Subsurface tumor progression investigated by noninvasive optical second harmonic tomography

Abstract

Nonlinear optical imaging with femtosecond (10 ؊15 -second) laser technology was used to evaluate the subsurface tumor progression in control, dysplasia, and cancerous 7,12-dimethylbenz[a]anthracene-treated hamster cheek pouch mucosa tissues. Two-dimensional images of hamster cheek pouch mucosa tissues were obtained by scanning the second harmonic signal at various sagittal and axial positions. The spatial mapping of the second harmonic signals showed depth differentiation between normal, dysplasia, and a more advanced cancerous state. This nonlinear optical method offers a noninvasive in situ imaging tool to the medical community.The medical community is searching for new and noninvasive in situ methods to better characterize the mucosal tissues of the cervix, colon, ear, nose, throat, oral cavity, and esophagus, where disease starts at the epithelial layer a few hundred micrometers beneath the surface. Second harmonic generation (SHG) imaging is a nonlinear optical technique that produces images of subsurface structures, rendering in situ histological information of tissues without surgery. The imageforming signal depends on the morphological symmetry of the molecules and the local intra-and intercellular matrices of the tissue. This symmetry dependence is derived from the secondorder, nonlinear optical susceptibility 2 tensor component that has been used to study phenomena both in the interior region and at surfaces or interfaces in various materials, including biological samples (1-6).SHG tomography can provide information about tissue structure in addition to that offered by other linear optical microscopy techniques, such as optical coherence tomography (OCT) and reflectance confocal microscopy (7,8). Both of these latter methods depend on the reflectance changes caused by local variations in the indices of refraction. Imaging techniques such as ultrasound and MRI with surface coils (100-m in spatial resolution) are limited in their ability to provide submicrometer resolutions that are comparable to linear and nonlinear optical microscopy (9, § ). The application of localized excitation makes it possible to use optical sectioning and image mapping in evaluating tissue in situ. This nonlinear optical technique allows imaging that is not restricted to signals emitted from fluorescent molecules. The illumination needs not correspond to a particular absorption band of a molecule; the wavelength can be selected to optimize excitation and imaging conditions. The SHG signal is proportional to the reflective coefficient and dependent on the reciprocal of quadratic powers of the index of refraction (6). This useful property provides a higher optical contrast for visualizing matrix structures than does reflectance microscopy such as OCT, in which the signal is proportional to the reflective coefficients.In this report, we demonstrate the applicability of the SHG imaging technique for the evaluation of subsurface tumor progression from normal to advanced cancer states. This technology provides a noninvasive h...

Two‐photon microscopy of deep intravital tissues and its merits in clinical research

“…By contrast, multiphoton microscopy in conjunction with NIR light allows for reduced photodamage and deeper sensing depth. These advantages of multiphoton microscopy, along with its z-sectioning, have been employed as an encouraging imaging method in studying of living cells (König et al, 1996;Piston, 1999), mapping ligand-gated ion channel distributions (Denk, 1994), organelles and DNA imaging (Malak et al, 1997;König et al, 2000;Dedov et al, 2001), imaging mammalian embryos (Squirrell et al, 1999), skin probing (Masters et al, 1997;So et al, 1998), imaging of tumourous tissues (Guo et al, 1999), membrane imaging and visualizing of biomolecular array Moreaux et al, 2000;Campagnola & Loew, 2003). Fluorescence spectroscopy of NAD(P)H and flavoprotein under 2-photon absorption were furthermore addressed (Huang et al, 2002).…”

In vivo multiphoton fluorescence imaging: A novel approach to oral malignancy

“…The dependence of the SHG signal on tissue structure and local symmetry has been used for macroscopic mapping [20,21] . SHG was observed in type I collagen by nanosecond laser irradiation over a broad spectral region [21].…”

“…Nonlinear susceptibility is an optical property characteristic of chiral molecules. The microcrystalline structure of collagen [21][22][23][24] makes it capable of SHG [19,20]. Several researchers have reported endogenous second harmonic generation in biological tissues.…”

“…Cross-beam scanning SHG microscopy was studied in transmission geometry to show detailed variation of collagenous filaments in rat tail tendon [19]. The dependence of the SHG signal on tissue structure and local symmetry has been used for macroscopic mapping [20,21] . SHG was observed in type I collagen by nanosecond laser irradiation over a broad spectral region [21].…”

Automated Motile Cell Capture and Analysis with Optical Traps

“…Since SHG is dependent on the scale and the form of molecular ordering within the tissue Zipfel et al, 2003) and collagen is highly organized in the tendon, the method yields high contrast signals in that tissue. SHG has been shown to produce optically sectioned images of the plasma membrane (Campagnola et al, 2002;Peleg et al, 1999), tumor development in hamster cheek pouch mucosa (Guo et al, 1999), collagen in RAFT tissue models (Zoumi et al, 2002), and sarcomeres with the C. elegans nematode (Campagnola and Loew, 2003;Fig. 3).…”