School of Physics and Astronomy , Cardiff University , The Parade , Cardiff CF24 3AA , United Kingdom.
Division of Cancer and Genetics, School of Medicine , Cardiff University , Heath Park , Cardiff CF14 4XN , United Kingdom.
Anal Chem. 2019 Feb 19;91(4):2813-2821. doi: 10.1021/acs.analchem.8b04706. Epub 2019 Jan 29.
Quantifying the chemical composition of unstained intact tissue and cellular samples with high spatio-temporal resolution in three dimensions would provide a step change in cell and tissue analytics critical to progress the field of cell biology. Label-free optical microscopy offers the required resolution and noninvasiveness, yet quantitative imaging with chemical specificity is a challenging endeavor. In this work, we show that hyperspectral coherent anti-Stokes Raman scattering (CARS) microscopy can be used to provide quantitative volumetric imaging of human osteosarcoma cells at various stages through cell division, a fundamental component of the cell cycle progress resulting in the segregation of cellular content to produce two progeny. We have developed and applied a quantitative data analysis method to produce volumetric three-dimensional images of the chemical composition of the dividing cell in terms of water, proteins, DNAP (a mixture of proteins and DNA, similar to chromatin), and lipids. We then used these images to determine the dry masses of the corresponding organic components. The attribution of proteins and DNAP components was validated using specific well-characterized fluorescent probes, by comparison with correlative two-photon fluorescence microscopy of DNA and mitochondria. Furthermore, we map the same chemical components under perturbed conditions, employing a drug that interferes directly with cell division (Taxol), showing its influence on cell organization and the masses of proteins, DNAP, and lipids.
以高时空分辨率量化未经染色的完整组织和细胞样本的化学成分，将为细胞和组织分析领域带来重大进展，这对细胞生物学的发展至关重要。无标记光学显微镜提供了所需的分辨率和非侵入性，但具有化学特异性的定量成像仍然是一项具有挑战性的任务。在这项工作中，我们表明，超光谱相干反斯托克斯拉曼散射（CARS）显微镜可用于通过细胞分裂对人骨肉瘤细胞的各个阶段进行定量体积成像，细胞分裂是细胞周期进展的基本组成部分，导致细胞内容物的分离以产生两个后代。我们已经开发并应用了一种定量数据分析方法，以根据水、蛋白质、DNAP（一种类似于染色质的蛋白质和 DNA 的混合物）和脂质来生成分裂细胞的化学组成的体积三维图像。然后，我们使用这些图像来确定相应有机成分的干质量。通过与 DNA 和线粒体的相关双光子荧光显微镜比较，使用特定的、特征明确的荧光探针验证了蛋白质和 DNAP 成分的归属。此外，我们在受到干扰的条件下绘制相同的化学成分，使用直接干扰细胞分裂的药物（紫杉醇），显示其对细胞组织和蛋白质、DNAP 和脂质质量的影响。
