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Abstract:We have combined Fourier-domain optical coherence tomography (FD-OCT) with a closed-loop adaptive optics (AO) system using a Hartmann-Shack wavefront sensor and a bimorph deformable mirror. The adaptive optics system measures and corrects the wavefront aberration of the human eye for improved lateral resolution (~4

Rationale: The cardiac conduction system (CCS) consists of distinct components including the sinoatrial node, atrioventricular node, His bundle, bundle branches, and Purkinje fibers. Despite an essential role for the CCS in heart development and function, the CCS has remained challenging to interrogate because of inherent obstacles including small cell numbers, large cell-type heterogeneity, complex anatomy, and difficulty in isolation. Single-cell RNA-sequencing allows for genome-wide analysis of gene expression at single-cell resolution. Objective: Assess the transcriptional landscape of the entire CCS at single-cell resolution by single-cell RNA-sequencing within the developing mouse heart. Methods and Results: Wild-type, embryonic day 16.5 mouse hearts (n=6 per zone) were harvested and 3 zones of microdissection were isolated, including: Zone I—sinoatrial node region; Zone II—atrioventricular node/His region; and Zone III—bundle branch/Purkinje fiber region. Tissue was digested into single-cell suspensions, cells isolated, mRNA reverse transcribed, and barcoded before high-throughput sequencing and bioinformatics analyses. Single-cell RNA-sequencing was performed on over 22 000 cells, and all major cell types of the murine heart were successfully captured including bona fide clusters of cells consistent with each major component of the CCS. Unsupervised weighted gene coexpression network analysis led to the discovery of a host of novel CCS genes, a subset of which were validated using fluorescent in situ hybridization as well as whole-mount immunolabeling with volume imaging (iDISCO+) in 3 dimensions on intact mouse hearts. Further, subcluster analysis unveiled isolation of distinct CCS cell subtypes, including the clinically relevant but poorly characterized transitional cells that bridge the CCS and surrounding myocardium. Conclusions: Our study represents the first comprehensive assessment of the transcriptional profiles from the entire CCS at single-cell resolution and provides a characterization in the context of development and disease.

We have developed an improved adaptive optics - optical coherence tomography (AO-OCT) system and evaluated its performance for in vivo imaging of normal and pathologic retina. The instrument provides unprecedented image quality at the retina with isotropic 3D resolution of 3.5 x 3.5 x 3.5 microm(3). Critical to the instrument's resolution is a customized achromatizing lens that corrects for the eye's longitudinal chromatic aberration and an ultra broadband light source (Delta lambda=112 nm lambda(0)= approximately 836 nm). The eye's transverse chromatic aberrations is modeled and predicted to be sufficiently small for the imaging conditions considered. The achromatizing lens was strategically placed at the light input of the AO-OCT sample arm. This location simplifies use of the achromatizing lens and allows straightforward implementation into existing OCT systems. Lateral resolution was achieved with an AO system that cascades two wavefront correctors, a large stroke bimorph deformable mirror (DM) and a micro-electromechanical system (MEMS) DM with a high number of actuators. This combination yielded diffraction-limited imaging in the eyes examined. An added benefit of the broadband light source is the reduction of speckle size in the axial dimension. Additionally, speckle contrast was reduced by averaging multiple B-scans of the same proximal patch of retina. The combination of improved micron-scale 3D resolution, and reduced speckle size and contrast were found to significantly improve visibility of microscopic structures in the retina.