SIMPLE, LOW-COST, ULTRA-RAPID SPECTRAL IMAGING PLATFORM

第一阶段-光谱成像包括测量光谱在 图像的每一个像素，都可以成为分子病理学的重要工具， 目前的应用包括多色荧光和光谱核型。这个 首席调查员已将其应用扩展到多色免疫组织化学， 显示它至少可以分辨3种颜色，即使它们是共同定位的。《校长》 研究人员还展示了它在分析苏木精和伊红染色中的用途 病理标本，显示其光谱区分 良性和恶性细胞。然而，目前的技术，如傅里叶变换干涉术和可调谐滤光器，要么昂贵，要么速度慢，要么两者兼而有之。中科院建议： 开发一种新的光谱成像平台，用于明场显微镜，将 既便宜又灵活。使用匹配滤波技术，它将能够 分析具有全光谱分辨率的场景，只需要2或3个，而不是 每个场20到100帧。在第一阶段，CRI将组装一个原型并演示 它的光谱分辨率和成像速度。作为原理证明，它将被用来定量分离免疫组织化学染色的玻片中的两个色原，并 从苏木素和曙红染色的乳房等复杂场景中收集光谱 癌症标本。在第二阶段，将添加匹配的过滤功能并对其进行测试 应用于多色免疫组织化学和Brightfield原位杂交。它也将是 适用于基于MALDI/MS的自动直接激光捕获显微解剖 蛋白质组学。 第二阶段-在第一阶段，CRI将建立一个原型新型光谱成像平台。在……里面 第二阶段，技术开发，特别是软件工具的开发 为了充分利用光谱成像，将继续进行。的三个应用领域 还将强调对癌症研究和临床实践的极大兴趣： 1)多色免疫组织化学；2)Brightfield多色原位杂交 3)激光捕获显微切割，用于基因组学、表达谱和 蛋白质组学。在组织化学中，可能很难检测和定量染色原 沉积，特别是当使用一种以上的颜色并且通常的反染色是 现在时。明场(透射式)原位杂交是一种很有前途的技术， 但如果没有多色光谱工具，它就无法与传统的、但更少的竞争 临床上方便，以鱼为基础的化验。有了这一领域的领导者，CRI将 光谱成像与多色透射式原位杂交相结合 (Trish)，并记录其临床用途。最后，激光捕获显微解剖 是从显微镜载玻片中获取纯细胞群体的中心方法。 不幸的是，这一过程可能非常繁琐，而且需要自动化。 我们打算使用光谱成像来定位苏木素和伊红染色的玻片上的适当区域，并利用这些信息来自动化激光显微解剖。 进程。一种结合了这种诊断和准备能力的工具应该 在诊所和实验室都找到一个位置。 建议的商业应用： 该光谱照明器可部署在标准病理成像显微镜中以 协助病理学家分析典型标本。因此，它可以取代传统的 这些显微镜中的照明源，并结合适当的软件， 可用于提供各种光谱辅助功能。这些将包括分析 以及免疫组织化学和原位杂交研究的定量，接口 配备激光显微解剖设备，并可能提供计算机辅助诊断支持 常规的组织病理学应用。照明系统可以以几种形式销售 方式：要么直接由CRI；要么与显微镜制造商合作；要么合作 与其他综合病理成像工作站供应商合作。

Phase I - Spectral imaging involves the measurement of an optical spectrum at every pixel of an image and can be an important tool for molecular pathology, current uses including multicolor fluorescence and spectral karyotyping. The Principal Investigator has extended its use to multicolor immunohistochemistry, showing it can resolve at least 3 colors even if they co-localize. The Principal Investigator has also shown its use in analyzing Hematoxylin and Eosin-stained pathology specimens, demonstrating its ability to spectrally discriminate between benign and malignant cells. However, current technologies, such as Fourier-transform interferometry and tunable filters, are expensive, slow or both. CRI proposes to develop a novel spectral imaging platform for use with brightfield microscopy that will be inexpensive and flexible. Using the technique of matched filtering, it will be able to analyze a scene with full spectral resolution, while requiring only 2 or 3 rather than 20 to 100 frames per field. In Phase I, CRI will assemble a prototype and demonstrate its spectral resolving power and imaging speed. As proof-of-principle, it will be used to quantitatively separate two chromogens in immunohistochemically stained slides, and to collect spectra from complex scenes such as Hematoxylin and Eosin-stained breast cancer specimens. In Phase II, matched filtering capability will be added and tested for use in multicolor immunohistochemistry and brightfield in-situ hybridization. It will also be adapted to automatically direct laser-capture microdissection for MALDI/MS-based proteomics. Phase II - In Phase I, CRI will build a prototype novel spectral imaging platform. In Phase II, development of the technology and in particular, of software tools to fully exploit spectral imaging, will continue. Three application areas of great interest for cancer research and clinical practice will also be emphasized: 1) multicolor immunohistochemistry; 2) brightfield multicolor in-situ hybridization and 3) laser-capture microdissection for input for genomics, expression profiling and proteomics. In histochemistry, it can be difficult to detect and quantitate chromogen deposition, especially if more than one color is used and usual counterstaining is present. Brightfield (transmission) in-situ hybridization is a promising technique, but without multicolor spectral tools, it cannot compete with conventional, but less clinically convenient, FISH-based assays. With leaders in this field, CRI will combine spectral imaging with multicolor transmission in-situ hybridization (TRISH) and document its clinical utility. Finally, laser-capture microdissection is a central method for harvesting pure cell populations from microscope slides. Unfortunately, the procedure can be extremely tedious and is in need of automation. We intend to employ spectral imaging to locate appropriate regions on Hematoxylin and Eosin-stained slides and to use this information to automate the laser micro-dissection process. An instrument combining such diagnostic and preparative capabilities should find a place in the clinic as well as in the laboratory. PROPOSED COMMERCIAL APPLICATION: The spectral illuminator can be deployed in standard pathology imaging microscopes to assist pathologists in analyzing typical specimens. As such, it can replace traditional illumination sources in these microscopes, and in combination with appropriate software, can be used to provide various spectrally assisted functions. These will include analysis and quantitation of immunohistochemical and in-situ hybridization-based studies, interfacing with laser-microdissection device and possibly computer-aided diagnostic support for regular histopathology applications. The illuminator system may be marketed in several ways: either by CRI directly; in cooperation with microscope manufacturers; or in partnership with other purveyors of integrated pathology imaging workstations.