基于 NADH 荧光的大鼠脑冷冻固定质量评价方法。

Quality evaluation method for rat brain cryofixation on the basis of NADH fluorescence.

机构信息

Britton Chance Center for Biomedical Photonics, Wuhan National Laboratory for Optoelectronics-Huazhong University of Science and Technology, Wuhan, 430074, P. R. China.

Key Laboratory of Biomedical Photonics of Ministry of Education, Department of Biomedical Engineering, Huazhong University of Science and Technology, Wuhan, 430074, P. R. China.

出版信息

Adv Exp Med Biol. 2013;789:435-440. doi: 10.1007/978-1-4614-7411-1_58.

DOI:10.1007/978-1-4614-7411-1_58

PMID:23852526

Abstract

The goal of biological samples' cryofixation is to trap a metabolic state as it exists in vivo by rapidly stopping internal reactions. However, obtaining perfect quality of cryofixation for large and high hypermetabolism organ/tissue (such as brain, heart) remains a challenge. The aim of this study was to develop and display a comprehensive and direct method to evaluate cryofixation's process and quality. Here, we adopt a delicate combination of homemade cryo-imaging system with a rat cardiac arrest model that can control cryofixation time optionally. we successfully evaluate the cryofixation time-related nicotinamide adenine dinucleotide (NADH) fluorescence pattern of several coronal sections in rat's brain that suffered from directional funnel cryofixation procedure. Through quantitative analysis of the distribution map of NADH fluorescence, we could obtain a relationship between cryofixation time and well cryofixation volume and then could deduce the cryofixation rates and quality at different time points. Our results also demonstrated that dissection of the temporal muscle of rat could significantly optimize the classical direct funnel cryofixation protocol.

摘要

生物样本的深冷固定的目标是通过快速停止内部反应来捕捉体内存在的代谢状态。然而，对于大体积和高代谢组织/器官（如脑、心脏），获得高质量的深冷固定仍然是一个挑战。本研究的目的是开发和展示一种全面、直接的方法来评估深冷固定的过程和质量。在这里，我们采用了自制的 cryo-imaging 系统与大鼠心脏骤停模型的精细组合，可以根据需要控制深冷固定时间。我们成功地评估了定向漏斗深冷固定过程中大鼠脑的几个冠状切片的深冷固定时间相关烟酰胺腺嘌呤二核苷酸（NADH）荧光模式。通过 NADH 荧光分布图谱的定量分析，我们可以获得深冷固定时间与良好深冷固定体积之间的关系，然后可以推断出不同时间点的深冷固定速率和质量。我们的结果还表明，大鼠颞肌的解剖可以显著优化经典的直接漏斗深冷固定方案。