Label-Free Assessment of Key Biological Autofluorophores: Material Characteristics and Opportunities for Clinical Applications.

Autofluorophores are endogenous fluorescent compounds that naturally occur in the intra and extracellular spaces of all tissues and organs. Most have vital biological functions - like the metabolic cofactors NAD(P)H and FAD+, as well as the structural protein collagen. Others are considered to be waste products - like lipofuscin and advanced glycation end products - which accumulate with age and are associated with cellular dysfunction. Due to their natural fluorescence, these materials have great utility for enabling non-invasive, label-free assays with direct ties to biological function. Numerous technologies, with different advantages and drawbacks, are applied to their assessment, including fluorescence lifetime imaging microscopy, hyperspectral microscopy, and flow cytometry. Here, the applications of label-free autofluorophore assessment are reviewed for clinical and health-research applications, with specific attention to biomaterials, disease detection, surgical guidance, treatment monitoring, and tissue assessment - fields that greatly benefit from non-invasive methodologies capable of continuous, in vivo characterization.

Multimodality imaging reveals angiogenic evolution in vivo during calvarial bone defect healing.

The healing of calvarial bone defects is a pressing clinical problem that involves the dynamic interplay between angiogenesis and osteogenesis within the osteogenic niche. Although structural and functional vascular remodeling (i.e., angiogenic evolution) in the osteogenic niche is a crucial modulator of oxygenation, inflammatory and bone precursor cells, most clinical and pre-clinical investigations have been limited to characterizing structural changes in the vasculature and bone. Therefore, we developed a new multimodality imaging approach that for the first time enabled the longitudinal (i.e., over four weeks) and dynamic characterization of multiple in vivo functional parameters in the remodeled vasculature and its effects on de novo osteogenesis, in a preclinical calvarial defect model. We employed multi-wavelength intrinsic optical signal (IOS) imaging to assess microvascular remodeling, intravascular oxygenation (SO2), and osteogenesis; laser speckle contrast (LSC) imaging to assess concomitant changes in blood flow and vascular maturity; and micro-computed tomography (µCT) to validate volumetric changes in calvarial bone. We found that angiogenic evolution was tightly coupled with calvarial bone regeneration and corresponded to distinct phases of bone healing, such as injury, hematoma formation, revascularization, and remodeling. The first three phases occurred during the initial two weeks of bone healing and were characterized by significant in vivo changes in vascular morphology, blood flow, oxygenation, and maturity. Overall, angiogenic evolution preceded osteogenesis, which only plateaued toward the end of bone healing (i.e., four weeks). Collectively, these data indicate the crucial role of angiogenic evolution in osteogenesis. We believe that such multimodality imaging approaches have the potential to inform the design of more efficacious tissue-engineering calvarial defect treatments.

Vascular phenotyping of the invasive front in breast cancer using a 3D angiogenesis atlas.

OBJECTIVE: Vascular remodeling at the invasive tumor front (ITF) plays a critical role in progression and metastasis of triple negative breast cancer (TNBC). Therefore, there is a crucial need to characterize the vascular phenotype (i.e. changes in the structure and function of vasculature) of the ITF and tumor core (TC) in TNBC. This requires high-resolution, 3D structural and functional microvascular data that spans the ITF and TC (i.e. ∼4-5 mm from the tumor's edge). Since such data are often challenging to obtain with most conventional imaging approaches, we employed a unique "3D whole-tumor angiogenesis atlas" derived from orthotopic xenografts to characterize the vascular phenotype of the ITF and TC in TNBC. METHODS: First, high-resolution (8 µm) computed tomography (CT) images of "whole-tumor" microvasculature were acquired from eight orthotopic TNBC xenografts, of which three tumors were excised at post-inoculation day 21 (i.e. early-stage) and five tumors were excised at post-inoculation day 35 (i.e. advanced-stage). These 3D morphological CT data were combined with soft tissue contrast from MRI as well as functional data generated in silico using image-based hemodynamic modeling to generate a multi-layered "angiogenesis atlas". Employing this atlas, blood vessels were first spatially stratified within the ITF (i.e. ≤1 mm from the tumor's edge) and TC (i.e. >1 mm from the tumor's edge) of each tumor xenograft. Then, a novel method was developed to visualize and characterize microvascular remodeling and perfusion changes in terms of distance from the tumor's edge. RESULTS: The angiogenesis atlas enabled the 3D visualization of changes in tumor vessel growth patterns, morphology and perfusion within the ITF and TC. Early and advanced stage tumors demonstrated significant differences in terms of their edge-to-center distributions for vascular surface area density, vascular length density, intervessel distance and simulated perfusion density (p â‰ª 0.01). Elevated vascular length density, vascular surface area density and perfusion density along the circumference of the ITF was suggestive of a preferential spatial pattern of angiogenic growth in this tumor cohort. Finally, we demonstrated the feasibility of differentiating the vascular phenotypes of ITF and TC in these TNBC xenografts. CONCLUSIONS: The combination of a 3D angiogenesis atlas and image-based hemodynamic modeling heralds a new approach for characterizing the role of vascular remodeling in cancer and other diseases.

