Fast axial scanning with remote focusing for multiphoton microscopy of cardiac tissue

Session

Session 4 - New Technologies: Recent advances from Acquisition to Analysis

Authors

Giedrė Astrauskaitė (1), Lewis Williamson (1), Sharika Mohanan (1), Steven Milan Moreno (1), Ryo Kinegawa (1), Erin Boland (1), Eline Huethorst (1), Godfrey Smith (1), Caroline Muellenbroich (1)

Affiliations

1. University of Glasgow

Keywords

multiphoton microscopy, remote focusing, cardiac electrophysiology

Abstract text

In the heart, electrical signals or action potentials (AP) cause the muscle to contract systematically and allow for the macroscopic function of a pump. If the heart undergoes a myocardial infarction, electrically inert scar tissue disrupts the natural electrical pathways which can lead to arrythmias. Therefore, understanding AP propagation in healthy and infarcted animal heart models is an important step to develop successful diagnostic and therapeutic tools for cardiac diseases.

Two-photon fluorescence microscopy (2PM), offers optical sectioning, cellular resolution, and high tissue penetration depth due to the use of infrared wavelengths [1] and, together with voltage sensitive dyes, the possibility to track action potentials. Along the long axis of cardiomyocytes, conduction velocity has been resolved with 2PM down to a depth of 500 µm in longitudinal planes in the rabbit ventricular wall in intact, Langendorff-perfused hearts [2-4]. However, the slower (30 cm/s) transmural cardiac conduction has not yet been investigated with 2PM. Conventional microscopes are not capable of rapid vertical scans, as traditional axial refocusing methods are slow or cause mechanical disturbance of the sample.

Remote focusing (RF) [5] allows fast refocusing by introducing a remote objective and a lightweight actuated remote mirror in its focal plane. Consequently, an optical copy of the sample can be axially probed by rapidly translating the mirror. Voice coil actuators are capable of axial actuation at several hundred of Hertz and therefore afford the necessary temporal resolution to follow the propagation of electrical wavefronts transmurally. It is possible to maintain cellular spatial resolution over a large dynamic range and the technique is compatible with 2PM. Few studies employing RF in a 2PM systems have been published [6-8], predominantly in neural tissue. Nevertheless, the brain is significantly less scattering compared to cardiac preparations [9]. RF was used also with 3PM [10] to image 600 µm deep in mouse cortex, albeit only at rates of 7 Hz.

Here we present the implementation of a compact remote focusing module compatible with retrofitting to a commercial 2PM specifically developed for functional cardiac imaging. The system is capable of 300 Hz axial scanning over a range of 200 µm in cardiac tissue and is validated against slower conventional 3D scanning in cardiac structural imaging. Using viable rabbit ventricular slices, we present preliminary data to validate the use of remote focusing to track AP propagation transmurally. We believe that this method will allow us to investigate quantitatively and at cellular resolution how scar tissue impacts cardiac conduction.

References

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