Label-Free Quantitative In Vitro Live Cell Imaging with Digital Holographic Microscopy
Label-Free Quantitative In Vitro Live Cell Imaging with Digital Holographic Microscopy
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DOI：
10.1007/11663_2019_6
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发表时间：
2019
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通讯作者：
B. Kemper;Andreas Bauwens;D. Bettenworth;M. Götte;B. Greve;L. Kastl;S. Ketelhut;P. Lenz;S. Mues;J. Schnekenburger;Angelika Vollmer
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作者：
B. Kemper;Andreas Bauwens;D. Bettenworth;M. Götte;B. Greve;L. Kastl;S. Ketelhut;P. Lenz;S. Mues;J. Schnekenburger;Angelika Vollmer
利用光学显微镜对活细胞培养物进行无标记定量体外成像是生命科学中各个研究领域的重要工具。数字全息显微镜（DHM）提供非接触式、微创的定量相衬成像，并可作为一个模块集成在普通的研究显微镜中。由于定量相位图像的数值再现，从单个数字全息图实现了多焦点成像。对记录的定量相衬图像的评估允许提取用于简化的对象跟踪和图像分割的数据。数字自动对焦的特殊DHM功能避免了机械焦点重新对准。由于定量DHM相位成像是基于检测透射中的光程长度变化，因此该方法仅需要用于物体照明的低光强度，这最小化了与样品的相互作用。因此，微创长期延时调查定量监测细胞形态，运动和增殖的动态变化是可访问的。此外，积分细胞折射率，这是相关的细胞内溶质浓度以及细胞体积和干质量，是可用的。本章首先介绍了DHM活细胞观察和程序的生物物理参数的提取定量DHM相衬图像。物理基础已经奠定后，在体外活细胞分析的几个选定的应用程序进行了说明。这包括悬浮细胞和球形细胞内细胞器的表征以及对渗透刺激、药物、毒素、纳米材料和遗传修饰的细胞反应的定量。随后的段落说明了DHM如何应用于量化细胞运动性、迁移和贴壁细胞培养物的形态。最后，基于细胞厚度测定的表型，细胞生长，增殖和伤口愈合的动态多模态成像，以及在病原体毒性测试和细胞纳米材料相互作用的表征中的应用得到了证明。
Label-free quantitative in vitro imaging of living cell cultures with light microscopy is an important tool for various research fields in the life sciences. Digital holographic microscopy (DHM) provides contactless, minimally invasive quantitative phase contrast imaging and can be integrated as a module in common research microscopes. Due to the numerical reconstruction of quantitative phase images, multi-focus imaging is achieved from a single digital hologram. The evaluation of the recorded quantitative phase contrast images allows the extraction of data for simplified object tracking and image segmentation. The special DHM feature of numerical autofocusing avoids mechanical focus realignment. As quantitative DHM phase imaging is based on the detection of optical path length changes in transmission, the method only requires low light intensities for object illumination which minimizes the interaction with the sample. Thus, minimally invasive long-term time-lapse investigations for quantitative monitoring of dynamic changes of cell morphology, motility, and proliferation are accessible. In addition, the integral cellular refractive index, which is related to intracellular solute concentrations as well as cellular volume and dry mass, is available. The chapter starts with an introduction to DHM for live cell observation and procedures for the extraction of biophysical parameters from quantitative DHM phase contrast images. After the physical basis has been laid out, several selected applications of in vitro live cell analysis are described. This includes the characterization of suspended cells and spherical intracellular organelles as well as the quantification of the cellular response to osmotic stimulation, drugs, toxins, nanomaterials, and genetic modifications. Subsequent paragraphs illustrate how DHM can be applied to quantify cell motility, migration, and the morphology of adherent cell cultures. Finally, phenotyping based on cell thickness determination, dynamic multimodal imaging of cellular growth, proliferation, and wound healing in vitro as well as applications in toxicity testing of pathogens and the characterization of cell nanomaterial interactions are demonstrated.