Label free measurement of retinal blood cell flux, velocity, hematocrit and capillary width in the living mouse eye

Abstract

Measuring blood cell dynamics within the capillaries of the living eye provides crucial information regarding the health of the microvascular network. To date, the study of single blood cell movement in this network has been obscured by optical aberrations, hindered by weak optical contrast, and often required injection of exogenous fluorescent dyes to perform measurements. Here we present a new strategy to non-invasively image single blood cells in the living mouse eye without contrast agents. Eye aberrations were corrected with an adaptive optics camera coupled with a fast, 15 kHz scanned beam orthogonal to a capillary of interest. Blood cells were imaged as they flowed past a near infrared imaging beam to which the eye is relatively insensitive. Optical contrast of cells was optimized using differential scatter of blood cells in the split-detector imaging configuration. Combined, these strategies provide label-free, non-invasive imaging of blood cells in the retina as they travel in single file in capillaries, enabling determination of cell flux, morphology, class, velocity, and rheology at the single cell level.

Adaptive optics imaging of the retinal microvasculature

“…Individual platelets, which are low contrast and in humans only 2.5–3 μm in diameter, have been resolved in rodents using AOSLO by reconstruction of high‐speed capillary cross‐sections . Data from this group are shown in Figure and demonstrate the potential for accurate characterisation of red cell shape and packing arrangement as well.…”

“…This allows extremely rapid (kilohertz) measurement of the flow profile across a single vessel; smaller vessels are precluded due to difficulties in compensating for motion of the eye. More recent strategies have extended this approach to individual capillaries in anaesthetised rodents, allowing the counting of individual red cells, white cells and platelets as well as accurate measurements of velocity, cell shape and lumen diameter . Example data from this method are shown in Figure , courtesy of Dr Jesse Schallek's laboratory, University of Rochester.…”

“…By freezing the AOSLO scanning raster on a particular vessel as described above, the cross‐sectional velocity of small retinal vessels can be measured and its evolution tracked rapidly over time. The method is best suited to vessels in the 30–80 μm range due to the significant, incessant movement of the eye; however, it can in principle be applied to individual capillaries . In many of the larger vessels in the body, idealised approximations of fluid mechanics hold and a parabolic velocity profile is expected.…”

“…These departures from idealised models of fluid mechanics may reflect changes in viscosity, and/or easier cell aggregation at slower flow speeds and as lumen size becomes comparable to cell size . The method has also been used to study complex flow dynamics such as mixing of flow downstream from a venous confluence; this phenomenon has been replicated in a number of studies …”

Capillary red blood cell velocimetry by phase-resolved optical coherence tomography

“…In addition, the proposed data processing method is robust and automatic, significantly simplifying volumetric blood flow speed measurements. Further, it is worth mentioning that compared to MPM and other layer-scanning-based optical capillary velocimetry methods [22], the proposed method is much faster for volumetric imaging as only a 2D XY scan is required for 3D imaging.…”