Cryo-correlative Light Research Articles

Molecular Visualization of Neuronal TDP43 Pathology In Situ.

Nuclear exclusion and cytoplasmic accumulation of the RNA-binding protein TDP43 are characteristic of amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD). Despite this, the origin and ultrastructure of cytosolic TDP43 deposits remain unknown. Accumulating evidence suggests that abnormal RNA homeostasis can drive pathological TDP43 mislocalization, enhancing RNA misprocessing due to loss of nuclear TDP43 and engendering a cycle that ends in cell death. Here, we show that adding small monovalent oligonucleotides successfully recapitulates pathological TDP43 mislocalization and aggregation in iPSC-derived neurons (iNeurons). By employing a multimodal in situ cryo-correlative light and electron microscopy pipeline, we examine how RNA influences the localization and aggregation of TDP43 in near-native conditions. We find that mislocalized TDP43 forms ordered fibrils within lysosomes and autophagosomes in iNeurons as well as in patient tissue, and provide the first high-resolution snapshots of TDP43 aggregates in situ. In so doing, we provide a cellular model for studying initial pathogenic events underlying ALS, FTLD, and related TDP43-proteinopathies.

Imaging intracellular components in situ using super-resolution cryo-correlative light and electron microscopy.

Super-resolution cryo-correlative light and electron microscopy (SRcryoCLEM) is emerging as a powerful method to enable targeted in situ structural studies of biological samples. By combining the high specificity and localization accuracy of single-molecule localization microscopy (cryoSMLM) with the high resolution of cryo-electron tomography (cryoET), this method enables accurately targeted data acquisition and the observation and identification of biomolecules within their natural cellular context. Despite its potential, the adaptation of SRcryoCLEM has been hindered by the need for specialized equipment and expertise. In this chapter, we outline a workflow for cryoSMLM and cryoET-based SRcryoCLEM, and we demonstrate that, given the right tools, it is possible to incorporate cryoSMLM into an established cryoET workflow. Using Vimentin as an exemplary target of interest, we demonstrate all stages of an SRcryoCLEM experiment: performing cryoSMLM, targeting cryoET acquisition based on single-molecule localization maps, and correlation of cryoSMLM and cryoET datasets using scNodes, a software package dedicated to SRcryoCLEM. By showing how SRcryoCLEM enables the imaging of specific intracellular components in situ, we hope to facilitate adoption of the technique within the field of cryoEM.

Precise targeting for 3D cryo-correlative light and electron microscopy volume imaging of tissues using a FinderTOP

Cryo-correlative light and electron microscopy (cryoCLEM) is a powerful strategy to high resolution imaging in the unperturbed hydrated state. In this approach fluorescence microscopy aids localizing the area of interest, and cryogenic focused ion beam/scanning electron microscopy (cryoFIB/SEM) allows preparation of thin cryo-lamellae for cryoET. However, the current method cannot be accurately applied on bulky (3D) samples such as tissues and organoids. 3D cryo-correlative imaging of large volumes is needed to close the resolution gap between cryo-light microscopy and cryoET, placing sub-nanometer observations in a larger biological context. Currently technological hurdles render 3D cryoCLEM an unexplored approach. Here we demonstrate a cryoCLEM workflow for tissues, correlating cryo-Airyscan confocal microscopy with 3D cryoFIB/SEM volume imaging. Accurate correlation is achieved by imprinting a FinderTOP pattern in the sample surface during high pressure freezing, and allows precise targeting for cryoFIB/SEM volume imaging.

HOPE-SIM, a cryo-structured illumination fluorescence microscopy system for accurately targeted cryo-electron tomography

Cryo-focused ion beam (cryo-FIB) milling technology has been developed for the fabrication of cryo-lamella of frozen native specimens for study by in situ cryo-electron tomography (cryo-ET). However, the precision of the target of interest is still one of the major bottlenecks limiting application. Here, we have developed a cryo-correlative light and electron microscopy (cryo-CLEM) system named HOPE-SIM by incorporating a 3D structured illumination fluorescence microscopy (SIM) system and an upgraded high-vacuum stage to achieve efficiently targeted cryo-FIB. With the 3D super resolution of cryo-SIM as well as our cryo-CLEM software, 3D-View, the correlation precision of targeting region of interest can reach to 110 nm enough for the subsequent cryo-lamella fabrication. We have successfully utilized the HOPE-SIM system to prepare cryo-lamellae targeting mitochondria, centrosomes of HeLa cells and herpesvirus assembly compartment of infected BHK-21 cells, which suggests the high potency of the HOPE-SIM system for future in situ cryo-ET workflows.

