Signal and measurement considerations for human translation of diffuse in vivo flow cytometry

Abstract

. Significance: “Diffuse in vivo flow cytometry” (DiFC) is an emerging technology for fluorescence detection of rare circulating cells directly in large deep-seated blood vessels in mice. Because DiFC uses highly scattered light, in principle, it could be translated to human use. However, an open question is whether fluorescent signals from single cells would be detectable in human-scale anatomies. Aim: Suitable blood vessels in a human wrist or forearm are at a depth of to 4 mm. The aim of this work was to study the impact of DiFC instrument geometry and wavelength on the detected DiFC signal and on the maximum depth of detection of a moving cell. Approach: We used Monte Carlo simulations to compute fluorescence Jacobian (sensitivity) matrices for a range of source and detector separations (SDS) and tissue optical properties over the visible and near infrared spectrum. We performed experimental measurements with three available versions of DiFC (488, 640, and 780 nm), fluorescent microspheres, and tissue mimicking optical flow phantoms. We used both computational and experimental data to estimate the maximum depth of detection at each combination of settings. Results: For the DiFC detection problem, our analysis showed that for deep-seated blood vessels, the maximum sensitivity was obtained with NIR light (780 nm) and 3-mm SDS. Conclusions: These results suggest that—in combination with a suitable molecularly targeted fluorescent probes—circulating cells and nanosensors could, in principle, be detectable in circulation in humans.

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

“…We consider each measurement of I with a source and detector as a SD measurement (Sec 6.1). Previous DiFC work has considered these types of measurements; 20 however, in this work, we introduce a DiFC measurement type based on a combination of SDs to form a DR (Sec. 6.3).…”

“…This work presents simulation results based on a Monte-Carlo (MC) model run in Monte-Carlo Extreme (MCXLAB 2020 running in MATLAB 2023a on an NVIDIA RTX 4090) 23 . For all simulations, optical properties meant to represent λ of 810 nm were used 20 . These were an absorption coefficient (μa) of 0.002 mm−1, a scattering coefficient (μs) of 7 mm−1, an anisotropy factor (g) of 0.9, and an index of refraction (n) of 1.37.…”

“…These Φ’s were used with Eq. (15) to yield the fluorescence Jacobian (W), 20 a key parameter used in all the simulation results as described in Appendix B.…”

“…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 .…”

“…Small misalignments (∼1 mm) do not significantly affect the signal quality because of the wide sensitivity volume of diffuse light. We recently showed that individual cell fluorescence signals might be detectable up to 4 mm deep using near-infrared (NIR) fluorophores and a source–detector distance (ρ) of ∼3 mm, increased depth and larger relative non-specific background signals presents challenges for detection of weakly labeled CTCs 20 . Hence methodology for reduction of the background signal and its contributed noise, such as dual slope or DR described in Sec.…”