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The complexity of current problems in biophysics and cell biology increasing require chemical and physical-based methods to glean meaningful solutions. Central to this approach is the development of new technologies. Research efforts in my lab are directed at further developing nonlinear optical spectroscopy and microscopy methods to address problems related to structural protein arrays (e.g. collagen, and acto-myosin complexes). One long-term goal is to disseminate new tools for high resolution clinical imaging applications that are more powerful than current methods. We are also developing new optical methods for nano/microfabrication of tissue engineering scaffolds. The group currently works on the following three projects: 1) Second Harmonic Generation (SHG) imaging of tissues We are developing SHG imaging microscopy as an “optical biopsy” tool to differentiate normal and diseases states. Since SHG is highly sensitive to the supramolecular structure of structural protein arrays, this imaging modality is a powerful approach to this problem. A central theme of this project is to develop a thorough understanding of the underlying chemical and physical properties that give rise to the SHG. This information then guides us to the appropriate optical signatures in assessing diseased tissues. We are currently focusing on muscular and skeletal disorders including aging and Osteogenesis Imperfecta, respectively. Through quantitative SHG measurements, we have observed that this technique provides clear delineation in these pathologies, and suggests its use as a clinical diagnostic tool. 2) Multiphoton excited (MPE) nano/microfabrication of tissue engineering scaffolds A major challenge in tissue engineering is the fabrication of multiscale (i.e. Extra Cellular Matrix-cell-tissue) scaffolds that provide the topographic and biochemical ECM cues found in native tissue such as skin. To solve this problem, we use MPE photochemistry to fabricate synthetic ECMs directly from structural proteins including collagen, fibronectin, and laminin. The approach is analogous to that of two-photon excited fluorescence microscopy, but rather than imaging plane by plane, we exploit the multiphoton confinement of the excitation to the focal plane to nano/microfabricate 3-D structures. The project entails development of new optical instrumentation to replicate the complex structure of the native ECM, physical and chemical characterization of the scaffolds, as well as developing a fundamental understanding of the topographic and biochemical factors that are responsible for cell differentiation and new tissue synthesis. 3) MPE Intracellular Nanofabrication A new project uses multiphoton excited photochemistry to fabricate structures inside live cells as a means of studying spatially localized signaling events. We can fabricate walls, channels, and corridors directly from cytoplasmic proteins. Our results indicate that these structures form effective barriers to diffusion. In collaboration with Dr. Vladimer Rodionov’s group, we will use this method to examine microtubule dynamics in fibroblasts.
Honors and Awards Phillip Morris Scholarship, 1985-1986 Kent Fellow, Yale, 1986-1990 JILA Fellowship, University of Colorado, 1992-1995
Selected Publications P.J. Campagnola,*M.-de Wei, A. Lewis, and L.M. Loew, “High resolution non-linear optical microscopy of living cells by second harmonic generation” Biophys. J 77, 3341, (1999). P.J. Campagnola,* A. R. Howell, D. Delguidas, G.A. Epling, J.D. Pitts, and S.L. Goodman, “3- Dimensional sub-micron polymerization of acrylamide by multi-photon excitation of xanthene dyes,” Macromolecules 33, 1511-1513 (2000). J. D. Pitts, P.J. Campagnola,* G.A. Epling, and S.L. Goodman, “Reaction efficiencies for sub-micron multi-photon freeform fabrications of proteins and polymers with applications in sustained release. Macromolecules 33, 1514-1523 (2000). H. A. Clark , P. J. Campagnola, * J. P. Wuskell, A. Lewis, and L. M. Loew “Second Harmonic Generation Properties of Fluorescent Polymer Encapsulated Gold Nanoparticles Studied by High Resolution Non-Linear Optical Microscopy” JACS (ed comm), 