence Correlation Spectroscopy. 2007. J. Phys. Chem. C 111: 1695-1708.
• Baumgart T, Hammond A, Sengupta P, Hess S, Holowka D, Baird B, Webb WW. 2007. Large-scale Fluid/Fluid Phase Separation of Proteins and Lipids in Giant Plasma Membrane Vesicles,” PNAS 104: 3165-3170.
• Hess ST, Gould TJ, Gudheti MV, Maas SA, Mills KD, Zimmerberg J. 2007. Dynamic Clustered Distribution of Hemagglutinin Resolved at 40 nm in Living Cell Membranes Discriminates Between Raft Theories. PNAS 104: 17370-5.

  For more information: Download PDF

• Hess ST, Girirajan TPK, Mason MD, Ultra-High Resolution Imaging by Fluorescence Photoactivation Localization Microscopy, Biophysical Journal 91: 4258-4272.

  For more information: Download PDF

• Hess ST, Kumar M, Verma A, Farrington J, Kenworthy A, Zimmerberg J. 2005. Quantitative electron microscopy and fluorescence spectroscopy of the membrane distribution of influenza hemagglutinin. J Cell Bio 169:965-976
• Hess ST, Heikal AA, Webb WW. 2004. Fluorescence photoconversion kinetics in novel green fluorescent protein pH sensors (pHluorins). J Phys Chem B 108:10138-10148
• Zimmerberg J, Kumar M, Verma A, Farrington J, Roth M, Kenworthy A, Hess ST. 2004. Studying spatial distributions of influenza hemagglutinin on the plasma membrane of fibroblasts: A work in progress. Macromolecular Symposia 219:17-23
• Baumgart T, Hess ST, Webb WW. 2003. Imaging Coexisting Fluid Domains in Biomembrane Models Coupling Curvature and Line Tension. Nature 425:821-824

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• Hess ST, Sheets ED, Wagenknecht-Wiesner A, Heikal AA. 2003. Quantitative analysis of the fluorescence properties of intrinsically fluorescent proteins in living cells. Biophys J 85:2566-2580
• Hess ST, Webb WW. 2002. Focal volume optics and experimental artifacts in confocal fluorescence correlation spectroscopy. Biophys J 83:2300-2317
• Hess ST, Huang S, Heikal AA, Webb WW. 2002. Biological and chemical applications of fluorescence correlation spectroscopy. Biochemistry 41:697-705
• Heikal AA, Hess ST, Sheets ED, Webb WW. 2002. Mutation-photophysics Relationship in Intrinsically Fluorescent Proteins. In: Femtochemistry and Femtobiology: Ultrafast Dynamics in Molecular Science. Douhal A, Santamaria J (Eds). World Scientific Publishing Co Pte Ltd, Singapore
• Heikal AA, Hess ST, Webb WW. 2001. Multiphoton molecular spectroscopy and excited-state dynamics of enhanced green fluorescent protein (EGFP): acid-base specificity. Chem Phys 274:37-55
• Heikal AA, Hess ST, Baird GS, Tsien RY, Webb WW. 2000. Molecular spectroscopy and dynamics of intrinsically fluorescent proteins: Coral red (dsRed) and yellow (Citrine). Proc Natl Acad Sci USA 97:11996-12001. See also correction in same volume, p:14831
• Albota M, Beljonne D, Brédas J-L, Ehrlich J, Fu J-Y, Heikal A, Hess S, Kogej T, Levin M, Marder S, McCord-Maughon D, Perry J, Röckel H, Rumi M, Subramaniam G, Webb W, Wu X-L, Xu C. 1998. Design of organic molecules with large two-photon absorption cross sections. Science 281:1653-1656
• McCambridge JD, Rizzo ND, Hess ST, Wang JQ, Ling XS, Prober DE. 1997. Pinning and vortex lattice structure in NbTi alloy multilayers. IEEE Trans Appl Supercon 7:1134-1137
• Hess ST, Blake JD, Blake RD. 1994. Wide variations in neighbor-dependent substitution rates. J Mol Biol 236:1022-1033
• Marx KA, Hess ST, Blake RD. 1994. Alignment of (dA) (dT) homopolymer tracts in gene flanking sequences suggests nucleosomal periodicity in D Discoideum DNA. J Biomol Struct Dyn 12:235-246
• Marx KA, Hess ST, Blake RD. 1993. Characteristics of the large (dA)(dT) homopolymer tracts in D discoideum gene flanking and intron sequences. J Biomol Struct Dyn 11:57-66
• Blake RD, Hess ST. 1992. The pattern of substitution mutation in different nearest-neighbor environments of the human genome. Comput Chem 16:165-170
• Blake RD, Hess ST, Nicholson-Tuell J. 1992. The influence of nearest neighbors on the rate and pattern of spontaneous point mutations. J Mol Evol 34:189-200