Molecular architecture of LRRK2 in association with cellular organelles

Mutations in leucine-rich repeat kinase 2 (LRRK2) lead to increased risk of Parkinson's disease. LRRK2 is a large multidomain protein that regulates vesicular trafficking, cytoskeleton dynamics, and lysosomal function. However, the exact mechanism of how LRRK2 carries out these various functionalities remains elusive. Elucidating the localization of LRRK2 and mapping its association with microtubules and cellular vesicles will shed light on the mechanism of action of the protein in normal and disease states. Combining cryo-correlative light and electron microscopy, cryo-FIB milling, cryo-electron tomography, subtomogram analysis and integrative modeling we aim to characterize various functional states of LRRK2 on cellular organelles. We first applied this workflow to determine the molecular architecture of LRRK2 as it associates with microtubules. In this study we aim to characterize association of LRRK2 with microtubules and cellular vesicles in human brain microglial cells. We aim to characterize WT as well as PD causing mutations and study the effects of kinase inhibitors on its association with cellular organelles. This study will provide important insight into how LRRK2 operates in its native and disease state.

Reconstitution of kinetochore motility and microtubule dynamics reveals a role for a kinesin-8 in establishing end-on attachments.

During mitosis, individual microtubules make attachments to chromosomes via a specialized protein complex called the kinetochore to faithfully segregate the chromosomes to daughter cells. Translocation of kinetochores on the lateral surface of the microtubule has been proposed to contribute to high fidelity chromosome capture and alignment at the mitotic midzone, but has been difficult to observe in vivo because of spatial and temporal constraints. To overcome these barriers, we used total internal reflection fluorescence (TIRF) microscopy to track the interactions between microtubules, kinetochore proteins, and other microtubule-associated proteins in lysates from metaphase-arrested Saccharomyces cerevisiae. TIRF microscopy and cryo-correlative light microscopy and electron tomography indicated that we successfully reconstituted interactions between intact kinetochores and microtubules. These kinetochores translocate on the lateral microtubule surface toward the microtubule plus end and transition to end-on attachment, whereupon microtubule depolymerization commences. The directional kinetochore movement is dependent on the highly processive kinesin-8, Kip3. We propose that Kip3 facilitates stable kinetochore attachment to microtubule plus ends through its abilities to move the kinetochore laterally on the surface of the microtubule and to regulate microtubule plus end dynamics.

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

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.

Electron cryo-tomography reveals the subcellular architecture of growing axons in human brain organoids.

During brain development, axons must extend over great distances in a relatively short amount of time. How the subcellular architecture of the growing axon sustains the requirements for such rapid build-up of cellular constituents has remained elusive. Human axons have been particularly poorly accessible to imaging at high resolution in a near-native context. Here, we present a method that combines cryo-correlative light microscopy and electron tomography with human cerebral organoid technology to visualize growing axon tracts. Our data reveal a wealth of structural details on the arrangement of macromolecules, cytoskeletal components, and organelles in elongating axon shafts. In particular, the intricate shape of the endoplasmic reticulum is consistent with its role in fulfilling the high demand for lipid biosynthesis to support growth. Furthermore, the scarcity of ribosomes within the growing shaft suggests limited translational competence during expansion of this compartment. These findings establish our approach as a powerful resource for investigating the ultrastructure of defined neuronal compartments.

