Flow cytometer in the infrared: inexpensive modifications to a commercial instrument

The application of molecules that fluoresce in the infrared (IR) region to measure cell products would be enhanced by a flow cytometer capable of measuring them. To our knowledge, none exist at this time. Accordingly, we have developed such an instrument.

A Becton Dickinson LSR flow cytometer was modified to include a small 785-nm IR diode laser the size of a C cell battery with 44-mW output power. The instrument was modified further to accommodate this laser in addition to a 405-nm solid-state laser, a 488-nm air-cooled argon laser, and a 658-nm solid-state laser. Because the IR laser is dangerous to the eye, the laser beams were viewed for optical alignment using a CCD camera and video monitor. An avalanche photodiode was used in place of a photomultiplier tube because its detection sensitivity in the IR region is superior.

To assess performance, scatter and fluorescence measurements were made using microspheres that fluoresce in the IR region, and human leukocytes were stained with CD45 biotin followed by a streptavidin conjugated with an IR dye. An avalanche photodiode was 2.3 to 2.8 times more sensitive than a photomultiplier tube for detecting IR fluorescence. Cells stained with CD45 biotin and avidin conjugated with an IR dye could easily be resolved and their fluorescence quantified; there was virtually no autofluorescence. In addition, a lipophilic membrane dye that emits in the IR region was studied. HL60 cells were stained with this dye and they exhibited bright fluorescence intensity.

A commercial instrument could be modified to accommodate an IR laser for exciting dyes that fluoresce in the IR region. This new capability will extend the range of fluorescence that can be measured by flow cytometry.