Mesoscopic Optical Imaging of Whole Mouse Heart

Francesco Giardini; Erica Lazzeri; Camilla Olianti; Giada Beconi; Irene Costantini; Ludovico Silvestri; Elisabetta Cerbai; Francesco S. Pavone; Leonardo Sacconi

doi:10.3791/62795

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Method Article

Mesoscopic Optical Imaging of Whole Mouse Heart

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DOI:

10.3791/62795

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October 14th, 2021

Francesco Giardini^*¹ , Erica Lazzeri^*¹ , Camilla Olianti^*¹ , Giada Beconi¹ , Irene Costantini¹^,² , Ludovico Silvestri¹^,³^,⁴ , Elisabetta Cerbai¹^,⁵ , Francesco S. Pavone¹^,³^,⁴ , Leonardo Sacconi¹^,³^,⁶

¹European Laboratory for Non-Linear Spectroscopy, ²Department of Biology, University of Florence, ³National Institute of Optics, National Research Council, ⁴Department of Physics and Astronomy, University of Florence, ⁵Department of Neurosciences, Psychology, Drugs and Child Health, University of Florence, ⁶Institute for Experimental Cardiovascular Medicine, Faculty of Medicine, University of Freiburg

^* These authors contributed equally

Summary

We report a method for mesoscopic reconstruction of the whole mouse heart by combining new advancements in tissue transformation and staining with the development of an axially scanned light-sheet microscope.

Abstract

Both genetic and non-genetic cardiac diseases can cause severe remodeling processes in the heart. Structural remodeling, such as collagen deposition (fibrosis) and cellular misalignment, can affect electrical conduction, introduce electromechanical dysfunctions and, eventually, lead to arrhythmia. Current predictive models of these functional alterations are based on non-integrated and low-resolution structural information. Placing this framework on a different order of magnitude is challenging due to the inefficacy of standard imaging methods in performing high-resolution imaging in massive tissue. In this work, we describe a methodological framework that allows imaging of whole mouse hearts with micrometric resolution. The achievement of this goal has required a technological effort where advances in tissue transformation and imaging methods have been combined. First, we describe an optimized CLARITY protocol capable of transforming an intact heart into a nanoporous, hydrogel-hybridized, lipid-free form that allows high transparency and deep staining. Then, a fluorescence light-sheet microscope able to rapidly acquire images of a mesoscopic field of view (mm-scale) with the micron-scale resolution is described. Following the mesoSPIM project, the conceived microscope allows the reconstruction of the whole mouse heart with micrometric resolution in a single tomographic scan. We believe that this methodological framework will allow clarifying the involvement of the cytoarchitecture disarray in the electrical dysfunctions and pave the way for a comprehensive model that considers both the functional and structural data, thus enabling a unified investigation of the structural causes that lead to electrical and mechanical alterations after the tissue remodeling.

Introduction

Structural remodeling associated with cardiac diseases can affect electrical conduction and introduce electromechanical dysfunctions of the organ¹^,². Current approaches used to predict functional alterations commonly employ MRI and DT-MRI to obtain an overall reconstruction of fibrosis deposition, vascular tree, and fiber distribution of the heart, and they are used to model preferential action potential propagation (APP) paths across the organ³^,⁴. These strategies can provide a beautiful overview of the heart organization. However, their spatial resol....