Biomedical Engineering Reference
In-Depth Information
8
SHG and Optical Clearing
8.1 Introduction ......................................................................................169
8.2 Physical Background ........................................................................ 170
8.3 Monte Carlo Simulations.................................................................172
8.4 Mechanism of Optical Clearing in Muscle...................................173
8.5 Optical Clearing in Collagenous Tissues......................................177
8.6 Optical Clearing in Skin..................................................................180
8.7 Optical Clearing in Kidney Tissue.................................................182
8.8 Alternative Investigation of Optical Clearing: Artificial
Environment......................................................................................184
8.9 Retention of SHG Polarization Signatures through Optical
Clearing ..............................................................................................185
8.10 Final Remarks....................................................................................187
References......................................................................................................187
Oleg Nadiarnykh
VU University
Paul J. Campagnola
University of
Wisconsin—Madison
8.1 introduction
There has been ever-increasing interest in the development of high-resolution optical imaging of the tis-
sue structure for biological and biomedical applications, both
in vitro
and
in vivo
. A special emphasis is
being made on minimally and noninvasive clinical imaging systems for diagnostics and monitoring of
cancers and other disorders. The most recent additions to the clinical tools are optical diffusion tomog-
raphy methods based on the measurement of scattering, absorption, and fluorescence from the tissue
over a depth of a few centimeters [1]. The main drawback of these methods is their little specificity due to
low resolution (~1 mm) that is inadequate for analysis of the microscopic tissue structure, for example,
remodeled collagen fibril/fiber assembly in the vicinity of tumors [2,3], disrupted myosin fibers and
sarcomeric patterns in muscle disorders [4,5], and various localized dynamic or static inhomogeneities
associated with disease states. Much better resolution is available through optical coherence tomogra-
phy (OCT, 1-2 μm lateral, 3-10 μm axial) and nonlinear optical (NLO) modalities: multiphoton (MPM)
and second-harmonic generation (SHG) microscopy (~0.5 μm). Although these techniques rely on near-
infrared (IR) wavelengths (700-1000 nm), imaging depths in tissues are still limited to ~0.5-1 mm at
best due to high multiple scattering of the excitation and even stronger scattering of emission signals
(shorter wavelengths), thus restricting imageable penetration depths for
in vivo
applications. We have
shown that the primary filter effect dominates for NLO processes in this spectral region [6]. We note
that OCT is carried out at 1300 nm, enabling increased depth, although still lacking true cellular reso-
lution. The biological window ends at approximately 1300 nm, where increased absorption from water
overtones becomes significant and the limiting factor.
One of the promising solutions for this intrinsic depth limitation is the optical clearing method,
where a high refractive index hyperosmotic agent is added to the tissue to increase its transparency and
to improve signal detection. The most accepted mechanism is via refractive index matching that reduces
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