We present two new methods used to determine the optical properties in ovarian cancer, idiopathic... more We present two new methods used to determine the optical properties in ovarian cancer, idiopathic pulmonary fibrosis and osteoarthritis. The bulk optical properties are determined by a combination of on axis attenuation, goniometry, and Monte Carlo simulations. The resulting wavelength dependent values delineates the importance of both μs’ and the fractal dimension. We also present a new method to determine μs’ on the SHG microscope. By measuring the depth dependence of the SHG emission directionality and performing Monte Carlo simulations, both the relative fibril size distribution and reduced scattering coefficient can be simultaneously determined without the need for bulk measurements.
Here, we examine ovarian cancer extracellular matrix (ECM) modification by measuring the waveleng... more Here, we examine ovarian cancer extracellular matrix (ECM) modification by measuring the wavelength dependence of optical scattering measurements and quantitative second-harmonic generation (SHG) imaging metrics in the range of 800-1100 nm in order to determine fibrillary changes in ex vivo normal ovary, type I, and type II ovarian cancer. Mass fractals of the collagen fiber structure is analyzed based on a power law correlation function using spectral dependence measurements of the reduced scattering coefficient μs′ where the mass fractal dimension is related to the power. Values of μs′ are measured using independent methods of determining the values of μs and g by on-axis attenuation measurements using the Beer-Lambert Law and by fitting the angular distribution of scattering to the Henyey-Greenstein phase function, respectively. Quantitativespectral SHG imaging on the same tissues determines FSHG/BSHG creation ratios related to size and harmonophore distributions. Both techniques...
Living cells function through the spatial compartmentalization of thousands of distinct proteins ... more Living cells function through the spatial compartmentalization of thousands of distinct proteins serving a multitude of diverse biochemical needs. Correlative super-resolution (SR) fluorescence and electron microscopy (EM) has emerged as a pathway to directly view nanoscale protein relationships to the underlying global ultrastructure, but has traditionally suffered from tradeoffs of structure preservation, fluorescence retention, resolution, and field of view. We developed a platform for three-dimensional correlative cryogenic SR and focused ion beam milled block-face EM across entire vitreously frozen cells that addresses these issues by preserving native ultrastructure and enabling independent SR and EM workflow optimization. Application to a variety of biological systems revealed a number of unexpected protein-ultrastructure relationships and underscored the value of a comprehensive multimodal view of ultrastructural variability across whole cells.
Journal of Toxicology and Environmental Health, 1977
This study was undertaken to examine the relative embryotoxicity in chick embryos of photochemica... more This study was undertaken to examine the relative embryotoxicity in chick embryos of photochemically reacted and unreacted diluted automotive exhaust emissions from a system equipped with a catalytic converter. Clean air controls and H2SO4 aerosol controls equivalent in concentration to those found in the catalytic exhaust atmosphere were also studied. From day 1 through day 14 of development, continuous exposure to nonirradiated exhause resulted in decreased survival, lowered embryonic weight, a small increase in heart/body weight ratio, and altered hematocrit and serum enzyme activities (LDH and GOT). Irradiated exhaust had little effect on survival or on embryonic weight but resulted in a higher liver/body weight ratio as well as altered hematocrit and serum enzyme activities. Interactions or cumulative effects of different compositions of exhaust atmospheres may play a role in differing biological responses between unreacted and irradiated exhaust. Sulfuric acid aerosol had a minimal effect on survival and resulted in only a slight decrease in embryonic weight and serum LDH activity, with no other apparent effects. In previous studies where the catalytic converter was not used, more pronounced effects on survival, increased heart/body weigh ratio, elevated serum GPT activity, and liver discoloration were observed. Thus, the introduction of an oxidizing catalytic converter appeared to alleviate some but not all of the embryotoxic effects of automotive exhaust.
