Fluorescence Lifetime Imaging Microscopy (FLIM) reveals spatial-metabolic changes in 3D breast cancer spheroids.

Cancer cells are mechanically sensitive to physical properties of the microenvironment, which can affect downstream signaling to promote malignancy, in part through the modulation of metabolic pathways. Fluorescence Lifetime Imaging Microscopy (FLIM) can be used to measure the fluorescence lifetime of endogenous fluorophores, such as the metabolic co-factors NAD(P)H and FAD, in live samples. We used multiphoton FLIM to investigate the changes in cellular metabolism of 3D breast spheroids derived from MCF-10A and MD-MB-231 cell lines embedded in collagen with varying densities (1 vs. 4 mg/ml) over time (Day 0 vs. Day 3). MCF-10A spheroids demonstrated spatial gradients, with the cells closest to the spheroid edge exhibiting FLIM changes consistent with a shift towards oxidative phosphorylation (OXPHOS) while the spheroid core had changes consistent with a shift towards glycolysis. The MDA-MB-231 spheroids had a large shift consistent with increased OXPHOS with a more pronounced change at the higher collagen concentration. The MDA-MB-231 spheroids invaded into the collagen gel over time and cells that traveled the farthest had the largest changes consistent with a shift towards OXPHOS. Overall, these results suggest that the cells in contact with the extracellular matrix (ECM) and those that migrated the farthest had changes consistent with a metabolic shift towards OXPHOS. More generally, these results demonstrate the ability of multiphoton FLIM to characterize how spheroids metabolism and spatial metabolic gradients are modified by physical properties of the 3D ECM.

Multiscale anisotropy analysis of second-harmonic generation collagen imaging of human pancreatic cancer.

Pancreatic ductal adenocarcinoma (PDAC) is one of the deadliest cancers with a minority (< 10%) of patients surviving five years past diagnosis. This could be improved with the development of new imaging modalities for early differentiation of benign and cancerous fibrosis. This study intends to explore the application of a two-photon microscopy technique known as second harmonic generation to PDAC using the 2D Wavelet Transform Modulus Maxima (WTMM) Anisotropy method to quantify collagen organization in fibrotic pancreatic tissue. Forty slides from PDAC patients were obtained and eight images were captured per each tissue category on each slide. Brownian surface motion and white noise images were generated for calibration and testing of a new variable binning approach to the 2D WTMM Anisotropy method. The variable binning method had greater resistance to wavelet scaling effects and white noise images were found to have the lowest anisotropy factor. Cancer and fibrosis had greater anisotropy factors (Fa) at small wavelet scales than normal and normal adjacent tissue. At a larger scale of 21 µm this relationship changed with normal tissue having a higher Fa than all other tissue groups. White noise is the best representative image for isotropy and the 2D WTMM anisotropy method is sensitive to changes induced in collagen by PDAC.

Beneficial impacts of neuromuscular electrical stimulation on muscle structure and function in the zebrafish model of Duchenne muscular dystrophy.

Neuromuscular electrical stimulation (NMES) allows activation of muscle fibers in the absence of voluntary force generation. NMES could have the potential to promote muscle homeostasis in the context of muscle disease, but the impacts of NMES on diseased muscle are not well understood. We used the zebrafish Duchenne muscular dystrophy (dmd) mutant and a longitudinal design to elucidate the consequences of NMES on muscle health. We designed four neuromuscular stimulation paradigms loosely based on weightlifting regimens. Each paradigm differentially affected neuromuscular structure, function, and survival. Only endurance neuromuscular stimulation (eNMES) improved all outcome measures. We found that eNMES improves muscle and neuromuscular junction morphology, swimming, and survival. Heme oxygenase and integrin alpha7 are required for eNMES-mediated improvement. Our data indicate that neuromuscular stimulation can be beneficial, suggesting that the right type of activity may benefit patients with muscle disease.

Visualization and analysis of whole depot adipose tissue neural innervation.

Little is known about the diversity and function of adipose tissue nerves, due in part to the inability to effectively visualize the tissue's diverse nerve subtypes and the patterns of innervation across an intact depot. The tools to image and quantify adipose tissue innervation are currently limited. Here, we present a method of tissue processing that decreases tissue thickness in the z-axis while leaving cells intact for subsequent immunostaining. This was combined with autofluorescence quenching techniques to permit intact whole tissues to be mounted on slides and imaged by confocal microscopy, with a complementary means to perform whole tissue neurite density quantification after capture of tiled z-stack images. Additionally, we demonstrate how to visualize nerve terminals (the neuro-adipose nexus) in intact blocks of adipose tissue without z-depth reduction. We have included examples of data demonstrating nerve subtypes, neurovascular interactions, label-free imaging of collagen, and nerve bundle digital cross-sections.

