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Ultrafast imaging of cardiomyocyte contractions by combining scanning ion conductance microscopy with a microelectrode array Stefan Simeonov, and Tilman E Schäffer Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.9b01092 • Publication Date (Web): 04 Jun 2019 Downloaded from http://pubs.acs.org on June 5, 2019
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Analytical Chemistry
Ultrafast Imaging of Cardiomyocyte Contractions by Combining Scanning Ion Conductance Microscopy with a Microelectrode Array Stefan Simeonov and Tilman E. Schäffer* Institute of Applied Physics, University of Tübingen, Auf der Morgenstelle 10, 72076 Tübingen, Germany *E-mail:
[email protected] ABSTRACT Beating cardiomyocytes undergo fast morphodynamics during the contraction-relaxation cycle. However, imaging these morphodynamics with a high spatial and temporal resolution is difficult, owing to a lack of suitable techniques. Here, we combine scanning ion conductance microscopy (SICM) with a microelectrode array (MEA) to image the 3D topography of cardiomyocytes during a contractionrelaxation cycle with 1 µm spatial and 1 ms time resolution. We record the vertical motion of cardiomyocytes at many locations across a cell by SICM and synchronize these data using the simultaneously recorded action potential by the MEA as a time reference. This allows us to reconstruct the time-resolved 3D morphology of cardiomyocytes during a full contraction-relaxation cycle with a raw data rate of 200 µs/frame and to generate spatially resolved images of contractile parameters (maximum displacement, time delay, asymmetry factor). We use the MEA-SICM setup to visualize the effect of blebbistatin, a myosin II inhibitor, on the morphodynamics of contractions. Further, we find an upper limit of 0.02 % for cell volume changes during an action potential. The results show that MEASICM provides an ultrafast imaging platform for investigating the functional interplay of cardiomyocyte electrophysiology and mechanics.
Keywords: MEA, SICM, nanopipette, scanning probe microscopy, electrophysiology, HL-1 cells
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INTRODUCTION Contracting cardiomyocytes undergo significant morphological changes throughout each contractionrelaxation cycle, which is the fundamental determinant of the contractile function of the heart muscle.1 Each cycle is initiated by an action potential,2 comprised of active and passive ionic flux through the cell membrane.3 The excitation of action potentials and the occurrence of contractions are closely connected.4 The electrophysiology of cardiomyocyte contractions has been studied extensively using, for example, the patch clamp technique,5,6 voltage sensitive dyes,7 or Ca2+ indicators.8 A microelectrode array (MEA)9 can record multiple electrophysiological signals extracellularly and non-invasively. MEAs have found application in cardiovascular research,10 pharmacology,11 signaling of neurons,12 and neuronal networks.10,13,14 To quantify the mechanics of cardiomyocyte contractions, a myriad of techniques has been developed. Examples are optical video microscopy,15,16 uniaxial tensile experiments,17,18 three-dimensional flexible polymeric micropillars,19 traction force microscopy,20 atomic force microscopy (AFM),21-23 and recently surface plasmon resonance microscopy.24 Investigating cardiomyocytes in the context of both electrophysiology and mechanics requires integrative techniques. A MEA-based technique has been developed for detecting the contraction of cardiomyocytes via electrical impedance.25 A combined MEA-AFM setup has been developed for simultaneously recording electrophysiological, morphological, and mechanical changes during contraction of rat embryonic cells at single positions on the cells.26,27 However, the application of an external mechanical force by the AFM tip may pose a physiological stimulus that affects cell behavior1,28,29 or may even damage the cell.30 Scanning ion conductance microscopy (SICM)31 can image live cells without mechanical contact and has become a powerful and versatile tool for non-invasive and high-resolution imaging.32-34 SICM has been used for morphological analysis in cardiovascular research35-39 and for measurement of mechanical40-43 and electrophysiological44,45 properties of live cells. Here, we report the development of a combined MEA-SICM setup for simultaneous recording of extracellular field potential (EFP) and cell morphology. Using this setup, we were able, for the first time, to reconstruct in 3D the surface morphology and velocity during a full contraction-relaxation cycle of HL-1 cells with a time resolution of 1 ms and a spatial resolution of 1 µm. We generated spatially resolved images of contractile parameters (maximum displacement, time delay, asymmetry 2 ACS Paragon Plus Environment
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Analytical Chemistry
factor) over the whole cell surface and during the full contraction-relaxation cycle. Finally, we analyzed the effect of blebbistatin, a myosin II inhibitor that suppresses cellular contractile activity, on the morphodynamics.
EXPERIMENTAL SECTION MEA-SICM setup The experimental setup consisted of a self-built, tip-scanning SICM system mounted on top of a MEA recording system (MEA2100-Lite, Multi Channel Systems MCS GmbH, Reutlingen, Germany), and was interfaced with an optical microscope (Ti-S, Nikon Corporation, Chiyoda, Japan). The MEA high pass filter was set to 0.1 Hz (2nd order) and the low pass filter to 3.5 kHz (4th order). The SICM system consisted of a 25 µm z-scanner (P-753.21C, Physik Instrumente, Karlsruhe, Germany) in combination with a 200 µm xy-scanner (P-527.3CL, Physik Instrumente) for vertical and lateral positioning of the nanopipette, respectively. A patch clamp amplifier (Axopatch 200B, Molecular Devices, Sunnyvale, CA) was used for measuring the ion current. The low pass filter of the amplifier was set to 10 kHz. The noise level of the ion current did not increase (increase