Multiphoton Microscopy in the Biomedical Sciences XVII, 2017
Remodeling of the extracellular matrix in human ovarian cancer, can be reflected in increased col... more Remodeling of the extracellular matrix in human ovarian cancer, can be reflected in increased collagen concentration, changes in alignment and/or up-regulation of different collagen isoforms, including Col III. Using fibrillar gel models, we demonstrate that Col I and Col III can be quantitatively distinguished by 3 distinct SHG polarization specific metrics: i) determination of helical pitch angle via the single axis molecular model, ii) dipole alignment via anisotropy, and iii) chirality via SHG circular dichroism (SHG-CD). These sub-resolution differentiations are possible due to differences in the α helix angles of the two isoforms, which co-mingle in the same fibrils. We also investigated the mechanism of the SHG-CD response and show that unlike conventional CD, it is dominated by electric dipole interactions and is consistent with the two state SHG model. We further applied these 3 polarization resolved analyses to human normal, high risk, benign tumors, and malignant human ovarian tissues. We found that these tissues could all be differentiated by these metrics, where high grade tissues had analogous α-helical pitch angles to the in the Col I/Col III gel model. This confirms literature suggestions based on immunofluorescence and gene expression that Col III is up-regulated in high grade ovarian cancers. The different tissues also displayed differing anisotropies, indicating the fibril assemblies are distinct and likely do not result from remodeling of existing collagen but synthesis of new collagen. Importantly, these SHG polarization methods provide structural information not otherwise possible and can serve as label-free biomarkers for ovarian and other cancers.
Diagnosis and Treatment of Diseases in the Breast and Reproductive System, 2017
Ovarian cancer remains the most deadly gynecological cancer with a poor aggregate survival rate. ... more Ovarian cancer remains the most deadly gynecological cancer with a poor aggregate survival rate. To improve upon this situation, we utilized collagen-specific Second Harmonic Generation (SHG) imaging microscopy and optical scattering measurements to probe structural differences in the extracellular matrix of normal stroma, benign tumors, endometrioid tumors, and low and high-grade serous (LGS and HGS) tumors. The SHG signatures of the emission directionality and conversion efficiency as well as the optical scattering are related to the organization of collagen on the sub-micron size. The wavelength dependence of these readouts adds additional characterization of the size and distribution of collagen fibrils/fibers relative to the interrogating wavelengths. We found strong wavelength dependent dependencies of these metrics that were different between the different tumors that are related to respective structural attributes in the collagen organization. These sub-resolution determinations are consistent with the dualistic classification of type I and II serous tumors. However, type I endometrioid tumors have strongly differing ECM architecture than the serous malignancies. Moreover, our analyses are further consistent with LGS and benign tumors having similar etiology. We identified optimal wavelengths for the SHG metrics as well as optical scattering measurements. The SHG metrics and optical scattering measurements were then used to form a linear discriminant model to classify the tissues, and we obtained high accuracy (~90%) between the tissue types. This delineation is superior to current clinical performance and has potential applicability in supplementing histological analysis, understanding the etiology, as well as development of an in vivo screening tool.
Biological tissues have complex 3D collagen fiber architecture that cannot be fully visualized by... more Biological tissues have complex 3D collagen fiber architecture that cannot be fully visualized by conventional second harmonic generation (SHG) microscopy due to electric dipole considerations. We have developed a multi-view SHG imaging platform that successfully visualizes all orientations of collagen fibers. This is achieved by rotating tissues relative to the excitation laser plane of incidence, where the complete fibrillar structure is then visualized following registration and reconstruction. We evaluated high frequency and Gaussian weighted fusion reconstruction algorithms, and found the former approach performs better in terms of the resulting resolution. The new approach is a first step toward SHG tomography.
Ovarian cancer remains the most deadly gynecological cancer with a poor aggregate survival rate; ... more Ovarian cancer remains the most deadly gynecological cancer with a poor aggregate survival rate; however, the specific rates are highly dependent on the stage of the disease upon diagnosis. Current screening and imaging tools are insufficient to detect early lesions and are not capable of differentiating the subtypes of ovarian cancer that may benefit from specific treatments. As an alternative to current screening and imaging tools, we utilized wavelength dependent collagen-specific Second Harmonic Generation (SHG) imaging microscopy and optical scattering measurements to probe the structural differences in the extracellular matrix (ECM) of normal stroma, benign tumors, endometrioid tumors, and low and high-grade serous tumors. The SHG signatures of the emission directionality and conversion efficiency as well as the optical scattering are related to the organization of collagen on the sub-micron size scale and encode structural information. The wavelength dependence of these reado...
