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.
Center for Biomedical Imaging Technology, Department of Physiology, University of Connecticut Health Center, Farmington, Connecticut 06030, USA.
Paul J
PJ
Campagnola
Leslie M
LM
Loew
D000200:Action Potentials / Q000502:physiology; D002462:Cell Membrane / Q000502:physiology* / Q000648:ultrastructure*; D002477:Cells / Q000166:cytology* / Q000648:ultrastructure; D007089:Image Enhancement / Q000295:instrumentation* / Q000379:methods*; D008564:Membrane Potentials / Q000502:physiology; D018613:Microscopy, Confocal / Q000295:instrumentation* / Q000379:methods*; D011506:Proteins / Q000648:ultrastructure*
D011506:Proteins