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.

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.

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 () mutant and a longitudinal design to elucidate the consequences of NMES on muscle health.

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.

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.

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.

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

: Spatial frequency domain imaging (SFDI) is a diffuse optical measurement technique that can quantify tissue optical absorption (μ) and reduced scattering () on a pixel-by-pixel basis. Measurements of μ 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 μ and as well as oxyhemoglobin and deoxyhemoglobin concentrations in biological tissue.

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.

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.

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.

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.

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.

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.

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 β 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 β is not well described by a two-state model based on known absorption bands.

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.

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.

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.