Amnis® imaging flow cytometry platforms discriminate cells in flow objectively and statistically based on their appearance. This capability can therefore be used for any experiment that results in changes to the cell’s image.
FRET using the ImageStream® X 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 a longer wavelength. The efficiency of the transfer is extremely sensitive to the separation distance between the fluorophores; therefore, when emission is detected at the longer wavelength, the conclusion is that the fluorophores were within approximately 10 nm 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® X allows measurement of the spatial location of the protein interaction within the cell or between cell conjugates even for rare subpopulations. Distinguishing intracellular 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. Browse our Amnis® Imaging Flow Cytometers portfolio