All-optical framework for the correlative imaging of cardiac meso-scale cytoarchitecture and multi-scale electrical conduction

Project Information

Optoheart

Grant agreement ID: 842893

DOI

10.3030/842893

Project closed

EC signature date 16 April 2019

Start date 1 September 2019

End date 31 August 2021

Funded under

• EXCELLENT SCIENCE - Marie Skłodowska-Curie Actions

Total cost

€ 212 933,76

EU contribution

€ 212 933,76

Coordinated by

UNIVERSITY OF GLASGOW
United Kingdom

Periodic Reporting for period 1 - Optoheart (All-optical framework for the correlative imaging of cardiac meso-scale cytoarchitecture and multi-scale electrical conduction)

Reporting period: 2019-09-01 to 2021-08-31

Summary of the context and overall objectives of the project

Millions of people are affected by abnormal heartbeats, or arrhythmias. Advances in understanding the precise conditions which promote the formation of arrhythmias will be vital to develop treatment strategies. Cardiac muscle cells need to act synchronously to an initiating electrical impulse to allow blood to be efficiently pumped around the body with each beat. Importantly, cardiac electric conduction is a tissue level phenomenon based on current propagation across billions of cells whose electro-mechanical function of contraction is intricately intertwined with their structural arrangement. In pathological conditions prone to develop arrhythmias, changes in tissue structure (consider scars and electrically inert tissue) caused by the disease have dire implications on the healthy function of cardiac conduction.
Light-sheet microscopy (LSM) has proven a useful tool in bioimaging to image whole organs with high frame rates at cellular resolution and, in combination with tissue clearing methods, is often employed to reconstruct the cyto-architecture over entire organs. Inherently to LSM, however, residual opaque objects, always present to some extent even in extremely well optically cleared samples, cause stripe artifacts, which, in the best case, severely affect image homogeneity and, in the worst case, completely obscure features of interest. Since whole organ datasets now routinely comprise several terabytes, automated tools to count, trace, or segment features of interest are needed to extract meaningful insights. It is therefore necessary to devise technical solutions to increase fidelity in imaging and relax computational demands on the algorithms used to turn data into knowledge.
In this study, we will develop innovative microscopy technology to measure cardiac tissue structure over the whole intact mouse heart and create maps of non-conducting tissue in health and disease. These structural maps of conducting and non-conducting tissue will inform previously obtained measurements of cardiac electric activity and allow to relate cardiac function back to its structure. This will provide us with vital information not only for our understanding of fundamental heart electrophysiology which will be translated to new medical treatments to for cardiac arrhythmias.

Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far

Light sheet fluorescence microscopy (LSFM) is a well-established technique for volumetric imaging of large samples with high speed and good resolution and is compatible with tissue optical clearing techniques that enhance light penetration in highly scattering media and improve image quality by reducing optical aberrations. We built two technical innovations for high-fidelity reconstructions of clarified mouse organs using light-sheet microscopy, i) an AOD system to remove striping artefacts in way that is compatible with confocal line detection (panel A) and ii) an autofocusing system (panel B) which takes advantage of the phase detection principle to allow for fast and accurate automatic refocusing in all wide-field type microscope techniques. The obtained data was contributed to existing mouse brain atlases. To adapt to the COVID pandemic, several literature review-based publications have been produced including i) methods for the removal and suppression of striping artifacts in light sheet microscopy, ii) optical technology used for the investigation of cardiac electrophysiology (manuscript accepted at Frontiers in Physiology) and iii) a position paper on the use of computer-assisted design in microscopy technology development (manuscript under revision). The results achieved during this project have been presented to the scientific community at several national conferences (Janelia Farm workshop for early career researchers, the Bath Light incubator, Biophysical society annual meeting and the SUPA annual gathering 2019) and through 3 publications. Furthermore, these achievements were disseminated to the general public at the Explorathon (aka European Researcher’s night) 2019 and through social media. For example, a twitter tutorial on the autofocusing publication lead to 9389 impressions (times people saw this tweet) and 195 engagements (times people interacted with this tweet, checked 08/10/2021).

Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far)

The light-sheet microscope and its technological innovations to improve image quality have been published in several scientific articles demonstrating its novelty and impact. In Silvestri et al (Silvestri, L., Müllenbroich, M. C., et al. (2021), Universal autofocus for quantitative volumetric microscopy of whole mouse brains. Nature Methods, 18(8), 953-958. [1]), a sample-agnostic real-time autofocus method for widefield microscopy was presented, which reliably removes defocus-induced aberrations and allows to reconstruct an intact and entire organ with subcellular resolution.
Furthermore, it was noticed that images acquired with on-photon excitation presented significant illumination inhomogeneity in the form of striping artefacts, mostly due to absorption and scattering from occlusions within the cleared sample. In Ricci et al (Ricci, Pietro, et al. "Fast multi-directional DSLM for confocal detection without striping artifacts." Biomedical Optics Express 11.6 (2020): 3111-3124., [2]), we published demonstrated the suppression of these striping artefacts using acousto optical deflectors (AODs) to dynamically pivot the exciting light sheet. Crucially, this method preserves its compatibility with confocal line detection for improved background reduction. These achievements pave the way to more quantitative structural studies in cleared tissue samples.
Based on this expertise, we published a review paper (Ricci, Pietro, et al. "Removing striping artifacts in light-sheet fluorescence microscopy: A review." Progress in Biophysics and Molecular Biology (2021), [3]) in which we outline the advantages, performances and limitations of stripe artefact reduction in light-sheet microscopy.

Technological advancements in light-sheet microscopy. Adapted from [1-3].