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

Abstract

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.

Visualization of Germination Proteins in Putative Bacillus cereus Germinosomes

“…In our study, the primary objective was to ascertain whether the GerRB-SGFP2 fusion protein can be visualized and localized in B. cereus dormant spores, and our work showed that what is almost certainly a germinosome can be visualized by its GerRB-SGFP2 fluorescence in almost a third of B. cereus spores ( Figure 2 H). However, the ~30% of B. cereus spores exhibiting a germinosome was much lower than that for germinosomes in B. subtilis spores [ 25 , 26 , 29 ]. Reasons for this large difference in apparent levels of germinosomes are not clear, but there are a number of possibilities including the following.…”

“…In B. subtilis spores, GRs are localized in 1-3 IM foci, termed a germinosome, for which GerD acts as a scaffold for GR assembly [21,24]. Recent work by our laboratory, using super-resolution three-dimensional structured illumination microscopy (3D-SIM) as well as annular Rescan Confocal Microscopy has allowed us to quantify germinosomes in single spores as well as to monitor germinosome dynamics upon germinant addition to wild-type B. subtilis spores [25,26].…”

Structural, Metabolic and Evolutionary Comparison of Bacterial Endospore and Exospore Formation

“…The germinosome comprises germination receptors, scaffolding proteins and other components, such as the SpoVA protein involved in DPA transport ( Griffiths et al, 2011 ). Fluorescence visualization studies reveal that the germinosome localizes at one pole of the dormant endospore, then disperses as germination progresses ( Troiano et al, 2015 ; Breedijk et al, 2020 ; Wang et al, 2020 ). The germination cascade is then followed by degradation of the spore cell wall by lytic enzymes, increasing permeability and allowing Ca 2+ -DPA complexes and ions to escape the endospore in order to be replaced with water from the environment ( Griffiths et al, 2011 ; Wang et al, 2015 ).…”

Structured illumination microscopy and image scanning microscopy: a review and comparison of imaging properties

“…They call the method rescan confocal microscopy. Breedijk et al proposed using annular illumination to improve the transverse resolution [61].…”