Remodeling of the collagen architecture in the extracellular matrix (ECM) has been implicated in ... more Remodeling of the collagen architecture in the extracellular matrix (ECM) has been implicated in ovarian cancer. To quantify these alterations we implemented a form of 3D texture analysis to delineate the fibrillar morphology observed in 3D Second Harmonic Generation (SHG) microscopy image data of normal (1) and high risk (2) ovarian stroma, benign ovarian tumors (3), low grade (4) and high grade (5) serous tumors, and endometrioid tumors (6). We developed a tailored set of 3D filters which extract textural features in the 3D image sets to build (or learn) statistical models of each tissue class. By applying k-nearest neighbor classification using these learned models, we achieved 83-91% accuracies for the six classes. The 3D method outperformed the analogous 2D classification on the same tissues, where we suggest this is due the increased information content. This classification based on ECM structural changes will complement conventional classification based on genetic profiles an...
We examine the optical second harmonic response in the UV, visible, and infrared regions from Ag(... more We examine the optical second harmonic response in the UV, visible, and infrared regions from Ag(1 11) immersed in aqueous electrolyte. We find that the second harmonic rotational anisotropy, which has been shown to be sensitive to the electrode crystalline structure, shows a marked wavelength dependence which is consistent with the electronic band structure for Ag(11 1). The anisotropy is highly sensitive to electrochemical potential induced changes in the surface electronic properties when the second harmonic frequency is in the UV region. This is in sharp contrast to the potential dependence of the second harmonic response in the visible or infrared region which arises entirely from the isotropic susceptibility elements.
Here we experimentally show that second-harmonic generation (SHG) imaging is not sensitive to col... more Here we experimentally show that second-harmonic generation (SHG) imaging is not sensitive to collagen fibers oriented parallel to the direction of laser propagation and, as a consequence, can potentially miss important structural information. As an alternative approach, we demonstrate the use of reflective micro-prisms to enable multi-view SHG imaging of mouse tail tendon by redirecting the focused excitation and collection of subsequent emission. Our approach data corroborates the theoretical treatment on vanishing and nonvanishing orientations, where fibers along the laser direction are largely transparent by SHG. In strong contrast, the two-photon excited fluorescence of dye-labeled collagen fibers is isotropic and is not subject to this constraint. We utilized Pearson correlation to quantify differences in fluorescent and backward detected SHG images of the tendon fiber structure, where the SHG and TPEF were highly statistically correlated (0.6-0.8) for perpendicular excitation but were uncorrelated for excitation parallel to the fiber axis. The results suggest that improved imaging of 3D collagen structure is possible with multi-view SHG microscopy.
Multi-wavelength SHG imaging microscopy is used to quantitatively probe alterations of the supram... more Multi-wavelength SHG imaging microscopy is used to quantitatively probe alterations of the supramolecular structure of collagen in the ECM of human ovarian cancer further developing the potential of collagen as a biomarker.
Purpose-The collagen structure throughout the patella has not been thoroughly investigated by 3D ... more Purpose-The collagen structure throughout the patella has not been thoroughly investigated by 3D imaging, where the majority of the exiting data comes from histological cross sections. It is important to have a better understanding of the architecture in normal tissues, where this could then be applied to imaging of diseased states. Methods-To address this shortcoming, we investigated the combined use of collagen specific Second Harmonic Generation (SHG) imaging and measurement of bulk optical properties to characterize collagen fiber orientations of the histologically defined zones of bovine articular cartilage. Forward and backward SHG intensities from sections from superficial, middle and deep zones were collected as a function of depth and analyzed by Monte Carlo simulations to extract the SHG creation direction, which is related to the fibrillar assembly. Results-Our results revealed differences in SHG forward-backward response between the three zones, where these are consistent with a previously developed model of SHG emission. Some of the findings are consistent with that from other modalities; however, SHG analysis showed the middle zone had the most organized fibril assembly. While not distinct, we also report bulk optical property values for these different zones within the patella.
