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Take your ImageStream to an entirely new level of performance
The benefits of EDF technology for your ImageStream system
- Improve precision
- Simplify analysis
- Reduce acquisition time
- Enhance discrimination
- Increase resolution
- Enable new applications
FISH In Suspension (FISHIS®)
Counting FISH spots accurately requires that all spots be in focus. Since spots may be located anywhere in the nucleus, maintaining focus throughout the cell is critical. EDF technology expands the depth of focus over the entire cell to make FISH in flow a reality.
Human male PBMC cells were stained with an X chromosome probe and imaged in standard and EDF modes. In standard mode, the FISH spots are detected but can be fuzzy and out of focus, impairing accurate chromosomal counts. In EDF mode, the spots are uniformly in focus throughout the depth of the cell. Automated FISH spot counting accuracy with EDF is dramatically improved. In standard mode, just 72 percent of the in-focus cells were characterized as having the expected single spot, while with EDF mode, 98 percent of all cells exhibited a single FISH spot.
Improved Counting of Nuclear Foci
Radiation-induced DNA strand breaks can be identified by staining
phosphorylated γ-H2AX. The ImageStream with EDF technology
enables precise counting of the foci within the nucleus to assess damage
on an individual cell level.
Irradiated H1299 cells were labeled with AlexaFluor 488 to stain γ-H2AX and DAPI to stain
the nucleus. The standard image panel and the EDF image panel show brightfield (column 1), DAPI (column 2), AF488 (column 3) and a composite of AF488 and DAPI (column 4). The resolution obtained with EDF mode reveals significantly more distinct spots, providing a more precise and accurate spot
count measurement.
Enhanced Discrimination of Nuclear Translocation
EDF technology enhances the discrimination of cellular features and improves precision in the quantitative analysis of cell imagery. EDF eliminates the need to apply a focus gate to the cell image data. For analyses that are sensitive to focus quality, such as NF-κB translocation, removing this subjective step from the analysis improves the overall reliability of statistical results.
The experiment shown here was designed to measure the translocation of NF-κB from
cytoplasm to nucleus. A panel showing untreated cells and LPS-stimulated cells is shown as imaged in
standard mode and EDF mode. The degree of translocation is quantitated by the degree of similarity
between the NF-κB image (FITC stain) and the nuclear image (DRAQ5 stain), which is calculated for each
cell. As shown in the histograms and indicated by the Rd values, EDF improves the discrimination
between the untreated and LPS-stimulated populations.
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Visual Confirmation of Leukocyte Cell Classification by Nuclear Morphology
In traditional microscopic analysis, nuclear morphology is the gold standard for leukocyte classification. EDF improves the resolution of nuclear detail, thereby providing the means to confirm immunophenotypic classification.
Human whole blood was stained with FITC anti-CD45 (green) and DRAQ5 nuclear dye (red)
and run on the ImageStream in standard and EDF imaging modes. The five major cell types are cleanly differentiated, and representative images of each are shown. EDF dramatically improves the imaging of nuclear chromatin structure and nuclear lobicity.
Increased Throughput
In addition to keeping the whole cell in focus, the EDF extended depth of field option allows the ImageStream to be run with a larger core diameter, thereby increasing throughput.
In standard mode, the ImageStream is run with a 10μm core. EDF
enables the use of a 16μm core with virtually no loss of cell focus, effectively
increasing the throughput by up to 3X.
Image the Whole Cell in Focus -- How EDF Technology Works
EDF uses a combination of specialized optics and unique image processing algorithms to project all structures within the cell into one crisp plane of focus. Figure 1 shows the effect of progressive defocus on a test pattern of 2μ bars. Figure 2 shows how the same pattern imaged with EDF technology maintains excellent image quality over the entire focus range.
Ordering Information
EDF Extended Depth of Field Option
The EDF option to the ImageStream system includes all required modifications to the instrument and IDEAS software, installation, testing, documentation and user training. The EDF option can be included with a new ImageStream system or installed as a field upgrade.
Catalog no. 200200