Spectral characterization and unmixing of intrinsic contrast in intact normal and diseased gastric tissues using hyperspectral two-photon microscopy

Background: Living tissues contain a range of intrinsic fluorophores and sources of second harmonic generation which provide contrast that can be exploited for fresh tissue imaging. Microscopic imaging of fresh tissue samples can circumvent the cost and time associated with conventional histology. Further, intrinsic contrast can provide rich information about a tissue\u27s composition, structure and function, and opens the potential for in-vivo imaging without the need for contrast agents. Methodology/Principal Findings: In this study, we used hyperspectral two-photon microscopy to explore the characteristics of both normal and diseased gastrointestinal (GI) tissues, relying only on their endogenous fluorescence and second harmonic generation to provide contrast. We obtained hyperspectral data at subcellular resolution by acquiring images over a range of two-photon excitation wavelengths, and found excitation spectral signatures of specific tissue types based on our ability to clearly visualize morphology. We present the two-photon excitation spectral properties of four major tissue types that are present throughout the GI tract: epithelium, lamina propria, collagen, and lymphatic tissue. Using these four excitation signatures as basis spectra, linear unmixing strategies were applied to hyperspectral data sets of both normal and neoplastic tissue acquired in the colon and small intestine. Our results show that hyperspectral unmixing with excitation spectra allows segmentation, showing promise for blind identification of tissue types within a field of view, analogous to specific staining in conventional histology. The intrinsic spectral signatures of these tissue types provide information relating to their biochemical composition. Conclusions/Significance: These results suggest hyperspectral two-photon microscopy could provide an alternative to conventional histology either for in-situ imaging, or intraoperative \u27instant histology\u27 of fresh tissue biopsies. © 2011 Grosberg et al

Spectral Characterization and Unmixing of Intrinsic Contrast in Intact Normal and Diseased Gastric Tissues Using Hyperspectral Two-Photon Microscopy

Living tissues contain a range of intrinsic fluorophores and sources of second harmonic generation which provide contrast that can be exploited for fresh tissue imaging. Microscopic imaging of fresh tissue samples can circumvent the cost and time associated with conventional histology. Further, intrinsic contrast can provide rich information about a tissue's composition, structure and function, and opens the potential for in-vivo imaging without the need for contrast agents.In this study, we used hyperspectral two-photon microscopy to explore the characteristics of both normal and diseased gastrointestinal (GI) tissues, relying only on their endogenous fluorescence and second harmonic generation to provide contrast. We obtained hyperspectral data at subcellular resolution by acquiring images over a range of two-photon excitation wavelengths, and found excitation spectral signatures of specific tissue types based on our ability to clearly visualize morphology. We present the two-photon excitation spectral properties of four major tissue types that are present throughout the GI tract: epithelium, lamina propria, collagen, and lymphatic tissue. Using these four excitation signatures as basis spectra, linear unmixing strategies were applied to hyperspectral data sets of both normal and neoplastic tissue acquired in the colon and small intestine. Our results show that hyperspectral unmixing with excitation spectra allows segmentation, showing promise for blind identification of tissue types within a field of view, analogous to specific staining in conventional histology. The intrinsic spectral signatures of these tissue types provide information relating to their biochemical composition.These results suggest hyperspectral two-photon microscopy could provide an alternative to conventional histology either for in-situ imaging, or intraoperative 'instant histology' of fresh tissue biopsies

Rectal Optical Markers for In-vivo Risk Stratification of Premalignant Colorectal Lesions.