ABSTRACT SHG imaging microscopy and tissue spectroscopy are used to quantitatively study structur... more ABSTRACT SHG imaging microscopy and tissue spectroscopy are used to quantitatively study structural changes in the collagen organization in the ECM in ovarian cancer using in in vitro models and ex vivo human tissues.
Patients with idiopathic fibrosis (IPF) have poor long-term survival as there are limited diagnos... more Patients with idiopathic fibrosis (IPF) have poor long-term survival as there are limited diagnostic/ prognostic tools or successful therapies. Remodeling of the extracellular matrix (ECM) has been implicated in IPF progression; however, the structural consequences on the collagen architecture have not received considerable attention. Here, we demonstrate that second harmonic generation (SHG) and multiphoton fluorescence microscopy can quantitatively differentiate normal and IPF human tissues. For SHG analysis, we developed a classifier based on wavelet transforms, principle component analysis, and a K-nearest-neighbor algorithm to classify the specific alterations of the collagen structure observed in IPF tissues. The resulting ROC curves obtained by varying the numbers of principal components and nearest neighbors yielded accuracies of >95%. In contrast, simpler metrics based on SHG intensity and collagen coverage in the image provided little or no discrimination. We also characterized the change in the elastin/collagen balance by simultaneously measuring the elastin autofluorescence and SHG intensities and found that the IPF tissues were less elastic relative to collagen. This is consistent with known mechanical consequences of the disease. Understanding ECM remodeling in IPF via nonlinear optical microscopy may enhance our ability to differentiate patients with rapid and slow progression and, thus, provide better prognostic information.
We present two new methods used to determine the optical properties in ovarian cancer, idiopathic... more We present two new methods used to determine the optical properties in ovarian cancer, idiopathic pulmonary fibrosis and osteoarthritis. The bulk optical properties are determined by a combination of on axis attenuation, goniometry, and Monte Carlo simulations. The resulting wavelength dependent values delineates the importance of both μs’ and the fractal dimension. We also present a new method to determine μs’ on the SHG microscope. By measuring the depth dependence of the SHG emission directionality and performing Monte Carlo simulations, both the relative fibril size distribution and reduced scattering coefficient can be simultaneously determined without the need for bulk measurements.
Here, we examine ovarian cancer extracellular matrix (ECM) modification by measuring the waveleng... more Here, we examine ovarian cancer extracellular matrix (ECM) modification by measuring the wavelength dependence of optical scattering measurements and quantitative second-harmonic generation (SHG) imaging metrics in the range of 800-1100 nm in order to determine fibrillary changes in ex vivo normal ovary, type I, and type II ovarian cancer. Mass fractals of the collagen fiber structure is analyzed based on a power law correlation function using spectral dependence measurements of the reduced scattering coefficient μs′ where the mass fractal dimension is related to the power. Values of μs′ are measured using independent methods of determining the values of μs and g by on-axis attenuation measurements using the Beer-Lambert Law and by fitting the angular distribution of scattering to the Henyey-Greenstein phase function, respectively. Quantitativespectral SHG imaging on the same tissues determines FSHG/BSHG creation ratios related to size and harmonophore distributions. Both techniques...
Living cells function through the spatial compartmentalization of thousands of distinct proteins ... more Living cells function through the spatial compartmentalization of thousands of distinct proteins serving a multitude of diverse biochemical needs. Correlative super-resolution (SR) fluorescence and electron microscopy (EM) has emerged as a pathway to directly view nanoscale protein relationships to the underlying global ultrastructure, but has traditionally suffered from tradeoffs of structure preservation, fluorescence retention, resolution, and field of view. We developed a platform for three-dimensional correlative cryogenic SR and focused ion beam milled block-face EM across entire vitreously frozen cells that addresses these issues by preserving native ultrastructure and enabling independent SR and EM workflow optimization. Application to a variety of biological systems revealed a number of unexpected protein-ultrastructure relationships and underscored the value of a comprehensive multimodal view of ultrastructural variability across whole cells.
