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We introduce wFLFM, an approach that enhances the resolution of Fourier light-field microscopy (FLFM) through a hybrid wide-field image. The system exploits the intrinsic compatibility of image formation between the on-axis FLFM elemental image and the wide-field image, allowing for minimal instrumental and computational complexity. The numerical and experimental results of wFLFM present a two- to three-fold improvement in the lateral resolution without compromising the 3D imaging capability in comparison with conventional FLFM.

 

The rapid development of scientific CMOS (sCMOS) technology has greatly advanced optical microscopy for biomedical research with superior sensitivity, resolution, field-of-view, and frame rates. However, for sCMOS sensors, the parallel charge-voltage conversion and different responsivity at each pixel induces extra readout and pattern noise compared to charge-coupled devices (CCD) and electron-multiplying CCD (EM-CCD) sensors. This can produce artifacts, deteriorate imaging capability, and hinder quantification of fluorescent signals, thereby compromising strategies to reduce photo-damage to live samples. Here, we propose a content-adaptive algorithm for the automatic correction of sCMOS-related noise (ACsN) for fluorescence microscopy. ACsN combines camera physics and layered sparse filtering to significantly reduce the most relevant noise sources in a sCMOS sensor while preserving the fine details of the signal. The method improves the camera performance, enabling fast, low-light and quantitative optical microscopy with video-rate denoising for a broad range of imaging conditions and modalities.

 

Development of safer drugs based on epigenetic modifiers, e.g., histone deacetylase inhibitors (HDACi), requires better understanding of their effects on cardiac electrophysiology. Using RNAseq data from the genotype-tissue-expression database (GTEx), we created models that link the abundance of acetylation enzymes (HDAC/SIRT/HATs), and the gene expression of ion channels (IC) via select cardiac transcription factors (TFs) in male and female adult human hearts (left ventricle, LV). Gene expression data (transcripts per million, TPM) from GTEx donors (21–70 y.o.) were filtered, normalized and transformed to Euclidian space to allow quantitative comparisons in 84 female and 158 male LVs. Sex-specific partial least-square (PLS) regression models, linking gene expression data for HDAC/SIRT/HATs to TFs and to ICs gene expression, revealed tight co-regulation of cardiac ion channels by HDAC/SIRT/HATs, with stronger clustering in the male LV. Co-regulation of genes encoding excitatory and inhibitory processes in cardiac tissue by the acetylation modifiers may help explain their predominantly net-neutral effects on cardiac electrophysiology. ATP1A1 , encoding for the Na/K pump, represented an outlier—with orthogonal regulation by the acetylation modifiers to most of the ICs. The HDAC/SIRT/HAT effects were mediated by strong (+) TF regulators of ICs, e.g., MEF2A and TBX5 , in both sexes. Furthermore, for male hearts, PLS models revealed a stronger (+/-) mediatory role on ICs for NKX25 and TGF1B/KLF4 , respectively, while RUNX1 exhibited larger (-) TF effects on ICs in females. Male-trained PLS models of HDAC/SIRT/HAT effects on ICs underestimated the effects on some ICs in females. Insights from the GTEx dataset about the co-expression and transcriptional co-regulation of acetylation-modifying enzymes, transcription factors and key cardiac ion channels in a sex-specific manner can help inform safer drug design. 

