Image Flow Cytometry
How do you combine the speed and precision of flow cytometry with spatially resolved information from microscopy? With image flow cytometry. Common applications are, for instance, translocation studies of transcription factors, spatially resolved analyzes of phagocytosed particles, or FRET experiments of target proteins in defined cellular compartments. Our 1-camera (CCD) Amnis ImageStream ISX Mark II system (Merck-Millipore) is equipped with 3 lasers (405nm, 488nm, 642nm) and a total of 6 channels (including optional transmitted light channel and side scatter). The optical arrangement of the lasers is co-linear. A 40x objective (NA 0.75, field of view 60μm) is installed. A maximum of 4,000 events per second can be recorded at 8-bit depth. At the lowest flow rate, the pixel resolution is 0.5 x 0.5 μm. Sample concentration should be 107-108 cells or particles per ml. The acquisition volume is low (~1μl/min), thus small sample volumes are sufficient. Low number of samples are measured from siliconized 0.5-2ml reaction tubes, whereas larger samples sets can be analyzed directly from 96-well plates using the build-in autosampler. In the latter case, we recommend fixing the cells and measuring them on the device at low speed overnight. The analysis software IDEAS is available on site. The ISX quickly generates dozens of gigabytes of data, and measurement as well as evaluation strategies must be planned carefully. Several 30-core processors are available for computing the data. A high-throughput micronucleus test has been established on this instrument (see Bekeschus et al., Environ., Mol. Mutagen 2018).
Contact: Dr. Sander Bekeschus, sander.bekeschusinp-greifswaldde