Instrumentation

FACSDiVA

Description

This is one two high-speed sorters located at the VRC Flow Cytometry. Currently this instrument is configured with three lasers (488nm Argon-Inova 90 laser, 595nm dye laser and a 408nm Krypton laser) and fourteen fluorescent detectors (16 parameter instrument). FACSDiVa Optical Light Path Figure 5 (PDF)

Figure 5. FACSDiVa Optical Light Path

These instruments are among the most advanced, state of the art flow cytometer currently available. With three laser capability, advanced optics and new generation of mixed gas tunable lasers, these instruments provide enormous flexibility in terms of applications as well as unprecedented sensitivity. The FACSDiVa configurations will be the instrument of choice for the development of new applications, for applications involving dyes requiring nonstandard wavelengths for excitation. In addition this flexibility can be targeted toward the analysis and sorting of highly complex cell populations and cells with very low expression of the antigen of interest. Figure 6 shows the complex filter system used to measure excitation energy of the common fluorochromes used in this system. The FACSDiVa uses DiVa software, equipped with intra-laser compensation and high speed sorting capabilities, which controls all of the sorting parameters. Many high speed multi-laser sorts are now done routinely with throughputs near 20,000 cells/second to 40,000 cells/second and sorted fractions exhibiting > 99% purity upon post sort analysis. However, not all experiments are suited for high-speed sorting, cells stimulated to become apoptotic, dendritic cells, and microglia, for example, are too fragile for high-speed conditions and are sorted under lower pressure conditions. FACSDiVa Optical Light Path Figure 6 (PDF)

Figure 6. FACSDiVa Optical Light Path

Cell sorting

One of the properties of the FACSDiVa is the ability to electronically deflect cells with preset, defined properties into a separate collection tube. For cell purification, flow cytometry is especially well suited for applications requiring high purity. Because multiple fluorochromes (e.g. up to 14 distinct fluorescent probes reacting with different cell associated molecules) can be assessed simultaneously, cell sorting by flow cytometry can separate complex mixtures of cells on the basis of multiple marker expression. In addition, a red diode laser mounted across the sort streams allows the operator to quickly define an accurate droplet delay. This device is called Accudrop® and can also be used to monitor the sort in real time and make sort correction when needed.

The FACSDiVa is also equipped with a Autoclone®, which will allow the tracking of specific information on cells sorted into a 96 well microtiter tray or in other tray configurations. The bitmapped sorting capability of the FACSDiVa digital electronics allows the operator to set up sophisticated sort decisions using logical gates combined with Boolean gating.

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