In Vivo Visualization of Intravascular Patrolling Immune Cells in the Primate Eye.

Purpose: Insight into the immune status of the living eye is essential as we seek to understand ocular disease and develop new treatments. The nonhuman primate (NHP) is the gold standard preclinical model for therapeutic development in ophthalmology, owing to the similar visual system and immune landscape in the NHP relative to the human. Here, we demonstrate the utility of phase-contrast adaptive optics scanning light ophthalmoscope (AOSLO) to visualize immune cell dynamics on the cellular scale, label-free in the NHP.

Photoreceptor loss does not recruit neutrophils despite strong microglial activation.

In response to central nervous system (CNS) injury, tissue resident immune cells such as microglia and circulating systemic neutrophils are often first responders. The degree to which these cells interact in response to CNS damage is poorly understood, and even less so, in the neural retina which poses a challenge for high resolution imaging in vivo. In this study, we deploy fluorescence adaptive optics scanning light ophthalmoscopy (AOSLO) to study fluorescent microglia and neutrophils in mice.

High-resolution structural and functional retinal imaging in the awake behaving mouse.

The laboratory mouse has provided tremendous insight to the underpinnings of mammalian central nervous system physiology. In recent years, it has become possible to image single neurons, glia and vascular cells in vivo by using head-fixed preparations combined with cranial windows to study local networks of activity in the living brain. Such approaches have also succeeded without the use of general anesthesia providing insights to the natural behaviors of the central nervous system.

In Vivo Capillary Structure and Blood Cell Flux in the Normal and Diabetic Mouse Eye.

Purpose: To characterize the early structural and functional changes in the retinal microvasculature in response to hyperglycemia in the Ins2Akita mouse.

Methods: A custom phase-contrast adaptive optics scanning light ophthalmoscope was used to image retinal capillaries of 9 Ins2Akita positive (hyperglycemic) and 9 Ins2Akita negative (euglycemic) mice from postnatal weeks 5 to 18. A 15 kHz point scan was used to image capillaries and measure red blood cell flux at biweekly intervals; measurements were performed manually.

Imaging the dynamics of individual processes of microglia in the living retina .

Microglia are an essential population of resident immune cells in the central nervous system (CNS) and retina. These microscopic cells possess sub-cellular processes that make them challenging to image due to limited resolution and contrast. The baseline behavior of microglial processes in the living retina has been poorly characterized, and yet are essential to understanding how these cells respond under conditions of health, development, stress and disease.

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

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.

imaging of corneal nerves and cellular structures in mice with Gabor-domain optical coherence microscopy.

Gabor-domain optical coherence microscopy (GDOCM) demonstrated corneal imaging with cellular resolution and differentiation in mice over a field of view of 1 mm. Contact and non-contact imaging was conducted on six healthy and six hyperglycemic C57BL/6J mice. Cellular resolution in the 3D GDOCM images was achieved after motion correction.

Origin of cell contrast in offset aperture adaptive optics ophthalmoscopy.

Offset aperture and split detector imaging are variants of adaptive optics scanning ophthalmoscopy recently introduced to improve the image contrast of retinal cells. Unlike conventional confocal scanning ophthalmoscopy, these approaches collect light laterally decentered from the optical axis. A complete explanation of how these methods enhance contrast has not been described.

Imaging Retinal Activity in the Living Eye.

Retinal function has long been studied with psychophysical methods in humans, whereas detailed functional studies of vision have been conducted mostly in animals owing to the invasive nature of physiological approaches. There are exceptions to this generalization, for example, the electroretinogram. This review examines exciting recent advances using in vivo retinal imaging to understand the function of retinal neurons.

Retinal ischemia induces α-SMA-mediated capillary pericyte contraction coincident with perivascular glycogen depletion.

Increasing evidence indicates that pericytes are vulnerable cells, playing pathophysiological roles in various neurodegenerative processes. Microvascular pericytes contract during cerebral and coronary ischemia and do not relax after re-opening of the occluded artery, causing incomplete reperfusion. However, the cellular mechanisms underlying ischemia-induced pericyte contraction, its delayed emergence, and whether it is pharmacologically reversible are unclear.

