Super-resolution confocal cryo-CLEM with cryo-FIB milling for in situ imaging of Deinococcus radiodurans

Abstract

Studying bacterial cell envelope architecture with electron microscopy is challenging due to the poor preservation of microbial ultrastructure with traditional methods. Here, we established and validated a super-resolution cryo-correlative light and electron microscopy (cryo-CLEM) method, and combined it with cryo-focused ion beam (cryo-FIB) milling and scanning electron microscopy (SEM) volume imaging to structurally characterize the bacterium Deinococcus radiodurans. Subsequent cryo-electron tomography (cryo-ET) revealed an unusual diderm cell envelope architecture with a thick layer of peptidoglycan (PG) between the inner and outer membranes, an additional periplasmic layer, and a proteinaceous surface S-layer. Cells grew in tetrads, and division septa were formed by invagination of the inner membrane (IM), followed by a thick layer of PG. Cytoskeletal filaments, FtsA and FtsZ, were observed at the leading edges of constricting septa. Numerous macromolecular complexes were found associated with the cytoplasmic side of the IM. Altogether, our study revealed several unique ultrastructural features of D. radiodurans cells, opening new lines of investigation into the physiology and evolution of the bacterium.

Keywords: Cell envelope architecture; Cryo-CLEM; Cryo-ET; Cryo-FIB; Cryo-super resolution microscopy; Microbial ultrastructure.

Graphical abstract

Fig. 1

Cryo-workflow. A) Bacterial cells were frozen on Finder EM grids with Vitrobot Mark IV. B) Low magnification grid atlases were collected under cryogenic conditions on the ZEISS LSM 900 confocal microscope equipped with an Airyscan 2 detector to identify regions of interest (ROIs). Subsequently, super-resolution fLM images were taken of cells identified in the ROIs. C) The grid was transferred into a cryo-FIB-SEM ZEISS Crossbeam 550 where targets were relocated by correlating SEM with LM images using ZEN Connect. Division sites were targeted using cryo- FIB-SEM volume imaging. 200 ​nm lamellae were milled. D) Cryotomograms of the lamellae were collected on a 300 ​kV Titan Krios TEM.

Fig. 2

Fluorescence LM atlas of a Finder EM grid under cryogenic conditions. Cells were stained with FM4-64 membrane dye, plunge-frozen and maintained cryogenically for imaging. A) A low magnification grid atlas was collected in reflection mode. Regions of interest (ROIs) are boxed in yellow. B) Once targets were identified, z-stacks were collected using the Airyscan 2 detector in super-resolution confocal mode. The maximum intensity projections (MIPs) of four representative z-stacks are shown on the right. Scale bar for A, 100 ​μm. Scale bar for B, 2 ​μm. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Fig. 3

Correlation and images of final lamellae using fLM, SEM and TEM. The EM grid was transferred to the ZEISS Crossbeam 550 and targets were relocated by correlating the aligned map of LM images with SEM images. A) After stage registration, medium magnification SEM images (boxed in green) were collected around the targets and were fine-aligned to the corresponding reflection mode images of the LM map in ZEN Connect. Milled areas are boxed in yellow. B) A low magnification TEM grid atlas. Milling patterns and Finder grid features were used for orientation and target identification (yellow boxes). C) Four representative SEM images of final lamellae. D) Correlation of lamellae with the MIP images (FM4-64 channel) from fLM using ZEN Connect. E) Medium magnification images of the lamellae from the TEM used to correlate subsequent cryotomogram collection. Scale bar for A-B, 100 ​μm. Scale bar for C-E, 5 ​μm. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Fig. 4

3D volume imaging vs cryo-ET for detecting subcellular structures. A) Cryo-FIB-SEM volume imaging revealed the overall cell shape of dividing cells. The cell envelope architecture was poorly discerned. Numerous macromolecular complexes (MC) associated with the IM were clearly visible. Storage granules (SG) were associated with segregating DNA (dashed line). Scale bar, 500 ​nm. B) A slice through the center of a cryotomogram generated using SIRT-like reconstruction and Topaz-Denoise algorithm clearly revealed the PG (∼40 ​nm) and an invaginating IM during cell division. Some MC along the IM (black arrows) were also apparent. Scale bar, 200 ​nm.

Fig. 5

Cell envelope architecture of D. radiodurans revealed by cryo-ET. A) Maximum intensity projection image of ROI1 from Fig. 2. Scale bar, 2 ​μm. B) SEM image of the lamella. Scale bar, 2 ​μm. C) TEM image of the lamella with target area boxed in yellow. Scale bar, 1 ​μm. D) 20 ​nm tomographic slice through the target cell showing the inner membrane (IM), outer membrane (OM), surface layer (S-layer), and peptidoglycan (PG). E) Segmentation of the cell envelope showing the IM in red, OM in blue, PG in green, and S-layer in purple. F) Side (XY) and top (YZ) views of the S-layer revealed a hexagonal diffraction pattern (FT) with 9.7 ​nm spacing. G) Density profile of the cell envelope revealed periplasmic space of ∼90 ​nm with ∼40 ​nm thick PG (green), an additional periplasmic layer (gray), and an OM to S-layer distance of ∼20 ​nm. Scale bar, 200 ​nm for D and E; 50 ​nm for F. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Fig. 6

Ultrastructure of dividing D. radiodurans cells revealed by cryo-ET. A) Maximum intensity projection image of ROI2. Scale bar, 2 ​μm. B) SEM image of lamella post cryo-FIB milling. Scale bar, 2 ​μm. C) TEM image of the lamella with target area boxed in yellow. Scale bar, 1 ​μm. D) 20 ​nm tomographic slice through a target cell showing the inner membrane (IM), peptidoglycan (PG), outer membrane (OM), surface layer (S-layer), storage granules (SG), macromolecular complexes (MC), and FtsA/Z filaments. E) Segmentation of the target cell showing IM in red, OM in blue, SG in light blue, MC in yellow, and FtsA/Z in green. Scale bar, 200 ​nm. F) Images of MC associated with the IM. G) FtsA/Z at the leading edges of constricting IM during cell division. H) YZ view of FtsA/Z filaments along the IM with a model diagram shown in color. Scale bar for F–H, 50 ​nm. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)