The Nikon C1si combines high spectral resolution, high sensitivity, and high acquisition speed

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. q 2006 International Society for Analytical Cytology Key terms: confocal microscopy; spectral imaging; spectral unmixing; fluorescence; Nikon; calibration Confocal fluorescence microscopes were originally developed as a means to produce 3D images with greater information content, by minimizing the reduction in signal to noise caused by out of focus fluorescence common in widefield images. This is accomplished by illuminating and acquiring images through confocal or conjugate pinholes, thereby restricting the in-focus optical section thickness. Stacks of images could then be combined into volume renderings of exquisite clarity. The first confocal microscopes were developed by scientists in their own laboratories seeking solutions to imaging problems encountered in their research (1,2). Commercialization of the technology has expanded the capabilities of confocal microscopes to generate data in multiple fluorescence channels, in time series, and in series of temporally resolved stacks of images acquired at multiple stage coordinates, no...

Reliability of confocal microscopy spectral imaging systems: Use of multispectral beads

“…Using a bandpass of 2.5 nm the peak values were less than 1 nm off the reported values (1,2,5,6,22). Spectral profiles of the two green beads with peaks of 512 and 525 nm were obtained as expected (Table 1 and Fig.…”

“…This equipment has the capacity to obtain unique fluorescence spectra of specific dye molecules that will increase our understanding of biological fluorescence emission process (9)(10)(11)(12)(13)(14)(15)(16). The popularity of this new field in biology has been described in a special issue of Cytometry that includes both review articles and specific application articles (17)(18)(19)(20)(21)(22)(23)(24)(25)(26). To insure that this equipment is functioning properly, new and improved tests have been developed to evaluate spectral performance on these instruments (1,2,5,6).…”

“…It is difficult to evaluate the spectra above 650 nm with the MIDL as the bands rapidly disappear (1,22). Some of these peaks between 650 and 800 nm are observable on a cold lamp.…”

“…Light was delivered to the sample with the 80/20 reflector and a 633 PlanApo 1.4NA objective. The multianode detector was able to obtain eight simultaneous bins of each 10.7 nm (22,24,31).…”

“…Spectral scans were acquired with 5 nm wide bins. The unit was used to demonstrate unmixing of the two fluorochomes and the reproducibility of the MIDL lamp to guaranteed 1 nm accuracy (22).…”