- Ph.D., University of Pennsylvania, 1980
Two-photon excitation (TPE) of fluorescence occurs when two photons are absorbed simultaneously by a fluorophore with excitation energy equal to the sum of the two photons' energies. TPE has many applications in fluorescence microscopy stemming from the deeper penetration of the specimen allowed by using longer wavelength excitation and the possibility of restricting excitation to a small three-dimensional region. Ultraviolet excitation at 350nm, which is strongly absorbed by cells, can be replaced by simultaneous excitation with two 700nm photons, which are transmitted much better by cells. Because the simultaneous absorption of two photons has probability proportional to the square of the photon density, the probability of absorption in TPE falls off rapidly as the distance increases from the focal point of a focused beam thus concentrating the excitation in the focal plane. In order for TPE to happen with efficiency useful for fluorescence microscopy the excitation light must be concentrated in time and space. A laser scanning confocal microscope provides the concentration in space. A pulsed laser provides the concentration in time. The laser we use provides pulses of approximately 100fs duration. A laser scanning TPE microscope produces a three-dimensional region of excitation which is similar in size to the region imaged in laser scanning confocal microscopy but TPE has the advantage of not exciting the out of focus planes. We have assembled laser scanning TPE microscopes using our laser and converting three different laser scanning confocal microscopes. We are currently awaiting a Zeiss 510 NLO laser scanning confocal which will be a big improvement.
Fluorescence resonance energy transfer (FRET) is the transfer of energy from an excited fluorophore, the donor, to another molecule, the acceptor, by a dipole-dipole interaction. FRET is not mediated by a photon. The efficiency of FRET falls off rapidly as the distance between the molecules increases from 1nm to 10nm. FRET is therefore a molecular scale ruler that is capable of indicating the distance between one molecule labeled with the donor and another molecule labeled with the acceptor. FRET may be detected by the loss of donor emission when the acceptor is present or by the increase in acceptor emission if the acceptor is fluorescent. Binding and dimerization studies are practical applications of FRET especially since the Green Fluorescent Protein (GFP) and its mutants provide some donor-acceptor pairs that may be attached to the proteins of interest via gene splicing and introduced into the target cells where the chimeric proteins are produced by the cells own metabolic machinery. A less modern but often effective method is to label fixed cells with antibodies to the proteins of interest, one antibody conjugated to the FRET donor, say fluorescein, and another antibody conjugated to the acceptor, say rhodamine.
Fluorescence recovery after photobleaching (FRAP) is used to measure the lateral mobility of fluorescent or fluorescently labelled cell membrane components. All the fluorescence in a small region of the membrane is rapidly bleached (destroyed) by an intense laser beam. The lateral motion (diffusion and/or flow) of the fluorescent probe from outside the bleached region into the bleached region causes the fluorescence in the bleached region to increase. The time course of recovery of the fluorescence can be used to determine the diffusion coefficient of the probe. The diffusion coefficient reflects many biologically important membrane properties such as composition and organization of membrane lipids, and interactions of membrane proteins with each other and with cytoskeletal proteins. I have developed software to analyze FRAP recovery curves to extract diffusion coefficients for two diffusing components. To evaluate the precision of those diffusion coefficients and the reliability of distinguishing one component from two component diffusion, I automated the creation and analysis of many simulated FRAP curves. These simulations allow evaluation of the quality of the results, and the conclusions from the simulations can be used to estimate the confidence that one can have in the results of analyzing comparable real world experiments.
Caged compounds are compounds that normally express biological activity but in which the biological activity is inhibited by a photo-labile chemical bond (cage); UV light breaks the bond (opens the cage) and restores biological activity. I have designed a microscope illuminator which allows simultaneous observation of the specimen fluorescence excited by visible light and irradiation of a defined region of the specimen by UV light releasing the caged compound. This allows high temporal resolution in the observation of the results of the effects of the compound.
- Liang, X.H., Kleeman, L.K. Jiang, H.H., Gordon, G., Goldman, J.E. Berry, G., Herman, B., and Levine, B. 1998. Protection against fatal Sindbis virus encephalitis by Beclin, a novel Bcl-2-interacting protein. J. Virology., 72(11): 8586-8596.
- Mahajan, N., Linder, K., Berry, G., Gordon, G., Heim, R., and Herman, B. 1998. Bcl-2 and Bax interactions in individual mitochondria probed with mutant green fluorescent proteins and fluorescence resonance energy transfer. Nature Biotechnology. 16(6):547-52.
- Herman, B., Wang, X.F., Wodnicki, P., Periasamy, A., Mahajan, N., Berry, G., and Gordon, G. 1998. Fluorescence Lifetime Imaging Microscopy. Applied Fluorescence in Chemistry, Biology, and Medicine. June 1998.
- Gordon, G.W., Berry, G., Liang, X.H., Levine, B., and Herman, B. 1998. Quantitative fluorescence resonance energy transfer (FRET) measurements using fluorescence microscopy. Biophysical J., 74(5).
- Jacobson, K. A., Moore, S. E., Yang, B., Doherty, P., Gordon, G. W., and Walsh, F. S. 1997. Cellular determinants of the lateral mobility of neural cell adhesion molecules. Biochim. Biophys. Acta 1330:138-144.
- Herman, B., Wodnicki, P., Kwon, S., Periasamy, A., Gordon, G.W., Mahajan, N., and Wang, X.F. 1997. Recent developments in monitoring calcium and protein interactions in cells using fluorescence lifetime microscopy. J. Fluores. 7:85-91.
- Periasamy, A., Wodnicki, P., Wang, X.F., Kwon, S., Gordon, G., and Herman, B. 1996. Time resolved fluorescence lifetime imaging microscopy (TRFLIM) using a picosecond pulsed tunable dye laser system. Rev. Sci. Instr. 67(10): 3722-3731.