# Recent Advances in Bacterial Persistence Mechanisms
## 5 Recent Progress of Technologies on Persister Studies
### From the Single-Cell Level  
Considering the low frequency and heterogeneity of persisters in the bacterial population, single-cell sequencing is another promising technique to study persisters. Previously, it has been used for antibiotic resistance studies [7; 24; 68; 94]. Specifically, single-bacteria RNA-seq exhibits great potential in persister phenotypic exploration [95], since it allows for investigation under different environments and provides huge data to better understand the developmental trajectory of the persistent bacteria. A new technology named BacDrop, which was based on the microdroplet technique for bacterial single-cell RNA-seq, enables multiplexing and massively parallel sequencing [96]. The author applied BacDrop to evaluate the heterogeneity in _K. pneumoniae_, depicting single-cell responses to various antibiotics. Regarding meropenem, most of the strong heterogeneous transcriptomic responses driven by different stress responses were observed, which were hidden behind bulk RNA-seq results before. [PERSON] et al. developed single-cell Raman spectroscopy based on D2O to evaluate the biochemical properties of _E. coli_ and its persisters [88]. Persister formation was induced by ampicillin of a high concentration. A significant difference in main cell components and metabolites was found between Raman spectral bands of persisters and untreated _E. coli_. According to the D2O absorption, the persister showed higher metabolic activity than untreated _E. coli_. It was consistent with the experiment conducted using a similar technique to explore the metabolic activity of persisters in mycobacteria [97]. These results demonstrated that persister cells showed distinct metabolic activity in the presence of the antibiotic. Compared to mass spectrometry, which is often used in proteome and metabolome analysis [98], Raman spectroscopy does not destroy samples, allowing for further downstream analyses [95]. Similarly, since most microdevices used are of a closed system nature, making the recovery and subsequent investigation of persister cells difficult, a directly accessible femtoliter droplet array was developed for persister collection and identification at the single-cell level [94; 99]. Moreover, this array could also be used as a single-cell drug efflux assay to screen for pump inhibitors [99]. The fluorescent technique is a classical method in scientific research, which can track the spatial information and quantitative traits of the target in real time. Based on fluorescence dilution, the dynamics of _S. aureus_ can be monitored at the single-cell level, providing evidence for the intracellular _S. aureus_ of a non-growing phenotype [7]. The _S. aureus_ was transformed with tetracycline-inducible green fluorescent protein (GFP), which allowed the monitoring of the decrease in GFP signal in individual cells to track cell lysis after the removal of the inducing agent. Persisters can be observed at a single-cell resolution, utilizing single-cell microscopy coupled with a glass-bottom dish and a nutrient agarose pad [100]. The result suggested that when transferred to fresh broth during the stationary phase, the persister showed a longer lag time, which suggested this subpopulation would still be in a non-dividing state for a long time. Time-lapse microscopy is one of the most ancient ways of observing cell replicative rates at the single-cell level [101]. Recently, using microfluidics coupled to fluorescence microscopy, researchers have tracked and monitored wild-type _E. coli_ during stationary growth to analyze persister formation at the single-cell level [68]. The other microfluidics-based techniques include the linear-colony device, which enables the study of individual cells [102]. Compared to nutrient agarose pads used in single-cell microscopy, linear-colony microfluidics can be changed instantly, making it easier to track cells throughout the entire experiment.