The ImageStream system’s CCD camera produces six images of each cell, including darkfield, brightfield, and up to four fluorescent colors. Each image is used to calculate over 40 features, so a six-image assay results in ~250 morphometric and photometric features per cell.

ImageStream system extracts far more information per cell than flow cytometers with 10 or more colors and can perform a wide variety of fluorescence localization tasks that cannot be done by traditional flow cytometry. Compared to a microscope, the ImageStream system has far higher throughput, bringing rare event detection and robust population statistics to image-based assays.

PMT’s are used in the autofocus and flow speed detection subsystems, but all cell data are collected using the system’s custom CCD camera.

TDI is a method of CCD camera operation that greatly increases sensitivity, even at very high imaging rates. Essentially, TDI is a means of electronically panning the camera to track the cells in the flow stream. As the cells move down the field of view, the photoelectrons on the CCD chip are shifted down the chip. This process is synchronized by measuring the velocity of the cells and constantly adjusting the shift rate of the photoelectrons to match. The end result is that light from each cell can be collected for ~10 milliseconds rather than just a few microseconds and without blurring.

The ImageStream system has fluorescence sensitivity comparable to modern flow cytometers in all colors and is significantly better than flow cytometry for smaller objects like bacteria, yeast, and sub-cellular organelles.

With TDI operation, all objects in the flow core are imaged without the need for a triggering system.

The magnification of the system is ~36X and image detail is comparable to 40-60X microscopy, as judged by eye. The optical system employs a 0.75 numerical aperture objective lens and is diffraction-limited. The image pixel size is 0.5 x 0.5 microns, so a 10 micron diameter cell covers approximately 300 pixels.

The imaging field of view is 48 microns wide, so the largest recommended object size is about 35 microns wide. The field of view is very tall and flow forces tend to orient long objects with the field, so the main consideration is object width.

No. The cell imaging rate is independent of the number of probes imaged in the assay.

No. The ImageStream system’s spectral detection bands are fixed. However, the system is compatible with a wide variety of fluorescent dyes commonly used in both flow cytometry and microscopy, thanks to a powerful laser, high NA objective lens, red-sensitive camera, and highly effective crosstalk compensation algorithms. Click here to view the detection bands and associated dyes.

The ImageStream system currently uses a single excitation laser, a 488 nm solid state unit with 200 mW of power. The system will be available with up to three lasers in late 2006.  In addition to the standard 488 nm unit, an optional 658 nm and your choice of either a 405 nm or 375 nm solid state laser will be available.

No. The fluidic system is designed to be extremely laminar, non-pulsatile, and to prevent cell tumbling.

The narrowest orifice in the system is 250 um and all fluids are pumped via motor-driven syringe pumps, so clogging problems are rare. To ensure trouble-free operation, Amnis recommends that samples be filtered through a 40 or 70 micron nylon mesh before running on the instrument .

The SpeedBead reagent is a suspension of small polystyrene microspheres that is run at the same time as the cell sample. The SpeedBead reagent is critical to ImageStream calibration as well as to numerous real-time systems including velocity detection and autofocus. The SpeedBead reagent costs less than two US dollars per sample.

The ImageStream system uses special cleanser, debubbler, and sterilizer solutions, which are all available from Amnis at a nominal cost.

Amnis recommends the use of either distilled water or phosphate-buffered saline as sheath fluids. The ImageStream system’s fluidics are conceptually similar to those of a flow cytometer but the actual design is quite different and will not function properly with commercially available sheath fluids intended for flow cytometers.

Yes. The fluid pressures and shear forces within the ImageStream system are very low. Please keep in mind that the system has no provision for controlled incubation.

Most protocols for flow cytometry can be adapted to the ImageStream system with relatively minor adjustments in cell concentration, sample volume, and probe titration.

A 10,000 cell file requires roughly 500 MB for both raw and processed data file storage.

The best configuration is a storage server connected to the ImageStream system and IDEAS® workstations via a gigabit network. However, our users employ a variety of data storage schemes, including archiving of data files to DVD disks.

No. Amnis recommends that each site have one or two advanced users, but these users can readily train others. ImageStream operation can be learned in a matter of hours. Users can become functional with the IDEAS data analysis software in a day or two.

IDEAS can export calculated feature values in plain text format to a file or to the Windows clipboard. Feature values can also be exported to an FCS file, though many flow cytometry packages lack the ability to handle the data ranges associated with image-based feature calculations. Graphs, statistics, images, and other data components can be copied to the Windows clipboard.

IDEAS will run on most modern PC’s running Windows 2000 or XP. A minimum of one gigabyte of RAM, video resolution of at least 1280 x 1024, a DVD burner for data archiving, and gigabit ethernet for data transfer are recommended. Up to four gigabytes of RAM can be installed in standard 32 bit Pentium-based computers and will facilitate the analysis of larger data files (more than 10,000 cells) and/or the analysis of multiple data files at the same time. IDEAS can also take advantage of higher video resolution settings to display more graphs and images at a time.

The INSPIRE instrument control software runs on the ImageStream system’s embedded Windows PC. The IDEAS data analysis software package is also a Windows application. IDEAS may run on Intel-based Apple hardware running Windows, but this has not been tested and is not supported.