Cellular image analysis and imaging by flow cytometry.

Imaging 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 fluorescence in situ hybridization (FISH) probes in human peripheral blood mononuclear cells. The FISH application will be enhanced further by the integration of extended depth-of-field imaging technology with the current optical system.

Extended depth of field imaging for high speed cell analysis.

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 ImageStream system whereby high resolution, multimode imagery from thousands of cells is collected in less than a minute with focus maintained over a 16 microm focal range. METHODS: A prototype EDF ImageStream system incorporating a Wavefront Coded 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) were used to compare standard and Wavefront Coded-based EDF imaging approaches for automated chromosome enumeration. RESULTS: Qualitative visual inspection of bead imagery reveals that the prototype ImageStream system with EDF maintains focus quality over a 16 microm focus range. Quantitative analysis shows the extended depth field collection mode has approximately ten-fold less variation in focus-sensitive feature values when compared with standard imaging. Automated chromosome enumeration from imagery of Jurkat cells probed using the FISHIS protocol is significantly more accurate using EDF imaging. CONCLUSIONS: The use of EDF techniques may significantly enhance the quantitation of cell imagery, particularly in applications such as FISH, where small discrete signals must be detected over a wide focal range within the cell.

Sensitivity measurement and compensation in spectral imaging.

BACKGROUND: The ImageStream system combines advances in CCD technologies with a novel optical architecture for high sensitivity and multispectral imaging of cells in flow. The sensitivity and dynamic range as well as a methodology for spectral compensation of imagery is presented. METHODS: Multicolored fluorescent beads were run on the ImageStream and a flow cytometer. Four single color fluorescent control samples of cells were run to quantify spectral overlap. An additional sample, labeled with all colors was run and compensated in six spectral channels. RESULTS: Analysis of empirical data for sensitivity and dynamic range matched theoretical predictions. The ImageStream system demonstrated fluorescence sensitivity comparable to a PMT-based flow cytometer. A methodology for addressing spectral overlap, individual pixel anomalies, and multiple imaging modalities was demonstrated for spectral compensation of K562 cells. Imagery is shown pre- and post-compensation. CONCLUSIONS: Unlike intensity measurements made with conventional flow cytometers, object size impacts both dynamic range and fluorescence sensitivity in systems that utilize pixilated detection. Simultaneous imaging of alternate modalities can be employed to increase fluorescent sensitivity. Effective compensation of complex multimode imagery spanning six spectral bands is accomplished in a semi-automated manner.

Quantitative measurement of nuclear translocation events using similarity analysis of multispectral cellular images obtained in flow.

Nuclear translocation of NF-kappaB initiates transcription of numerous genes, many of which are critical to host defense. Fluorescent image-based methods that quantify this event have historically utilized adherent cells with large cytoplasm-to-nuclear area ratios. However, many immunologically relevant cells are naturally non-adherent and have small cytoplasm-to-nuclear area ratios. Using the ImageStream imaging flow cytometer, we have developed a novel method that measures nuclear translocation in large populations using cross-correlation analysis of nuclear and NF-kappaB images from each cell. This approach accurately measures NF-kappaB translocation in cells with small cytoplasmic areas in dose- and time-dependent manners. Further, NF-kappaB translocation was accurately measured in a subset of cells contained in a mixed population and the technique was successfully employed to measure IRF-7 translocation in plasmacytoid dendritic cells (PDC) obtained from human peripheral blood. The techniques described here provide an objective and statistically robust method for measuring cytoplasmic to nuclear molecular translocation events in a variety of immunologically relevant cell types with characteristically low cytoplasm-to-nuclear area ratios.

Distinguishing modes of cell death using the ImageStream multispectral imaging flow cytometer.

BACKGROUND: Here we demonstrate the ability of the ImageStream 100 Multispectral Imaging Cytometer to discriminate between live, necrotic, and early and late apoptotic cells, using unique combinations of photometric and morphometric features. METHODS: Live, necrotic, and early and late apoptotic cells were prepared and analyzed by immunofluorescence microscopy, conventional flow cytometry, and imaging flow cytometry, both as single populations and as a heterogeneous mixture of cells. RESULTS: Live (annexin V(-), 7-AAD(-)) and early apoptotic (annexin V(+), 7-AAD(-)) cells were readily identifiable using either conventional or ImageStream based flow cytometric methods. However, inspection of multispectral images of cells demonstrated that the annexin V(+), 7-AAD(+) population contained both necrotic and late-stage apoptotic cells. Although these cells could not be distinguished using conventional flow cytometric techniques, they were separable using unique combinations of photometric and morphometric measures available using ImageStream technologies. CONCLUSIONS: Using multispectral imagery, morphologically distinct cell populations can be distinguished using features not available with conventional flow cytometers. In particular, the ability to couple morphometric with photometric measures makes it possible to distinguish live cells from cells in the early phases of apoptosis, as well as late apoptotic cells from necrotic cells.