Immersion-Based Clearing and Autofluorescence Quenching in Myocardial Tissue

IF 2.4 4区医学 Q3 HEMATOLOGY

Microcirculation Pub Date : 2025-11-05 DOI:10.1111/micc.70034

Victoria E. Sturgess, Nadia K. Korovesis, Domingo E. Uceda, Ali Citalan-Madrid, Eric V. Vu, Katherine Stangis, Françoise Van den Bergh, Salman I. Essajee, Binyamin Jacobovitz, Gregory B. Sands, Daniel A. Lawrence, Johnathan D. Tune, Daniel A. Beard, Geoffrey G. Murphy, C. Alberto Figueroa

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引用次数: 0

Abstract

Objective

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.

Methods

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.

Results

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.

Conclusions

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.

Abstract Image

查看原文本刊更多论文

心肌组织浸泡清除和自身荧光猝灭。

目的：最近在光学显微镜和组织制备方面的创新使得复杂微血管网络的三维可视化成为可能。组织清除技术提高光穿透和扩展成像深度。通常，基于灌注的方法用于血管标记和组织清除。然而，基于浸泡的方法在处理大型动物模型的组织时提供了增强的实用性。方法：我们提出了一种基于浸泡的心肌组织微血管标记和组织清除方案，使用番茄凝集素和CUBIC（清晰，无阻碍的脑/体成像鸡尾酒和计算分析），证明了成像深度高达150 μm的成功。本方案优化了大鼠和猪心肌组织的脱脂和猝灭阶段。使用信噪比（SNR）和平均z片强度的自动分析来评估图像质量。结果：CUBIC Reagent I孵育时间为24 h，图像质量最佳。猝灭剂TrueVIEW、甘氨酸和台锥蓝对信噪比值没有显著影响。TrueBlack和苏丹黑B与没有淬灭剂孵育的对照组相比，显示出成像深度降低的趋势。总体而言，大鼠心肌组织的信噪比高于猪组织样本。结论：该方案为优化浸入式心肌组织清除方法提供了严格的基础。未来的研究将量化解剖和拓扑冠状动脉微血管来比较疾病状态。

本文章由计算机程序翻译，如有差异，请以英文原文为准。

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来源期刊

Microcirculation 医学-外周血管病

CiteScore

5.00

自引率

4.20%

发文量

审稿时长

6-12 weeks

期刊介绍： The journal features original contributions that are the result of investigations contributing significant new information relating to the vascular and lymphatic microcirculation addressed at the intact animal, organ, cellular, or molecular level. Papers describe applications of the methods of physiology, biophysics, bioengineering, genetics, cell biology, biochemistry, and molecular biology to problems in microcirculation. Microcirculation also publishes state-of-the-art reviews that address frontier areas or new advances in technology in the fields of microcirculatory disease and function. Specific areas of interest include: Angiogenesis, growth and remodeling; Transport and exchange of gasses and solutes; Rheology and biorheology; Endothelial cell biology and metabolism; Interactions between endothelium, smooth muscle, parenchymal cells, leukocytes and platelets; Regulation of vasomotor tone; and Microvascular structures, imaging and morphometry. Papers also describe innovations in experimental techniques and instrumentation for studying all aspects of microcirculatory structure and function.