Multiscale anisotropy analysis of second-harmonic generation collagen imaging of mouse skin.

SIGNIFICANCE: Morphological collagen signatures are important for tissue function, particularly in the tumor microenvironment. A single algorithmic framework with quantitative, multiscale morphological collagen feature extraction may further the use of collagen signatures in understanding fundamental tumor progression. AIM: A modification of the 2D wavelet transform modulus maxima (WTMM) anisotropy method was applied to both digitally simulated collagen fibers and second-harmonic-generation imaged collagen fibers of mouse skin to calculate a multiscale anisotropy factor to detect collagen fiber organization. APPROACH: The modified 2D WTMM anisotropy method was initially validated on synthetic calibration images to establish the robustness and sensitivity of the multiscale fiber organization tool. Upon validation, the algorithm was applied to collagen fiber organization in normal wild-type skin, melanoma stimulated skin, and integrin α10KO skin. RESULTS: Normal wild-type skin collagen fibers have an increased anisotropy factor at all sizes scales. Interestingly, the multiscale anisotropy differences highlight important dissimilarities between collagen fiber organization in normal wild-type skin, melanoma stimulated, and integrin α10KO skin. At small scales (∼2 to 3 µm), the integrin α10KO skin was vastly different than normal skin (p-value ∼ 10 - 8), whereas the melanoma stimulated skin was vastly different than normal at large scales (∼30 to 40 µm, p-value ∼ 10 - 15). CONCLUSIONS: This objective computational collagen fiber organization algorithm is sensitive to collagen fiber organization across multiple scales for effective exploration of collagen morphological alterations associated with melanoma and the lack of α10 integrin binding.

Multiphoton Microscopy for the Characterization of Cellular Behavior on Naturally Derived Polysaccharide Tissue Constructs With Irregular Surfaces for the Development of Platform Biomaterials.

Over the past decade, the use of polymers as platform materials for biomedical applications including tissue engineering has been of rising interest. Recently, the use of naturally derived polysaccharides as 3-D scaffolds for tissue regeneration has shown promising material characteristics; however, due to complexities in composition, morphology, and optical properties, adequate spatial and temporal characterization of cellular behavior in these materials is lacking. Multiphoton microscopy has emerged as a viable tool for performing such quantification by permitting greater imaging depth while simultaneously minimizing un-favorable scattering and producing high-resolution optical cross sections for non-invasive analysis. Here we describe a method using endogenous contrast of cellulose nanofibers (CNF) using Second Harmonic Generation (SHG), combined with 2-photon fluorescence of Cell Tracker Orange for spatial and longitudinal imaging of cellular proliferation. Cell Tracker Orange is an ideal fluorophore to avoid the broad CNF autofluorescence allowing for segmentation of cells using a semi-automatic routine. Individual cells were identified using centroid locations for 3D cell proliferation. Overall, the methods presented are viable for investigation of cellular interactions with polysaccharide candidate biomaterials.

OpenSFDI: an open-source guide for constructing a spatial frequency domain imaging system.

Significance: Spatial frequency domain imaging (SFDI) is a diffuse optical measurement technique that can quantify tissue optical absorption (µa) and reduced scattering (µs') on a pixel-by-pixel basis. Measurements of µa at different wavelengths enable the extraction of molar concentrations of tissue chromophores over a wide field, providing a noncontact and label-free means to assess tissue viability, oxygenation, microarchitecture, and molecular content. We present here openSFDI: an open-source guide for building a low-cost, small-footprint, three-wavelength SFDI system capable of quantifying µa and µs' as well as oxyhemoglobin and deoxyhemoglobin concentrations in biological tissue. The companion website provides a complete parts list along with detailed instructions for assembling the openSFDI system.

Aim: We describe the design of openSFDI and report on the accuracy and precision of optical property extractions for three different systems fabricated according to the instructions on the openSFDI website.

Approach: Accuracy was assessed by measuring nine tissue-simulating optical phantoms with a physiologically relevant range of µa and µs' with the openSFDI systems and a commercial SFDI device. Precision was assessed by repeatedly measuring the same phantom over 1 h.