122, 10234 (2000). Terasaki, M., Campagnola, P., Rolls, M.M., Stein, P., Ellenberg, J., Hinkle, B., and Slepchenko, B. A new model for nuclear envelope breakdown. Mol. Biol. Cell, 12, 503-510 (2001). P. J. Campagnola, H. A. Clark, W. A. Mohler, A. Lewis, and L. M. Loew, “Second Harmonic Imaging Microscopy of Living Cells” J. Biomedical Optics, 6, 277-286 (2001). P. J. Campagnola*, A.C. Millard, M. Terasaki, P.E. Hoppe, C. J. Malone, and W.A. Mohler, “3-Dimesional High-Resolution Second Harmonic Generation Imaging of Endogenous Structural Proteins in Biological Tissues” Biophys J. 81, 493-508 (2002). J. D. Pitts, A. R. Howell, I. Banerjee, J. Wang, S. L. Goodman, and P. J. Campagnola* New photoactivators for multi-photon excited 3 dimensional sub-micron crosslinking of proteins. Photochem Photobiol, 76 135-144 (2002). M. Sridhar, S. Basu, V. L. Scranton, and P.J. Campagnola,* “Construction of a multipurpose non-linear optical microscope for multi-photon excited optical fabrication” Rev. Sci. Instrum, 74, 3474-3477 (2003). S. Basu and P.J. Campagnola,* “Enzymatic Activity of Alkaline Phosphatase inside Protein and Polymer Structures Fabricated via Multi-photon Excitation” Biomacromolecules, 5, 572-579 (2004). R.M. Brown, Jr, A.C. Millard, and P.J. Campagnola,*”Macromolecular Structure of Cellulose Studied by Second Harmonic Generation Imaging Microscopy” Optic Lett 22, 2207-2209 (2003). P.J. Campagnola and L.M. Loew, “Exact change required: Second harmonic imaging microscopy visualizes distinct biomolecular arrays in live cells tissues and organisms” Nature Biotech, 11, 1356-1360, (2003). S. Basu and P.J . Campagnola “Properties of Crosslinked Protein Matrices for Tissue Engineering Applications Synthesized by Multiphoton Excitation” J. Mat. Biomed. Res., 71A, 359-368 (2004). S. Basu, V. Rodionov, M. Terasaki, and P.J. Campagnola “Multiphoton Excited Microfabrication in Live Cells via Rose Bengal Crosslinking of Cytoplasmic Proteins” Optics Lett.,30. 159-161 (2005) S. Basu, C.W. Wolgemuth, and P.J. Campagnola, “Measurement of Normal and Anomalous Diffusion of Dyes within Protein Structures Fabricated via Multi-photon Excited Crosslinking,” Biomacromoleucles,5, 2347-2357 (2004). Basu S, Cunningham LP, Pins G, Bush K, Toboada R, Howell AR, Wang J, Campagnola PJ. “Multi-photon Excited Fabrication of Collagen Matrices Crosslinked by a Modified Benzophenone Dimer: Bioactivity and Enzymatic Degradation”. Biomacromolecules,6, 1465-1474 (2005). S. Plotnikov, V. Juneja, A. Isaacson, W.A. Mohler, and P. J. Campagnola, Optical Clearing for Improved Contrast in Second Harmonic Generation Imaging of Skeletal Muscle, Biophys. J.,90, 328-339 (2006) S. Plotnikov, A.C. Millard, P.J. Campagnola, and W.A. Mohler, “Characterization of the myosin-based source for second-harmonic generation from muscle sarcomeres.” Biophys J., 90, 693-703 (2006). G. D. Pins, K. A. Bush, L. P. Cunningham, and P.J. Campagnola, “Multiphoton Excited Fabricated Nano and MicroPatterned Extracellular Matrix Proteins Direct Cellular Morphology”, J Biomed. Mat. Res. In press.
Invited Reviews L.M. Loew, P.J. Campagnola, A. Lewis, and J.P. Wuskell “Confocal and Non-linear Optical Imaging of Potentiometric Dyes” Methods in Cell Biology, 70, 429-453, (2002) A.C. Millard, P.J. Campagnola, W.A. Mohler, A. Lewis, and L.M. Loew, “Second Harmonic Imaging of Living Cells”, to appear in Methods in Enzymology. P.J. Campagnola,* A.C. Millard, and W.A. Mohler, “Second Harmonic Generation Imaging Microscopy of Endogenous Structural Proteins” Methods, 29, 97 – 109 (2002). William A. Mohler and P.J. Campagnola,* “Nonlinear optical spectroscopy and imaging of structural proteins in living tissues” Optics and Photonics News, 14, 40-45 (2003). P. J. Campagnola, “Second Harmonic Generation Imaging Microscopy of Structural Protein Arrays in Tissue, “Handbook of biological nonlinear optical microscopy" to be publi1zshed by the Oxford University press. Patents P.J. Campagnola and S.L. Goodman, “Freeform Nanofabrication using multi-photon excitation” U.S. #6316153 S.L. Goodman and P.J. Campagnola “ Freeform fabrication using multi-photon excitation US #6713772 |
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