The epigenetic landscape and the responses to pharmacological epigenetic regulators in each human are unique. Classes of epigenetic writers and erasers, such as histone acetyltransferases, HATs, and histone deacetylases, HDACs, control DNA acetylation/deacetylation and chromatin accessibility, thus exerting transcriptional control in a tissue- and person-specific manner. Rapid development of novel pharmacological agents in clinical testing—HDAC inhibitors (HDACi)—targets these master regulators as common means of therapeutic intervention in cancer and immune diseases. The action of these epigenetic modulators is much less explored for cardiac tissue, yet all new drugs need to be tested for cardiotoxicity. To advance our understanding of chromatin regulation in the heart, and specifically how modulation of DNA acetylation state may affect functional electrophysiological responses, human-induced pluripotent stem-cell-derived cardiomyocyte (hiPSC-CM) technology can be leveraged as a scalable, high-throughput platform with ability to provide patient-specific insights. This review covers relevant background on the known roles of HATs and HDACs in the heart, the current state of HDACi development, applications, and any adverse cardiac events; it also summarizes relevant differential gene expression data for the adult human heart vs. hiPSC-CMs along with initial transcriptional and functional results from using this new experimental platform to yield insights on epigenetic control of the heart. We focus on the multitude of methodologies and workflows needed to quantify responses to HDACis in hiPSC-CMs. This overview can help highlight the power and the limitations of hiPSC-CMs as a scalable experimental model in capturing epigenetic responses relevant to the human heart. 

Optical techniques for recording and manipulating cellular electrophysiology have advanced rapidly in just a few decades. These developments allow for the analysis of cardiac cellular dynamics at multiple scales while largely overcoming the drawbacks associated with the use of electrodes. The recent advent of optogenetics opens up new possibilities for regional and tissue-level electrophysiological control and hold promise for future novel clinical applications. This article, which emerged from the international NOTICE workshop in 2018 1 , reviews the state-of-the-art optical techniques used for cardiac electrophysiological research and the underlying biophysics. The design and performance of optical reporters and optogenetic actuators are reviewed along with limitations of current probes. The physics of light interaction with cardiac tissue is detailed and associated challenges with the use of optical sensors and actuators are presented. Case studies include the use of fluorescence recovery after photobleaching and super-resolution microscopy to explore the micro-structure of cardiac cells and a review of two photon and light sheet technologies applied to cardiac tissue. The emergence of cardiac optogenetics is reviewed and the current work exploring the potential clinical use of optogenetics is also described. Approaches which combine optogenetic manipulation and optical voltage measurement are discussed, in terms of platforms that allow real-time manipulation of whole heart electrophysiology in open and closed-loop systems to study optimal ways to terminate spiral arrhythmias. The design and operation of optics-based approaches that allow high-throughput cardiac electrophysiological assays is presented. Finally, emerging techniques of photo-acoustic imaging and stress sensors are described along with strategies for future development and establishment of these techniques in mainstream electrophysiological research. 

Optogenetic methods for pacing of cardiac tissue can be realized by direct genetic modification of the cardiomyocytes to express light-sensitive actuators, such as channelrhodopsin-2, ChR2, or by introduction of light-sensitized non-myocytes that couple to the cardiac cells and yield responsiveness to optical pacing. In this study, we engineer three-dimensional “spark cells” spheroids, composed of ChR2-expressing human embryonic kidney cells (from 100 to 100,000 cells per spheroid), and characterize their morphology as function of cell density and time. These “spark-cell” spheroids are then deployed to demonstrate site-specific optical pacing of human stem-cell-derived cardiomyocytes (hiPSC-CMs) in 96-well format using non-localized light application and all-optical electrophysiology with voltage and calcium small-molecule dyes or genetically encoded sensors. We show that the spheroids can be handled using liquid pipetting and can confer optical responsiveness of cardiac tissue earlier than direct viral or liposomal genetic modification of the cardiomyocytes, with 24% providing reliable stimulation of the iPSC-CMs within 6 h and >80% within 24 h. Moreover, our data show that the spheroids can be frozen in liquid nitrogen for long-term storage and transportation, after which they can be deployed as a reagent on site for optical cardiac pacing. In all cases, optical stimulation was achieved at relatively low light levels (<0.15 mW/mm 2 ) when 5 ms or longer pulses were used. Our results demonstrate a scalable, cost-effective method with a cryopreservable reagent to achieve contactless optical stimulation of cardiac cell constructs without genetically modifying the myocytes, that can be integrated in a robotics-amenable workflow for high-throughput drug testing.