Results: The openSFDI systems had an error of 0 ± 6 % in µa and -2 ± 3 % in µs', compared to a commercial SFDI system. Bland-Altman analysis revealed the limits of agreement between the two systems to be ± 0.004 mm - 1 for µa and -0.06 to 0.1 mm - 1 for µs'. The openSFDI system had low drift with an average standard deviation of 0.0007 mm - 1 and 0.05 mm - 1 in µa and µs', respectively.

Conclusion: The openSFDI provides a customizable hardware platform for research groups seeking to utilize SFDI for quantitative diffuse optical imaging.

Probing ECM remodeling in idiopathic pulmonary fibrosis via second harmonic generation microscopy analysis of macro/supramolecular collagen structure.

Idiopathic pulmonary fibrosis (IPF) is a progressive disease with poor prognosis with short lifespan following diagnosis as patients have limited effective treatment options. A fundamental limitation is a lack of knowledge of the underlying collagen alterations in the disease, as this could lead to better diagnostics, prognostics, and measures of treatment efficacy. While the fibroses is the primary presentation of the disease, the collagen architecture has not been well studied beyond standard histology. Here, we used several metrics based on second harmonic generation (SHG) microscopy and optical scattering measurements to characterize the subresolution collagen assembly in human IPF and normal lung tissues. Using SHG directional analysis, we found that while collagen synthesis is increased in IPF, the resulting average fibril architecture is more disordered than in normal tissue. Wavelength-dependent optical scattering measurements lead to the same conclusion, and both optical approaches are consistent with ultrastructural analysis. SHG circular dichroism revealed significant differences in the net chirality between the fibrotic and normal collagen, where the former has a more randomized helical structure. Collectively, the measurements reveal significant changes in the collagen macro/supramolecular structure in the abnormal fibrotic collagen, and we suggest these alterations can serve as biomarkers for IPF diagnosis and progression.

Stromal alterations in ovarian cancers via wavelength dependent Second Harmonic Generation microscopy and optical scattering.

BACKGROUND: 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. METHOD: 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. RESULTS: 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 readouts adds additional characterization of the size and distribution of collagen fibrils/fibers relative to the interrogating wavelengths. We found a strong wavelength dependence of these metrics that are related to significant structural differences in the collagen organization and are consistent with the dualistic classification of type I and II serous tumors. Moreover, type I endometrioid tumors have strongly differing ECM architecture than the serous malignancies. The SHG metrics and optical scattering measurements were used to form a linear discriminant model to classify the tissues, and we obtained high accuracy (>90%) between high-grade serous tumors from the other tissue types. High-grade serous tumors account for ~70% of ovarian cancers, and this delineation has potential clinical applications in terms of supplementing histological analysis, understanding the etiology, as well as development of an in vivo screening tool. CONCLUSIONS: SHG and optical scattering measurements provide sub-resolution information and when combined provide superior diagnostic power over clinical imaging modalities. Additionally the measurements are able to delineate the different subtypes of ovarian cancer and may potentially assist in treatment protocols. Understanding the altered collagen assembly can supplement histological analysis and provide new insight into the etiology. These methods could become an in vivo screening tool for earlier detection which is important since ovarian malignancies can metastasize while undetectable by current clinical imaging resolution.

3D texture analysis for classification of second harmonic generation images of human ovarian cancer.

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 and can serve as an additional biomarker. Moreover, the texture analysis algorithm is quite general, as it does not rely on single morphological metrics such as fiber alignment, length, and width but their combined convolution with a customizable basis set.

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

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.

Articular cartilage zonal differentiation via 3D Second-Harmonic Generation imaging microscopy.

PURPOSE: The collagen structure throughout the patella has not been thoroughly investigated by 3D imaging, where the majority of the existing data come 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 of 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. CONCLUSIONS: Collectively, these results provide quantitative measurements of structural changes at both the fiber and fibril assembly of the different cartilage zones and reveals structural information not possible by other microscope modalities. This can provide quantitative insight to the collagen fiber network in normal cartilage, which may ultimately be developed as a biomarker for osteoarthritis.

Second harmonic generation microscopy analysis of extracellular matrix changes in human idiopathic pulmonary fibrosis.

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.

Experimental and simulation study of the wavelength dependent second harmonic generation of collagen in scattering tissues.

