FtsZ is a tubulin ortholog that is highly conserved in bacterial species. This GTPase is an essential protein that polymerizes at the mid-cell, recruits the division machinery, and may generate constrictive forces necessary for cytokinesis.
FtsZ polymerizes into a midplane ring structure (the Z-ring) early in the cell cycle before recruiting the rest of the divisome. In Caulobacter crescentus
, FtsZ also recruits proteins that direct cell elongation and cellular polarity.
Given its importance in cell division, the Z-ring structure is the subject of active investigation, but its subcellular and subwavelength dimensions prevent visualization with conventional fluorescence measurements.
Short FtsZ protofilaments were observed at constriction sites in C. crescentus
by cryo-electron tomography (ET),
indicating that the Z-ring structure is not simply a smooth, closed-ring-like structure, but a collection of overlapping protofilaments. Super-resolution (SR) optical imaging techniques are therefore ideally suited for the investigation of the “superstructure”, or assembly, formed from the FtsZ filaments.
In single-molecule fluorescence (SMF) imaging, the emission from spatially isolated fluorophores yields high-accuracy information about the position of that fluorophore because the image of a 3–5 nm fluorescent protein (FP) is a reasonable approximation to the point-spread function (PSF) of the microscope.
While rigorously a single molecule is an emitting dipole, in practice FP fusions are orientationally mobile, so that the diffraction-limited single-molecule spot in a wide-field fluorescence image can be numerically fit to estimate the xy
position of the emitter. This technique permits sub-diffraction-limited information to be obtained from diffraction-limited images when single molecules are spaced apart by more than the optical diffraction limit. Applied to live bacterial cells, wide-field SMF microscopy has enabled high-resolution investigations of biomolecule dynamics. For instance, bursts of fluorescence have identified low-level gene expression,
and single-molecule tracking experiments have measured the diffusion coefficient of free, cytoplasmic protein and the velocity of motion of polymerized protein.
Additionally, freely diffusing cytoplasmic proteins can be captured using very fast acquisition times, however when employing longer acquisition times these fast-moving molecules are not resolved, biasing the camera toward molecules which form quasi-static structures which are stable on the time scale of the imaging.
Herein, live-cell SMF imaging is used to characterize the polymerization/depolymerization dynamics of the protein FtsZ, in three spatial dimensions.
With densely packed fluorescent labels, super-resolution (SR) images can be reconstructed from movies where sparse subsets of the molecules are sequentially localized in each movie frame. Importantly, the active emitter density in any frame can be controlled at a low level by photoswitching, photoactivation, cellular dynamics, chemical control, transient dark states, etc.[8–13]
Single-molecule SR imaging based on photoactivated localization microscopy (PALM) has recently been applied to investigations of the FtsZ superstructure in Escherichia coli,
demonstrating that the Z-ring of E. coli
is a loose bundle of protofilaments.
An alternative SR fluorescence imaging technique, stimulated emission depletion (STED) microscopy, has yielded consistent results for FtsZ rings in fixed Bacillus subtilis
cells stained with an anti-FtsZ antibody.
A variety of SR techniques have been applied to biomolecular structures in cells. Wide-field cellular imaging is generally limited to the thin surface layer next to the coverslip by the need to use a total-internal reflection (TIRF) microscope geometry, though thicker cells have been examined with Bessel beams and two-photon selective excitation.
Due to their thin (~micron-diameter) size, the entirety of bacterial cells can be imaged in wide-field without TIRF.
Still, most experiments have been limited to a two-dimensional projection of the three-dimensional sample. Methods to extend wide-field, single-molecule-based SR imaging to three dimensions include astigmatism,[17,18]
and double-helix point-spread function microscopy.
Here, we use optical astigmatism to gain axial (z) information for each single-molecule fit.[17,18]
The astigmatism is introduced via a weak cylindrical lens in the emission pathway of the wide-field single-molecule microscope. In the presence of this lens, the detected PSF from a single-molecule is asymmetric, and in addition to localizing the molecule in the xy
plane based on the center of the PSF, the degree of PSF asymmetry indicates the axial position of the molecule. For fluorescent protein fusions in C. crescentus
cells, the ability of the astigmatic single-molecule microscope to resolve axial molecular positions with <100 nm z
localization precision promises to shed light on macromolecular structure in three dimensions.