Biomedical Engineering Reference
In-Depth Information
TABLE 14.3
Lateral Resolution of THG Microscopy in Live Human Skin and Fixed Human Skin
Live Human Skin (NA 1.2)
Fixed Human Skin (NA 0.9)
Resolution (nm) Resolution (nm)
Depth (μm) SHG THG Depth (μm) SHG THG
0 554.5 453.7 75 554 453
100 613.4 500.8 150 608 501
200 762.9 622.8 225 913 752
300 921.7 752.5 300 1287 1051
Theoretical 369 307.5 Theoretical 500 401
Source:
Adapted from Tsai, TH et al. 2006, Optical signal degradation study in fixed human skin using confocal micros-
copy and higher-harmonic optical microscopy,
Opt. Express
, 14(2), 749-758. With permission of Optical Society of America.
14.4 conclusion
In this chapter, the combined SHG/THG microscopy, the so-called higher-harmonic generation
microscopy or multiharmonic generation microscopy, has been introduced, including the princi-
ple, system setup, and biomedical applications. Instead of using a Ti:S laser for combined SHG/2PF
microscopy, a Cr:F laser with a wavelength located within 1200-1300 nm is used for combined SHG/
THG microscopy. Based on the Cr:F excitation, the wavelength of THG can be shifted into the visible
range to facilitate the efficient detection of THG signals. The appropriate wavelength of Cr:F excitation
(1230 nm) can reduce both the scattering and absorption in the bio-tissues, which can help to increase
the imaging penetrability and reduce the photodamages. Different from 2PF microscopy, only virtual-
level transition is involved in THG microscopy. Since both the SHG and THG processes obey the energy
conservation and no energy is deposited in the interacted tissues, the combined SHG/THG microscopy
can be said to be noninvasive. Utilizing the different characteristics of SHG and THG microscopy, vari-
ous image contrasts can be provided by SHG and THG microscopy, respectively, to reveal lots of valu-
able information in the bio-tissues. Based on the noninvasiveness nature of the combined SHG/THG
microscopy, this imaging modality is very suitable for
in vivo
and clinical investigation, especially the
long-term observation. So far, the combined SHG/THG microscopy has been applied to the
ex vivo
and
in vivo
investigation of different standard animal models such as zebrafish and mouse. Furthermore,
this imaging modality is also used to perform the clinical diagnosis of human tissues like human skin
and human oral mucosa. Owing to the interface sensitivity that THG microscopy possesses, THG
microscopy can be used as a generally purposed microscopy to provide histological information of the
tissues. In the combined SHG/THG microscopy, THG microscopy plays a similar role to 2PF micros-
copy to localize the SHG signals and identify the contrast sources of the SHG signals. However, com-
pared with 2PF microscopy, no issues about photodamages and photobleaching have to be taken into
consideration in THG microscopy even when the illumination power is around 100 mW. Thus, the
combined SHG/THG microscopy is noninvasive and much desirable for clinical applications. In the
latter parts of this chapter, some results on the clinical trials of SHG/THG imaging of human skin have
been discussed. Making use of the strong SHG contrasts provided by collagen fibers in the dermis and
the THG contrasts from cellular structures, the combined SHG/THG microscopy can help to reveal
both the connective tissue distribution in the dermis and the cellular morphology in the epidermis.
In addition, since THG contrasts can be enhanced by melanin and hemoglobin in the skin through
real-level resonance, THG microscopy can provide additional information on the pigmentation dis-
tribution and blood flow. All previous studies thus show that SHG and THG microscopy are with the
capability to reveal the diagnostic characteristics of various skin diseases such as nevus, melanoma,
and BCC. With the diagnostic significance, its noninvasiveness nature, >300-μm imaging penetrabil-
ity, and submicron spatial resolution, the combined SHG/THG microscopy is an undoubted desirable
