Abstract
Understanding how bacteria grow and divide requires insight into both the molecular-level dynamics of ultrastructure and the chemistry of the constituent components. Atomic force microscopy (AFM) can provide near molecular resolution images of biological systems but typically provides limited chemical information. Conversely, while super-resolution optical microscopy allows localization of particular molecules and chemistries, information on the molecular context is difficult to obtain. Here, we combine these approaches into STORMForce (stochastic optical reconstruction with atomic force microscopy) and the complementary SIMForce (structured illumination with atomic force microscopy), to map the synthesis of the bacterial cell wall structural macromolecule, peptidoglycan, during growth and division in the rod-shaped bacterium Bacillus subtilis. Using “clickable” d-amino acid incorporation, we fluorescently label and spatially localize a short and controlled period of peptidoglycan synthesis and correlate this information with high-resolution AFM of the resulting architecture. During division, septal synthesis occurs across its developing surface, suggesting a two-stage process with incorporation at the leading edge and with considerable in-filling behind. During growth, the elongation of the rod occurs through bands of synthesis, spaced by ∼300 nm, and corresponds to denser regions of the internal cell wall as revealed by AFM. Combining super-resolution optics and AFM can provide insights into the synthesis processes that produce the complex architectures of bacterial structural biopolymers.
Highlights
Understanding how bacteria grow and divide requires insight into both the molecular-level dynamics of ultrastructure and the chemistry of the constituent components
We converted the inverted optical microscope beneath a JPK Nanowizard III Atomic force microscopy (AFM) into a Stochastic optical reconstruction microscopy (STORM) through the addition of a 70 mW 642 nm laser as a light source, routed to the microscope via an optical fiber to remove noise associated with open optics that may couple to the AFM, and an EM-charge coupled device (CCD) Hamamatsu camera to collect the single molecule optical signal associated with stochastic blinking of fluorophores (Figure 1A and Methods)
To confirm data obtained with STORMForce, and to improve throughput, we developed an approach for correlative structured illumination microscopy (SIM) with AFM (SIMForce)
Summary
Understanding how bacteria grow and divide requires insight into both the molecular-level dynamics of ultrastructure and the chemistry of the constituent components. While super-resolution optical microscopy allows localization of particular molecules and chemistries, information on the molecular context is difficult to obtain We combine these approaches into STORMForce (stochastic optical reconstruction with atomic force microscopy) and the complementary SIMForce (structured illumination with atomic force microscopy), to map the synthesis of the bacterial cell wall structural macromolecule, peptidoglycan, during growth and division in the rod-shaped bacterium Bacillus subtilis. Image overlay was initially performed through use of fiducial markers (see Methods), but it was found that in samples with relatively dense sample dispersion, alignment was possible by direct overlay (“dead reckoning” based on colocation of the two imaging modalities) and adjustment to ensure good correlation across the resulting STORMForce image
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
