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Applications of second-harmonic generation imaging microscopy in ovarian and breast cancer.

Tilbury K, Campagnola PJ - Perspect Medicin Chem (2015)

Bottom Line: Striking changes in collagen architecture are associated with these epithelial cancers, and SHG can image these changes with great sensitivity and specificity with submicrometer resolution.We also describe methods that exploit the SHG physical underpinnings that are effective in delineating normal and malignant tissues.The coherence and corresponding phase-matching process of SHG results in emission directionality (forward to backward), which is related to sub-resolution fibrillar assembly.

View Article: PubMed Central - PubMed

Affiliation: Department of Biomedical Engineering, University of Wisconsin-Madison, Madison, WI, USA.

ABSTRACT
In this perspective, we discuss how the nonlinear optical technique of second-harmonic generation (SHG) microscopy has been used to greatly enhance our understanding of the tumor microenvironment (TME) of breast and ovarian cancer. Striking changes in collagen architecture are associated with these epithelial cancers, and SHG can image these changes with great sensitivity and specificity with submicrometer resolution. This information has not historically been exploited by pathologists but has the potential to enhance diagnostic and prognostic capabilities. We summarize the utility of image processing tools that analyze fiber morphology in SHG images of breast and ovarian cancer in human tissues and animal models. We also describe methods that exploit the SHG physical underpinnings that are effective in delineating normal and malignant tissues. First we describe the use of polarization-resolved SHG that yields metrics related to macromolecular and supramolecular structures. The coherence and corresponding phase-matching process of SHG results in emission directionality (forward to backward), which is related to sub-resolution fibrillar assembly. These analyses are more general and more broadly applicable than purely morphology-based analyses; however, they are more computationally intensive. Intravital imaging techniques are also emerging that incorporate all of these quantitative analyses. Now, all these techniques can be coupled with rapidly advancing miniaturization of imaging systems to afford their use in clinical situations including enhancing pathology analysis and also in assisting in real-time surgical determination of tumor margins.

No MeSH data available.


Related in: MedlinePlus

Depth-dependent SHG F/B measurements of normal and high-grade serous ovarian cancer. Best fits using Monte Carlo simulations and independently measured μs and g resulted in 93% and 77% forward-directed SHG in normal and cancer respectively. Adapted from Ref. 30
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f4-pmc-7-2015-021: Depth-dependent SHG F/B measurements of normal and high-grade serous ovarian cancer. Best fits using Monte Carlo simulations and independently measured μs and g resulted in 93% and 77% forward-directed SHG in normal and cancer respectively. Adapted from Ref. 30

Mentions: The fibrillar gels used in the previous studies are essentially nonscattering, and FSHG/BSHG can be determined directly. However, this is not possible in real tissues. In tissue imaging, the measured SHG signal is a convolution of the initially emitted directionality (FSHG/BSHG) and the subsequent scattering of these photons at λSHG. Scattering in tissues is described by the scattering coefficient μs and scattering anisotropy g. The former is the inverse of the distance a photon will propagate before undergoing a collision and changing direction, and therefore is a measure of density. The scattering anisotropy g (unrelated to the SHG anisotropy β) describes the directionality of the scattering and varies from 0 to 1, with higher values corresponding to greater organization. Differentiation of the coherent SHG directionality FSHG/BSHG and the incoherent components (scattering) is an important consideration for clinical applications because both may be used to differentiate normal from malignant tissues that have changes in fibril size and/or organization.59 Campagnola and colleagues have developed a technique to extract FSHG/BSHG through a combination of 3D SHG imaging of measured forward to backward ratio (F/B) as a function of depth into the tissue, independent measurement of μs and g, and Monte Carlo simulations based on these parameters to model the propagation of the SHG photons. This combined approach affords decoupling of the SHG creation from scattering.29,59–61 As shown in Figure 4, the optimal Monte Carlo simulations revealed FSHG best fit values of 93% and 77% for the normal and high-grade ovarian cancer, respectively.29 We stress that this quantity arises from the sub-resolution fibril structure, and the best fit values are consistent with predictions based on our phase-matching model in conjunction with the corresponding transmission electron microscopy (TEM) images.29,55 Both normal and malignant ovarian stroma have fibrils that are much smaller in diameter (∼60 nm) than λSHG. However, the malignant stroma has more regularly packed fibrils where this condition is analogous to quasi-phase-matching, resulting in more efficient backward SHG.62 In sum, this analysis cannot resolve the small fibrils, but the coherence of the SHG permits inferences of organization on this sub-resolution size scale. SHG directionality measurements have also been extended to breast cancer, but the analyses have been inconclusive.63 This may be because the measurements were taken on thin histological slides and are susceptible to reflections and are also highly sensitive to any slight changes in thickness. In contrast, our combined imaging and simulation method requires tissues of 50–100 μm in thickness to properly account for scattering and are less sensitive to experimental artifacts. In addition, breast tissues are more heterogeneous than the ovarian stroma, which is comprised predominantly of dense collagen. Therefore, additional studies are required to obtain robust knowledge of the SHG directionality alterations associated with normal and malignant breast stroma.


