Label-free imaging of immune cell dynamics in the living retina using adaptive optics

Abstract

Our recent work characterized the movement of single blood cells within the retinal vasculature (Joseph et al. 2019) using adaptive optics ophthalmoscopy. Here, we apply this technique to the context of acute inflammation and discover both infiltrating and tissue-resident immune cells to be visible without any labeling in the living mouse retina using near-infrared light alone. Intravital imaging of immune cells can be negatively impacted by surgical manipulation, exogenous dyes, transgenic manipulation and phototoxicity. These confounds are now overcome, using phase contrast and time-lapse videography to reveal the dynamic behavior of myeloid cells as they interact, extravasate and survey the mouse retina. Cellular motility and differential vascular responses were measured noninvasively and in vivo across hours to months at the same retinal location, from initiation to the resolution of inflammation. As comparable systems are already available for clinical research, this approach could be readily translated to human application.

Imaging the structure and dynamic activity of retinal microglia and macrophage-like cells in the living human eye

“…Non-confocal AOSLO imaging of mice has allowed label-free imaging of microglia and their motility 37,38 . Some of these investigations in mice have used fluorescent markers to identify resident microglia and differentiate them from infiltrating immune cells as well as image retinal inflammation in an endotoxin induced uveitis model 39 where heterogenous immune cells, neutrophil and monocyte populations and their motility were imaged. They also showed neutrophils rolling along the venular endothelium and infiltrating monocytes and macrophages present both in vessels and extravasated into retinal tissue 39 .…”

“…Some of these investigations in mice have used fluorescent markers to identify resident microglia and differentiate them from infiltrating immune cells as well as image retinal inflammation in an endotoxin induced uveitis model 39 where heterogenous immune cells, neutrophil and monocyte populations and their motility were imaged. They also showed neutrophils rolling along the venular endothelium and infiltrating monocytes and macrophages present both in vessels and extravasated into retinal tissue 39 . One of the limitations of human imaging is that we do not have the flexibility to selectively label cells, so we lack the capability to differentiate activated resident microglia from infiltrating macrophages.…”

Development of a Preclinical Laser Speckle Contrast Imaging Instrument for Assessing Systemic and Retinal Vascular Function in Small Rodents

“… 24 , 75 – 77 Although automated analysis workflows have simplified the lengthy and laborious process of determining cell velocities with AO-SLO, the limited FOV still requires flow analysis to be performed on a vessel-by-vessel basis. 76 A side-by-side comparison of LSCI and AO-SLO would enable LSCI-based measurements of speckle intensity to be related directly to flow velocity (at least within the individual animal) and would allow quantitative measurements of flow in vessels across the whole retina without montaging.…”

Macrophage-like Cells Are Increased in Patients with Vision-Threatening Diabetic Retinopathy and Correlate with Macular Edema

“…OCT-A is important for this analysis because the vascular plexus is used for registration of multiple scans. MLCs possess properties associated with macrophages including mobility, a ramified morphology [ 9 ], and correspondence to Cx3cr1 + cells (a macrophage marker) in mice [ 10 ]. Furthermore, we recently showed that clinical MLCs correspond to microglia, vitreal hyalocytes, and perivascular macrophages at steady state, and additionally include classical monocytes and monocyte-derived macrophages during neuroinflammation [ 11 ].…”