A live-cell super-resolution technique demonstrated by imaging germinosomes in wild-type bacterial spores.

Time-lapse fluorescence imaging of live cells at super-resolution remains a challenge, especially when the photon budget is limited. Current super-resolution techniques require either the use of special exogenous probes, high illumination doses or multiple image acquisitions with post-processing or combinations of the aforementioned. Here, we describe a new approach by combining annular illumination with rescan confocal microscopy. This optics-only technique generates images in a single scan, thereby avoiding any potential risks of reconstruction related artifacts. The lateral resolution is comparable to that of linear structured illumination microscopy and the axial resolution is similar to that of a standard confocal microscope. As a case study, we present super-resolution time-lapse imaging of wild-type Bacillus subtilis spores, which contain low numbers of germination receptor proteins in a focus (a germinosome) surrounded by an autofluorescent coat layer. Here, we give the first evidence for the existence of germinosomes in wild-type spores, show their spatio-temporal dynamics upon germinant addition and visualize spores coming to life.