In vivo phenotyping of the microvasculature in necrotizing enterocolitis with multicontrast optical imaging.

OBJECTIVE: Necrotizing enterocolitis (NEC) is the most prevalent gastrointestinal emergency in premature infants and is characterized by a dysfunctional gut microcirculation. Therefore, there is a dire need for in vivo methods to characterize NEC-induced changes in the structure and function of the gut microcirculation, that is, its vascular phenotype. Since in vivo gut imaging methods are often slow and employ a single-contrast mechanism, we developed a rapid multicontrast imaging technique and a novel analyses pipeline for phenotyping the gut microcirculation. METHODS: Using an experimental NEC model, we acquired in vivo images of the gut microvasculature and blood flow over a 5000 × 7000 µm2 field of view at 5 µm resolution via the following two endogenous contrast mechanisms: intrinsic optical signals and laser speckles. Next, we transformed intestinal images into rectilinear "flat maps," and delineated 1A/V gut microvessels and their perfusion territories as "intestinal vascular units" (IVUs). Employing IVUs, we quantified and visualized NEC-induced changes to the gut vascular phenotype. RESULTS: In vivo imaging required 60-100 s per animal. Relative to the healthy gut, NEC intestines showed a significant overall decrease (i.e. 64-72%) in perfusion, accompanied by vasoconstriction (i.e. 9-12%) and a reduction in perfusion entropy (19%)within sections of the vascular bed. CONCLUSIONS: Multicontrast imaging coupled with IVU-based in vivo vascular phenotyping is a powerful new tool for elucidating NEC pathogenesis.

VascuViz: a multimodality and multiscale imaging and visualization pipeline for vascular systems biology.

Despite advances in imaging, image-based vascular systems biology has remained challenging because blood vessel data are often available only from a single modality or at a given spatial scale, and cross-modality data are difficult to integrate. Therefore, there is an exigent need for a multimodality pipeline that enables ex vivo vascular imaging with magnetic resonance imaging, computed tomography and optical microscopy of the same sample, while permitting imaging with complementary contrast mechanisms from the whole-organ to endothelial cell spatial scales. To achieve this, we developed 'VascuViz'-an easy-to-use method for simultaneous three-dimensional imaging and visualization of the vascular microenvironment using magnetic resonance imaging, computed tomography and optical microscopy in the same intact, unsectioned tissue. The VascuViz workflow permits multimodal imaging with a single labeling step using commercial reagents and is compatible with diverse tissue types and protocols. VascuViz's interdisciplinary utility in conjunction with new data visualization approaches opens up new vistas in image-based vascular systems biology.

Advances in translational imaging of the microcirculation.

The past few decades have seen an explosion in the development and use of methods for imaging the human microcirculation during health and disease. The confluence of innovative imaging technologies, affordable computing power, and economies of scale have ushered in a new era of "translational" imaging that permit us to peer into blood vessels of various organs in the human body. These imaging techniques include near-infrared spectroscopy (NIRS), positron emission tomography (PET), and magnetic resonance imaging (MRI) that are sensitive to microvascular-derived signals, as well as computed tomography (CT), optical imaging, and ultrasound (US) imaging that are capable of directly acquiring images at, or close to microvascular spatial resolution. Collectively, these imaging modalities enable us to characterize the morphological and functional changes in a tissue's microcirculation that are known to accompany the initiation and progression of numerous pathologies. Although there have been significant advances for imaging the microcirculation in preclinical models, this review focuses on developments in the assessment of the microcirculation in patients with optical imaging, NIRS, PET, US, MRI, and CT, to name a few. The goal of this review is to serve as a springboard for exploring the burgeoning role of translational imaging technologies for interrogating the structural and functional status of the microcirculation in humans, and highlight the breadth of current clinical applications. Making the human microcirculation "visible" in vivo to clinicians and researchers alike will facilitate bench-to-bedside discoveries and enhance the diagnosis and management of disease.

