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

Increased Inter-Colony Fusion Rates Are Associated with Reduced COI Haplotype Diversity in an Invasive Colonial Ascidian Didemnum vexillum

Considerable progress in our understanding of the population genetic changes associated with biological invasions has been made over the past decade. Using selectively neutral loci, it has been established that reductions in genetic diversity, reflecting founder effects, have occurred during the establishment of some invasive populations. However, some colonial organisms may actually gain an ecological advantage from reduced genetic diversity because of the associated reduction in inter-colony conflict. Here we report population genetic analyses, along with colony fusion experiments, for a highly invasive colonial ascidian, Didemnum vexillum. Analyses based on mitochondrial cytochrome oxidase I (COI) partial coding sequences revealed two distinct D. vexillum clades. One COI clade appears to be restricted to the probable native region (i.e., north-west Pacific Ocean), while the other clade is present in widely dispersed temperate coastal waters around the world. This clade structure was supported by 18S ribosomal DNA (rDNA) sequence data, which revealed a one base-pair difference between the two clades. Recently established populations of D. vexillum in New Zealand displayed greatly reduced COI genetic diversity when compared with D. vexillum in Japan. In association with this reduction in genetic diversity was a significantly higher inter-colony fusion rate between randomly paired New Zealand D. vexillum colonies (80%, standard deviation ±18%) when compared with colonies found in Japan (27%, standard deviation ±15%). The results of this study add to growing evidence that for colonial organisms reductions in population level genetic diversity may alter colony interaction dynamics and enhance the invasive potential of newly colonizing species

Media 3: Simultaneous multiplane in vivo nonlinear microscopy using spectral encoding

Originally published in Optics Letters on 15 July 2012 (ol-37-14-2967

Media 1: Simultaneous multiplane in vivo nonlinear microscopy using spectral encoding

Originally published in Optics Letters on 15 July 2012 (ol-37-14-2967

Electrical stimulus artifact cancellation and neural spike detection on large multi-electrode arrays.

Simultaneous electrical stimulation and recording using multi-electrode arrays can provide a valuable technique for studying circuit connectivity and engineering neural interfaces. However, interpreting these measurements is challenging because the spike sorting process (identifying and segregating action potentials arising from different neurons) is greatly complicated by electrical stimulation artifacts across the array, which can exhibit complex and nonlinear waveforms, and overlap temporarily with evoked spikes. Here we develop a scalable algorithm based on a structured Gaussian Process model to estimate the artifact and identify evoked spikes. The effectiveness of our methods is demonstrated in both real and simulated 512-electrode recordings in the peripheral primate retina with single-electrode and several types of multi-electrode stimulation. We establish small error rates in the identification of evoked spikes, with a computational complexity that is compatible with real-time data analysis. This technology may be helpful in the design of future high-resolution sensory prostheses based on tailored stimulation (e.g., retinal prostheses), and for closed-loop neural stimulation at a much larger scale than currently possible

Analysis of responses of neurons in a neighborhood of the stimulating electrode.

<p><b>A</b> Spatial configuration: stimulating electrode (blue/yellow annulus) and four neurons on its vicinity. Soma of green neuron and axon of pink neuron overlap with stimulating electrode. <b>B</b> Activation curves (solid lines) along with human-curated and algorithm inferred spike probabilities (gray and colored circles, respectively) of all the four cells. Stimulation elicited activation of green and pink neurons; however, the two other neurons remained inactive. <b>C</b> Raster plots for the activated cells, with responses sorted by stimulation strength in the y axis. Human and algorithm inferred latencies are in good agreement (gray and colored circles, respectively). Here, direct somatic activation of the green neuron leads to lower-latency and lower-threshold activation than of the pink neuron, which is activated through its axon.</p

Visual inspection of traces reveals the difficulty of the problem.

<p>First column: templates of spiking neurons. Second to fourth columns: responses of one (<b>A</b>) or two (<b>B</b>) cells to electrical stimulation at increasing stimulation amplitudes as recorded in the stimulating electrode (first rows) or a neighboring, non-stimulating electrode (third rows). If the stimulation artifact is known (gray traces) it can be subtracted from raw traces to produce a baseline (second and fourth rows) amenable for template matching: traces with spike(s) (colored) match, on each electrode, either a translation of a template (<b>A</b> and <b>B</b>) or the sum of different translations of two or more templates (<b>B</b>). As reflected by the activation curves (fifth column) for strong enough stimuli spiking occurs with probability close to one, consistent with the absence of black traces in the rightmost columns.</p

Summary of relevant notation.

<p>Summary of relevant notation.</p