Molecular medicine now requires molecular pathology. While fluorescence has traditionally been used for high-resolution multiplexed molecular imaging, clinical practitioners prefer non-fluorescent multicolor methods. However, in brightfield, typically, only one color is used at a time, which precludes assessment of co-expression on a cell-by-cell basis. Similar constraints apply to brightfield in-situ hybridization techniques. Double- and triple-staining procedures are rarely performed in non-research settings not only because the wet chemistry can be difficult, but also because it can be challenging or impossible to determine visually where and to what extent different chromogens may physically overlap. Spectral imaging can be useful in this context. Two methods of acquiring spectral images are described, along with their application to multicolor immunohistochemistry and transmission in-situ hybridization (TRISH): 1) liquid crystal tunable filters; and 2) a novel, spectrally agile light source. This source emits white light of any desired color temperature, or single 10-nm wavelength bands in the range 420 to 700 nm, or any combination of wavelengths with individual intensity control. Both methods are allied with a grayscale camera and appropriate algorithms to analyze multicolor samples of clinical significance. Spectrally unmixed images clearly separate signals linked to different chromogens, even with spectral and spatial overlap. Intriguing challenges in matching mathematical algorithms to these specific problems remain: how many bands are enough? What are the optimal unmixing procedures? What automated tools can be applied to speed and simplify the procedures?

Date Published: 2 July 2003
PDF: 7 pages
Proc. SPIE 4959, Spectral Imaging: Instrumentation, Applications, and Analysis II, (2 July 2003); doi: 10.1117/12.485550

Published in SPIE Proceedings Vol. 4959:
Spectral Imaging: Instrumentation, Applications, and Analysis II
Richard M. Levenson; Gregory H. Bearman; Anita Mahadevan-Jansen, Editor(s)