Multiplexing with multispectral imaging: From mice to microscopy
Multiplexing with multispectral imaging: From mice to microscopy
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DOI：
10.1093/ilar.49.1.78
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发表时间：
2008-01-01
期刊：
影响因子：
2.5
通讯作者：
Backer, Marina V.
中科院分区：
文献类型：
--
作者：
Levenson, Richard M.;Lynch, David T.;Backer, Marina V.
生物模型的设计和应用日益复杂，以及新型荧光探针的出现，对分子成像系统提出了新的要求，以提供增强的灵敏度、可靠的定量以及解析多个同时信号的能力。由于普遍存在的自发荧光信号（主要来自皮肤和肠道）的存在，灵敏度受到限制，特别是在可见光谱范围内，需要将其与目标荧光团的信号分开。基于荧光的成像还受到可见光区域和较小程度的近红外 (NIR) 区域组织的吸收和散射特性的影响。然而，典型动物模型（通常是小鼠）的小尺寸通常允许检测到甚至从相对较深的位置产生的足够的光，以允许捕获具有可接受的信噪比的信号。多光谱成像能够将自发荧光与标记荧光分开，与传统方法相比，灵敏度可提高 300 倍，同时提高定量准确性。在近红外区域，自发荧光虽然仍然很重要，但问题不大。然而，从多个荧光团中解开信号的任务仍然存在。多光谱成像可以分离五个或更多荧光团，每个信号单独定量和可视化。临床前小动物成像通常伴随着体内阶段之前和之后的显微镜分析。这可能涉及固定或冷冻组织的组织培养操作和/或组织学检查。由于在灵敏度、定量和多重方面具有相同的优势，基于显微镜的多光谱技术形成了体内成像的极好补充。
Increasing sophistication in the design and application of biological models as well as the advent of novel fluorescent probes have led to new demands on molecular imaging systems to deliver enhanced sensitivity, reliable quantitation, and the ability to resolve multiple simultaneous signals. Sensitivity is limited, especially in the visible spectral range, by the presence of ubiquitous autofluorescence signals (mostly arising from the skin and gut), which need to be separated from those of targeted fluorophores. Fluorescence-based imaging is also affected by absorbing and scattering properties of tissue in both the visible and to a lesser extent the near-infrared (NIR) regions. However, the small size of typical animal models (usually mice) often permits the detection of enough light arising even from relatively deep locations to allow the capture of signals with an acceptable signal-to-noise ratio. Multispectral imaging, through its ability to separate autofluorescence from label fluorescence, can increase sensitivity as much as 300 times compared to conventional approaches, and concomitantly improve quantitative accuracy. In the NIR region, autofluorescence, while still significant, poses less of a problem. However, the task of disentangling signals from multiple fluorophores remains. Multispectral imaging allows the separation of five or more fluorophores, with each signal quantitated and visualized separately. Preclinical small animal imaging is often accompanied by microscopic analysis, both before and after the in vivo phase. This can involve tissue culture manipulations and/or histological examination of fixed or frozen tissue. Due to the same advantages in sensitivity, quantitation, and multiplexing, microscopy-based multispectral techniques form an excellent complement to in vivo imaging.