论文标题
中红外光热荧光原位杂交用于功能分析和遗传鉴定单细胞
Mid-Infrared Photothermal-Fluorescence in Situ Hybridization for Functional Analysis and Genetic Identification of Single Cells
论文作者
论文摘要
需要对具有单细胞分辨率和高吞吐量的微生物的同时识别和代谢分析，以回答“谁在复杂的微生物群落中吃什么，何时和何处”的问题。在这里，我们提出了一个中红外光热荧光原位杂交（MIP-FISH）平台，该平台可以直接桥接基因型和表型。通过对MIP成像的多次改进，可以对掺入单个细菌细胞蛋白的同位素标记化合物的敏感检测变得可能是可能的，而同时用rRNA靶向的探针同时检测了鱼类标记的探针，从而使分析细胞的鉴定能够鉴定。在概念验证实验中，我们表明，由于$^{13} $ c原子的合并，蛋白质酰胺I区域的清晰频谱变化源自$^{13} $ c-c-labelled-葡萄糖，可以通过mip-fish来利用ip-fish以区分和识别$^{13} $ c-labell babell exteremem semples semplem semplect in n off complect human ob gut gut gut。所提供的方法为微生物学的单细胞结构 - 功能分析开辟了新的机会。
Simultaneous identification and metabolic analysis of microbes with single-cell resolution and high throughput is necessary to answer the question of "who eats what, when, and where" in complex microbial communities. Here, we present a mid-infrared photothermal-fluorescence in situ hybridization (MIP-FISH) platform that enables direct bridging of genotype and phenotype. Through multiple improvements of MIP imaging, the sensitive detection of isotopically-labelled compounds incorporated into proteins of individual bacterial cells became possible, while simultaneous detection of FISH labelling with rRNA-targeted probes enabled the identification of the analyzed cells. In proof-of-concept experiments, we showed that the clear spectral red shift in the protein amide I region due to incorporation of $^{13}$C atoms originating from $^{13}$C-labelled-glucose can be exploited by MIP-FISH to discriminate and identify $^{13}$C-labelled bacterial cells within a complex human gut microbiome sample. The presented methods open new opportunities for single-cell structure-function analyses for microbiology.
