HLS-Scanning small laser beam flow cytometry:New concepts for microparticle analysis

Cytomics Analytical, Llc, West Lafayette IN

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Abstract

? DESCRIPTION (provided by applicant): Flow cytometry has been a core technology in biological and medical sciences for nearly 50 years having been initiated by Mack Fulwyler in 1965 and driven by the Herzenberg laboratory into the field of immunology as well as many other fields. However the core principles have changed little over that time. While the limits of the technology has advanced from 1 to a current maximum of 18 fluorescent colors and two scatter signals, the greater majority of instruments operate somewhere in the lower-middle of this technology range. The fundamental principles of the technology have remained the same over this time and only measures total cellular signals based on each cell as it passes the detection point. This provides a rich data source, but with low content with regard to each parameter collected. Each cell has a single value for the entire cell for each variable collected. The contrast with a technology such as confocal microscopy is clear. The latter produces tremendous data content, but mostly based on a fewer cells with inability to operate on suspended cells-the key value of flow cytometry. Separating cells using cellular properties to obtain population information is difficult in imaging, and physical separation is virtually impossible. It is true that high content screening systems can collect a lot of data on a lot of cells, but the manipulation of those populations is not as sophisticated as multiparameter flow cytometry. The present innovation produces both high content and high cell data streams but importantly with significant increase in resolution of small particles because of the scanning technology. Our approach is generated by many years of leading edge development of disk reading and writing technologies at the highest possible resolution. DVD discs for example have pits in the 10 nm range. By transferring some of these ideas onto a microfluidic system using laser diameters at similar sizes used in Blu-ray we have opened up an entirely new opportunity and have defined this as micro-scanning imaging confocal flow cytometry. The result is a tremendously rich data content on cells flowing at approximately 1 m/s with a scan rate of 1MHz to achieve very high resolution data. A major opportunity now exists to rewrite the information collected from blood cells and in particular microparticles which are too small to be analyzed by current flow cytometers. When realized, the proposed technology will be able to collect many channels of fluorescence as well as detailed morphological data on tiny particles opening up a new chapter in advanced micro particle analysis in whole blood or plasma as well as other biological samples.