In vivo label-free measurement of lymph flow velocity and volumetric flow rates using Doppler optical coherence tomography

Abstract

Direct in vivo imaging of lymph flow is key to understanding lymphatic system function in normal and disease states. Optical microscopy techniques provide the resolution required for these measurements, but existing optical techniques for measuring lymph flow require complex protocols and provide limited temporal resolution. Here, we describe a Doppler optical coherence tomography platform that allows direct, label-free quantification of lymph velocity and volumetric flow rates. We overcome the challenge of very low scattering by employing a Doppler algorithm that operates on low signal-to-noise measurements. We show that this technique can measure lymph velocity at sufficiently high temporal resolution to resolve the dynamic pulsatile flow in collecting lymphatic vessels.

Lymphatic function measurements influenced by contrast agent volume and body position

“…Previous measurements after no injection found a similar frequency (3.0 ± 0.3/min) to that following a 4-μl and 20-μl injection (17). Similarly, volumetric flow with no injection was found to be approximately 0.03 ± 0.01 μl/hr, which is comparable to the 4-μl measurement (8).…”

“…The mean volumetric flow was lower in mice injected with 4 μl compared with 20 μl, with the minimum flow ( Figure 5, A and C, respectively) and peak flow ( Figure 5, B and D, respectively) both showing this difference. Minimum flow occurs between contractions, while peak flow occurs immediately following a contraction (17). Volumetric flow rate is quantified from DOCT measurements by using the instantaneous cross-sectional vessel area and instantaneous velocity averaged over the vessel cross section ( Figure 5, E and F and Supplemental Videos 7 and 8, respectively).…”

“…This allows for the assessment of wall movement, permitting quantification of contraction frequency and ejection fraction (EF; percentage of volume change per contraction) (9,16). DOCT is unique in that, contrary to the NIRF and fluorescence imaging of lymphatic contraction, no dye is needed to measure the output of volumetric flow (17). There are many differences between experimental parameters used for these techniques, including mouse position, volume of dye injected, invasiveness of the preparation, and data analysis (Table 1).…”

“…This research focus has catalyzed the advancement of techniques used to assess lymphatic function (7), including near-infrared fluorescence (NIRF) imaging, direct fluorescence imaging of lymphatic contraction, and Doppler optical coherence tomography (DOCT) (8)(9)(10). NIRF imaging is used both preclinically and clinically to visualize how injected NIR contrast agents -frequently the FDA-approved molecule indocyanine green (ICG) -move though lymphatic vessels (11)(12)(13).…”

In vivo label-free lymphangiography of cutaneous lymphatic vessels in human burn scars using optical coherence tomography

“…A validation study in human skin by comparing OCT against other imaging techniques using contrast agents will be needed to further characterize the accuracy of OCT-based imaging of lymphatic vessels and the errors in the measurement of vessel density. In addition, the incorporation of the measurement of lymph flow velocity [33] into the imaging of the vessel morphology would expand the utility of our method.…”