<title>Sensitive imaging of spectrally overlapping flourochromes using the LSM 510 META</title>

Abstract

Multi-color fluorescence microscopy has become a popular way to discriminate between multiple proteins, organelles or functions in a single cell or animal and can be used to approximate the physical relationships between individual proteins within the cell, for instance, by using Fluorescence Resonance Energy Transfer (FRET). However, as researchers attempt to gain more information from single samples by using multiple dyes or fluorescent proteins (FPs), spectral overlap between emission signals can obscure the data. Signal separation using glass filters is often impractical for many dye combinations. In cases where there is extensive overlap between fluorochromes, separation is often physically impossible or can only be achieved by sacrificing signal intensity. Here we test the performance of a new, integrated laser scanning system for multispectral imaging, the Zeiss LSM 510 META. This system consists of a sensitive multispectral imager and online linear unmixing functions integrated into the system software. Below we describe the design of the META device and show results from tests of the linear unmixing experiments using fluorochromes with overlapping emission spectra. These studies show that it is possible to expand the number of dyes used in multicolor applications.