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The inherent constraints on resolution, speed and field of view have hindered the development of high-speed, three-dimensional microscopy techniques over large scales. Here, we present a multiplane line-scan imaging strategy, which uses a series of axially distributed reflecting slits to probe different depths within a sample volume. Our technique enables the simultaneous imaging of an optically sectioned image stack with a single camera at frame rates of hundreds of hertz, without the need for axial scanning. We demonstrate the applicability of our system to monitor fast dynamics in biological samples by performing calcium imaging of neuronal activity in mouse brains and voltage imaging of cardiomyocytes in cardiac samples.

Summary Precise measurement of action potentials (APs) is needed to observe electrical activity and cellular communication within cardiac tissue. Voltage-sensitive dyes (VSDs) are traditionally used to measure cardiac APs; however, they require acute chemical addition that prevents chronic imaging. Genetically encoded voltage indicators (GEVIs) enable long-term studies of APs without the need of chemical additions, but current GEVIs used in cardiac tissue exhibit poor kinetics and/or low signal to noise (SNR). Here, we demonstrate the use of Archon1, a recently developed GEVI, in hiPSC-derived cardiomyocytes (CMs). When expressed in CMs, Archon1 demonstrated fast kinetics comparable with patch-clamp electrophysiology and high SNR significantly greater than the VSD Di-8-ANEPPS. Additionally, Archon1 enabled monitoring of APs across multiple cells simultaneously in 3D cardiac tissues. These results highlight Archon1's capability to investigate the electrical activity of CMs in a variety of applications and its potential to probe functionally complex in vitro models, as well as in vivo systems., Graphical Abstract, Highlights • Genetic sensor Archon1 reports membrane voltage in hiPSC-derived cardiomyocytes • Archon1 monitors action potentials in 2D and 3D cardiac tissue with high sensitivity • Archon1 repeatedly monitored voltage in the same cells and over extended time periods • Voltage dynamics of multiple cells were recorded simultaneously with Archon1, Biotechnology; Bioelectronics; Technical Aspects of Cell Biology; Electronic Materials

Living tissue-engineered heart valves (TEHV) would be a major benefit for children who require a replacement with the capacity for growth and biological integration. A persistent challenge for TEHV is accessible human cell source(s) that can mimic native valve cell phenotypes and matrix remodeling characteristics that are essential for long-term function. Mesenchymal stem cells derived from bone marrow (BMMSC) or adipose tissue (ADMSC) are intriguing cell sources for TEHV, but they have not been compared with pediatric human aortic valve interstitial cells (pHAVIC) in relevant 3D environments. In this study, we compared the spontaneous and induced multipotency of ADMSC and BMMSC with that of pHAVIC using different induction media within three-dimensional (3D) bioactive hybrid hydrogels with material modulus comparable to that of aortic heart valve leaflets. pHAVIC possessed some multi-lineage differentiation capacity in response to induction media, but limited to the earliest stages and much less potent than either ADMSC or BMMSC. ADMSC expressed cell phenotype markers more similar to pHAVIC when conditioned in basic fibroblast growth factor (bFGF) containing HAVIC growth medium, while BMMSC generally expressed similar extracellular matrix remodeling characteristics to pHAVIC. Finally, we covalently attached bFGF to PEG monoacrylate linkers and further covalently immobilized in the 3D hybrid hydrogels. Immobilized bFGF upregulated vimentin expression and promoted the fibroblastic differentiation of pHAVIC, ADMSC, and BMMSC. These findings suggest that stem cells retain a heightened capacity for osteogenic differentiation in 3D culture, but can be shifted toward fibroblast differentiation through matrix tethering of bFGF. Such a strategy is likely important for utilizing stem cell sources in heart valve tissue engineering applications.