Friedrich Schiller University Jena, Institute of Physical Chemistry and Abbe Center of Photonics, Member of the Leibniz Center for Photonics in Infection Research, 07743 Jena, Germany.
Leibniz Institute of Photonic Technology, Member of Leibniz Research Alliance Leibniz Health Technologies, Member of the Leibniz Center for Photonics in Infection Research, 07745 Jena, Germany.
Analyst. 2023 Nov 6;148(22):5627-5635. doi: 10.1039/d3an00902e.
Major drawbacks of direct mid-infrared spectroscopic imaging of single cells in an aqueous buffer are strong water absorption, low resolution typically above 10 μm, and Mie scattering effects. This study demonstrates how an indirect detection principle can overcome these drawbacks using the optical photothermal infrared (O-PTIR) technique for high-resolution discrete wavenumber imaging and fingerprint spectroscopy of cultivated cells as a model system in a simple liquid sample chamber. The O-PTIR spectra of six leukemia- and cancer-derived cell lines showed main IR bands near 1648, 1547, 1447, 1400, 1220, and 1088 cm. Five spectra of approximately 260 single cells per cell type were averaged, the O-PTIR data set was divided into leukemia-derived cells (THP-1, HL 60, Jurkat, and Raji) and cancer cells (HeLa and HepaRG), and partial least squares linear discriminant analysis (PLS-LDA) was applied in the spectral range 800-1800 cm to train three classification models. A leukemia cancer cell model showed an accuracy of 90.0%, the HeLa HepaRG cell model had an accuracy of 95.4%, and the model for the distinction of leukemia cells had an accuracy of 75.4%. IR bands in linear discriminants (LDs) of the models were correlated with second derivative spectra that resolved more than 25 subbands. The IR and second derivative spectra of proteins, DNA, RNA and lipids were collected as references to confirm band assignments. O-PTIR images of single cells at a 200 nm step size were acquired at 1086, 1548, and 1746 cm to visualize the nucleic acid, protein, and lipid distribution, respectively. Variations in subcellular features and in the lipid-to-protein and nucleic acid-to-protein ratios were identified that were consistent with biomolecular information in LDs. In conclusion, O-PTIR can provide high-quality spectra and images with submicron resolution of single cells in aqueous buffers that offer prospects in high-content screening applications.
直接在水缓冲液中对单个细胞进行中红外光谱成像的主要缺点是强水吸收、典型的高分辨率超过 10 μm 和米氏散射效应。本研究展示了如何使用光学光热红外（O-PTIR）技术克服这些缺点，该技术用于高分辨率离散波数成像和指纹光谱学，以培养细胞作为简单液体样品室中的模型系统。六种白血病和癌症衍生细胞系的 O-PTIR 光谱在 1648、1547、1447、1400、1220 和 1088 cm 附近显示主要 IR 带。对每种细胞类型的大约 260 个单个细胞的 5 个光谱进行平均，将 O-PTIR 数据集分为白血病衍生细胞（THP-1、HL 60、Jurkat 和 Raji）和癌细胞（HepaRG 和 HeLa），并在 800-1800 cm 的光谱范围内应用偏最小二乘线性判别分析（PLS-LDA）来训练三个分类模型。白血病-癌症细胞模型的准确性为 90.0%，HepaRG-HeLa 细胞模型的准确性为 95.4%，白血病细胞区分模型的准确性为 75.4%。模型中线性判别（LD）的 IR 带与分辨出超过 25 个子带的二阶导数光谱相关。收集蛋白质、DNA、RNA 和脂质的 IR 和二阶导数光谱作为参考，以确认带分配。以 200 nm 的步长在 1086、1548 和 1746 cm 处获取单个细胞的 O-PTIR 图像，分别可视化核酸、蛋白质和脂质的分布。确定了亚细胞特征以及脂质-蛋白质和核酸-蛋白质比的变化，这些变化与 LD 中的生物分子信息一致。总之，O-PTIR 可以提供具有亚微米分辨率的单个细胞的高质量光谱和图像，为高内涵筛选应用提供了前景。
