Autofluorescence based fluorescence lifetime imaging microscopy (AF-FLIM) techniques have come a long way from early studies on cancer characterization and have now been widely employed in several cellular and animal studies covering a wide range of diseases. The majority of research in autofluorescence imaging (AFI) study metabolic fluxes in live biological samples. However, tissues from clinical or scientific studies are often chemically fixed for preservation and stabilization of tissue morphology. Fixation is particularly crucial for enzymatic, functional, or histopathology studies. Interpretations of metabolic imaging such as optical redox intensity imaging and AF-FLIM, have often been viewed as potentially unreliable in a fixed sample due to lack of studies in this field. In this study, we carefully evaluate the possibility of extracting microenvironment information in fixed tissues using reduced nicotinamide adenine dinucleotide (phosphate) (NAD(P)H) endogenous fluorescence. The ability to distinguish changes such as metabolism and pH using intrinsic fluorescence in fixed tissues has great pathological value. In this work, we show that the lifetime based metabolic contrast in a sample is preserved after chemical fixation. The fluorescence lifetime of a sample increases with an additive fixative like formaldehyde; however, the fixed tissues retain metabolic signatures even after fixation. This study presents an opportunity to successfully image archived unstained histopathology tissues, and generate useful AF-FLIM signatures. We demonstrate the capability to draw metabolic interpretations in fixed tissues even after long periods of storage.

中文翻译：

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固定生物组织中的 NAD(P)H 荧光寿命测量。

基于自发荧光的荧光寿命成像显微镜 (AF-FLIM) 技术从癌症表征的早期研究中取得了长足的进步，现在已广泛应用于涵盖多种疾病的多项细胞和动物研究中。大多数自发荧光成像 (AFI) 研究研究活体生物样本中的代谢通量。然而，来自临床或科学研究的组织通常被化学固定以保存和稳定组织形态。固定对于酶促、功能或组织病理学研究尤为重要。由于缺乏该领域的研究，代谢成像（例如光学氧化还原强度成像和 AF-FLIM）的解释通常被认为在固定样本中可能不可靠。在这项研究中，我们仔细评估了使用还原烟酰胺腺嘌呤二核苷酸（磷酸盐）（NAD（P）H）内源性荧光提取固定组织中微环境信息的可能性。在固定组织中使用固有荧光区分代谢和 pH 等变化的能力具有很大的病理学价值。在这项工作中，我们表明样本中基于寿命的代谢对比在化学固定后得以保留。样品的荧光寿命随着甲醛等附加固定剂的增加而增加；然而，固定的组织即使在固定后仍保留代谢特征。这项研究提供了一个机会，可以成功地对存档的未染色组织病理学组织进行成像，并生成有用的 AF-FLIM 特征。