129 Stephen W. Paddock (ed.), Confocal Microscopy: Methods and Protocols, Methods in Molecular Biology, vol. 1075, DOI 10.1007/978-1-60761-847-8_5, © Springer Science+Business Media New York 2014 Chapter 5 Clearing Up the Signal: Spectral Imaging and Linear Unmixing in Fluorescence Microscopy Timo Zimmermann, Joanne Marrison, Karen Hogg, and Peter O’Toole Abstract The ongoing progress in fluorescence labeling and in microscope instrumentation allows the generation and the imaging of complex biological samples that contain increasing numbers of fluorophores. For the correct quantitative analysis of datasets with multiple fluorescence channels, it is essential that the signals of the different fluorophores are reliably separated. Due to the width of fluorescence spectra, this cannot always be achieved using the fluorescence filters in the microscope. In such cases spectral imaging of the fluorescence data and subsequent linear unmixing allows the separation even of highly overlapping fluoro- phores into pure signals. In this chapter, the problems of fluorescence cross talk are defined, the concept of spectral imaging and separation by linear unmixing is described, and an overview of the microscope types suitable for spectral imaging are given. Key words Spectral imaging, Linear unmixing, Image analysis, Fluorescence cross talk, Multichannel imaging 1 Introduction The introduction of fluorescent dyes for microscopy and their combination with immunochemistry provided important stimuli for light microscopy after decades of relative stagnation in regard to new developments. The possibility to exclusively visualize highly specific intracellular structures in distinctive colors against a dark background has changed our visual perception as well as our understanding of cellular mechanisms. The replacement of photo- graphic film by highly sensitive monochromatic CCD cameras that generate digital images that can be easily merged into dramatic multicolor images helped to establish fluorescence light micros- copy as the method of choice for cell biological imaging. Fluorescence imaging was also ideally suited for a groundbreaking new technology, confocal microscopy, which emerged as a power- ful tool for biological imaging at the end of the 1980s and allowed the generation of highly resolved three-dimensional datasets of