Levenson Richard M, Mansfield James R
CRI Inc, Woburn, MA, USA.
Cytometry A. 2006 Aug 1;69(8):748-58. doi: 10.1002/cyto.a.20319.
Multispectral imaging (MSI) is currently in a period of transition from its role as an exotic technique to its being offered in one form or another by all the major microscopy manufacturers. This is because it provides solutions to some of the major challenges in fluorescence-based imaging, namely ameliorating the consequences of the presence of autofluorescence and the need to easily accommodate relatively high levels of signal multiplexing. MSI, which spectrally characterizes and computationally eliminates autofluorescence, enhances the signal-to-background dramatically, revealing otherwise obscured targets. While this article concentrates on examples derived from liquid-crystal tunable filter-based technology, the intent is to showcase the advantages of multispectral imaging in general. Some technologies used to generate multispectral images are compatible with only particular optical configurations, such as point-scanning laser confocal microscopy. Band-sequential approaches, such as those afforded by liquid-crystal tunable filters (LCTFs), can be conveniently coupled with a variety of imaging modalities, which, in addition to fluorescence microscopy, include brightfield (nonfluorescent) microscopy as well as small-animal, noninvasive in-vivo imaging. Brightfield microscopy is the chosen format for histopathology, which relies on immunohistochemistry to provide molecularly resolved clinical information. However, in contrast to fluorescent labels, multiple chromogens, if they spatially overlap, are much harder to separate and quantitate, unless MSI approaches are used. In-vivo imaging is a rapidly growing field with applications in basic biology, drug discovery, and clinical medicine. The sensitivity of fluorescence-based in-vivo imaging, as with fluorescence microscopy, can be limited by the presence of significant autofluorescence, a limitation which can be overcome through the utilization of MSI.
多光谱成像（MSI）目前正处于一个转型期，从一项奇特的技术转变为所有主要显微镜制造商都以某种形式提供的技术。这是因为它为基于荧光的成像中的一些主要挑战提供了解决方案，即改善自发荧光存在的后果以及轻松适应相对高水平信号复用的需求。MSI通过光谱表征并通过计算消除自发荧光，显著提高了信噪比，揭示了原本模糊不清的目标。虽然本文主要关注基于液晶可调滤光片技术的示例，但目的是展示多光谱成像的总体优势。一些用于生成多光谱图像的技术仅与特定的光学配置兼容，例如点扫描激光共聚焦显微镜。波段顺序方法，如液晶可调滤光片（LCTF）提供的方法，可以方便地与多种成像方式相结合，除了荧光显微镜外，还包括明场（非荧光）显微镜以及小动物无创体内成像。明场显微镜是组织病理学的首选形式，它依赖免疫组织化学来提供分子水平解析的临床信息。然而，与荧光标记不同，多种显色剂如果在空间上重叠，除非使用MSI方法，否则很难分离和定量。体内成像作为一个快速发展的领域，在基础生物学、药物发现和临床医学中都有应用。与荧光显微镜一样，基于荧光的体内成像的灵敏度可能会受到大量自发荧光的限制，而利用MSI可以克服这一限制。
