Formalin fixation and paraffin embedding interfere with preservation of optical metabolic assessments based on endogenous NAD(P)H and FAD two photon excited fluorescence.
Formalin fixation and paraffin embedding interfere with preservation of optical metabolic assessments based on endogenous NAD(P)H and FAD two photon excited fluorescence.
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福尔马林固定和石蜡包埋会干扰基于内源性 NAD(P)H 和 FAD 双光子激发荧光的光学代谢评估的保存。
DOI：
10.1101/2023.06.16.545363
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发表时间：
2023
期刊：
影响因子：
--
通讯作者：
Georgakoudi,Irene
中科院分区：
文献类型：
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作者：
Sánchez-Hernández,Adriana;Polleys,ChristopherM;Georgakoudi,Irene
内源性 NAD(P)H 和 FAD 双光子激发荧光 (TPEF) 图像为各种活体标本提供具有高空间分辨率的功能代谢信息。固定后代谢功能光学指标的保存将有助于评估多种疾病背景下代谢变化影响的研究。然而，缺乏对福尔马林固定、石蜡包埋和切片对光学代谢读数保存影响的可靠评估。在这里，我们评估了针对新鲜切除的小鼠口腔上皮和相应的块状和切片固定组织的 NAD(P)H 和 FAD TPEF 检测而优化的激发/发射设置下的强度和寿命图像。我们发现注视会影响整体强度以及所获取图像的强度波动。因此，鳞状上皮的光学氧化还原比（定义为FAD/(NAD(P)H + FAD)）的深度依赖性变化在固定后不会保留。这与 755nm 激发光谱的显着变化一致，这些变化揭示了固定时的加宽以及石蜡包埋和切片时的额外扭曲。对针对 NAD(P)H TPEF 检测优化的激发/发射设置获取的荧光寿命图像的分析表明，固定改变了观察到的荧光的长寿命和长寿命强度分数。这些参数以及较短的 TPEF 寿命在包埋和切片时会发生显着改变。因此，我们的研究强调，福尔马林固定、石蜡包埋和切片过程中形成的自发荧光产物与 NAD(P)H 和 FAD 发射高度重叠，并限制了利用此类组织评估代谢活性的潜力。
Endogenous NAD(P)H and FAD two-photon excited fluorescence (TPEF) images provide functional metabolic information with high spatial resolution for a wide range of living specimens. Preservation of metabolic function optical metrics upon fixation would facilitate studies which assess the impact of metabolic changes in the context of numerous diseases. However, robust assessments of the impact of formalin fixation, paraffin embedding, and sectioning on the preservation of optical metabolic readouts are lacking. Here, we evaluate intensity and lifetime images at excitation/emission settings optimized for NAD(P)H and FAD TPEF detection from freshly excised murine oral epithelia and corresponding bulk and sectioned fixed tissues. We find that fixation impacts the overall intensity as well as the intensity fluctuations of the images acquired. Accordingly, the depth-dependent variations of the optical redox ratio (defined as FAD/(NAD(P)H + FAD)) across squamous epithelia are not preserved following fixation. This is consistent with significant changes in the 755 nm excited spectra, which reveal broadening upon fixation and additional distortions upon paraffin embedding and sectioning. Analysis of fluorescence lifetime images acquired for excitation/emission settings optimized for NAD(P)H TPEF detection indicate that fixation alters the long lifetime of the observed fluorescence and the long lifetime intensity fraction. These parameters as well as the short TPEF lifetime are significantly modified upon embedding and sectioning. Thus, our studies highlight that the autofluorescence products formed during formalin fixation, paraffin embedding and sectioning overlap highly with NAD(P)H and FAD emission and limit the potential to utilize such tissues to assess metabolic activity.