Perspectives
SRM and SMT are rapidly developing technologies that are expected to allow major advances in understanding bacterial cell biology in the future. 1) Live-STED microscopy has the potential to image fluorescently labeled molecules during complex bacterial processes with up to 5-times better resolution than conventional live cell microscopy (Stockhammer, 2020). 2) New developments and optimizations of fluorescent probes (e.g., concerning on-/off-switching properties, brightness), especially for MINFLUX nanoscopy, will increase the versatility and flexibility of the method, e.g., in multicolor and live imaging as well as SMT (Remmel et al., 2023). 3) Combining MINFLUX nanoscopy with a PAINT-labeling approach may enable parallel imaging of three or more molecules of interest (Ostersehlt et al., 2022). 4) Advanced labeling approaches of molecules may also open up unforeseen methodological options. E.g., the internal ALFA tag in Y. enterocolitica YopD (see above) can be bound by fluorescent nanobodies added extracellularly during a bacterial cell infection, allowing the kinetics of T3SS pore assembly and disassembly to be visualized in living bacteria and host cells (Rudolph et al., 2022).
Finally, the ability of MINFLUX tracking to directly observe the movements of single molecules promises new insights into the structure-function relationship of complex molecular processes in living bacterial and host cells. Particularly the spatiotemporal resolution that could be achieved with this technology was something that could not have been dreamed of not so long ago (Deguchi et al., 2023, Wolff et al., 2023).