Applications of second-harmonic generation imaging microscopy in ovarian and breast cancer.

Tilbury K, Campagnola PJ - Perspect Medicin Chem (2015)

Depth-dependent SHG F/B measurements of normal and high-grade serous ovarian cancer. Best fits using Monte Carlo simulations and independently measured μs and g resulted in 93% and 77% forward-directed SHG in normal and cancer respectively. Adapted from Ref. 30
© Copyright Policy - open-access
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4403703&req=5

f4-pmc-7-2015-021: Depth-dependent SHG F/B measurements of normal and high-grade serous ovarian cancer. Best fits using Monte Carlo simulations and independently measured μs and g resulted in 93% and 77% forward-directed SHG in normal and cancer respectively. Adapted from Ref. 30
Mentions: The fibrillar gels used in the previous studies are essentially nonscattering, and FSHG/BSHG can be determined directly. However, this is not possible in real tissues. In tissue imaging, the measured SHG signal is a convolution of the initially emitted directionality (FSHG/BSHG) and the subsequent scattering of these photons at λSHG. Scattering in tissues is described by the scattering coefficient μs and scattering anisotropy g. The former is the inverse of the distance a photon will propagate before undergoing a collision and changing direction, and therefore is a measure of density. The scattering anisotropy g (unrelated to the SHG anisotropy β) describes the directionality of the scattering and varies from 0 to 1, with higher values corresponding to greater organization. Differentiation of the coherent SHG directionality FSHG/BSHG and the incoherent components (scattering) is an important consideration for clinical applications because both may be used to differentiate normal from malignant tissues that have changes in fibril size and/or organization.59 Campagnola and colleagues have developed a technique to extract FSHG/BSHG through a combination of 3D SHG imaging of measured forward to backward ratio (F/B) as a function of depth into the tissue, independent measurement of μs and g, and Monte Carlo simulations based on these parameters to model the propagation of the SHG photons. This combined approach affords decoupling of the SHG creation from scattering.29,59–61 As shown in Figure 4, the optimal Monte Carlo simulations revealed FSHG best fit values of 93% and 77% for the normal and high-grade ovarian cancer, respectively.29 We stress that this quantity arises from the sub-resolution fibril structure, and the best fit values are consistent with predictions based on our phase-matching model in conjunction with the corresponding transmission electron microscopy (TEM) images.29,55 Both normal and malignant ovarian stroma have fibrils that are much smaller in diameter (∼60 nm) than λSHG. However, the malignant stroma has more regularly packed fibrils where this condition is analogous to quasi-phase-matching, resulting in more efficient backward SHG.62 In sum, this analysis cannot resolve the small fibrils, but the coherence of the SHG permits inferences of organization on this sub-resolution size scale. SHG directionality measurements have also been extended to breast cancer, but the analyses have been inconclusive.63 This may be because the measurements were taken on thin histological slides and are susceptible to reflections and are also highly sensitive to any slight changes in thickness. In contrast, our combined imaging and simulation method requires tissues of 50–100 μm in thickness to properly account for scattering and are less sensitive to experimental artifacts. In addition, breast tissues are more heterogeneous than the ovarian stroma, which is comprised predominantly of dense collagen. Therefore, additional studies are required to obtain robust knowledge of the SHG directionality alterations associated with normal and malignant breast stroma.

Bottom Line: Striking changes in collagen architecture are associated with these epithelial cancers, and SHG can image these changes with great sensitivity and specificity with submicrometer resolution.We also describe methods that exploit the SHG physical underpinnings that are effective in delineating normal and malignant tissues.The coherence and corresponding phase-matching process of SHG results in emission directionality (forward to backward), which is related to sub-resolution fibrillar assembly.

View Article: PubMed Central - PubMed

Affiliation: Department of Biomedical Engineering, University of Wisconsin-Madison, Madison, WI, USA.

ABSTRACT
In this perspective, we discuss how the nonlinear optical technique of second-harmonic generation (SHG) microscopy has been used to greatly enhance our understanding of the tumor microenvironment (TME) of breast and ovarian cancer. Striking changes in collagen architecture are associated with these epithelial cancers, and SHG can image these changes with great sensitivity and specificity with submicrometer resolution. This information has not historically been exploited by pathologists but has the potential to enhance diagnostic and prognostic capabilities. We summarize the utility of image processing tools that analyze fiber morphology in SHG images of breast and ovarian cancer in human tissues and animal models. We also describe methods that exploit the SHG physical underpinnings that are effective in delineating normal and malignant tissues. First we describe the use of polarization-resolved SHG that yields metrics related to macromolecular and supramolecular structures. The coherence and corresponding phase-matching process of SHG results in emission directionality (forward to backward), which is related to sub-resolution fibrillar assembly. These analyses are more general and more broadly applicable than purely morphology-based analyses; however, they are more computationally intensive. Intravital imaging techniques are also emerging that incorporate all of these quantitative analyses. Now, all these techniques can be coupled with rapidly advancing miniaturization of imaging systems to afford their use in clinical situations including enhancing pathology analysis and also in assisting in real-time surgical determination of tumor margins.

No MeSH data available.


Related in: MedlinePlus