Cited by 1,326Advanced Fluorescence Microscopy TechniquesSpectroscopy Techniques in Biomedical and Chemical ResearchCell Image Analysis Techniques

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

Campagnola, Paul J, Loew, Leslie M·2003·Nature Biotechnology

DOI 10.1038/nbt894

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Abstract

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 a…

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Authors: Campagnola, P. J., & Loew, L. M.
Affiliations: UConn Health (US)
Corresponding author: Loew, Leslie M
ORCID: Campagnola, Paul J
Loew, Leslie M
Venue: Nature Biotechnology
Publisher: Springer Science and Business Media LLC
Published: 2003-10-31
Volume: 21
Issue: 11
Pages: 1356-1360
ISSN: 0733-222X (ISSN-L)
1087-0156
1546-1696
DOI: 10.1038/nbt894
OpenAlex: W2077572701
Cited by: 1,326
References: 29
Citations by year: 2026: 37 · 2025: 42 · 2024: 43 · 2023: 50 · 2022: 55 · 2021: 55 · 2020: 67 · 2019: 79 · 2018: 55 · 2017: 67 · 2016: 73 · 2015: 79 · 2014: 61 · 2013: 77 · 2012: 72
FWCI: 3.10
Type: journal article
Language: en
Open access: closed
Licence: www.springer.com/tdm (tdm)
Locations: Nature Biotechnology (publishedVersion)
PubMed (publishedVersion)
Funders: National Institutes of Health (5r21rr013472-02)
Office of Naval Research
National Institute of Biomedical Imaging and Bioengineering (R01EB00196)
National Center for Research Resources (R21 RR13472)
Topics: Life Sciences › Biochemistry, Genetics and Molecular Biology › Biophysics › Advanced Fluorescence Microscopy Techniques
Life Sciences › Biochemistry, Genetics and Molecular Biology › Biophysics › Spectroscopy Techniques in Biomedical and Chemical Research
Life Sciences › Biochemistry, Genetics and Molecular Biology › Biophysics › Cell Image Analysis Techniques
Keywords: second harmonic generation, nonlinear optical microscopy, collagen imaging, microtubules, myosin, membrane potential, neuronal electrical activity, in vivo imaging
MeSH terms: Action Potentials, Cell Membrane, Cells, Image Enhancement, Membrane Potentials, Proteins, Microscopy, Confocal

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

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