Andrew J. Radosevich

In vivo 3D morphology of astrocyte–vasculature interactions in the somatosensory cortex: implications for neurovascular coupling

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.

Hyperspectral in vivo two-photon microscopy of intrinsic contrast

In vivo two-photon imaging of intrinsic contrast can provide valuable information about structural tissue elements such as collagen and elastin and fluorescent metabolites such as nicotinamide adenine dinucleotide. Yet low signal and overlapping emission spectra can make it difficult to identify and delineate these species in vivo. We present a novel approach that combines excitation scanning with spectrally resolved emission two-photon microscopy, allowing distinct structures to be delineated based on their characteristic spectral fingerprints. The amounts of intrinsic fluorophores present in each voxel can also be evaluated. We demonstrate our method using in vivo imaging of nude mouse skin.

Modulation of light-enhancement to symbiotic algae by light-scattering in corals and evolutionary trends in bleaching.

Calcium carbonate skeletons of scleractinian corals amplify light availability to their algal symbionts by diffuse scattering, optimizing photosynthetic energy acquisition. However, the mechanism of scattering and its role in coral evolution and dissolution of algal symbioses during “bleaching” events are largely unknown. Here we show that differences in skeletal fractal architecture at nano/micro-lengthscales within 96 coral taxa result in an 8-fold variation in light-scattering and considerably alter the algal light environment. We identified a continuum of properties that fall between two extremes: (1) corals with low skeletal fractality that are efficient at transporting and redistributing light throughout the colony with low scatter but are at higher risk of bleaching and (2) corals with high skeletal fractality that are inefficient at transporting and redistributing light with high scatter and are at lower risk of bleaching. While levels of excess light derived from the coral skeleton is similar in both groups, the low-scatter corals have a higher rate of light-amplification increase when symbiont concentration is reduced during bleaching, thus creating a positive feedback-loop between symbiont concentration and light-amplification that exposes the remaining symbionts to increasingly higher light intensities. By placing our findings in an evolutionary framework, in conjunction with a novel empirical index of coral bleaching susceptibility, we find significant correlations between bleaching susceptibility and light-scattering despite rich homoplasy in both characters; suggesting that the cost of enhancing light-amplification to the algae is revealed in decreased resilience of the partnership to stress.

Can OCT be sensitive to nanoscale structural alterations in biological tissue

Exploration of nanoscale tissue structures is crucial in understanding biological processes. Although novel optical microscopy methods have been developed to probe cellular features beyond the diffraction limit, nanometer-scale quantification remains still inaccessible for in situ tissue. Here we demonstrate that, without actually resolving specific geometrical feature, OCT can be sensitive to tissue structural properties at the nanometer length scale. The statistical mass-density distribution in tissue is quantified by its autocorrelation function modeled by the Whittle-Mateŕn functional family. By measuring the wavelength-dependent backscattering coefficient μb(λ) and the scattering coefficient μs, we introduce a technique called inverse spectroscopic OCT (ISOCT) to quantify the mass-density correlation function. We find that the length scale of sensitivity of ISOCT ranges from ~30 to ~450 nm. Although these sub-diffractional length scales are below the spatial resolution of OCT and therefore not resolvable, they are nonetheless detectable. The sub-diffractional sensitivity is validated by 1) numerical simulations; 2) tissue phantom studies; and 3) ex vivo colon tissue measurements cross-validated by scanning electron microscopy (SEM). Finally, the 3D imaging capability of ISOCT is demonstrated with ex vivo rat buccal and human colon samples.

Modeling Light Scattering in Tissue as Continuous Random Media Using a Versatile Refractive Index Correlation Function

Optical interactions with biological tissue provide powerful tools for study, diagnosis, and treatment of disease. When optical methods are used in applications involving tissue, scattering of light is an important phenomenon. In imaging modalities, scattering provides contrast, but also limits imaging depth, so models help optimize an imaging technique. Scattering can also be used to collect information about the tissue itself providing diagnostic value. Therapies involving focused beams require scattering models to assess dose distribution. In all cases, models of light scattering in tissue are crucial to correctly interpreting the measured signal. Here, we review a versatile model of light scattering that uses the Whittle-Matérn correlation family to describe the refractive index correlation function Bn(rd). In weakly scattering media such as tissue, Bn(rd) determines the shape of the power spectral density from which all other scattering characteristics are derived. This model encompasses many forms such as mass fractal and the Henyey-Greenstein function as special cases. We discuss normalization and calculation of optical properties including the scattering coefficient and anisotropy factor. Experimental methods using the model are also described to quantify tissue properties that depend on length scales of only a few tens of nanometers.

