Biomedical Engineering Reference
In-Depth Information
4
SHG Microscopy and Its
Comparison with THG,
CARS, and Multiphoton
Excited Fluorescence
Imaging
4.1 Introduction ........................................................................................ 81
4.2 Physical Origin and Contrast Mechanism of SHG and the
Other NLO Microscopies ..................................................................82
SHG Photophysics • TPEF Photophysics • THG
Photophysics • CARS and SRS Photophysics
4.3 Instrumentation of NLO Microscopes............................................89
4.4 Representative Biological Applications of SHG Imaging
and Its Comparison with Other NLO Techniques........................92
Representative SHG Examples and Unique Aspects • TPEF and
SHG Comparison • THG and SHG Comparison • CARS, SRS, and
SHG Comparison
4.5 Summary..............................................................................................97
References........................................................................................................97
Xiyi Chen
LaserGen Corporation
Paul J. Campagnola
University of
Wisconsin—Madison
4.1 introduction
Second-harmonic generation (SHG) is a nonlinear effect that was discovered in 1961 by Franken, Hill,
and Weinreich [1] where frequency-doubled 347 nm light was generated when intense 694 nm light
from a ruby laser was focused on a quartz sample. Subsequently, with the development of short-pulse
lasers that produce much higher instantaneous light intensity and improved methods to grow uniaxial
birefringent crystals with large nonlinear susceptibility, SHG has been used as an effective approach for
new wavelength generation. This greatly extends scientific researchers' capability as most primary laser
sources are in the visible/near-infrared spectrum, whereas the optical absorption of most materials is at
bluer wavelengths.
Soon after SHG was discovered in crystals, it began to find useful applications in biological research.
In 1971, Fine and Hansen reported that SHG can be produced from collagenous tissues [2]. Freund then
extended this by implementing SHG into a stage scanning microscope to image rat tail tendon at ~50
micron resolution. Subsequently, Lewis et al. showed that image cell membranes that had been labeled
voltage-sensitive dyes produced SHG contrast [3]. Later, Campagnola et  al. implemented SHG into a
laser scanning microscope, first for cellular imaging and then for imaging tissues [4-6].
81
 
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