Sam Hess


  • B.S., Physics, Yale University, 1995
  • M.S., Physics, Cornell University, 1998
  • Ph.D., Physics, Cornell University, 2002

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)

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.

Selected Publications

  • “Molecular Imaging with Neural Training of Identification Algorithm (MINuTIA),” A.J. Nelson and S.T. Hess, Microscopy Research and Technique (in press).
  • “Antimicrobial Agent Triclosan Disrupts Mitochondrial Structure, Revealed by Super-resolution Microscopy, and Inhibits Mast Cell Signaling via Calcium Modulation Toxicology and Applied Pharmacology,” Lisa M Weatherly, Andrew J. Nelson, Juyoung Shim, Abigail M. Riitano, Erik D. Gerson, Andrew J. Hart, Jaime de Juan-Sanz, Timothy A. Ryan, Roger Sher, Samuel T. Hess, Julie A. Gosse, Toxicololgy and Applied Pharmacology 349: 39-54 (2018).
  • “Total internal reflection fluorescence based multiplane localization microscopy enables super-resolved volume imaging,” P.P. Mondal, S.T. Hess, Applied Physics Letters 110 (21), 211102 (2017).
  • “The Role of Probe Photophysics in Localization-Based Superresolution Microscopy,” Francesca Pennacchietti, Travis Gould, and Samuel T. Hess, Biophysical Journal 113 (9) 2037–2054 (2017).
  • “A Cross Beam Excitation Geometry for Localization Microscopy,” Matthew Valles and Samuel T. Hess, iScience Notes DOI: (2017).
  • “Spectral Fluorescence Photoactivation Localization Microscopy,” Michael Mlodzianoski, M. S. Gunewardene, and Samuel T. Hess, PLoS One 11(3): e0147506 (2016).
  • “Super Resolution Fluorescence Localization Microscopy,” Michael J. Mlodzianoski, Matthew M. Valles, Samuel T. Hess, in Encyclopedia of Cell Biology, Ed. Ralph Bradshaw, Philip Stahl and John Heuser & Sergio Grinstein (2016).
  • “Antimicrobial Agent Triclosan is a Proton Ionophore Uncoupler of Mitochondria in Living Rat and Human Mast Cells and in Primary Human Keratinocytes,” Lisa M. Weatherly, Juyoung Shim, Hina N. Hashmi, Rachel H. Kennedy, Samuel T. Hess, and Julie A. Gosse, Journal of Applied Toxicology Jul 23. doi: 10.1002/jat.3209 (2015).
  • “Dances with Membranes: Breakthroughs from Super-Resolution Imaging,” Nikki M. Curthoys, Matthew Parent, Michael Mlodzianoski, Andrew J. Nelson, Jennifer Lilieholm, Michael B. Butler, Matthew Valles, and Samuel T. Hess, in Current Topics in Membranes, Ed. Anne Kenworthy (2015).
  • “Nanoscale Imaging of Caveolin-1 Membrane Domains in vivo,” Kristin A. Gabor, Dahan Kim, Carol H. Kim, and Samuel T. Hess, PLoS One 10(2): e0117225 (2015).
  • “Combining Total Internal Reflection Sum Frequency Spectroscopy Spectral Imaging and Confocal Fluorescence Microscopy,” Edward S. Allgeyer, Sarah M. Sterling, Mudalige S. Gunewardene, Samuel T. Hess, David J. Neivandt, and Michael D. Mason, Langmuir 31 (3): 987-994 (2015).
  • “High-Speed Fluorescence Photoactivation Localization Microscopy,” Andrew J. Nelson, Mudalige S. Gunewardene, and Samuel T. Hess, Proc. SPIE 9169, Nanoimaging and Nanospectroscopy II, 91690P (28 August 2014); doi: 10.1117/12.2064271
  • “In cellulo evaluation of phototransformation quantum yields in fluorescent proteins used as markers for single-molecule localization microscopy,” S. Avilov, R. Berardozzi, M.S. Gunewardene, V. Adam, S.T. Hess, and D. Bourgeois, PLoS One. 9(6):e98362 (2014).
