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).