Journal of Toxicology and Environmental Health, 1977
This study was undertaken to examine the relative embryotoxicity in chick embryos of photochemica... more This study was undertaken to examine the relative embryotoxicity in chick embryos of photochemically reacted and unreacted diluted automotive exhaust emissions from a system equipped with a catalytic converter. Clean air controls and H2SO4 aerosol controls equivalent in concentration to those found in the catalytic exhaust atmosphere were also studied. From day 1 through day 14 of development, continuous exposure to nonirradiated exhause resulted in decreased survival, lowered embryonic weight, a small increase in heart/body weight ratio, and altered hematocrit and serum enzyme activities (LDH and GOT). Irradiated exhaust had little effect on survival or on embryonic weight but resulted in a higher liver/body weight ratio as well as altered hematocrit and serum enzyme activities. Interactions or cumulative effects of different compositions of exhaust atmospheres may play a role in differing biological responses between unreacted and irradiated exhaust. Sulfuric acid aerosol had a minimal effect on survival and resulted in only a slight decrease in embryonic weight and serum LDH activity, with no other apparent effects. In previous studies where the catalytic converter was not used, more pronounced effects on survival, increased heart/body weigh ratio, elevated serum GPT activity, and liver discoloration were observed. Thus, the introduction of an oxidizing catalytic converter appeared to alleviate some but not all of the embryotoxic effects of automotive exhaust.
Multiphoton Microscopy in the Biomedical Sciences XVII, 2017
Remodeling of the extracellular matrix in human ovarian cancer, can be reflected in increased col... more Remodeling of the extracellular matrix in human ovarian cancer, can be reflected in increased collagen concentration, changes in alignment and/or up-regulation of different collagen isoforms, including Col III. Using fibrillar gel models, we demonstrate that Col I and Col III can be quantitatively distinguished by 3 distinct SHG polarization specific metrics: i) determination of helical pitch angle via the single axis molecular model, ii) dipole alignment via anisotropy, and iii) chirality via SHG circular dichroism (SHG-CD). These sub-resolution differentiations are possible due to differences in the α helix angles of the two isoforms, which co-mingle in the same fibrils. We also investigated the mechanism of the SHG-CD response and show that unlike conventional CD, it is dominated by electric dipole interactions and is consistent with the two state SHG model. We further applied these 3 polarization resolved analyses to human normal, high risk, benign tumors, and malignant human ovarian tissues. We found that these tissues could all be differentiated by these metrics, where high grade tissues had analogous α-helical pitch angles to the in the Col I/Col III gel model. This confirms literature suggestions based on immunofluorescence and gene expression that Col III is up-regulated in high grade ovarian cancers. The different tissues also displayed differing anisotropies, indicating the fibril assemblies are distinct and likely do not result from remodeling of existing collagen but synthesis of new collagen. Importantly, these SHG polarization methods provide structural information not otherwise possible and can serve as label-free biomarkers for ovarian and other cancers.