Imaging single-cell blood flow in the smallest to largest vessels in the living retina.

Tissue light scatter limits the visualization of the microvascular network deep inside the living mammal. The transparency of the mammalian eye provides a noninvasive view of the microvessels of the retina, a part of the central nervous system. Despite its clarity, imperfections in the optics of the eye blur microscopic retinal capillaries, and single blood cells flowing within.

Capillary pericytes express α-smooth muscle actin, which requires prevention of filamentous-actin depolymerization for detection.

Recent evidence suggests that capillary pericytes are contractile and play a crucial role in the regulation of microcirculation. However, failure to detect components of the contractile apparatus in capillary pericytes, most notably α-smooth muscle actin (α-SMA), has questioned these findings. Using strategies that allow rapid filamentous-actin (F-actin) fixation (i.

Vision science and adaptive optics, the state of the field.

Adaptive optics is a relatively new field, yet it is spreading rapidly and allows new questions to be asked about how the visual system is organized. The editors of this feature issue have posed a series of question to scientists involved in using adaptive optics in vision science. The questions are focused on three main areas.

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

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.

Imaging translucent cell bodies in the living mouse retina without contrast agents.

The transparency of most retinal cell classes typically precludes imaging them in the living eye; unless invasive methods are used that deploy extrinsic contrast agents. Using an adaptive optics scanning light ophthalmoscope (AOSLO) and capitalizing on the large numerical aperture of the mouse eye, we enhanced the contrast from otherwise transparent cells by subtracting the left from the right half of the light distribution in the detector plane. With this approach, it is possible to image the distal processes of photoreceptors, their more proximal cell bodies and the mosaic of horizontal cells in the living mouse retina.

Morphology and topography of retinal pericytes in the living mouse retina using in vivo adaptive optics imaging and ex vivo characterization.

Purpose: To noninvasively image retinal pericytes in the living eye and characterize NG2-positive cell topography and morphology in the adult mouse retina.

Methods: Transgenic mice expressing fluorescent pericytes (NG2, DsRed) were imaged using a two-channel, adaptive optics scanning laser ophthalmoscope (AOSLO). One channel imaged vascular perfusion with near infrared light.

Retinal intrinsic optical signals in a cat model of primary congenital glaucoma.

Purpose: To examine the impact of reduced inner retinal function and breed on intrinsic optical signals in cats.

Methods: Retinal intrinsic optical signals were recorded from anesthetized cats with a modified fundus camera. Near infrared light (NIR, 700-900 nm) was used to illuminate the retina while a charge-coupled device (CCD) camera captured the NIR reflectance of the retina.

Blood contrast agents enhance intrinsic signals in the retina: evidence for an underlying blood volume component.

Purpose: To examine the extent to which neurovascular coupling contributes to stimulus-evoked intrinsic signals in the retina.

Methods: The retinas of five adult cats were examined in vivo. Animals were anesthetized and paralyzed for imaging stability.

Noninvasive functional imaging of the retina reveals outer retinal and hemodynamic intrinsic optical signal origins.

We have adapted intrinsic signal optical imaging of neural activity to the noninvasive functional imaging of the retina. Results to date demonstrate the feasibility and potential of this new method of functional assessment of the retina. In response to visual stimuli, we have imaged reflectance changes in the retina that are robust and spatially colocalized to the sites of stimulation.

Stimulus-evoked intrinsic optical signals in the retina: spatial and temporal characteristics.

Purpose: To characterize the properties of stimulus-evoked retinal intrinsic signals and determine the underlying origins.

Methods: Seven adult cats were anesthetized and paralyzed to maximize imaging stability. The retina was stimulated with a liquid crystal display (LCD) integrated into a modified fundus camera (Topcon, Tokyo, Japan).

Stimulus-evoked intrinsic optical signals in the retina: pharmacologic dissection reveals outer retinal origins.

Purpose: To elucidate the anatomic origins of stimulus-evoked intrinsic optical signals in the mammalian retina by using selective pharmacologic blockade of specific retinal layers.

Methods: Four adult cats were used to investigate the stimulus-evoked intrinsic signals. The retinas were visually stimulated with a liquid crystal display (LCD) integrated into a modified fundus camera.