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Biophys J, January 2002, p. 493-508, Vol. 82, No. 1

Three-Dimensional High-Resolution Second-Harmonic Generation Imaging of Endogenous Structural Proteins in Biological Tissues

Paul J. Campagnola,*Dagger Andrew C. Millard,*Dagger Mark Terasaki,*Dagger Pamela E. Hoppe,§ Christian J. Malone, and William A. Mohlerdagger Dagger

 *Department of Physiology and  dagger Department of Genetics and Developmental Biology and  Dagger Center for Biomedical Imaging Technology, University of Connecticut Health Center, Farmington, Connecticut 06030;  §Department of Genetics, Washington University School of Medicine, St. Louis, Missouri 63110; and  Laboratory of Molecular Biology, University of Wisconsin, Madison, Wisconsin 53706 USA

We find that several key endogenous protein structures give rise to intense second-harmonic generation (SHG)---nonabsorptive frequency doubling of an excitation laser line. Second-harmonic imaging microscopy (SHIM) on a laser-scanning system proves, therefore, to be a powerful and unique tool for high-resolution, high-contrast, three-dimensional studies of live cell and tissue architecture. Unlike fluorescence, SHG suffers no inherent photobleaching or toxicity and does not require exogenous labels. Unlike polarization microscopy, SHIM provides intrinsic confocality and deep sectioning in complex tissues. In this study, we demonstrate the clarity of SHIM optical sectioning within unfixed, unstained thick specimens. SHIM and two-photon excited fluorescence (TPEF) were combined in a dual-mode nonlinear microscopy to elucidate the molecular sources of SHG in live cells and tissues. SHG arose not only from coiled-coil complexes within connective tissues and muscle thick filaments, but also from microtubule arrays within interphase and mitotic cells. Both polarization dependence and a local symmetry cancellation effect of SHG allowed the signal from species generating the second harmonic to be decoded, by ratiometric correlation with TPEF, to yield information on local structure below optical resolution. The physical origin of SHG within these tissues is addressed and is attributed to the laser interaction with dipolar protein structures that is enhanced by the intrinsic chirality of the protein helices.

Biophys J, January 2002, p. 493-508, Vol. 82, No. 1
© 2002 by the Biophysical Society   0006-3495/02/01/493/16  $2.00


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