Biomedical Engineering Reference
In-Depth Information
14
Combined SHG/THG
Imaging
14.1 Excitation Laser Sources for SHG/THG Microscopy .................309
Selection of Wavelength • Reduction of
Photodamage • Improvement of Penetrability
14.2 Cr:F-Based SHG/THG Microscopy ............................................... 312
Principle of SHG and THG • 3D Spatial Resolution • System Setup
14.3 Biomedical Applications.................................................................. 319
Imaging Contrasts of SHG and THG • Animal Models:
Zebrafish • Animal Models: Mouse • Human Tissues •
Clinical Applications
14.4 Conclusion .........................................................................................343
References..................................................................................................... 344
Szu-Yu Chen
National Taiwan University
Chi-Kuang Sun
National Taiwan University
Currently, two-photon fluorescence (2PF) microscopy is the most common technique used in combina-
tion with second harmonic generation (SHG) microscopy. Making use of the various endogenous fluo-
rophores found in bio-tissues, 2PF microscopy can provide more cellular, morphological, and molecular
information regarding these tissues in addition to the SHG-revealed information; moreover, it can also
help localize the SHG signals and identify the contrast sources of these signals. However, since 2PF
microscopy is a fluorescence-based technique, issues of photodamage and photobleaching due to mul-
tiphoton absorption are always of concern in the imaging process. To avoid such fluorescence-induced
concerns, third harmonic generation (THG) microscopy, a third-order nonlinear optical microscopy,
could be used instead of 2PF microscopy. THG microscopy is well known to have interface sensitivity
and can be used as a general-purpose type of microscopy to provide structural information of the tissues.
Based on the characteristics of virtual-level transition and energy conservation, which are the same as
in SHG microscopy, with a combination of SHG with THG, the problems of fluorescence-induced pho-
todamage and photobleaching could be avoided so as to reduce the invasiveness. Meanwhile, owing to
its higher-order nonlinearity, a higher spatial resolution can be achieved with THG microscopy. In this
chapter, combined SHG/THG microscopy will be introduced, including the principles, system setup,
and the biomedical applications accomplished.
14.1 excitation Laser Sources for SHG/tHG Microscopy
14.1.1 Selection of Wavelength
For studies in bio-tissues based on two-photon microscopic, combining SHG, and 2PF microscopies,
femtosecond mode-locked Ti:sapphire (Ti:S) lasers are the most commonly used standard laser sources.
Ti:S lasers can be broadly tuned from 700 to 1000 nm and have the ability to excite a wide range of
fluorophores. By using a Ti:S laser, a more efficient excitation of SHG can be achieved in the bio-tissues
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