Abstract
Spectral imaging is a natural extension of the capabilities of confocal microscopes. The first confocal spectral imaging (CSI) instruments were able to acquire spectral data that allowed the emissions of overlapping fluorescent probes to be assigned to data channels representing a spectrum rather than a range of emission wavelengths. This marked a significant improvement over what could be done by channel series with standard confocal microscopes. However the performance of these earlier designs can fall short in one or more of the following areas; sensitivity, spectral resolution and reproducibility, acquisition speed, and unmixing accuracy. Nikon has recently introduced a new CSI instrument, C1si, that overcomes some of the more serious performance deficiencies of earlier designs through unique optical, electronic, and data handling advances. C1si uses a multianode photomultiplier tube (PMT) as the detector and typically acquires spectral data in a single scan. Sensitivity is enhanced over designs diffracting randomly polarized fluorescence by rotating the polarization of all emission photons to the S‐plane, the plane for which the diffraction grating is most efficient. Three diffraction gratings are provided supporting wavelength sampling increments of 2.5, 5, and 10 nm. Improvements have been made in the digitization process to increase detection efficiency as well. C1si is calibrated to a high enough standard that it is possible to share and reproduce data between instruments. The algorithm implemented in the EZ‐C1 software is able to accurately and repeatedly unmix fluorescent probes with emission peaks separated by as little as 5 nm. It is possible to unmix probes with emission peaks separated by 20 nm with a 10–1 brightness difference. Three probes can be unmixed with emission peaks contained within a 20 nm range. Acquisition is fast enough and the sensitivity is sufficient for C1si to acquire more than 100 frames of spectral time series data without serious photobleaching. © 2006 International Society for Analytical Cytology