Development of a high performance, compact Snapshot Hyperspectral Camera with light integrator array.The overall goal of this research is to develop a compact, high dynamic range, snapshot hyperspectral camerafor biomedical and biological applications. The technology is based on utilization of a TApered Light IntegratorArray for Imaging Spectrometry. For the purposes of this application and name simplification we call the systemTALIARIS. As the proposed development is a platform technology (complete system can be treated as a spectralcamera easily coupled to microscopes as well as to endoscopes, fundus cameras and other imaginginstruments) it can be used in fundamental cell signaling studies as well as medical diagnostics for exampleretinal imaging / cancer diagnostics. In this proposal though, the experimental focus will be limited to cell signalingin microscopic imaging.The proposed system offers faster frame rates and higher dynamic range (by 1+ orders of magnitude) than othercurrent methods for hyperspectral and multi-spectral imaging of living systems, while still permitting diffraction-limited resolution. Snapshot feature in combination with high light collection efficiency (based on unique, recentlypatented array of light integrators for spectral imaging) greatly enhances a range of possible biologicalexperiments (its characteristics include also low photobleaching, increased patient comfort at lower lightintensities, etc.). The TALIARIS instrument is fully parallel so data at all wavelengths will be collectedsimultaneously from the entire field of view. Thus this approach has a great potential to overcome limitations ofexisting hyperspectral modalities, and enables real time imaging of multiple signaling processes in livingsystems.Towards this goal, our first aim will focus on the development of a proof of concept TALIARIS system and aprototype of array of miniature light integrators. The TALIARIS will transform a 3D spatial-spectral object cubeto a 2D mapped image, which allows acquisition of entire 3D data cube in the snapshot mode. The core of thesystem is the custom-made array of miniature light integrators allowing collection of all light at the input andconcentrating it at smaller output areas to create void spaces needed for spectral information. For low noiseimaging, the proposed device will employ IRIS 15 camera from Photometrics (15Mpix sCMOS camera). TheTALIARIS system will be optimized for throughput and will permit imaging in real time at 30+ frames/second,and 16-bit dynamic range. The proposed system, we will provide datacube dimensions spanning from100x100x25 to 250x250x50 with spectral sampling of 5 nm over 470 - 670 spectral range. Spatial resolution andFOV are inherently connected with fore-optics and will depend on specific microscope objective being used inexperiments. TALIARIS will be validated in microscopic imaging to obtain effective spatial resolution of 0.5micron and 125 microns FOV.The second aim will focus on development of calibration and real-time linear unmixing procedures. This aim willlead to optimized performance in regard of resolution, spectral unmixing, and data collection for 3-dimensionalimaging (x, y, ï¬). This aim will also provide software tools capable of displaying both data cubes and pseudo-color unmixed images in real time. This last feature is critical for both live cell imaging as well as diagnosticapplications, as it provides an immediate feedback during real time observations. In Aim 2, we will also test theTALIARIS spectrometer against currently available spectral imaging systems in several cell imaging applications.These experiments will focus on tests of dynamic biological systems, which are routinely being used in the labof Dr. David Piston's (collaborator on the project). We will be able to validate the results from the TALIARISagainst Zeiss hyperspectral microscopic META imagers as well as other snapshot techniques like ImageMapping Spectrometers (IMS). System evaluations will utilize a variety of fluorophore combinations, starting withtwo-color pairs, moving to more complex combinations such as CFP/GFP/YFP/Fluo-4, and mCherry/SNARF-1/Fura-Red.
Public Health Relevance Statement: The project targets the development of a low cost, compact and high sensitivity Snapshot Hyperspectral
Camera called TALIARIS. Its, high light collection efficiency and high speed allows to rapidly advance the
investigation of live cells signaling, when labeled with multiple fluorescent contrasts. The instrument's
principle allows obtaining spectral information for entire image without scanning and thus improve signal
to noise ratio and limits photo-bleaching effects. It also enables more efficient investigation of transient
biological events. Technologies applied in the project and their low cost may potentially allow access of
larger group of scientists to spectral imaging instrumentation. The system has additional potential to be
used in diagnostic applications like retinal imaging and cancer diagnostics.
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