Research Focus

Many advances in biology and medicine are driven by the availability of new diagnostic tools. Our research focuses on the engineering of novel microscopy instrumentation and the application of these new tools to study biomedical problems. The problems tackled in my laboratory range from understanding the structure/function of single proteins, nature's smallest machines, to the devolpment of a new non-invasive optical method for cancer diagnosis. The available research topics in my laboratory can be categorized into molecular, cellular and tissue levels:

Molecular level projects

• Standing-Wave Total Internal Reflection Microscopy (SW-TIRM): Conventional optical microscopy has a resolution about 300 nm, limited by the diffraction of light. We are developing SW-TIRM that may fundamentally overcome the resolution barrier of optical microscopy and achieve resolution below 50 nm. This technique offers the potential to study the dynamics and function of cells and proteins with resolution approaching that of electron microscopes (In collaboration with Dr. E. Shyamsunder, Boston University).
• Single Molecule Dynamics: We are developing microscopic spectroscopy, imaging, and manipulation technologies to study proteins and biopolymers, such as DNA, on a single molecular level. Single molecule studies provide an understanding of how domain conformation changes regulate protein activities and functions. With our collaborators, we are studying the effect of DNA torsional forces on drug and protein binding and are studying the dynamics of l-exonuclease as it digests DNA. (In collaboration with Dr. P. Dedon, MIT and Dr. E. Gratton, UIUC).

Cellular level projects

• Cellular Mechanics and Mechanotransduction: In many of biological systems, such as bones, cartilages, and the vascular system, mechanical forces play important roles in regulating biological activities. We are developing micromanipulation techniques, optical and magnetic, to exert precise force on the cellular cytoskeleton. Further, we are developing imaging and spectroscopic methods to quantify cellular strains in 3-D, to monitor mechano-sensitive signaling events, and to study mechanical effects on gene expression. (In collaboration with Dr. R. Lee and Dr. R. Kamm, MIT)
• Intracellular Transport and Trafficking: A variety of intracellular transport and trafficking processes, such as endocytosis, exocytosis, and bacterial/viral invasion, are of fundamental biological importance. We are developing two new methods to quantify these processes. The first technique is two-photon single particle tracking. This technique allows the trajectory of a single particle to be monitored with nanometer spatial resolution and millisecond time resolution inside living cells. The second technique is two-photon fluorescence correlation spectroscopy that allows the quantification of the number density, the diffusion rate, and the flow rate of particles being transported inside cells.

Tissue level projects

• Functional Deep Tissue Imaging: There is a lack of non-invasive diagnostic techniques for imaging thick tissue biochemistry and morphology at sub-cellular resolution. Two-photon imaging allows the mapping of fluorophore distribution inside tissue down to a depth of over 500 mm providing sub-cellular level tissue morphological information. Spectroscopic measurement of endogenous fluorescence further allows us to quantify tissue biochemical and metabolic states. Spectroscopic instruments are developed to quantify the spectra and the lifetime of tissue fluorophores. (In collaboration with Dr. C. Dong, National Taiwan University, Dr. C. Buehler, Paul Scherrer Institut, and Dr. T. Hacewicz, Unilever Edgewater Laboratory).
• Two-Photon 3-D Image Cytometry: We are developing 3-D image cytometer based on a video-rate two-photon scanning microscopy. The high speed of this microscope allows the sampling of a large cell population and measures cellular properties high statistical precision. The use of a two-photon microscope further allows properties of individual cells to be assessed inside tissues in vivo. Two-photon 3-D cytometer has the potential for the detection of rare cellular events inside living animals. A promising application of this 3-D image cytometer is for the study of mitotic recombination events in tissues. (In collaboration with Dr. B. Engleward, MIT).
• Two-Photon Endoscopy: Based on two-photon deep tissue imaging technology, we are developing a miniaturized system for clinical diagnosis. We aim to distinguish healthy and pathological tissues based on their microscopic morphology and the variations in their endogenous fluorescence spectra. The development of this endoscopic instrument may enable non-invasive cancer diagnosis in dermal, gynecological, and gastrointestinal systems. (In collaboration with Dr. P. Kaplan, Unilever Edgewater Laboratory and Dr. S. Acker, Wisconsin Medical Center).