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 tissues 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 ‘instant histology’ of fresh tissue biopsies.

A predictive model of backscattering at subdiffusion length scales.

We provide a methodology for accurately predicting elastic backscattering radial distributions from random media with two simple empirical models. We apply these models to predict the backscattering based on two classes of scattering phase functions: the Henyey-Greenstein phase function and a generalized two parameter phase function that is derived from the Whittle-Matérn correlation function. We demonstrate that the model has excellent agreement over all length scales and has less than 1% error for backscattering at subdiffusion length scales for tissue-relevant optical properties. The presented model is the first available approach for accurately predicting backscattering at length scales significantly smaller than the transport mean free path.

Polarized Enhanced Backscattering Spectroscopy for Characterization of Biological Tissues at Subdiffusion Length Scales

Since the early 1980s, the enhanced backscattering (EBS) phenomenon has been well-studied in a large variety of nonbiological materials. Yet, until recently, the use of conventional EBS for the characterization of biological tissue has been fairly limited. In this study, we detail the unique ability of EBS to provide spectroscopic, polarimetric, and depth-resolved characterization of biological tissue using a simple backscattering instrument. We first explain the experimental and numerical procedures used to accurately measure and model the full azimuthal EBS peak shape in biological tissue. Next, we explore the peak shape and height dependencies for different polarization channels and spatial coherence of illumination. We then illustrate the extraordinary sensitivity of EBS to the shape of the scattering phase function using suspensions of latex microspheres. Finally, we apply EBS to biological tissue samples in order to measure optical properties and observe the spatial length scales at which backscattering is altered in early colon carcinogenesis.

Spatially resolved optical and ultrastructural properties of colorectal and pancreatic field carcinogenesis observed by inverse spectroscopic optical coherence tomography.

Abstract. Field carcinogenesis is the initial stage of cancer progression. Understanding field carcinogenesis is valuable for both cancer biology and clinical medicine. Here, we used inverse spectroscopic optical coherence tomography to study colorectal cancer (CRC) and pancreatic cancer (PC) field carcinogenesis. Depth-resolved optical and ultrastructural properties of the mucosa were quantified from histologically normal rectal biopsies from patients with and without colon adenomas (n=85) as well as from histologically normal peri-ampullary duodenal biopsies from patients with and without PC (n=22). Changes in the epithelium and stroma in CRC field carcinogenesis were separately quantified. In both compartments, optical and ultra-structural alterations were consistent. Optical alterations included lower backscattering (μb) and reduced scattering (μs′) coefficients and higher anisotropy factor g. Ultrastructurally pronounced alterations were observed at length scales up to ∼450  nm, with the shape of the mass density correlation function having a higher shape factor D, thus implying a shift to larger length scales. Similar alterations were found in the PC field carcinogenesis despite the difference in genetic pathways and etiologies. We further verified that the chromatin clumping in epithelial cells and collagen cross-linking caused D to increase in vitro and could be among the mechanisms responsible for the observed changes in epithelium and stroma, respectively.

Measurement of optical scattering properties with low-coherence enhanced backscattering spectroscopy

Low-coherence enhanced backscattering (LEBS) is a depth selective technique that allows noninvasive characterization of turbid media such as biological tissue. LEBS provides a spectral measurement of the tissue reflectance distribution as a function of distance between incident and reflected ray pairs through the use of partial spatial coherence broadband illumination. We present LEBS as a new depth-selective technique to measure optical properties of tissue in situ. Because LEBS enables measurements of reflectance due to initial scattering events, LEBS is sensitive to the shape of the phase function in addition to the reduced scattering coefficient (μ(s) (*)). We introduce a simulation of LEBS that implements a two parameter phase function based on the Whittle-Matérn refractive index correlation function model. We show that the LEBS enhancement factor (E) primarily depends on μ(s) (*), the normalized spectral dependence of E (S(n)) depends on one of the two parameters of the phase function that also defines the functional type of the refractive index correlation function (m), and the LEBS peak width depends on both the anisotropy factor (g) and m. Three inverse models for calculating these optical properties are described and the calculations are validated with an experimental measurement from a tissue phantom.