We report on the wavelength dependence of second harmonic generation (SHG) of collagen in scattering tissues over the wavelength range of 800-1200 nm. The study incorporates inclusion of the molecular hyperpolarizability ß of collagen and optical scattering, both of which are wavelength dependent. Using 3D SHG imaging and Monte Carlo simulations, we find the wavelength dependence of ß is not well described by a two-state model based on known absorption bands. We further find that longer wavelength excitation is inefficient as the reduction in scattering is overcome by the decreased ß far from resonance and the optimal excitation is within the 800-900 nm range. The impact is larger for backward collected SHG.

Differentiation of Col I and Col III isoforms in stromal models of ovarian cancer by analysis of second harmonic generation polarization and emission directionality.

A profound remodeling of the extracellular matrix occurs in many epithelial cancers. In ovarian cancer, the minor collagen isoform of Col III becomes upregulated in invasive disease. Here we use second harmonic generation (SHG) imaging microscopy to probe structural differences in fibrillar models of the ovarian stroma comprised of mixtures of Col I and III. The SHG intensity and forward-backward ratios decrease with increasing Col III content, consistent with decreased phasematching due to more randomized structures. We further probe the net collagen α-helix pitch angle within the gel mixtures using what is believed to be a new pixel-based polarization-resolved approach that combines and extends previous analyses. The extracted pitch angles are consistent with those of peptide models and the method has sufficient sensitivity to differentiate Col I from the Col I/Col III mixtures. We further developed the pixel-based approach to extract the SHG signal polarization anisotropy from the same polarization-resolved image matrix. Using this approach, we found that increased Col III results in decreased alignment of the dipole moments within the focal volume. Collectively, the SHG measurements and analysis all indicate that incorporation of Col III results in decreased organization across several levels of collagen organization. Furthermore, the findings suggest that the collagen isoforms comingle within the same fibrils, in good agreement with ultrastructural data. The pixel-based polarization analyses (both excitation and emission) afford determination of structural properties without the previous requirement of having well-aligned fibers, and the approaches should be generally applicable in tissue.

Precise, motion-free polarization control in Second Harmonic Generation microscopy using a liquid crystal modulator in the infinity space.

Second Harmonic Generation (SHG) microscopy coupled with polarization analysis has great potential for use in tissue characterization, as molecular and supramolecular structural details can be extracted. Such measurements are difficult to perform quickly and accurately. Here we present a new method that uses a liquid crystal modulator (LCM) located in the infinity space of a SHG laser scanning microscope that allows the generation of any desired linear or circular polarization state. As the device contains no moving parts, polarization can be rotated accurately and faster than by manual or motorized control. The performance in terms of polarization purity was validated using Stokes vector polarimetry, and found to have minimal residual polarization ellipticity. SHG polarization imaging characteristics were validated against well-characterized specimens having cylindrical and/or linear symmetries. The LCM has a small footprint and can be implemented easily in any standard microscope and is cost effective relative to other technologies.

Bone matrix osteonectin limits prostate cancer cell growth and survival.

There is considerable interest in understanding prostate cancer metastasis to bone and the interaction of these cells with the bone microenvironment. Osteonectin/SPARC/BM-40 is a collagen binding matricellular protein that is enriched in bone. Its expression is increased in prostate cancer metastases, and it stimulates the migration of prostate carcinoma cells. However, the presence of osteonectin in cancer cells and the stroma may limit prostate tumor development and progression. To determine how bone matrix osteonectin affects the behavior of prostate cancer cells, we modeled prostate cancer cell-bone interactions using the human prostate cancer cell line PC-3, and mineralized matrices synthesized by wild type and osteonectin-null osteoblasts in vitro. We developed this in vitro system because the structural complexity of collagen matrices in vivo is not mimicked by reconstituted collagen scaffolds or by more complex substrates, like basement membrane extracts. Second harmonic generation imaging demonstrated that the wild type matrices had thick collagen fibers organized into longitudinal bundles, whereas osteonectin-null matrices had thinner fibers in random networks. Importantly, a mouse model of prostate cancer metastases to bone showed a collagen fiber phenotype similar to the wild type matrix synthesized in vitro. When PC-3 cells were grown on the wild type matrices, they displayed decreased cell proliferation, increased cell spreading, and decreased resistance to radiation-induced cell death, compared to cells grown on osteonectin-null matrix. Our data support the idea that osteonectin can suppress prostate cancer pathogenesis, expanding this concept to the microenvironment of skeletal metastases.