  • “Localization-Based Superresolution Microscopy,” Andrew J. Nelson and Samuel T. Hess, Journal of Microscopy 254: 1-8 (2014).
  • “Precisely and accurately localizing single emitters in fluorescence microscopy: state-of-the-art and best practice,” Hendrik Deschout, Francesca Cella Zanacchi, Michael Mlodzianoski, Alberto Diaspro, Joerg Bewersdorf, Samuel T. Hess, and Kevin Braeckmans, Nature Methods 11: 253-256 (2014).
  • “Simultaneous Multicolor Imaging of Biological Structures with Fluorescence Photoactivation Localization Microscopy,” N.M. Curthoys, M.J. Mlodzianoski, D. Kim, and S.T. Hess, Journal of Visualized Experiments (82), e50680 (2013).
  • “Bleed-through correction for rendering and correlation analysis in multi-colour localization microscopy, “ Dahan Kim, Nikki M. Curthoys, Matthew T. Parent, and Samuel T. Hess, Journal of Optics 15: 094011 (2013).
  • “Super Resolution Microscopy Reveals that Caveolin-1 is required for antiviral immune response,” Kristin A Gabor, Chad R Stevens, Matthew J Pietraszewski, Travis J Gould, Siew Hong Lam, Zhiyuan Gong, Samuel T Hess, Carol H Kim, PLoS One 8: e68759 (2013).
  • “Actin Mediates the Nanoscale Membrane Organization of the Clustered Membrane Protein Influenza Hemagglutinin” Manasa V. Gudheti, Nikki M. Curthoys, Travis J. Gould, Dahan Kim, Kristin A. Gabor, Mudalige S. Gunewardene, Joshua Zimmerberg, Julie A. Gosse, Carol H. Kim, and Samuel T. Hess, Biophysical Journal, 104: 2182-92 (2013).
  • “Optical Nanoscopy: From Acquisition to Analysis,” Travis J. Gould, Samuel T. Hess, Joerg             Bewersdorf, Annual Review of Biomedical Engineering, 14: 231-54 (2012).
  • “Dynamic nanoscale organization of membrane protein complexes in mitochondrial micro-compartments of living cells,” T. Appelhans, C. Richter, V. Wilkens, S.T. Hess, J. Piehler and K. B. Busch, Nano Letters 12: 610-6 (2012).
  • “Superresolution imaging of multiple fluorescent proteins with highly overlapping emission spectra in living cells,” M.S. Gunewardene, F.V. Subach, T.J. Gould, G.P. Penoncello, M.V. Gudheti, V.V. Verkhusha, and S.T. Hess, Biophysical Journal 101: 1522-8 (2011).
  • “Elongated membrane zones boost interactions of diffusing proteins,” J. Zimmerberg and S.T. Hess, Cell 146: 501-3 (2011).
  • “Localization-Based Super-Resolution Light Microscopy,” Kristin A. Gabor, Mudalige S. Gunewardene, David Santucci, and Samuel T. Hess, Microscopy Today 19: 12-16 (2011).
  • “A small peptide modeled after the NRAGE repeat domain inhibits XIAP-TAB1-TAK1 signaling for NF-κB activation and apoptosis in P19 cells.” J.A. Rochira, N.N. Matluk, T.L. Adams, A.A. Karaczyn, L. Oxburgh, S.T. Hess, and J.M. Verdi, PLoS One 6:e20659 (2011).
  • “Imaging Biological Structures with Fluorescence Photoactivation Localization Microscopy,” T. J. Gould, V.V. Verkhusha, and S.T. Hess, Nature Protocols 4: 291-308 (2009).
  • “Red Lights, Camera, Photoactivation!” S.T. Hess, Nature Methods 6: 124-125 (2009).
  • “Imaging Molecular Positions and Anisotropies,” T. J. Gould, M.S. Gunewardene, M.V. Gudheti, V.V. Verkhusha, S.R. Yin, J.A. Gosse, and S.T. Hess, Nature Methods 5: 1027-30 (2008).
  • “A Quantitative Comparison of the Photophysical Properties of Select Quantum Dots and Organic Fluorophores,” T.J. Gould, J. Bewersdorf, and S.T. Hess, Z. Phys. Chem. 222: 833-849 (2008).
