Development of a high performance, compact Snapshot Hyperspectral Camera with light integrator array

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 camera for biomedical and biological applications. The technology is based on utilization of a TApered Light Integrator Array for Imaging Spectrometry. For the purposes of this application and name simplification we call the system TALIARIS. As the proposed development is a platform technology (complete system can be treated as a spectral camera easily coupled to microscopes as well as to endoscopes, fundus cameras and other imaging instruments) it can be used in fundamental cell signaling studies as well as medical diagnostics for example retinal imaging / cancer diagnostics. In this proposal though, the experimental focus will be limited to cell signaling in microscopic imaging. The proposed system offers faster frame rates and higher dynamic range (by 1+ orders of magnitude) than other current 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, recently patented array of light integrators for spectral imaging) greatly enhances a range of possible biological experiments (its characteristics include also low photobleaching, increased patient comfort at lower light intensities, etc.). The TALIARIS instrument is fully parallel so data at all wavelengths will be collected simultaneously from the entire field of view. Thus this approach has a great potential to overcome limitations of existing hyperspectral modalities, and enables real time imaging of multiple signaling processes in living systems. Towards this goal, our first aim will focus on the development of a proof of concept TALIARIS system and a prototype of array of miniature light integrators. The TALIARIS will transform a 3D spatial-spectral object cube to a 2D mapped image, which allows acquisition of entire 3D data cube in the snapshot mode. The core of the system is the custom-made array of miniature light integrators allowing collection of all light at the input and concentrating it at smaller output areas to create void spaces needed for spectral information. For low noise imaging, the proposed device will employ IRIS 15 camera from Photometrics (15Mpix sCMOS camera). The TALIARIS 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 from 100x100x25 to 250x250x50 with spectral sampling of 5 nm over 470 – 670 spectral range. Spatial resolution and FOV are inherently connected with fore-optics and will depend on specific microscope objective being used in experiments. TALIARIS will be validated in microscopic imaging to obtain effective spatial resolution of 0.5 micron and 125 microns FOV. The second aim will focus on development of calibration and real-time linear unmixing procedures. This aim will lead to optimized performance in regard of resolution, spectral unmixing, and data collection for 3-dimensional imaging (x, y, ). 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 diagnostic applications, as it provides an immediate feedback during real time observations. In Aim 2, we will also test the TALIARIS 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 lab of Dr. David Piston’s (collaborator on the project). We will be able to validate the results from the TALIARIS against Zeiss hyperspectral microscopic META imagers as well as other snapshot techniques like Image Mapping Spectrometers (IMS). System evaluations will utilize a variety of fluorophore combinations, starting with two-color pairs, moving to more complex combinations such as CFP/GFP/YFP/Fluo-4, and mCherry/SNARF- 1/Fura-Red.