High performance image mapping spectrometer (IMS) for snapshot hyperspectral imaging applications.

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.

Snapshot Hyperspectral Light-Sheet Imaging of Signal Transduction in Live Pancreatic Islets.

The observation of ionic signaling dynamics in intact pancreatic islets has contributed greatly to our understanding of both α- and ß-cell function. Insulin secretion from ß-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 ß-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 ß-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.

Snapshot 3D optical coherence tomography system using image mapping spectrometry.

A snapshot 3-Dimensional Optical Coherence Tomography system was developed using Image Mapping Spectrometry. This system can give depth information (Z) at different spatial positions (XY) within one camera integration time to potentially reduce motion artifact and enhance throughput. The current (x,y,λ) datacube of (85×356×117) provides a 3D visualization of sample with 400 µm depth and 13.4 µm in transverse resolution. Axial resolution of 16.0 µm can also be achieved in this proof-of-concept system. We present an analysis of the theoretical constraints which will guide development of future systems with increased imaging depth and improved axial and lateral resolutions.