Positive-Charge-Based Small Molecule Dyes for Gut Microbiota Fluorescent Imaging

As one of the research hotspots in recent years, gut microbiota have been proven to be closely related to host metabolism, nutrient absorption, and immune regulation. However, there are still many urgent issues in the research of gut microbiota, such as the localization and tracking of gut microbiota. In this research, two new fluorescent probes, EF and 6F, were developed by optimizing the structure of the positron salt small molecule probe F16. <i>In vitro</i> labeling experiments showed that EF and 6F can quickly label Gram-positive bacteria, <i>Staphylococcus aureus</i> and <i>Lactobacillus reuteri</i>, as well as Gram-negative bacteria, <i>Escherichia coli</i> and <i>Salmonella pullorum</i>. Meanwhile, EF and 6F have little bacterial toxicity and are used at a maximum concentration of 200 μM. Compared with EF, 6F has better hydrophilicity and stronger fluorescence characteristics in aqueous solutions, making it more suitable for imaging within gut microbiota populations. The results of <i>in vivo</i> imaging experiments indicate that EF and 6F can label and image the intestinal microbiota colonized by the mouse intestinal mucosal epithelium without causing any damage to intestinal tissue. Compared with commercially available MitoTracker dyes and fluorescein 5-isothiocyanate (FITC) dyes, EF and 6F exhibit better biocompatibility. Therefore, the compounds EF and 6F synthesized in this study are novel small molecule probes suitable for imaging gut microbiota, providing a better probe selection for exploring complex gut microbiota.