Diagnosis and Treatment of Diseases in the Breast and Reproductive System, 2017
Ovarian cancer remains the most deadly gynecological cancer with a poor aggregate survival rate. ... more Ovarian cancer remains the most deadly gynecological cancer with a poor aggregate survival rate. To improve upon this situation, we utilized collagen-specific Second Harmonic Generation (SHG) imaging microscopy and optical scattering measurements to probe structural differences in the extracellular matrix of normal stroma, benign tumors, endometrioid tumors, and low and high-grade serous (LGS and HGS) tumors. The SHG signatures of the emission directionality and conversion efficiency as well as the optical scattering are related to the organization of collagen on the sub-micron size. The wavelength dependence of these readouts adds additional characterization of the size and distribution of collagen fibrils/fibers relative to the interrogating wavelengths. We found strong wavelength dependent dependencies of these metrics that were different between the different tumors that are related to respective structural attributes in the collagen organization. These sub-resolution determinations are consistent with the dualistic classification of type I and II serous tumors. However, type I endometrioid tumors have strongly differing ECM architecture than the serous malignancies. Moreover, our analyses are further consistent with LGS and benign tumors having similar etiology. We identified optimal wavelengths for the SHG metrics as well as optical scattering measurements. The SHG metrics and optical scattering measurements were then used to form a linear discriminant model to classify the tissues, and we obtained high accuracy (~90%) between the tissue types. This delineation is superior to current clinical performance and has potential applicability in supplementing histological analysis, understanding the etiology, as well as development of an in vivo screening tool.
Biological tissues have complex 3D collagen fiber architecture that cannot be fully visualized by... more Biological tissues have complex 3D collagen fiber architecture that cannot be fully visualized by conventional second harmonic generation (SHG) microscopy due to electric dipole considerations. We have developed a multi-view SHG imaging platform that successfully visualizes all orientations of collagen fibers. This is achieved by rotating tissues relative to the excitation laser plane of incidence, where the complete fibrillar structure is then visualized following registration and reconstruction. We evaluated high frequency and Gaussian weighted fusion reconstruction algorithms, and found the former approach performs better in terms of the resulting resolution. The new approach is a first step toward SHG tomography.
Ovarian cancer remains the most deadly gynecological cancer with a poor aggregate survival rate; ... more Ovarian cancer remains the most deadly gynecological cancer with a poor aggregate survival rate; however, the specific rates are highly dependent on the stage of the disease upon diagnosis. Current screening and imaging tools are insufficient to detect early lesions and are not capable of differentiating the subtypes of ovarian cancer that may benefit from specific treatments. As an alternative to current screening and imaging tools, we utilized wavelength dependent collagen-specific Second Harmonic Generation (SHG) imaging microscopy and optical scattering measurements to probe the structural differences in the extracellular matrix (ECM) of normal stroma, benign tumors, endometrioid tumors, and low and high-grade serous tumors. The SHG signatures of the emission directionality and conversion efficiency as well as the optical scattering are related to the organization of collagen on the sub-micron size scale and encode structural information. The wavelength dependence of these reado...
Remodeling of the collagen architecture in the extracellular matrix (ECM) has been implicated in ... more Remodeling of the collagen architecture in the extracellular matrix (ECM) has been implicated in ovarian cancer. To quantify these alterations we implemented a form of 3D texture analysis to delineate the fibrillar morphology observed in 3D Second Harmonic Generation (SHG) microscopy image data of normal (1) and high risk (2) ovarian stroma, benign ovarian tumors (3), low grade (4) and high grade (5) serous tumors, and endometrioid tumors (6). We developed a tailored set of 3D filters which extract textural features in the 3D image sets to build (or learn) statistical models of each tissue class. By applying k-nearest neighbor classification using these learned models, we achieved 83-91% accuracies for the six classes. The 3D method outperformed the analogous 2D classification on the same tissues, where we suggest this is due the increased information content. This classification based on ECM structural changes will complement conventional classification based on genetic profiles an...
We examine the optical second harmonic response in the UV, visible, and infrared regions from Ag(... more We examine the optical second harmonic response in the UV, visible, and infrared regions from Ag(1 11) immersed in aqueous electrolyte. We find that the second harmonic rotational anisotropy, which has been shown to be sensitive to the electrode crystalline structure, shows a marked wavelength dependence which is consistent with the electronic band structure for Ag(11 1). The anisotropy is highly sensitive to electrochemical potential induced changes in the surface electronic properties when the second harmonic frequency is in the UV region. This is in sharp contrast to the potential dependence of the second harmonic response in the visible or infrared region which arises entirely from the isotropic susceptibility elements.