  • “Three-Dimensional sub-100 nm Resolution Fluorescence Microscopy of Thick Samples,” Manuel F. Juette, Travis J. Gould, Mark D. Lessard, Michael J. Mlodzianoski, Bhupendra S. Nagpure, Brian T. Bennett, Samuel T. Hess, and Joerg Bewersdorf, Nature Methods 5: 527-9 (2008).
  • “Dynamic Clustered Distribution of Hemagglutinin Resolved at 40 nm in Living Cell Membranes Discriminates Between Raft Theories,” S. T. Hess, T. J. Gould, M. V. Gudheti, S.A. Maas, K.D. Mills, and J. Zimmerberg, PNAS 104: 17370-5 (2007).
  • “Imaging and shape analysis of giant unilamellar vesicles (GUVs) as model plasma membranes: Effect of trans-DOPC (dielaidoyl phosphatidylcholine) on membrane properties,” M. V. Gudheti, M. Mlodzianoski, and S. T. Hess, Biophysical Journal 93: 1-13 (2007).
  • “Large-scale Fluid/Fluid Phase Separation of Proteins and Lipids in Giant Plasma Membrane Vesicles,” T. Baumgart, A. Hammond, P. Sengupta, S. Hess, D. Holowka, B. Baird, and W. W. Webb, PNAS 104: 3165-3170 (2007).
  • “Fluorescence Intermittency Limits Brightness in CdSe/ZnS Nanoparticles Quantified by Fluorescence Correlation Spectroscopy,” J. Rochira, M. Gudheti, T. Gould, R. Laughlin, J. Nadeau, and S. T. Hess, Journal of Physical Chemistry C 111: 1695-1708 (2007).
  • “Ultra-High Resolution Imaging by Fluorescence Photoactivation Localization Microscopy,” S. T. Hess, T.P.K. Girirajan, and M.D. Mason, Biophysical Journal 91: 4258-4272 (2006).
  • Hess S.T., Kumar, M., Verma, A., Farrington, J., Kenworthy, A., and Zimmerberg, J., “Quantitative Electron Microscopy and Fluorescence Spectroscopy of the Membrane Distribution of Influenza Hemagglutinin,” Journal of Cell Biology 169: 965-976 (2005).
  •  “Fluorescence Photoconversion Kinetics in Novel Green Fluorescent Protein pH Sensors (pHluorins),” S.T. Hess, A.A. Heikal, and W.W. Webb, J. Phys. Chem. B 108: 10138-10148 (2004).
  • “Imaging Coexisting Fluid Domains in Biomembrane Models Coupling Curvature and Line Tension,” T. Baumgart, S.T. Hess, and W.W. Webb, Nature 425: 821-824 (2003).
  • “Quantitative Analysis of the Fluorescence Properties of Intrinsically Fluorescent Proteins in Living Cells,” S. T. Hess, E.D. Sheets, A. Wagenknecht-Wiesner, and A. A. Heikal, Biophysical Journal 85: 2566-2580 (2003).
  •  “Focal Volume Optics and Experimental Artifacts in Confocal Fluorescence Correlation Spectroscopy,” S.T. Hess and W.W. Webb, Biophysical Journal 83: 2300-2317 (2002).
  • “Biological and Chemical Applications of Fluorescence Correlation Spectroscopy,” S.T. Hess, S. Huang, A.A. Heikal, and W.W. Webb, Biochemistry 41: 697-705 (2002).
  • “Multiphoton molecular spectroscopy and excited-state dynamics of enhanced green fluorescent protein (EGFP): acid-base specificity,” A.A. Heikal, S.T. Hess, W.W. Webb, Chemical Physics 274: 37-55 (2001).
  • “Molecular spectroscopy and dynamics of intrinsically fluorescent proteins: Coral red (dsRed) and yellow (Citrine)”, A.A. Heikal, S.T. Hess, G.S. Baird, R.Y. Tsien, and W.W. Webb, Proc. Natl. Acad. Sci. USA 97: 11996-12001 (2000). See also correction in same volume, p. 14831.
  • “Design of Organic Molecules with Large Two-Photon Absorption Cross Sections,” M. Albota, D. Beljonne, J.-L. Brédas, J. Ehrlich, J.-Y. Fu, A. Heikal, S. Hess, T. Kogej, M. Levin, S. Marder, D. McCord-Maughon, J. Perry, H. Röckel, M. Rumi, G. Subramaniam, W. Webb, X.-L. Wu, and C. Xu, Science 281, 1653-1656 (1998).