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

高性能、紧凑型快拍高光谱相机与光积分器阵列的开发。 本研究的总体目标是研制一种结构紧凑、高动态范围、快拍的高光谱相机 用于生物医学和生物学应用。该技术基于锥形光积分器的利用 用于成像光谱的阵列。出于应用和名称简化的目的，我们将系统称为 塔里亚里斯。由于拟议的发展是一个平台技术（完整的系统可以被视为一个光谱 容易耦合到显微镜以及内窥镜、眼底相机和其他成像设备的相机 它可以用于基础细胞信号研究以及医学诊断，例如 视网膜成像/癌症诊断。然而，在这个提议中，实验的重点将限于细胞信号传导 在显微成像中。 所提出的系统提供了更快的帧速率和更高的动态范围（1+数量级）比其他 目前用于生命系统的高光谱和多光谱成像的方法，同时仍然允许衍射- 有限的分辨率。快照功能与高光收集效率相结合（基于独特的，最近 用于光谱成像的光积分器的专利阵列）极大地增强了一系列可能的生物学特性， 实验（其特征还包括低光漂白，在较低光下增加患者舒适度 强度等）。TALIARIS仪器是完全平行的，因此将收集所有波长的数据 从整个视野同时。因此，这种方法具有克服限制的巨大潜力。 现有的高光谱模式，并使真实的时间成像的多个信号过程中生活 系统. 为了实现这一目标，我们的首要目标将集中在开发概念验证TALIARIS系统和 微型光积分器阵列的原型。TALIARIS将把一个3D空间光谱物体立方体 转换为2D映射图像，允许在快照模式下采集整个3D数据立方体。的核心 系统是定制的微型光积分器阵列，允许在输入处收集所有光， 将其集中在较小的输出区域以产生光谱信息所需的空隙空间。针对低噪声 成像，申报器械将采用Photometrics的IRIS 15相机（15 Mpix sCMOS相机）。的 TALIARIS系统将针对吞吐量进行优化，并将允许以30+帧/秒的速度进行真实的实时成像， 16位动态范围拟议的系统，我们将提供数据立方体的尺寸跨越从 100 x100 x25至250 x250 x50，在470 - 670光谱范围内的5 nm光谱采样。空间分辨率和 FOV与前光学器件固有地连接，并且将取决于在其中使用的特定显微镜物镜。 实验TALIARIS将在显微成像中进行验证，以获得0.5的有效空间分辨率 微米和125微米FOV。 第二个目标将侧重于校准和实时线性解混程序的开发。这一目标将 导致在分辨率、光谱解混和三维数据收集方面优化性能 成像（x，y，x）。这一目标还将提供能够显示数据立方体和伪数据的软件工具。 在真实的时间中的彩色未混合图像。这最后一个特征对于活细胞成像以及诊断都是至关重要的。 应用，因为它在真实的时间观测期间提供即时反馈。在目标2中，我们还将测试 TALIARIS光谱仪与目前可用的光谱成像系统在几种细胞成像应用中的对比。 这些实验将侧重于动态生物系统的测试，这是在实验室中经常使用的 大卫活塞博士（该项目的合作者）。我们将能够验证来自TALIARIS的结果 与蔡司高光谱显微镜Meta成像仪以及其他快照技术（如Image 测绘光谱仪（IMS）。系统评估将利用各种荧光团组合，从 双色对，转向更复杂的组合，如CFP/GFP/YFP/Fluo-4和mCherry/SNARF- 1/Fura-红色。

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.