PDF(Pubmed)
Abstract：
BACKGROUND: Inclusion bodies (IBs) are well-known subcellular structures in bacteria where protein aggregates are collected. Various methods have probed their structure, but single-cell spectroscopy remains challenging. Atomic Force Microscopy-based Infrared Spectroscopy (AFM-IR) is a novel technology with high potential for the characterisation of biomaterials such as IBs.
RESULTS: We present a detailed investigation using AFM-IR, revealing the substructure of IBs and their variation at the single-cell level, including a rigorous optimisation of data collection parameters and addressing issues such as laser power, pulse frequency, and sample drift. An analysis pipeline was developed tailored to AFM-IR image data, allowing high-throughput, label-free imaging of more than 3500 IBs in 12,000 bacterial cells. We examined IBs generated in Escherichia coli under different stress conditions. Dimensionality reduction analysis of the resulting spectra suggested distinct clustering of stress conditions, aligning with the nature and severity of the applied stresses. Correlation analyses revealed intricate relationships between the physical and morphological properties of IBs.
CONCLUSIONS: Our study highlights the power and limitations of AFM-IR, revealing structural heterogeneity within and between IBs. We show that it is possible to perform quantitative analyses of AFM-IR maps over a large collection of different samples and determine how to control for various technical artefacts.
RESULTS: We present a detailed investigation using AFM-IR, revealing the substructure of IBs and their variation at the single-cell level, including a rigorous optimisation of data collection parameters and addressing issues such as laser power, pulse frequency, and sample drift. An analysis pipeline was developed tailored to AFM-IR image data, allowing high-throughput, label-free imaging of more than 3500 IBs in 12,000 bacterial cells. We examined IBs generated in Escherichia coli under different stress conditions. Dimensionality reduction analysis of the resulting spectra suggested distinct clustering of stress conditions, aligning with the nature and severity of the applied stresses. Correlation analyses revealed intricate relationships between the physical and morphological properties of IBs.
CONCLUSIONS: Our study highlights the power and limitations of AFM-IR, revealing structural heterogeneity within and between IBs. We show that it is possible to perform quantitative analyses of AFM-IR maps over a large collection of different samples and determine how to control for various technical artefacts.
摘要：
背景：包涵体（IB）是细菌中收集蛋白质聚集体的众所周知的亚细胞结构。各种方法探测了它们的结构，但是单细胞光谱学仍然具有挑战性。基于原子力显微镜的红外光谱（AFM-IR）是一种新型技术，具有很高的潜力，可用于表征IBs等生物材料。
结果：我们使用AFM-IR进行了详细的调查，揭示了IBs的子结构及其在单细胞水平上的变化，包括严格优化数据收集参数和解决激光功率等问题，脉冲频率,和样本漂移。针对AFM-IR图像数据开发了分析管道，允许高吞吐量，在12,000个细菌细胞中对3500多个IBs进行无标签成像。我们检查了在不同胁迫条件下在大肠杆菌中产生的IBs。对所得光谱的降维分析表明，应力条件具有明显的聚类，与所施加应力的性质和严重程度保持一致。相关分析揭示了IBs的物理和形态特性之间的复杂关系。
结论：我们的研究强调了AFM-IR的功效和局限性，揭示了IBs内部和之间的结构异质性。我们表明，可以对大量不同样品进行AFM-IR图的定量分析，并确定如何控制各种技术伪影。
结果：我们使用AFM-IR进行了详细的调查，揭示了IBs的子结构及其在单细胞水平上的变化，包括严格优化数据收集参数和解决激光功率等问题，脉冲频率,和样本漂移。针对AFM-IR图像数据开发了分析管道，允许高吞吐量，在12,000个细菌细胞中对3500多个IBs进行无标签成像。我们检查了在不同胁迫条件下在大肠杆菌中产生的IBs。对所得光谱的降维分析表明，应力条件具有明显的聚类，与所施加应力的性质和严重程度保持一致。相关分析揭示了IBs的物理和形态特性之间的复杂关系。
结论：我们的研究强调了AFM-IR的功效和局限性，揭示了IBs内部和之间的结构异质性。我们表明，可以对大量不同样品进行AFM-IR图的定量分析，并确定如何控制各种技术伪影。
