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Flow Cytometry
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"Single Particle High Resolution Spectral Analysis Flow Cytometry",
Gregory Goddard, John C. Martin, Mark Naivar, Peter M. Goodwin, Steven W. Graves, Robb Habbersett, John P. Nolan, James H. Jett, National Flow Cytometry Resource, Bioscience Division, Los Alamos National Laboratory, Cytometry Part A 69A:842–851
Copyright ©2006 International Society for Analytical Cytology
While conventional multi-parameter flow cytometers have proven highly successful, there are several types of analytical measurements that would benefit from a more comprehensive and flexible approach to spectral analysis including, but certainly not limited to spectral deconvolution of overlapping emission spectra, fluorescence resonance energy transfer measurements, metachromic dye analysis, free versus bound dye resolution, and Raman spectroscopy. The described system utilizes a diffraction grating to disperse the collected fluorescence and side-scattered light from cells or microspheres passing through the interrogation region over a rectangular charge-coupled-device image sensor. The flow cell and collection optics are taken from a conventional flow cytometer with minimal modifications to assure modularity of the system.
"Multispectral Cytometry: The Next Generation",
J. Paul Robinson, Biophotonics International, October 2004, 36-40
A multispectral flow cytometer is described in which a grating disperses signals from a fluorescence collector lens to a 32-channel Hamamatsu PMT. Detectors submit the data to the computational system, which determines hyperspectral curves for every particle and performs spectral unmixing.
"Multispectral Cytometry of Single Bio-particles Using a 32-Channel Detector",
J. Paul Robinson, Bartek Rajwa, Gerald Gregori, James Jones, Valery Patsekin, Proceedings of SPIE Vol. 5692
Detecting biological particles and subsequently identifying them in a very short period of time is highly desirable, but a very difficult task. There are several pathways for developing rapid detection systems. For example, one can reduce sample size to a very small volume, and amplify cellular components by PCR technology with a view to identifying antigen-specific molecules. Alternatively, antibody-based assays allow for detection and identification of a variety of well-characterized pathogens. The system proposed utilizes flow cytometry technology to rapidly detect spectral fingerprints or organisms. However, the current limit for simultaneously detectable fluorescence signals in flow cytometry is around 12-15. Making these measurements is very complex and the necessity for advanced spectral overlap calculations creates a number of difficult problems to solve in a 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. Further, analysis of complex data sets requires some completely new approaches, particularly in the area of multispectral analysis. This paper describes the key components and principles involved in building a next-generation instrument which can collect simultaneously 32 bands of fluorescence from a particle in less than 5 microseconds. This would allow the analysis of several thousand bioparticles per second. The flow cytometry system based on our new detector would be designed to be portable and low cost.
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