Abstract
Purpose :
Adaptive optics scanning light ophthalmoscopy (AOSLO) can image single blood cells in the retina. AOSLO can serve as a new platform to study the cellular components of whole blood in vivo. Here, we demonstrate an imaging-based method to study two rare blood cell types in the retinal circulation of living mice.
Methods :
Two sparse populations of white blood cells (WBCs) were imaged in the living retina: circulating GFP-labeled monocytes (CX3CR1, Jackson Labs stock 005582), and CD8+ T-cells harvested from a GFP donor mouse (stock 006567) and adoptively transferred into a healthy C57BL/6J (stock 00664) mouse via intraperitoneal injection. A single retinal arteriole and a single venule from each mouse were imaged with a custom-built AOSLO with reflectance (796 nm) and fluorescence (488 nm ex/520Δ35 nm em) capabilities. Circulating cells were imaged using a 15kHz point scanned obliquely across a vessel, which simultaneously images the red blood cells and fluorescent WBCs. Cell velocities were quantified using the automated Radon approach previously described (Joseph et al., 2019). Total imaged blood volume was approximated by flow speed x π x radius2 of the blood vessel. Fluorescent WBC populations were automatically counted and manually validated.
Results :
In CX3CR1+ mouse, we observed the passage of labeled monocytes at rates of 27.7 cells/min in the arteriole and 14.0 cells/min in the venule. Based on the estimated blood volume imaged, this represents monocyte density of 244 cells/µL in the arteriole and 95 cells/µL in the venule. These counts in vivo were within an order of magnitude of the expected density of monocytes in circulation (van Furth & Sluiter, 1986). In the T-cell recipient mouse, we detected an average of 328 cells/min in the arteriole and 204 cells/min in the venule. Scaled by the blood volume imaged relative to total blood volume, we estimate 1.01-1.61 million CD8+ T-cells in systemic circulation. The total number of cells injected into the peritoneum was estimated to be 10 million cells, indicating 10 to 16% of the cells entered systemic circulation.
Conclusions :
Here we demonstrate the ability to count and measure the speeds of monocytes and T-cells in high flow vessels in the body. Such capability provides the foundation for studying rare immune cells of interest that fight inflammation, infection and even mediate cutting-edge therapies that use adoptively transferred immune cells.
This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.