Zimmermann Timo
Advanced Light Microscopy Facility, European Molecular Biology Laboratory, Meyerhofstrasse 1,69117 Heidelberg, Germany.
Adv Biochem Eng Biotechnol. 2005;95:245-65. doi: 10.1007/b102216.
Fluorescence microscopy is an essential tool for modern biological research. The wide range of available fluorophores and labeling techniques allows the creation of increasingly complex multicolored samples. A reliable separation of the different fluorescence labels is required for analysis and quantitation, but it is complicated by the significant overlap of the emission spectra. This problem can be addressed on the acquisition and the processing side by the use of spectral imaging in conjunction with linear unmixing of the image data. This method allows the reliable separation of even strongly overlapping fluorescence signals and has become an important tool in colocalization and in FRET studies. In this chapter, the microscope techniques available for spectral imaging are presented and the theory of linear unmixing is explained. Possible limitations as well as approaches for image optimization are discussed to help to realize the full potential of this novel method. Biological applications that can be improved by spectral imaging and linear unmixing are presented.
荧光显微镜是现代生物学研究的重要工具。可用荧光团和标记技术的广泛多样性使得能够创建日益复杂的多色样本。分析和定量需要可靠地分离不同的荧光标记，但发射光谱的显著重叠使其变得复杂。通过结合光谱成像和图像数据的线性解混，可以在采集和处理方面解决这个问题。该方法能够可靠地分离甚至是高度重叠的荧光信号，已成为共定位和荧光共振能量转移（FRET）研究中的重要工具。在本章中，介绍了可用于光谱成像的显微镜技术，并解释了线性解混的理论。讨论了可能的局限性以及图像优化方法，以帮助充分发挥这种新方法的潜力。还介绍了可通过光谱成像和线性解混得到改进的生物学应用。
