Personnel
Sam Hess
University of Maine, Orono
Department of Physics
313 Bennett Hall
(207) 581-1036
sam.t.hess@umit.maine.edu
http://physics.umaine.edu/bios/
faculty/profindex.htm
Lab Members:
Manasa Gudheti, Postdoctoral Fellow
Jennifer Rochira, Graduate Student
Michael Mlodzianowski, Graduate Student
Mudalige (Siyath) Gunewardene, Graduate Student
Ryan Laughlin, Undergraduate
Bhupendra Nagpure, Undergraduate, College of the Atlantic
Research:
Development of Fluorescence Photophysics Tools for the study of Biomembrane Organization
Function and Lateral Organization of Biomembranes. How do biomembranes organize themselves to orchestrate biological functions such as signal transduction, protein trafficking, immune system response, and neurotransmission? Domains rich in cholesterol and sphingomyelin form under many conditions in biomembrane models, but do similar specialized microscopic membrane domains (microdomains) exist in living cells? If so, how and when do they form, and what are their size, shape, and contents? In cells infected with influenza virus, for example, one finds that the plasma membrane contains clusters of the viral fusion protein, Hemagglutinin (HA). How are HA domains used by the virus to gain entry into uninfected cells and to form new viruses, which will bud from the cell surface? To address these questions, we will use confocal microscopy, Fluorescence Recovery after Photobleaching, Fluorescence Resonance Energy Transfer, and Fluorescence Correlation Spectroscopy, coupled with numerical analysis and simulation of membrane mechanics and domain structure. We will attempt to resolve a long-standing controversy in the field of membrane biophysics in which numerous membrane models do not find consensus on the structure, size, and even existence of microdomains.
Single Molecule Fluorescence Photophysics of Quantum Dots and Fluorescent Proteins. Quantum dots and fluorescent proteins such as the green and red fluorescent proteins from Aequoria victoria and Discosoma sp. are being used increasingly for biological applications in living cells. However, optimization of such probes relies on understanding of their molecular photophysics. Fluorescence correlation spectroscopy can provide a wealth of information about the photophysical properties of single fluorescent molecules: fluorescence flicker, blinking, environmental sensitivity (for example to changes pH, ion concentrations, and temperature), photobleaching quantum yield, and intersystem crossing rates can be determined by solution measurements of such probes. Once such properties are determined, the signal-to-noise ratio for such probes can be optimized, while experimental artifacts, invasiveness, and toxicity to living cells can be minimized.
Focal Volume Optics and Experimental Artifacts in Confocal and Multiphoton Microscopy and Fluorescence Correlation Spectroscopy. Quantitative microscopy and spectroscopy relies on detailed knowledge of the observation volume, the volume in which fluorescence is both excited and detected. Numerical modeling of confocal optics has revealed conditions in which experimental artifacts will arise due to the non-Gaussian form of the observation volume. Experimental and theoretical results both show that under-filling the objective back aperture and minimizing the detector aperture or using two-photon excitation will result in the most nearly Gaussian observation volume and negligible experimental artifacts, but also a reduced signal-to-noise ratio. Thus, in order to obtain accurate fluorophore concentrations, diffusion coefficients, or other kinetic rate constants, users of FCS and quantitative microscopy must consider the trade-off between signal-to-noise and experimental artifacts.
Recent Publications:
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
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
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
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
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
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
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
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
Baumgart T, Hess ST, Webb WW. 2003. Imaging Coexisting Fluid Domains in Biomembrane Models Coupling Curvature and Line Tension. Nature 425:821-824
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, 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
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
Mukesh K, Hess S, Verma A, Farrington J, Kenworthy A, Roth M, Zimmerberg J. 2005. Lipid-protein interactions mediate influenza viral infection and membrane fusion. (in prep)
Hess ST, Heikal AA, Webb WW. 2005. Comparative Studies of Molecular Dynamics and Spectroscopy of Mutated Green Fluorescent Proteins. (in prep)
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