Background

In MRI of formalin-fixed tissue one of the problems is the dependence of tissue relaxation properties on formalin composition and composition of embedding medium (EM) used for scanning. In this study, we investigated molecular mechanisms by which the EM composition affects T2 relaxation directly and T1 relaxation indirectly.

Objective

To identify principal components of formaldehyde based EM and the mechanism by which they affect relaxation properties of fixed tissue.

Methods

We recorded high resolution ¹H NMR spectra of common formalin fixatives at temperatures in the range of 5 °C to 45 °C. We also measured T1 and T2 relaxation times of various organs of formalin fixed (FF) zebrafish at 7 T at 21 °C and 31 °C in several EM with and without fixative or gadolinium contrast agents.

Results

We showed that the major source of T2 variability is chemical exchange between protons from EM hydroxyls and water, mediated by the presence of phosphate ions. The exchange rate increases with temperature, formaldehyde concentration in EM and phosphate concentration in EM. Depending on which side of the coalescence the system resides, the temperature increase can lead to either shortening or prolongation of T2, or to no noticeable change at all when very close to the coalescence. Chemical exchange can be minimized by washing out from EM the fixative, the phosphate or both.

Conclusion

The dependence of T2 in fixed tissue on the fixative origin and composition described in prior literature could be attributed to the phosphate buffer accelerated chemical exchange among the fixative hydroxyls and the tissue water. More consistent results in the relaxation measurements could be obtained by stricter control of the fixative composition or by scanning fixed tissue in PBS without fixative.

中文翻译：

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水性包埋介质组成对固定组织松弛特性的影响

背景

在福尔马林固定的组织的MRI中，问题之一是组织松弛特性对福尔马林成分和用于扫描的包埋介质（EM）的成分的依赖性。在这项研究中，我们研究了EM组成直接影响T2弛豫和间接影响T1弛豫的分子机制。

客观的

识别基于甲醛的EM的主要成分及其影响固定组织松弛特性的机理。

方法

我们在5°C至45°C的温度范围内记录了普通福尔马林固定剂的高分辨率¹ H NMR光谱。我们还测量了几种含或不含固定剂或g造影剂的EM中福尔马林固定（FF）斑马鱼在7 T在21°C和31°C下各种器官的T1和T2弛豫时间。

结果

我们表明，T2变异性的主要来源是来自EM羟基和水的质子之间的化学交换，这是由磷酸根离子的存在介导的。交换速率随温度，EM中的甲醛浓度和EM中的磷酸盐浓度而增加。根据系统驻留在合并的哪一侧，温度升高可能导致T2缩短或延长，或者非常接近合并时根本不会引起明显变化。通过从EM洗掉固定剂，磷酸盐或两者，可以最大程度地减少化学交换。

结论

固定组织中T2对固定剂来源和组成的依赖关系可归因于现有文献中所述，这归因于磷酸盐缓冲液加速了固定剂羟基与组织水之间的化学交换。通过更严格地控​​制固定剂组合物或通过在不含固定剂的PBS中扫描固定的组织，可以获得在松弛测量中更一致的结果。