Here we experimentally show that second-harmonic generation (SHG) imaging is not sensitive to col... more Here we experimentally show that second-harmonic generation (SHG) imaging is not sensitive to collagen fibers oriented parallel to the direction of laser propagation and, as a consequence, can potentially miss important structural information. As an alternative approach, we demonstrate the use of reflective micro-prisms to enable multi-view SHG imaging of mouse tail tendon by redirecting the focused excitation and collection of subsequent emission. Our approach data corroborates the theoretical treatment on vanishing and nonvanishing orientations, where fibers along the laser direction are largely transparent by SHG. In strong contrast, the two-photon excited fluorescence of dye-labeled collagen fibers is isotropic and is not subject to this constraint. We utilized Pearson correlation to quantify differences in fluorescent and backward detected SHG images of the tendon fiber structure, where the SHG and TPEF were highly statistically correlated (0.6-0.8) for perpendicular excitation but were uncorrelated for excitation parallel to the fiber axis. The results suggest that improved imaging of 3D collagen structure is possible with multi-view SHG microscopy.
Multi-wavelength SHG imaging microscopy is used to quantitatively probe alterations of the supram... more Multi-wavelength SHG imaging microscopy is used to quantitatively probe alterations of the supramolecular structure of collagen in the ECM of human ovarian cancer further developing the potential of collagen as a biomarker.
Purpose-The collagen structure throughout the patella has not been thoroughly investigated by 3D ... more Purpose-The collagen structure throughout the patella has not been thoroughly investigated by 3D imaging, where the majority of the exiting data comes from histological cross sections. It is important to have a better understanding of the architecture in normal tissues, where this could then be applied to imaging of diseased states. Methods-To address this shortcoming, we investigated the combined use of collagen specific Second Harmonic Generation (SHG) imaging and measurement of bulk optical properties to characterize collagen fiber orientations of the histologically defined zones of bovine articular cartilage. Forward and backward SHG intensities from sections from superficial, middle and deep zones were collected as a function of depth and analyzed by Monte Carlo simulations to extract the SHG creation direction, which is related to the fibrillar assembly. Results-Our results revealed differences in SHG forward-backward response between the three zones, where these are consistent with a previously developed model of SHG emission. Some of the findings are consistent with that from other modalities; however, SHG analysis showed the middle zone had the most organized fibril assembly. While not distinct, we also report bulk optical property values for these different zones within the patella.
ABSTRACT SHG imaging microscopy and tissue spectroscopy are used to quantitatively study structur... more ABSTRACT SHG imaging microscopy and tissue spectroscopy are used to quantitatively study structural changes in the collagen organization in the ECM in ovarian cancer using in in vitro models and ex vivo human tissues.
Patients with idiopathic fibrosis (IPF) have poor long-term survival as there are limited diagnos... more Patients with idiopathic fibrosis (IPF) have poor long-term survival as there are limited diagnostic/ prognostic tools or successful therapies. Remodeling of the extracellular matrix (ECM) has been implicated in IPF progression; however, the structural consequences on the collagen architecture have not received considerable attention. Here, we demonstrate that second harmonic generation (SHG) and multiphoton fluorescence microscopy can quantitatively differentiate normal and IPF human tissues. For SHG analysis, we developed a classifier based on wavelet transforms, principle component analysis, and a K-nearest-neighbor algorithm to classify the specific alterations of the collagen structure observed in IPF tissues. The resulting ROC curves obtained by varying the numbers of principal components and nearest neighbors yielded accuracies of >95%. In contrast, simpler metrics based on SHG intensity and collagen coverage in the image provided little or no discrimination. We also characterized the change in the elastin/collagen balance by simultaneously measuring the elastin autofluorescence and SHG intensities and found that the IPF tissues were less elastic relative to collagen. This is consistent with known mechanical consequences of the disease. Understanding ECM remodeling in IPF via nonlinear optical microscopy may enhance our ability to differentiate patients with rapid and slow progression and, thus, provide better prognostic information.
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Papers by Kirby Campbell