# Immersion‐Based Clearing and Autofluorescence Quenching in Myocardial Tissue

## TL;DR

This paper introduces an immersion-based protocol for clearing and labeling myocardial tissue to enable deeper imaging of microvascular networks.

## Contribution

The study introduces an optimized immersion-based protocol for myocardial tissue clearing and labeling using CUBIC and tomato lectin.

## Key findings

- A 24-hour incubation with CUBIC Reagent I yielded optimal image quality in myocardial tissues.
- Quenching agents like TrueVIEW, Glycine, and Trypan Blue did not significantly affect signal-to-noise ratios.
- Rat myocardial tissues showed higher signal-to-noise ratios compared to pig tissues.

## Abstract

Recent innovations in optical microscopy and tissue preparation permit 3D visualization of complex microvascular networks. Tissue clearing techniques improve light penetration and extend imaging depth. Typically, perfusion‐based approaches are used for vascular labeling and tissue clearing. However, immersion‐based methodologies provide enhanced practicality when processing tissues from larger animal models.

We present an immersion‐based microvascular labeling and tissue clearing protocol for myocardial tissues using tomato lectin and CUBIC (Clear, Unobstructed Brain/Body Imaging Cocktails and Computational analysis), demonstrating success for imaging depths of up to 150 μm. This protocol optimized the delipidation and quenching stages for rat and pig myocardial tissues. Image quality was assessed using an automated analysis of signal‐to‐noise ratios (SNR) and average z‐slice intensities.

Optimal image quality was obtained with 24‐h CUBIC Reagent I incubation times. Quenching agents TrueVIEW, Glycine, and Trypan Blue did not significantly impact SNR values. TrueBlack and Sudan Black B showed trends of reduced imaging depth compared to controls without quencher incubation. Overall, rat myocardial tissues had higher SNRs than pig tissue samples.

This protocol provides a rigorous foundation for the optimization of immersion‐based approaches for myocardial tissue clearing. Future studies will quantify anatomical and topological coronary microvascular to compare disease states.