Label-Free Identification of Spore-Forming Bacteria Using Ultrabroadband Multiplex Coherent Anti-Stokes Raman Scattering Microspectroscopy
Label-Free Identification of Spore-Forming Bacteria Using Ultrabroadband Multiplex Coherent Anti-Stokes Raman Scattering Microspectroscopy
复制标题
使用超宽带多重相干反斯托克斯拉曼散射显微光谱法无标记鉴定孢子形成细菌
DOI：
10.1021/acs.jpcb.2c07291
复制
发表时间：
2023
期刊：
影响因子：
--
通讯作者：
Kano Hideaki
中科院分区：
文献类型：
--
作者：
Tanaka Kyosuke;Oketani Ryosuke;Terada Takeshi;Leproux Philippe;Morono Yuki;Kano Hideaki
孢子形成细菌积累吡啶二羧酸（DPA）以形成孢子，从而在极端环境中生存。振动光谱被广泛用于检测DPA和阐明细菌的存在，而营养细胞，另一种形式的孢子形成细菌，尚未被广泛研究。在此，我们应用相干反斯托克斯拉曼散射（汽车）显微镜光谱识别孢子和营养细胞没有染色或分子标记。通过DPA产生的强汽车信号鉴定孢子。此外，我们在汽车图像1735 cm-1处观察到营养细胞中的亮点。营养细胞中含有C=O键的分子种类，因为这种振动模式与羰基有关。候选分子种类之一是二酮庚二酸（DKP），DPA前体。通过比较营养细胞获得的光谱与DKP类似物（酮庚二酸）的光谱以及DFT计算获得的结果，验证了这一假设。结果表明，所观察到的营养细胞处于产孢过程中。汽车光谱可用于监测孢子的成熟和预形成。
Spore-forming bacteria accumulate dipicolinic acid (DPA) to form spores to survive in extreme environments. Vibrational spectroscopy is widely used to detect DPA and elucidate the existence of the bacteria, while vegetative cells, another form of spore-forming bacteria, have not been studied extensively. Herein, we applied coherent anti-Stokes Raman scattering (CARS) microscopy to spectroscopically identify both spores and vegetative cells without staining or molecular tagging. The spores were identified by the strong CARS signals due to DPA. Furthermore, we observed bright spots in the vegetative cells in the CARS image at 1735 cm–1. The vegetative cells contained molecular species with C=O bonds because this vibrational mode was associated with the carbonyl group. One of the candidate molecular species is diketopimelic acid (DKP), a DPA precursor. This hypothesis was verified by comparing the spectrum obtained by the vegetative cells with that of the DKP analogue (ketopimelic acid) and with the result obtained by DFT calculation. The results indicate that the observed vegetative cell is in the sporulation process. CARS spectra can be used to monitor the maturation and preformation of spores.