Abstract：
Holotomography (HT) is a cutting-edge fast live-cell quantitative label-free imaging technique. Based on the principle of quantitative phase imaging, it combines holography and tomography to record a three-dimensional map of the refractive index, used as intrinsic optical and quantitative imaging contrast parameter of biological samples, at a sub-micrometer spatial resolution. In this study HT has been employed for the first time to analyze the changes of fibroblasts differentiating towards myofibroblasts - recognized as the main cell player of fibrosis - when cultured in vitro with the pro-fibrotic factor, namely transforming growth factor-β1. In parallel, F-actin, vinculin, α-smooth muscle actin, phospho-myosin light chain 2, type-1 collagen, peroxisome proliferator-activated receptor-gamma coactivator-1α expression and mitochondria were evaluated by confocal laser scanning microscopy. Plasmamembrane passive properties and transient receptor potential canonical channels\' currents were also recorded by whole-cell patch-clamp. The fluorescence images and electrophysiological results have been compared to the data obtained by HT and their congruence has been discussed. HT turned out to be a valid approach to morphologically distinguish fibroblasts from well differentiated myofibroblasts while obtaining objective measures concerning volume, surface area, projection area, surface index and dry mass (i.e., the mass of the non-aqueous content inside the cell including proteins and subcellular organelles) of the entire cell, nuclei and nucleoli with the major advantage to monitor outer and inner features in living cells in a non-invasive, rapid and label-free approach. HT might open up new research opportunities in the field of fibrotic diseases. RESEARCH HIGHLIGHTS: Holotomography (HT) is a label-free laser interferometric imaging technology exploiting the intrinsic optical property of cells namely refractive index (RI) to enable a direct imaging and analysis of whole cells or intracellular organelles. HT turned out a valid approach to distinguish morphological features of living unlabeled fibroblasts from differentiated myofibroblasts. HT provided quantitative information concerning volume, surface area, projection area, surface index and dry mass of the entire fibroblasts/myofibroblasts, nuclei and nucleoli.
摘要：
全断层成像（HT）是一种先进的快速活细胞定量无标记成像技术。基于定量相位成像的原理,它结合了全息和层析成像来记录折射率的三维图，用作生物样品的固有光学和定量成像对比参数，亚微米空间分辨率。在这项研究中，HT首次用于分析成纤维细胞向肌成纤维细胞分化的变化-被认为是纤维化的主要细胞参与者-当与促纤维化因子在体外培养时，即转化生长因子-β1。并行,F-肌动蛋白,维古林,α-平滑肌肌动蛋白，磷酸肌球蛋白轻链2，1型胶原，通过共聚焦激光扫描显微镜评估过氧化物酶体增殖物激活受体-γ共激活因子-1α的表达和线粒体。全细胞膜片钳还记录了质膜被动特性和瞬时受体电位典型通道电流。将荧光图像和电生理结果与HT获得的数据进行了比较，并讨论了它们的一致性。HT被证明是一种有效的方法，可以在形态学上区分成纤维细胞和分化良好的肌成纤维细胞，同时获得有关体积的客观指标。表面积,投影面积,表面指数和干质量(即，细胞内的非水性内容物的质量，包括整个细胞的蛋白质和亚细胞器），细胞核和核仁的主要优点是在非侵入性的活细胞中监测外部和内部特征，快速和无标签的方法。HT可能在纤维化疾病领域开辟新的研究机会。研究重点：全息成像（HT）是一种无标记的激光干涉成像技术，利用细胞的固有光学特性，即折射率（RI），可以对整个细胞或细胞内细胞器进行直接成像和分析。HT证明了一种有效的方法来区分活的未标记成纤维细胞与分化的肌成纤维细胞的形态特征。HT提供了有关体积的定量信息，表面积,投影面积,整个成纤维细胞/肌成纤维细胞的表面指数和干重，细胞核和核仁.
