Remote focusing to follow action potential transmurally in acute rabbit cardiac slices

Astrauskaite, Giedre (2025) Remote focusing to follow action potential transmurally in acute rabbit cardiac slices. PhD thesis, University of Glasgow.

Abstract

Electrical signals or action potentials (APs) originate in the pacemaker cells of the heart and travel through the organ in an orchestrated fashion. This electrical conduction governs mechanical contraction, making the heart an efficient pump. During amyocardial infarction (MI), oxygen deprived cardiac cells are rendered electrically inert as the scar is formed. This impairs the natural conduction pathways and can lead to life-threatening arrythmias. Therefore, understanding AP propagation in relation to the complex 3-dimensional tissue morphology of healthy and infarcted animal models is a pivotal step to establish diagnostic and therapeutic tools.

To indirectly investigate cardiac electrophysiology in large-scale intact tissue, optical mapping with voltage sensitive dyes (VSDs) is commonly employed [1]. Nevertheless, AP propagation in distinct cellular layers in depth below the epicardium is inaccessible with optical mapping. Conversely, two-photon fluorescence microscopy (2PM) allows cellular resolution and large tissue penetration depth with inherent optical sectioning [2]. In the ventricular wall of the heart, two directions of conduction can be considered: longitudinal, along the long axis of the cell and transmural, from the endocardium to the epicardium. Along the long axis of the cardiomyocytes, with 2PM, action potentials have previously been resolved as deep as 500 µm in distinct cellular layers of Langendorff-perfused rabbit hearts [3]. However, conventional microscopes cannot facilitate axial scanning fast enough to resolve action potentials and thereforetransmural cardiac conduction has not yet been investigated with 2PM.

In remote refocusing (RF), a remote objective and a lightweight mirror in its focal plane are introduced in the optical system [4, 5]. Consequently, the 3-dimensional optical copy of the sample can be probed vertically by rapid remote mirror actuation. RF is compatible with high numerical aperture 2PM systems and offers the temporal resolution necessary to resolve transmural action potential propagation over a large refocusing range [4, 5]. Studies employing RF in multiphoton microscopes focus predominantly on deep tissue neural function [6, 7]. Nevertheless, the brain is significantly less scattering compared to cardiac preparations. Sarcomere length of cardiomyocytes was measured utilising RF, but only cardiac structure, not electrical function was investigated [8].

Therefore, the aim of this work is, firstly, to develop a 2-photon microscope system enhanced with RF for rapid axial scanning and capable of probing action potential propagation along both longitudinal and transmural directions within deep myocardium. Our implementation of a remote refocusing module retrofitted to a commercial Scientifica 2-photon microscope achieves 250 Hz axial scanning over a range of 100 µm while maintaining under 5 µm axial resolution. The necessary system power efficiency and dispersion optimisation [9] is discussed: 22 mW average power with 15.9% throughput and a close to transform-limited pulse duration of 156 fs at sample is allowed by the system. Secondly, the full experimental pipeline including the preparation, viability evaluation and imaging of the cardiac preparation is established. Acute rabbit ventricular slice model adapted from [10] and prepared from hearts that have previously been used for other experiments (in line with the University’s strategy for reduction of animal research [11]) was tailored for 2P-RF imaging at room temperature. Live-dead staining (triphenyl tetrazolium chloride) of N=6 slices revealed no hypoxic core and gave a qualitative verification of slice viability in the central region of the preparations; slice contractility and action potential properties were characterised with optical mapping (CellOPTIQ system). The slices exhibit single-peak contractile traces throughout the entire preparation when stimulated at 0.3 Hz. Furthermore, in N=6 slices, the action potential duration was approximately 2 times shorter after increasing the concentration (from 15 mM to 30 mM) of 2,3-butanedione monoxime (BDM), an electromechanical uncoupler necessary for 2-photon imaging. The variation of AP duration from slice to slice at 30 mM BDM was characterised. Finally, the preliminary data to validate the use of our 2P-remote focusing to investigate cardiac AP propagation transmurally is presented. The time to acquire z-y planes in myocardium is reduced from minutes (conventional z-stack acquisition) to seconds with 122 Hz RF. When the remote refocusing module is bypassed, we resolve AP traces in electrically stimulated slices with rapid longitudinal galvanometric mirror scanning with SNR > 7 over the range of 120 µm in depth. Importantly, with the RF unit in place, action potential peaks were visible over approximately 60 µm range with static remote refocusing with SNR > 4. Therefore, we believe that the 2P-RF system presented will allow to resolve transmural APs with rapid axial scanning and enable a quantitative investigation how scar tissue impacts cardiac conduction in post-MI preparations.

• Item Type: Thesis (PhD)
• Qualification Level: Doctoral
• Subjects: Q Science > QC Physics; Q Science > QH Natural history > QH301 Biology; T Technology > T Technology (General)
• Colleges/Schools: College of Science and Engineering > School of Physics and Astronomy
• Supervisor's Name: Muellenbroich, Dr. Caroline
• Date of Award: 2025
• Depositing User: Theses Team
• Unique ID: glathesis:2025-85244
• Copyright: Copyright of this thesis is held by the author.
• Date Deposited: 23 Jun 2025 14:40
• Last Modified: 23 Jun 2025 14:43
• Thesis DOI: 10.5525/gla.thesis.85244
• URI: https://theses.gla.ac.uk/id/eprint/85244

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