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Retinal microvascular and neuronal pathologies probed in vivo by adaptive optical two-photon fluorescence microscopy.

ABSTRACT: The retina, behind the transparent optics of the eye, is the only neural tissue whose physiology and pathology can be non-invasively probed by optical microscopy. The aberrations intrinsic to the mouse eye, however, prevent high-resolution investigation of retinal structure and function in vivo. Optimizing the design of a two-photon fluorescence microscope (2PFM) and sample preparation procedure, we found that adaptive optics (AO), by measuring and correcting ocular aberrations, is essential for resolving putative synaptic structures and achieving three-dimensional cellular resolution in the mouse retina in vivo. Applying AO-2PFM to longitudinal retinal imaging in transgenic models of retinal pathology, we characterized microvascular lesions with sub-capillary details in a proliferative vascular retinopathy model, and found Lidocaine to effectively suppress retinal ganglion cell hyperactivity in a retinal degeneration model. Tracking structural and functional changes at high-resolution longitudinally, AO-2PFM enables microscopic investigations of retinal pathology and pharmacology for disease diagnosis and treatment in vivo.

SUBMITTER: Zhang Q

PROVIDER: S-EPMC10089658 | biostudies-literature | 2023 Apr

REPOSITORIES: biostudies-literature

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Retinal microvascular and neuronal pathologies probed in vivo by adaptive optical two-photon fluorescence microscopy.

Zhang Qinrong Q Yang Yuhan Y Cao Kevin J KJ Chen Wei W Paidi Santosh S Xia Chun-Hong CH Kramer Richard H RH Gong Xiaohua X Ji Na N

eLife 20230411

The retina, behind the transparent optics of the eye, is the only neural tissue whose physiology and pathology can be non-invasively probed by optical microscopy. The aberrations intrinsic to the mouse eye, however, prevent high-resolution investigation of retinal structure and function in vivo. Optimizing the design of a two-photon fluorescence microscope (2PFM) and sample preparation procedure, we found that adaptive optics (AO), by measuring and correcting ocular aberrations, is essential for ...[more]

PMID: 37039777

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Project description:Pluripotent stem cell-derived organoid technologies have opened avenues to preclinical basic science research, drug discovery, and transplantation therapy in organ systems. Stem cell-derived organoids follow a time course similar to species-specific organ gestation in vivo. However, heterogeneous tissue yields, and subjective tissue selection reduce the repeatability of organoid-based scientific experiments and clinical studies. To improve the quality control of organoids, we introduced a live imaging technique based on two-photon microscopy to non-invasively monitor and characterize retinal organoids' (RtOgs') long-term development. Fluorescence lifetime imaging microscopy (FLIM) was used to monitor the metabolic trajectory, and hyperspectral imaging was applied to characterize structural and molecular changes. We further validated the live imaging experimental results with endpoint biological tests, including quantitative polymerase chain reaction (qPCR), single-cell RNA sequencing, and immunohistochemistry. With FLIM results, we analyzed the free/bound nicotinamide adenine dinucleotide (f/b NADH) ratio of the imaged regions and found that there was a metabolic shift from glycolysis to oxidative phosphorylation. This shift occurred between the second and third months of differentiation. The total metabolic activity shifted slightly back toward glycolysis between the third and fourth months and stayed relatively stable between the fourth and sixth months. Consistency in organoid development among cell lines and production lots was examined. Molecular analysis showed that retinal progenitor genes were expressed in all groups between days 51 and 159. Photoreceptor gene expression emerged around the second month of differentiation, which corresponded to the shift in the f/b NADH ratio. RtOgs between 3 and 6 months of differentiation exhibited photoreceptor gene expression levels that were between the native human fetal and adult retina gene expression levels. The occurrence of cone opsin expression (OPN1 SW and OPN1 LW) indicated the maturation of photoreceptors in the fourth month of differentiation, which was consistent with the stabilized level of f/b NADH ratio starting from 4 months. Endpoint single-cell RNA and immunohistology data showed that the cellular compositions and lamination of RtOgs at different developmental stages followed those in vivo.

Project description:SignificanceThe accurate large-scale mapping of cerebral microvascular blood flow velocity is crucial for a better understanding of cerebral blood flow (CBF) regulation. Although optical imaging techniques enable both high-resolution microvascular angiography and fast absolute CBF velocity measurements in the mouse cortex, they usually require different imaging techniques with independent system configurations to maximize their performances. Consequently, it is still a challenge to accurately combine functional and morphological measurements to co-register CBF speed distribution from hundreds of microvessels with high-resolution microvascular angiograms.AimWe propose a data acquisition and processing framework to co-register a large set of microvascular blood flow velocity measurements from dynamic light scattering optical coherence tomography (DLS-OCT) with the corresponding microvascular angiogram obtained using two-photon microscopy (2PM).ApproachWe used DLS-OCT to first rapidly acquire a large set of microvascular velocities through a sealed cranial window in mice and then to acquire high-resolution microvascular angiograms using 2PM. The acquired data were processed in three steps: (i) 2PM angiogram coregistration with the DLS-OCT angiogram, (ii) 2PM angiogram segmentation and graphing, and (iii) mapping of the CBF velocities to the graph representation of the 2PM angiogram.ResultsWe implemented the developed framework on the three datasets acquired from the mice cortices to facilitate the coregistration of the large sets of DLS-OCT flow velocity measurements with 2PM angiograms. We retrieved the distributions of red blood cell velocities in arterioles, venules, and capillaries as a function of the branching order from precapillary arterioles and postcapillary venules from more than 1000 microvascular segments.ConclusionsThe proposed framework may serve as a useful tool for quantitative analysis of large microvascular datasets obtained by OCT and 2PM in studies involving normal brain functioning, progression of various diseases, and numerical modeling of the oxygen advection and diffusion in the realistic microvascular networks.

Dataset Information

Retinal microvascular and neuronal pathologies probed in vivo by adaptive optical two-photon fluorescence microscopy.

Publications

Retinal microvascular and neuronal pathologies probed in vivo by adaptive optical two-photon fluorescence microscopy.

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