Emerging Applications

The powerful combination of quantitative image analysis and flow cytometry in a single platform creates exceptional new experimental capabilities. On this page you will find the latest applications under development.

Barcoding

Screening drug candidates requires the rapid analysis of large numbers of samples. Multiplexing samples on the ImageStream by mixing dozens of samples from a time course or dose response titration into one tube dramatically increases the sample throughput of the system. This is achieved through fluorescent cell barcoding with subsequent identification of each sample based on a pattern of fluorescent dye and staining intensity unique to each treatment/time point. Automated location of populations based on fluorescence intensity and color allows deconvolution of the barcoded samples after acquisition.

Here is an example of measuring the movement of NF-κB from the cytoplasm to the nucleus in response to varying doses and times of exposure to TNF-α. By labeling 64 separate samples with different combinations of 3 dyes (AlexaFluor 405, 488, and 660) at 4 intensity levels (43 = 64) it was possible to run one tube of the combined samples on the ImageStream and acquire a single file. After acquisition of over 600,000 images in 10-15 minutes, the color and intensity combinations of the samples were deconvoluted for further quantitative image analysis. Mean Similarity scores are reported in the spreadsheet and a heatmap has been applied. See the cell signaling application for more information about the translocation assay. The combination of high speed image acquisition and automated quantitative image analysis provided by the ImageStream system offers a major advancement in the field of drug discovery.

FRET using the ImageStream for detection of protein-protein interactions

In fluorescence microscopy, light at one wavelength is absorbed by a fluorophore and emitted at a longer wavelength. FRET (Fluorescence or Forster Resonance Energy Transfer) can occur when a second fluorophore is in very close contact such that it can accept the energy from the first fluorophore and emit the light at an even longer wavelength. The efficiency of the transfer is extremely sensitive to the separation distance between the fluorophores and therefore when the emission is detected at the longer wavelength the conclusion is that the fluorophores were within approximately 10 nanometers of each other. When two fluorophores (a donor and acceptor pair) are used to label two different proteins, the close proximity of the two proteins are inferred by the measurement of the FRET from the donor to the acceptor fluorophore. The combination of high speed image acquisition and automated quantitative image analysis with FRET on the ImageStream allows measurement of the spatial location of the protein interaction within the cell or between cell conjugates even for rare subpopulations. Distinguishing intracelluar location of FRET vs. location of the proteins when not exhibiting FRET behavior offers a major advancement in understanding signaling pathways.

In this experiment receptor 1 is labeled with PE (donor) and receptor 2 is labeled with AF647 (acceptor). Cells were stimulated or not-stimulated and images collected with 488nm laser excitation. The graph shows that FRET to the AF647 fluorophore can be detected in the stimulated sample.