Levenson Richard M, Lynch David T, Kobayashi Hisataka, Backer Joseph M, Backer Marina V
CRI Inc., Woburn, MA 01801, USA.
ILAR J. 2008;49(1):78-88. doi: 10.1093/ilar.49.1.78.
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.
生物模型设计与应用的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，即要具备更高的灵敏度、可靠的定量能力以及分辨多个同时存在信号的能力。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，尤其是在可见光谱范围内，这些自发荧光信号需要与靶向荧光团的信号区分开来。基于荧光的成像还受到组织在可见光以及在较小程度上近红外（NIR）区域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即使来自相对较深位置的足够光线，以实现具有可接受信噪比的信号捕获。多光谱成像通过其将自发荧光与标记荧光分离的能力，与传统方法相比，灵敏度可提高多达300倍，并同时提高定量准确性。在近红外区域，自发荧光虽然仍然显著，但问题较小。然而，从多个荧光团中分离信号的任务仍然存在。多光谱成像允许分离五个或更多荧光团，每个信号分别进行定量和可视化。临床前小动物成像通常在体内阶段前后都伴有显微镜分析。这可能涉及组织培养操作和/或对固定或冷冻组织的组织学检查。由于在灵敏度、定量和多路复用方面具有相同的优势，基于显微镜的多光谱技术成为体内成像的出色补充。
