The observation of ionic signaling dynamics in intact pancreatic islets has contributed greatly to our understanding of both α- and β-cell function. Insulin secretion from β-cells depends on the firing of action potentials and consequent rises of intracellular calcium activity ([Ca(2+)]i). Zinc (Zn(2+)) is cosecreted with insulin, and has been postulated to play a role in cell-to-cell cross talk within an islet, in particular inhibiting glucagon secretion from α-cells. Thus, measuring [Ca(2+)]i and Zn(2+) dynamics from both α- and β-cells will elucidate mechanisms underlying islet hormone secretion. [Ca(2+)]i and intracellular Zn(2+) can be measured using fluorescent biosensors, but the most efficient sensors have overlapping spectra that complicate their discrimination. Hyperspectral imaging can be used to distinguish signals from multiple fluorophores, but available hyperspectral implementations are either too slow to measure the dynamics of ionic signals or not suitable for thick samples. We have developed a five-dimensional (x,y,z,t,λ) imaging system that leverages a snapshot hyperspectral imaging method, image mapping spectrometry, and light-sheet microscopy. This system provides subsecond temporal resolution from deep within multicellular structures. Using a single excitation wavelength (488 nm) we acquired images from triply labeled samples with two biosensors and a genetically expressing fluorescent protein (spectrally overlapping with one of the biosensors) with high temporal resolution. Measurements of [Ca(2+)]i and Zn(2+) within both α- and β-cells as a function of glucose concentration show heterogeneous uptake of Zn(2+) into α-cells that correlates to the known heterogeneities in [Ca(2+)]i. These differences in intracellular Zn(2+) among α-cells may contribute to the inhibition in glucagon secretion observed at elevated glucose levels.
We describe the hyperspectral Image Mapping Spectrometer (IMS) which is able to acquire spectral images of the retina with no scanning components. We then utilize this technology to obtain absolute oxygen saturation measurements in four patients with retinal diseases. The IMS acquires spatial and spectral information of a scene in a single camera exposure time. The IMS is attached to the port of a Topcon TRC-50EX funduscopic camera in order to acquire images. The IMS has high spatial (350×350) and spectral sampling (40), and avoids motion artifacts associated with eye movements. Oximetry values are obtained by measuring the absorption of retinal blood. Four patients with different retinal diseases were imaged via the IMS in this study. Oxygen saturation is calculated using a least squares fit applied to the whole blood oximetry equation. Oxygen saturation was found to be 88.6% (±1.9) and 34.1% (±7.0) for the artery and vein, respectively, in a patient with non-exudative age-related macular degeneration (AMD) with borderline glaucoma, 100.0% (±0.0) and 50.4% (±8.2) in a patient with exudative AMD, 100.0% (±0.0) and 45.2% (±7.0) in a patient with retinitis pigmentosa, and 94.1% (±2.5) and 44.9% (±5.6) in a patient with chronic iridocyclitis. These values are consistent with previously published data. Based on spatial and spectral data, we created an oxygen saturation map with oxygenation data overlying retinal vasculature. Hyperspectral imaging may be a promising way to measure retinal oxygen saturation. IMS technology is advantageous in its ability to obtain images free of motion artifacts.
Snapshot hyperspectral imaging augments pixel dwell time and acquisition speeds over existing scanning systems, making it a powerful tool for fluorescence microscopy. While most snapshot systems contain fixed datacube parameters (x,y,λ), our novel snapshot system, called the lenslet array tunable snapshot imaging spectrometer (LATIS), demonstrates tuning its average spectral resolution from 22.66 nm (80x80x22) to 13.94 nm (88x88x46) over 485 to 660 nm. We also describe a fixed LATIS with a datacube of 200x200x27 for larger fieldof-view (FOV) imaging. We report <1 sec exposure times and high resolution fluorescence imaging with minimal artifacts. Tracking Using a High-Speed Hyperspectral Line-Scanning Microscope," PLoS One 8(5), e64320 (2013). 14. P. M. Kasili and T. Vo-Dinh, "Hyperspectral imaging system using acousto-optic tunable filter for flow cytometry applications," Cytometry A 69(8), 835-841 (2006).
A high performance, snapshot Image Mapping Spectrometer was developed that provides fast image acquisition (100 Hz) of 16 bit hyperspectral data cubes (210x210x46) over a spectral range of 515-842 nm. Essential details of the opto-mechanical design are presented. Spectral accuracy, precision, and image reconstruction metrics such as resolution are discussed. Fluorescently stained cell samples were used to directly compare the data obtained using newly developed and the reference image mapping spectrometer. Additional experimental results are provided to demonstrate the abilities of the new spectrometer to acquire highly-resolved, motion-artifact-free hyperspectral images at high temporal sampling rates.
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