Principles of Amnis Imaging Flow Cytometry.

Over the last decade imaging flow cytometry (IFC) has become an established technique, as evidenced by its use in over 500 peer-reviewed scientific articles. Nevertheless, it is still an emerging technique with an installed base of less than 5 % that of conventional flow cytometers. In parallel with its adoption, the technology has evolved rapidly, increasing in speed, sensitivity, and real-time data analysis capacity by over an order of magnitude since its introduction.

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.

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.

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.

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.