Selected Publications

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1. Buehler, C., Kim, K.H., Masters, B.R., So, P.T.C., Kochevar, I.E., Gratton, E., "Two-photon Deep Tissue Imaging," IEEE Engineering in Medicine and Biology Magazine, 18, 23-30 (1999).

2. Chen, Y., Muller, J.D., So, P.T.C., Gratton, E., "The Photon Counting Histogram in Fluorescence Fluctuation Spectroscopy," Biophys. J., 77, 553-67 (1999).

3. Farrer, R.A., Previte, M.J.R., Olson,C.E., Peyser, L.A., Fourkas, J.T., and So, P.T.C., "Single-molecule detection with a two-photon fluorescence microscope with fast-scanning capabilities and polarization sensitivity," Opt. Lett., 24, 1832-1834 (1999).

4. Kim, K.H., Buehler, C., So, P.T.C., "High Speed Two-photon Scanning Microscopy," Applied Optics, 38, 6004-6009 (1999).

5. Masters, B.R., So, P.T.C., Kim, K.H., Buehler, C., and Gratton, E., "Multiphoton excitation microscopy, confocal microscopy, and spectroscopy of living cells and tissues; functional metabolic imaging of human skin in vivo," Methods Enzymol., 307, 513-536 (1999).

6. Masters, B.R., So, P.T.C., Gratton, E., Gonnord, G., Corcuff, P., "Microscopy of Human Skin In Vivo: Multi-photon Excitation Microscopy and Confocal Microscopy," Microscopy and Microanalysis, 5, 282-289 (1999).

7. Masters, B.R., So, P.T.C. and Gratton, E., "Multi-photon Excitation Microscopy and Confocal Microscopy of Human Skin In Vivo," Comments on Molecular and Cellular Biophysics, 9, 379-405 (1999).

8. Riegler, M., Castagliuolo, I., So, P.T.C., Lotz, M., Wang, C., Wlk, M., Sogukoglu, T., Cosentini, E., Bischof, G., Hamilton, G., Teleky, B., Wenzl, E., Matthews, J.B., and Pothoulakis, C., "Effect of Substance P on Human Colonic Mucosa In Vitro," American Journal of Physiology: Gastrointestinal and Liver Physiology, 276, G1473-83, (1999).

9. Cragg, G. and So, P.T.C., "Standing-Wave Total Internal Reflection Microscopy," Opt. Lett., 25, 46-48 (2000).

10. Buehler, C., Dong, C.Y., So, P.T.C., and Gratton, E., "Time-resolved polarization imaging by pump-probe (stimulated emission) fluorescence microscopy," Biophys. J., 79, 536-549 (2000).

11. Frolov, A., Petrescu, A., Atshaves, B.P., So, P.T.C., Gratton, E., Serrero, G., and Schroeder, F., "High density lipoprotein-mediated cholesterol uptake and targeting to lipid droplets in intact L-cell fibroblasts. A single- and multiphoton fluorescence approach," J. Biol. Chem., 275, 12769-12780 (2000).

12. Gilbert, R.J., Hoffman, M., So, P.T.C., "Imaging of three-dimensional epithelial architecture and function in cultured CaCo2a monolayers with two-photon excitation microscopy," Micro. Res. Tech., 51, 204-210 (2000).

13. So, P.T.C., Dong, C.Y., Master, B.R., and Berland, K.M., "Two-Photon Excitation Fluorescence Microscopy," Ann. Rev. Biomedical Eng., 2, 399-429 (2000).

14. So, P.T.C., "Two-Photon Fluorescence Microscopy: A New Tool for Tissue Imaging and Spectroscopy," Histotechnology, 23, 221-228 (2000).

15. Masters, B.R., and So, P.T.C., "Confocal Microscopy and Two-Photon Excitation Microscopy of Human Skin In Vivo," Opt. Exp., 8, 2-10 (2001).

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