HemoSYS: A Toolkit for Image-based Systems Biology of Tumor Hemodynamics.

Abnormal tumor hemodynamics are a critical determinant of a tumor's microenvironment (TME), and profoundly affect drug delivery, therapeutic efficacy and the emergence of drug and radio-resistance. Since multiple hemodynamic variables can simultaneously exhibit transient and spatiotemporally heterogeneous behavior, there is an exigent need for analysis tools that employ multiple variables to characterize the anomalous hemodynamics within the TME. To address this, we developed a new toolkit called HemoSYS for quantifying the hemodynamic landscape within angiogenic microenvironments. It employs multivariable time-series data such as in vivo tumor blood flow (BF), blood volume (BV) and intravascular oxygen saturation (Hbsat) acquired concurrently using a wide-field multicontrast optical imaging system. The HemoSYS toolkit consists of propagation, clustering, coupling, perturbation and Fourier analysis modules. We demonstrate the utility of each module for characterizing the in vivo hemodynamic landscape of an orthotropic breast cancer model. With HemoSYS, we successfully described: (i) the propagation dynamics of acute hypoxia; (ii) the initiation and dissolution of distinct hemodynamic niches; (iii) tumor blood flow regulation via local vasomotion; (iv) the hemodynamic response to a systemic perturbation with carbogen gas; and (v) frequency domain analysis of hemodynamic heterogeneity in the TME. HemoSYS (freely downloadable via the internet) enables vascular phenotyping from multicontrast in vivo optical imaging data. Its modular design also enables characterization of non-tumor hemodynamics (e.g. brain), other preclinical disease models (e.g. stroke), vascular-targeted therapeutics, and hemodynamic data from other imaging modalities (e.g. MRI).

Tumor Ensemble-Based Modeling and Visualization of Emergent Angiogenic Heterogeneity in Breast Cancer.

There is a critical need for new tools to investigate the spatio-temporal heterogeneity and phenotypic alterations that arise in the tumor microenvironment. However, computational investigations of emergent inter- and intra-tumor angiogenic heterogeneity necessitate 3D microvascular data from 'whole-tumors' as well as "ensembles" of tumors. Until recently, technical limitations such as 3D imaging capabilities, computational power and cost precluded the incorporation of whole-tumor microvascular data in computational models. Here, we describe a novel computational approach based on multimodality, 3D whole-tumor imaging data acquired from eight orthotopic breast tumor xenografts (i.e. a tumor 'ensemble'). We assessed the heterogeneous angiogenic landscape from the microvascular to tumor ensemble scale in terms of vascular morphology, emergent hemodynamics and intravascular oxygenation. We demonstrate how the abnormal organization and hemodynamics of the tumor microvasculature give rise to unique microvascular niches within the tumor and contribute to inter- and intra-tumor heterogeneity. These tumor ensemble-based simulations together with unique data visualization approaches establish the foundation of a novel 'cancer atlas' for investigators to develop their own in silico systems biology applications. We expect this hybrid image-based modeling framework to be adaptable for the study of other tissues (e.g. brain, heart) and other vasculature-dependent diseases (e.g. stroke, myocardial infarction).

Heat transfer model for deep tissue injury: a step towards an early thermographic diagnostic capability.

