SynopsisImaging flow cytometry combines the statistical power and fluorescence sensitivity of standard flow cytometry with the spatial resolution and quantitative morphology of digital microscopy. The technique is a good fit for clinical applications by providing a convenient means for imaging and analyzing cells directly in bodily fluids. Examples are provided of the discrimination of cancerous from normal mammary epithelial cells and the high throughput quantitation of FISH probes in human peripheral blood mononuclear cells. The FISH application will be further enhanced by the integration of extended depth of field imaging technology with the current optical system. Keywordsimaging; flow; cytometry; fluorescence; brightfield; darkfield; multispectral Quantifying Cellular Structure in Health and DiseaseThe eukaryotic cell is a highly structured, three-dimensional object containing a wide range of molecular species. The size, shape, and structure of the cell, as well as the abundance, location, and co-location of any of these constituent biomolecules may be of significance in any given clinical situation or research application. For instance, in hematopoiesis, as cells differentiate and mature, different subsets of molecules are expressed that reflect a specialized functional capacity for that unique cell type (e.g., granulocytes vs. lymphocytes). In general the characterization of this array of constituent molecules by imaging or flow cytometry provides insight into the physiological function of any particular cell or alternatively, pathological changes that may have occurred or accrued. In clinical practice and in research settings, cellular evaluation by imaging technologies and flow cytometry provides significant information reflecting the particular cellular phenotype, both normal and pathological. Microscopy provides a wealth of information, but data acquisition rates are slow and analysis is generally subjective. In flow cytometry, data acquisition is rapid and better suited for the evaluation of pathologies present in low frequency, but the data are only intensity-based, thus lacking the morphology that truly lends credence to the analysis.In addition, the assessment and evaluation of cell samples by imaging and flow cytometric techniques is complicated by a number of factors. For instance, changes in a cell type or aCorresponding author for proofs and reprints:

Background: Fluoresence microscopy is an extremely useful tool to analyze the intensity, location and movement of fluorescently tagged molecules on, within or between cells. However, the technique suffers from slow image acquisition rates and limited depth of field. Confocal microscopy addresses the depth of field issue via ''optical sectioning and reconstruction'', but only by further reducing the image acquisition rate to repeatedly scan the cell at multiple focal planes. In this paper we describe a technique to perform high speed, extended depth of field (EDF) imaging using a modified ImageStreamÒ system whereby high resolution, multimode imagery from thousands of cells is collected in less than a minute with focus maintained over a 16 lm focal range. Methods: A prototype EDF ImageStream system incorporating a Wavefront Coded TM element was used to capture imagery from fluorescently labeled beads. Bead imagery was quantitatively analyzed using photometric and morphological features to assess consistency of feature values with respect to focus position. Jurkat cells probed for chromosome Y using a fluorescence in situ hybridization in suspension protocol (FISHIS TM ) were