KEYWORDS: Signal to noise ratio, In vivo imaging, Fluorescence, Flow cytometry, Tissues, Autofluorescence, Systems modeling, Optical testing, Near infrared, Monte Carlo methods
Diffuse in-vivo Flow Cytometry (DiFC) is a fluorescence sensing method to detect labeled circulating cells
in-vivo non-invasively. The Dual-Ratio (DR) is an optical measurement method that aims to suppress most noise
and enhance SNR in deep tissue regions. We investigate DR applied to Near-InfraRed (NIR) DiFC to improve
circulating cells’ maximum detectable depth and SNR. Results indicate that two critical conditions control
the success of DR DiFC. First, the fraction of noise that DR methods cannot cancel (i.e., non-multiplicative
factors) cannot dominate. Second, DR DiFC has an advantage regarding SNR if the spatial distribution of tissue
autofluorescence contributors is surface-weighted.
KEYWORDS: Signal to noise ratio, Fluorophores, Fluorescence, Tissues, Signal detection, Target detection, Near infrared, Interference (communication), In vivo imaging, Skin
SignificanceDiffuse in vivo flow cytometry (DiFC) is an emerging fluorescence sensing method to non-invasively detect labeled circulating cells in vivo. However, due to signal-to-noise ratio (SNR) constraints largely attributed to background tissue autofluorescence (AF), DiFC’s measurement depth is limited.AimThe dual ratio (DR)/dual slope is an optical measurement method that aims to suppress noise and enhance SNR to deep tissue regions. We aim to investigate the combination of DR and near-infrared (NIR) DiFC to improve circulating cells’ maximum detectable depth and SNR.ApproachPhantom experiments were used to estimate the key parameters in a diffuse fluorescence excitation and emission model. This model and parameters were implemented in Monte Carlo to simulate DR DiFC while varying noise and AF parameters to identify the advantages and limitations of the proposed technique.ResultsTwo key factors must be true to give DR DiFC an advantage over traditional DiFC: first, the fraction of noise that DR methods cannot cancel cannot be above the order of 10% for acceptable SNR. Second, DR DiFC has an advantage, in terms of SNR, if the distribution of tissue AF contributors is surface-weighted.ConclusionsDR cancelable noise may be designed (e.g., through the use of source multiplexing), and indications point to the AF contributors’ distribution being truly surface-weighted in vivo. Successful and worthwhile implementation of DR DiFC depends on these considerations, but results point to DR DiFC having possible advantages over traditional DiFC.
KEYWORDS: In vivo imaging, Near infrared, Signal to noise ratio, Flow cytometry, Luminescence, Blood, Tissues, In vitro testing, Optical filters, Fiber lasers
SignificanceDiffuse 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.AimWe describe the design of a NIR-DiFC instrument and demonstrate its use in optical flow phantoms in vitro and in mice in vivo.ApproachWe 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.ResultsNIR-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.ConclusionsNIR-DiFC allows use of emerging NIR contrast agents. Our work could pave the way for future use of NIR-DiFC in humans.
KEYWORDS: Tissue optics, Near infrared, Monte Carlo methods, Blood vessels, Signal detection, Sensors, Optical properties, Luminescence, In vivo imaging, Signal to noise ratio
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 ∼2 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.
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