BACKGROUND: Deep tissue injury (DTI) is a class of serious lesions which develop in the deep tissue layers as a result of sustained tissue loading or pressure-induced ischemic injury. DTI lesions often do not become visible on the skin surface until the injury reaches an advanced stage, making their early detection a challenging task. THEORY: Early diagnosis leading to early treatment mitigates the progression of the lesion and remains one of the priorities in clinical care. The aim of the study is to relate changes in tissue temperature with key physiological changes occurring at the tissue level to develop criteria for the detection of incipient DTIs. METHOD: Skin surface temperature distributions of the damaged tissue were analyzed using a multilayer tissue model. Thermal response of the skin surface to a cooling stress, was computed for deep tissue inflammation and deep tissue ischemia, and then compared with computed skin temperature of healthy tissue. RESULTS: For a deep lesion situated in muscle and fat layers, measurable skin temperature differences were observed within the first five minutes of thermal recovery period including temperature increases between 0.25 °C to 0.9 °C during inflammation and temperature decreases between -0.2 °C to -0.5 °C during ischemia. CONCLUSIONS: The computational thermal models can explain previously published thermographic findings related to DTIs and pressure ulcers. It is concluded that infrared thermography can be used as an objective, non-invasive and quantitative means of early DTI diagnosis. VIRTUAL SLIDES: The virtual slides for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/1461254346108378.

HEAT TRANSFER MODEL AND QUANTITATIVE ANALYSIS OF DEEP TISSUE INJURY.

Deep tissue injuries (DTI) are serious lesions which may develop in deep tissue layers as a result of sustained tissue loading or ischemic injury. These lesions may not become visible on the skin surface until the injury reaches an advanced stage making their early detection a challenging task. Early diagnosis leading to early treatment mitigates the progression of lesion and remains one of the priorities in management. The aim of this study is to examine skin surface temperature distributions of damaged tissue and develop criteria for the detection of incipient DTI. A multilayer quantitative heat transfer model of the skin tissue was developed using finite element based software COMSOL Multiphysics. Thermal response of the skin surface was computed during deep tissue inflammation and deep tissue ischemia and then compared with that of healthy tissue. In the presence of a DTI, an increase of about 0.5°C in skin surface temperatures was noticed during initial phase of deep tissue inflammation, which was followed by a surface temperature decrease of about 0.2°C corresponding to persistent deep tissue ischemia. These temperature differences are large enough to be detected by thermographic imaging. This study, therefore, also enhances the understanding of the previously detected thermographic quantitative changes associated with DTI.

Retroacetabular stress-shielding in THA.

We conducted a randomized clinical trial to compare periacetabular bone density changes after total hip arthroplasty using press-fit components with soft and hard liner materials. Bone density changes were assessed using quantitative computed tomography-assisted osteodensitometry. Twenty press-fit cups with alumina ceramic liners and 20 press-fit cups with highly cross-linked polyethylene liners were included; the nonoperated contralateral side was used as the control. Computed tomography scans were performed postoperatively and 1 year after the index operation. At the 1-year followup, we found no differences of periacetabular bone density changes between the alumina and polyethylene liner cohorts. However, we observed marked periacetabular cancellous bone density loss (up to -34%) in both cohorts. In contrast, we observed only moderate cortical bone density changes. The decrease of periacetabular cancellous bone density with retention of cortical bone density after THA suggests stress transfer to the cortical bone.

Prioritization of patients with rectal bleeding for urgent outpatient colonoscopy--a pilot study.

AIM: Rectal bleeding is a common symptom in general practice and may be associated with colorectal neoplasia. Waiting-lists for outpatient colonoscopies and first specialist appointments are long. The aim of our study was to determine the value of presenting signs and symptoms in prioritising patients with rectal bleeding for urgent colonoscopy. METHOD: Patients were asked to fill out a 'Bowel symptoms Assessment Questionnaire' at their first visit to a Colorectal Clinic. Patients were then assessed by clinicians who referred them for further investigations as appropriate. Factors from the questionnaire (e.g. age, family history, perianal symptoms, and so on) were analysed to assess correlation with colorectal cancer or neoplasia. These were analysed using logistic regression, SPSS Answertree and 2x2 tables. RESULTS: 105 patients completed the questionnaire. Thirty patients had colonoscopy/barium enema. Fifteen patients had colorectal cancer (CRC) or neoplasia detected. There was a significant increase in risk of developing CRC or neoplasia in patients above the age of 67 or if they had a positive family history of CRC or neoplasia. CONCLUSION: We conclude that patients with rectal bleeding, above the age of 67 or those with a positive family history are at a higher risk of neoplasia and should receive priority access to colonoscopy prior to first specialist assessment.