Near-infrared diffuse in vivo flow cytometry

Abstract

. Significance: 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. Aim: We describe the design of a NIR-DiFC instrument and demonstrate its use in optical flow phantoms in vitro and in mice in vivo . Approach: 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. Results: 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. Conclusions: NIR-DiFC allows use of emerging NIR contrast agents. Our work could pave the way for future use of NIR-DiFC in humans.

Dual-ratio approach for detection of point fluorophores in biological tissue

“…To estimate the potential performance of a DR DiFC instrument, we performed DiFC measurements in tissue-mimicking flow phantoms with NIR wavelengths (770 nm excitation and 810 nm emission) 20 . Briefly, we used Jade Green high intensity (Spherotech Inc., Lake Forest, Illinois, United States) cell-mimicking fluorescent microspheres (size ∼10 to 14 μm) for NIR SD measurements 3 . Microspheres were suspended at a concentration of 1×103 mL−1 in phosphate buffered saline and flowed through Tygon tubing (internal diameter of 0.25 mm; TGY-010-C, Small Parts Inc., Seattle, Washington, United States) embedded in a tissue-mimicking optical flow phantom made of high-density polyethylene.…”

“… 20 Briefly, we used Jade Green high intensity (Spherotech Inc., Lake Forest, Illinois, United States) cell-mimicking fluorescent microspheres (size to ) for NIR SD measurements. 3 Microspheres were suspended at a concentration of in phosphate buffered saline and flowed through Tygon tubing (internal diameter of 0.25 mm; TGY-010-C, Small Parts Inc., Seattle, Washington, United States) embedded in a tissue-mimicking optical flow phantom made of high-density polyethylene. Microsphere suspensions were pumped with a syringe pump (0-2209, Harvard Apparatus, Holliston, Massachusetts, United States) at a flow rate of or average flow velocity of to approximate linear velocities in the mouse leg femoral artery.…”

“…(c) Use of two sources (1 and 2) and two detectors (A and B) would permit four source and detector pairs separated by a source–detector distance (ρ). (d) Photograph of NIR DiFC system 3 on a diffusive flow phantom with fiber probes arranged perpendicular to the tubing direction. The dashed black line in (d) corresponds to the mid-plane shown in (c).…”

“…Diffuse in vivo flow cytometry (DiFC) is an emerging optical technique that enables fluorescence detection of rare circulating cells in the bloodstream in the optically diffusive medium. 1 – 4 Dual slope or dual ratio (DR) is a new diffuse optical technique that is designed to suppress noise in the optical signal and reduce sensitivity to superficial tissue regions. 5 – 8 A challenge of DiFC is the contamination of the target fluorescence signal from noise, which may be associated with background autofluorescence (AF).…”

Effect of pulsed laser parameters on photoacoustic flow cytometry efficiency in vitro and in vivo

“…Currently, there are several common approaches to the construction of in vivo cytometry systems, including photoacoustic flow cytometry (PAFC) [8][9][10][11], ultrafast scanning photoacoustic system [10,12,13], fluorescence [14][15][16][17][18], fluorescence imaging [19][20][21][22][23], scattering [24][25][26][27], diffuse fluorescence [7,[28][29][30][31], stimulated Raman scattering [32] among other cytometry methods.…”