Purpose: Colorectal cancer remains the second leading cause of cancer deaths in the U.S. despite being eminently preventable by colonoscopy via removal of premalignant adenomas. In order to more effectively reduce colorectal cancer mortality, improved screening paradigms are needed. Our group pioneered the use of low coherence enhanced backscattering (LEBS) spectroscopy to detect the presence of adenomas throughout the colon via optical interrogation of the rectal mucosa. In a previous ex-vivo biopsy study of 219 patients, LEBS demonstrated excellent diagnostic potential with 89.5% accuracy for advanced adenomas. The objective of the current cross-sectional study is to assess the viability of rectal LEBS in-vivo. Experimental Design: Measurements from 619 patients were taken using a minimally invasive 3.4 mm diameter LEBS probe introduced into the rectum via anoscope or direct insertion, requiring ~1 minute from probe insertion to withdrawal. The diagnostic LEBS marker was formed as a logistic regression of the optical reduced scattering coefficient μs∗ and mass density distribution factor D. Results: The rectal LEBS marker was significantly altered in patients harboring advanced adenomas and multiple non-advanced adenomas throughout the colon. Blinded and cross-validated test performance characteristics showed 88% sensitivity to advanced adenomas, 71% sensitivity to multiple non-advanced adenomas, and 72% specificity in the validation set. Conclusions: We demonstrate the viability of in-vivo LEBS measurement of histologically normal rectal mucosa to predict the presence of clinically relevant adenomas throughout the colon. The current work represents the next step in the development of rectal LEBS as a tool for colorectal cancer risk stratification

Skeletal Light-Scattering Accelerates Bleaching Response in Reef-Building Corals

Background At the forefront of ecosystems adversely affected by climate change, coral reefs are sensitive to anomalously high temperatures which disassociate (bleaching) photosynthetic symbionts (Symbiodinium) from coral hosts and cause increasingly frequent and severe mass mortality events. Susceptibility to bleaching and mortality is variable among corals, and is determined by unknown proportions of environmental history and the synergy of Symbiodinium- and coral-specific properties. Symbiodinium live within host tissues overlaying the coral skeleton, which increases light availability through multiple light-scattering, forming one of the most efficient biological collectors of solar radiation. Light-transport in the upper ~200 μm layer of corals skeletons (measured as ‘microscopic’ reduced-scattering coefficient, μ′S,m), has been identified as a determinant of excess light increase during bleaching and is therefore a potential determinant of the differential rate and severity of bleaching response among coral species. Results Here we experimentally demonstrate (in ten coral species) that, under thermal stress alone or combined thermal and light stress, low-μ′S,m corals bleach at higher rate and severity than high-μ′S,m corals and the Symbiodinium associated with low-μ′S,m corals experience twice the decrease in photochemical efficiency. We further modelled the light absorbed by Symbiodinium due to skeletal-scattering and show that the estimated skeleton-dependent light absorbed by Symbiodinium (per unit of photosynthetic pigment) and the temporal rate of increase in absorbed light during bleaching are several fold higher in low-μ′S,m corals. Conclusions While symbionts associated with low-μ′S,m corals receive less total light from the skeleton, they experience a higher rate of light increase once bleaching is initiated and absorbing bodies are lost; further precipitating the bleaching response. Because microscopic skeletal light-scattering is a robust predictor of light-dependent bleaching among the corals assessed here, this work establishes μ′S,m as one of the key determinants of differential bleaching response

Media 1: Two electric field Monte Carlo models of coherent backscattering of polarized light

Originally published in JOSA A on 01 November 2014 (josaa-31-11-2394

in vivo

Astrocytes are increasingly believed to play an important role in neurovascular coupling. Recent in vivo studies have shown that intracellular calcium levels in astrocytes correlate with reactivity in adjacent diving arterioles. However, the hemodynamic response to stimulation involves a complex orchestration of vessel dilations and constrictions that spread rapidly over wide distances. In this work, we study the three-dimensional cytoarchitecture of astrocytes and their interrelations with blood vessels down through layer IV of the mouse somatosensory cortex using in vivo two-photon microscopy. Vessels and astrocytes were visualized through intravenous dextran-conjugated fluorescein and cortically applied sulforhodamine 101 (SR101), respectively. In addition to exploring astrocyte density, vascular proximity, and microvascular density, we found that sheathing of subpial vessels by astrocyte processes was continuous along all capillaries, arterioles, and veins, comprising a highly interconnected pathway through which signals could feasibly be relayed over long distances via gap junctions. An inner SR101-positive sheath noted along pial and diving arterioles was determined to be nonastrocytic, and appears to represent selective SR101 staining of arterial endothelial cells. Our findings underscore the intimate relationship between astrocytes and all cortical blood vessels, and suggest that astrocytes could influence neurovascular regulation at a range of sites, including the capillary bed and pial arterioles