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Published on: January 21, 2013
Second-harmonic imaging microscopy of living cells
P J Campagnola1, H A Clark, W A Mohler
1Center for Biomedical Imaging Technology, University of Connecticut Health Center, 263 Farmington Avenue, Farmington, Connecticut 06030, USA.
Journal of Biomedical Optics
|August 23, 2001
Summary
Second harmonic generation (SHG) microscopy offers high-resolution imaging for cellular structures, complementing two-photon excited fluorescence (TPEF) with unique contrast mechanisms and high sensitivity for membrane potential and protein imaging.
Area of Science:
- Nonlinear optics
- Biomedical imaging
- Cellular microscopy
Background:
- Second harmonic generation (SHG) is a nonlinear optical imaging microscopy technique.
- SHG shares advantageous features with two-photon excited fluorescence (TPEF) microscopy, including optical sectioning and reduced photodamage.
- SHG offers a complementary contrast mechanism to TPEF, primarily detected in the transmitted light path.
Purpose of the Study:
- To develop and present Second Harmonic Generation (SHG) microscopy for high-resolution imaging.
- To highlight SHG's capabilities in imaging cellular membranes and intact tissues.
- To compare SHG with established techniques like TPEF and confocal microscopy.
Main Methods:
- Utilizing nonlinear optical principles for SHG microscopy.
- Employing optical sectioning for three-dimensional imaging of thick specimens.
- Detecting SHG signals in the transmitted light optical path.
Main Results:
- SHG provides high-resolution imaging of cellular membranes and intact tissues.
- SHG demonstrates high membrane specificity and sensitivity in reporting membrane potential.
- SHG can image highly ordered structural proteins without exogenous labels.
- SHG results in less photodamage to living tissue compared to confocal microscopy.
Conclusions:
- SHG microscopy is a powerful tool for high-resolution, low-photodamage imaging of biological specimens.
- SHG offers unique advantages for studying membrane properties and structural proteins.
- SHG serves as a valuable complement to TPEF microscopy in biological research.
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