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Second-harmonic imaging microscopy for visualizing biomolecular arrays in cells, tissues and organisms


Although the nonlinear optical effect known as second-harmonic generation (SHG) has been recognized since the earliest days of laser physics and was demonstrated through a microscope over 25 years ago, only in the past few years has it begun to emerge as a viable microscope imaging contrast mechanism for visualization of cell and tissue structure and function. Only small modifications are required to equip a standard laser-scanning two-photon microscope for second-harmonic imaging microscopy (SHIM). Recent studies of the three-dimensional in vivo structures of well-ordered protein assemblies, such as collagen, microtubules and muscle myosin, are beginning to establish SHIM as a nondestructive imaging modality that holds promise for both basic research and clinical pathology. Thus far the best signals have been obtained in a transmitted light geometry that precludes in vivo measurements on large living animals. This drawback may be addressed through improvements in the collection of SHG signals via an epi-illumination microscope configuration. In addition, SHG signals from certain membrane-bound dyes have been shown to be highly sensitive to membrane potential. Although this indicates that SHIM may become a valuable tool for probing cell physiology, the small signal size would limit the number of photons that could be collected during the course of a fast action potential. Better dyes and optimized microscope optics could ultimately lead to the imaging of neuronal electrical activity with SHIM.

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Figure 1: Endogenous SHG images of native mouse leg.
Figure 2: Endogenous SHG imaging of a living adult C. elegans nematode, showing two distinct axial slices.
Figure 3: Membrane potential sensitivity of SHG from di-4-ANEPPS on a voltage-clamped N1E-115 neuroblastoma cell.


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The authors would like to thank their collaborators Aaron Lewis, Andrew Millard, William Mohler, Heather Clark and David Boudreau for their contributions to this work. We gratefully acknowledge financial support under US Office of Naval Research grant no. N0014-98-1-0703, National Institute of Biomedical Imaging and Bioengineering grant no. R01EB00196 and National Center for Research Resources grant no. R21 RR13472.

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Correspondence to Leslie M Loew.

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Campagnola, P., Loew, L. Second-harmonic imaging microscopy for visualizing biomolecular arrays in cells, tissues and organisms. Nat Biotechnol 21, 1356–1360 (2003).

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