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.
