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

Abstract

Significance: Diffuse 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.Aim: The 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.Approach: Phantom 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. Results:Two 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.Conclusions: DR 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.

Two-color diffuse in vivo flow cytometer

“…20 In mice, suitable blood vessels are approximately 1 mm in depth, although we have showed that detection to 2 to 4 mm in tissue is feasible with suitable choice of wavelength and instrument geometry. 23,24 Hence, DiFC allows for non-invasive sampling of large peripheral blood volumes and detection of rare cells to, for instance, show that CTC numbers generally increase over the course of disease development in mouse metastasis models, but that they can fluctuate significantly over 24-h periods. 11,20,21 However, the DiFC systems we have developed thus far have been limited to detection of single fluorophores due to specially designed optical fiber bundles with integrated miniaturized filters and lenses that are not easily interchanged.…”

Measurement and analysis of rare circulating tumor cell dynamics with diffuse in vivo flow cytometry

“…Large vessels -for example in the tail or leg of a mouse -carry on the order of 100 L of blood per minute (Tan et al, 2019). In mice, suitable blood vessels are approximately 1 mm in depth (Tan et al, 2019), although we have showed that detection to 2-4 mm in tissue is feasible with suitable choice of wavelength and instrument geometry (Blaney et al, 2023;Ivich et al, 2022). Hence, DiFC allows for non-invasive sampling of large peripheral blood volumes and detection of rare cells to, for instance, show that CTC numbers generally increase over the course of disease development in mouse metastasis models, but that they can fluctuate significantly over 24-hour periods (Patil et al, 2019;Tan et al, 2019;Williams et al, 2020).…”

“…Ongoing experiments are investigating whether CTCs in mouse tumor models can be labeled with these molecular probes while circulating in vivo. Additionally, we are investigating alternative source-detector arrangements to optimize DiFC sensitivity and the use of machine learning to improve DiFC signal processing (Blaney et al, 2023).…”