Near-infrared diffuse in vivo flow cytometry

Abstract

Diffuse in vivo flow cytometry (DiFC) is an emerging technique for enumerating rare fluorescently labeled circulating cells noninvasively in the bloodstream. Thus far, we have reported red and blue-green versions of DiFC. Use of near-infrared (NIR) fluorescent light would in principle allow use of DiFC in deeper tissues and would be compatible with emerging NIR fluorescence molecular contrast agents. We describe the design of a NIR-DiFC instrument and demonstrate its use in optical flow phantoms in vitro and in mice in vivo. We developed an improved optical fiber probe design for efficient collection of fluorescence from individual circulating cells and efficient rejection of instrument autofluorescence. We built a NIR-DiFC instrument. We tested this with NIR fluorescent microspheres and cell lines labeled with OTL38 fluorescence contrast agent in a flow phantom model. We also tested NIR-DiFC in nude mice injected intravenously with OTL38-labeled L1210A cells. NIR-DiFC allowed detection of circulating tumor cells (CTCs) in flow phantoms with mean signal-to-noise ratios (SNRs) of 19 to 32 dB. In mice, fluorescently labeled CTCs were detectable with mean SNR of 26 dB. NIR-DiFC also exhibited orders significantly lower autofluorescence and false-alarm rates than blue-green DiFC. NIR-DiFC allows use of emerging NIR contrast agents. Our work could pave the way for future use of NIR-DiFC in humans.