We are requesting funds for a Compucyte iCys Laser Scanning Cytometer (LSC). The LSC is a relatively recently developed instrument that joins many of the advantages of traditional microscopy and flow cytometry, and significantly extends the limits of single cell analyses. Rather than randomly imaging an entire field like a microscope does, it selects, images and analyzes cells that meet certain adjustable criteria (such as size or fluorescence). Unlike conventional flow cytometry, the LSC is usually used to analyze cells fixed to horizontal surface, such as cultured cells on slides or in a variety of culture vessels. Live cells can be imaged and cytometrically analyzed for fluorescent signals, and a variety of types of fixed specimens can also be studied in this way. The instrument uses an automated nanostep X-Y stage to move samples through the objective lens, applying up to three different lasers to excite fluorescent emissions and quantitating these using an array of four photomultiplier tube (PMT) detectors. Thus, the LSC is essentially a powerful hybrid technology that combines imaging and cytometric analysis in a single platform. Importantly, with the LSC image becomes a parameter, and detailed microscopic pictures are stored and referenced directly to cytometric data. A highly appealing feature of the instrument that we plan to use extensively is its ability to gather quantitative fluorescence data from microscopic areas of fluorescently labeled tissue sections, and to display and analyze such data in a manner that is comparable in many cases to true cytometric data such as one obtains with traditional flow cytometry. We plan to house and operate the iCys LSC in our well established central Flow Cytometry Core Laboratory, where there will be ample expertise and oversight to ensure that the instrument is used to its full capacity. The Core Laboratory has an experienced senior faculty member (Dr. Steven Porcelli) as its supervisor, and employs three full time faculty level associates to direct operations and provide technical supervision. There is also a range of internal consultants with expertise in areas of major relevance to the development of applications that will use the LSC. The Core Laboratory has a well developed and carefully monitored financial plan, and receives substantial institutional support from the institution and from our NCI-funded Cancer Center to ensure its efficient and stable operation. By incorporating the iCys LSC into this core laboratory, a large body of investigators pursuing many diverse NIH-funded basic and translational research projects will gain convenient and affordable access to this important new technology. Public Health Relevance: This application requests funding that will allow our Flow Cytometry Core Laboratory to acquire a new analytical instrument called the Compucyte iCys Laser Scanning Cytometer. This instrument will provide many researchers at our institution with a powerful new way to analyze cells and tissues. The studies that will be done with this instrument will advance our understanding of a wide spectrum of areas with high relevance to biomedical science, including cancer biology, immunology, autoimmune diseases and infectious diseases.
