Cryogenic electron tomography (cryo-ET) combined with subtomogram averaging, allows in situ visualization and structure determination of macromolecular complexes at subnanometre resolution.... Show moreCryogenic electron tomography (cryo-ET) combined with subtomogram averaging, allows in situ visualization and structure determination of macromolecular complexes at subnanometre resolution. Cryogenic focused ion beam (cryo-FIB) micromachining is used to prepare a thin lamella-shaped sample out of a frozen-hydrated cell for cryo-ET imaging, but standard cryo-FIB fabrication is blind to the precise location of the structure or proteins of interest. Fluorescence-guided focused ion beam (FIB) milling at target locations requires multiple sample transfers prone to contamination, and relocation and registration accuracy is often insufficient for 3D targeting. Here, we present in situ fluorescence microscopy-guided FIB fabrication of a frozen-hydrated lamella to address this problem: we built a coincident three-beam cryogenic correlative microscope by retrofitting a compact cryogenic microcooler, custom positioning stage, and an inverted widefield fluorescence microscope (FM) on an existing FIB scanning electron microscope. We show FM controlled targeting at every milling step in the lamella fabrication process, validated with transmission electron microscope tomogram reconstructions of the target regions. The ability to check the lamella during and after the milling process results in a higher success rate in the fabrication process and will increase the throughput of fabrication for lamellae suitable for high-resolution imaging. Show less
Animals, plants, and fungi live in a microbe-dominated world. Investigating the interactions and processes at the host-microbe interface offers insight to how bacteria influence the development,... Show moreAnimals, plants, and fungi live in a microbe-dominated world. Investigating the interactions and processes at the host-microbe interface offers insight to how bacteria influence the development, health, and disease of the host. Optimization of existing imaging technologies and development of novel instrumentation will provide the tools needed to fully understand the dynamic relationship that occurs at the host-microbe interface throughout the lifetime of the host. In this review, we describe the current methods used in cryo-electron microscopy (cryo-EM) including cryo-fixation, sample processing, FIB-SEM, and cryotomography. Further, we highlight the new advances associated with these methods that open the cryo-EM discipline to large, complex multicellular samples, like symbiotic tissues. We describe the advantages and challenges associated with correlative imaging techniques and sample thinning methods like lift-out. By highlighting recent pioneering studies in the large-volume or symbiotic sample workflows, we provide insight into how symbiotic model systems will benefit from cryo-EM methods to provide artefact-free, near-native, macromolecular-scale resolution imaging at the host-microbe interface throughout the development and maintenance of symbiosis. Cryo-EM methods have brought a deep fundamental understanding of prokaryotic biology since its conception. We propose the application of existing and novel cryo-EM techniques to symbiotic systems is the logical next step that will bring an even greater understanding how microbes interact with their host tissues. Show less
The genus Plasmodium is a unicellular eukaryotic parasite that is the causative agent of malaria, which is transmitted by Anopheline mosquito. There are a total of three developmental stages in the... Show moreThe genus Plasmodium is a unicellular eukaryotic parasite that is the causative agent of malaria, which is transmitted by Anopheline mosquito. There are a total of three developmental stages in the production of haploid parasites in the Plasmodium life cycle: the oocyst stage in mosquitoes and the liver and blood stages in mammalian hosts. The Plasmodium oocyst stage plays an important role in the production of the first generation of haploid parasites. Nuclear division is the most important event that occurs during the proliferation of all eukaryotes. However, obtaining the details of nuclear division at the oocyst stage is challenging owing to difficulties in preparation. In this study, we used focused-ion-beam-milling combined with scanning-electron-microscopy to report the 3D architecture during nuclear segregations in oocyst stage. This advanced technology allowed us to analyse the 3D details of organelle segregation inside the oocyst during sporogony formation. It was revealed that multiple nuclei were involved with several centrosomes in one germ nucleus during sporozoite budding (endopolygeny). Our high-resolution 3D analysis uncovered the endopolygeny-like nuclear architecture of Plasmodium in the definitive host. This nuclear segregation was different from that in the blood stage, and its similarity to other apicomplexan parasite nuclear divisions such as Sarcocystis is discussed. Show less
