Multispectral Flow Cytometry: Next Generation Tools for Automated Classification

Flow cytometry has moved from a relatively simple technology 30 years ago, to a very sophisticated and high-speed detection technology today. However, the number of simultaneous fluorescence dyes that can be separated is limited by the difficulty in overlapping spectra and the complexity of resolving this spectral overlap problem. High-speed multianode PMTs may change this situation. The system we propose utilizes such a technology to allow full spectral analysis of cells and particles as they flow past the light source. Making these measurements is very complex and the necessity for advanced spectral overlap calculations creates a number of difficult problems to solve in a very short period of time. Next-generation instruments can either increase the number of detectors or modify the principles of collection. If the detector system were simplified, the overall cost and complexity of single-cell analytical systems might be reduced. This requires changes in both hardware and software that allow for the analysis of 30 or more spectral signals. Analysis of complex data sets requires some completely new analytical approaches, particularly in the area of multispectral analysis. This presentation discusses a next-generation instrument, which can collect simultaneously 32 bands of fluorescence from a particle in less than 5 microseconds. This opens new opportunities for analysis of bioparticles in a very fast and high content fashion.