Label-free three-dimensional imaging and quantitative analysis of living fibroblasts and myofibroblasts by holotomographic microscopy

IF 2 3区工程技术 Q2 ANATOMY & MORPHOLOGY

Microscopy Research and Technique Pub Date : 2024-07-10 DOI:10.1002/jemt.24648

Francesca Sbrana, Flaminia Chellini, Alessia Tani, Martina Parigi, Rachele Garella, Francesco Palmieri, Sandra Zecchi-Orlandini, Roberta Squecco, Chiara Sassoli

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引用次数: 0

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.

Abstract Image

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利用全息显微镜对活体成纤维细胞和肌成纤维细胞进行无标记三维成像和定量分析。

全息成像（HT）是一种尖端的快速活细胞定量无标记成像技术。它以定量相位成像原理为基础，结合全息成像和断层成像技术，以亚微米级的空间分辨率记录折射率的三维图，折射率是生物样本的内在光学和定量成像对比参数。在这项研究中，HT 被首次用于分析纤维母细胞向肌成纤维细胞分化过程中的变化，肌成纤维细胞被认为是纤维化的主要细胞角色，在体外与促纤维化因子（即转化生长因子-β1）一起培养。与此同时，共聚焦激光扫描显微镜还对 F-肌动蛋白、长春新碱、α-平滑肌肌动蛋白、磷酸肌球蛋白轻链 2、1 型胶原蛋白、过氧化物酶体增殖激活受体-γ 辅激活剂-1α 的表达和线粒体进行了评估。全细胞膜片钳还记录了质膜被动特性和瞬态受体电位通道电流。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，可从形态上区分成纤维细胞和分化良好的肌成纤维细胞，同时获得有关整个细胞、细胞核和核小体的体积、表面积、投影面积、表面指数和干质量（即细胞内非水性内容物的质量，包括蛋白质和亚细胞器）的客观测量值，其主要优点是以非侵入性、快速和无标记的方法监测活细胞的内外特征。HT 可能会为纤维化疾病领域带来新的研究机会。研究亮点： Holotomography（HT）是一种无标记激光干涉成像技术，它利用细胞固有的光学特性，即折射率（RI），对整个细胞或细胞内的细胞器进行直接成像和分析。HT 是区分活的未标记成纤维细胞和已分化的肌成纤维细胞形态特征的有效方法。HT 提供了有关整个成纤维细胞/肌成纤维细胞、细胞核和核小体的体积、表面积、投影面积、表面指数和干质量的定量信息。

本文章由计算机程序翻译，如有差异，请以英文原文为准。

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来源期刊

Microscopy Research and Technique 医学-解剖学与形态学

CiteScore

5.30

自引率

20.00%

发文量

233

审稿时长

4.7 months

期刊介绍： Microscopy Research and Technique (MRT) publishes articles on all aspects of advanced microscopy original architecture and methodologies with applications in the biological, clinical, chemical, and materials sciences. Original basic and applied research as well as technical papers dealing with the various subsets of microscopy are encouraged. MRT is the right form for those developing new microscopy methods or using the microscope to answer key questions in basic and applied research.