Structural basis for VIPP1 oligomerization and maintenance of thylakoid membrane integrity

Vesicle-inducing protein in plastids 1 (VIPP1) is essential for the biogenesis and maintenance of thylakoid membranes, which transform light into life. However, it is unknown how VIPP1 performs its vital membrane-remodeling functions. Here, we use cryo-electron microscopy to determine structures of cyanobacterial VIPP1 rings, revealing how VIPP1 monomers flex and interweave to form basket-like assemblies of different symmetries. Three VIPP1 monomers together coordinate a non-canonical nucleotide binding pocket on one end of the ring. Inside the ring's lumen, amphipathic helices from each monomer align to form large hydrophobic columns, enabling VIPP1 to bind and curve membranes. Invivo mutations in these hydrophobic surfaces cause extreme thylakoid swelling under high light, indicating an essential role of VIPP1 lipid binding in resisting stress-induced damage. Using cryo-correlative light and electron microscopy (cryo-CLEM), we observe oligomeric VIPP1 coats encapsulating membrane tubules within the Chlamydomonas chloroplast. Our work provides a structural foundation for understanding how VIPP1 directs thylakoid biogenesis and maintenance.

CorRelator: Interactive software for real-time high precision cryo-correlative light and electron microscopy

Cryo-correlative light and electron microscopy (CLEM) is a technique that uses the spatiotemporal cues from fluorescence light microscopy (FLM) to investigate the high-resolution ultrastructure of biological samples by cryo-electron microscopy (cryo-EM). Cryo-CLEM provides advantages for identifying and distinguishing fluorescently labeled proteins, macromolecular complexes, and organelles from the cellular environment. Challenges remain on how correlation workflows and software tools are implemented on different microscope platforms to support automated cryo-EM data acquisition. Here, we present CorRelator: an open-source desktop application that bridges between cryo-FLM and real-time cryo-EM/ET automated data collection. CorRelator implements a pixel-coordinate-to-stage-position transformation for flexible, high accuracy on-the-fly and post-acquisition correlation. CorRelator can be integrated into cryo-CLEM workflows and easily adapted to standard fluorescence and transmission electron microscope (TEM) system configurations. CorRelator was benchmarked under live-cell and cryogenic conditions using several FLM and TEM instruments, demonstrating that CorRelator reliably supports real-time, automated correlative cryo-EM/ET acquisition, through a combination of software-aided and interactive alignment. CorRelator is a cross-platform software package featuring an intuitive Graphical User Interface (GUI) that guides the user through the correlation process. CorRelator source code is available at: https://github.com/wright-cemrc-projects/corr.

Post-correlation on-lamella cryo-CLEM reveals the membrane architecture of lamellar bodies

Lamellar bodies (LBs) are surfactant-rich organelles in alveolar cells. LBs disassemble into a lipid-protein network that reduces surface tension and facilitates gas exchange in the alveolar cavity. Current knowledge of LB architecture is predominantly based on electron microscopy studies using disruptive sample preparation methods. We established and validated a post-correlation on-lamella cryo-correlative light and electron microscopy approach for cryo-FIB milled cells to structurally characterize and validate the identity of LBs in their unperturbed state. Using deconvolution and 3D image registration, we were able to identify fluorescently labeled membrane structures analyzed by cryo-electron tomography. In situ cryo-electron tomography of A549 cells as well as primary Human Small Airway Epithelial Cells revealed that LBs are composed of membrane sheets frequently attached to the limiting membrane through “T”-junctions. We report a so far undescribed outer membrane dome protein complex (OMDP) on the limiting membrane of LBs. Our data suggest that LB biogenesis is driven by parallel membrane sheet import and by the curvature of the limiting membrane to maximize lipid storage capacity.

Cryo-correlative light and electron microscopy workflow for cryo-focused ion beam milled adherent cells.

In situ cryo-electron tomography of cryo-focused ion beam (cryo-FIB) milled cells enables the study of cellular organelles in unperturbed conditions and close to the molecular resolution. However, due to the crowdedness of the cellular environment, the identification of individual macromolecular complexes either on organelles or inside the cytosol in cryo-electron tomograms is challenging. Cryo-correlative light and electron microscopy (cryo-CLEM) employs a fluorescently labeled feature of interest imaged by cryo-light microscopy that is correlated to cryo-electron microscopy maps of cryo-FIB milled lamellae using correlation markers discernable by both imaging methods. Here, we provide a protocol for a post-correlation on-lamella cryo-CLEM approach for localization of fluorescently labeled organelles of interest in cryo-lamellae after cryo-FIB milling and tomography of adherent plunge frozen cells.

Where in the cell is my protein?

The application of cryo-correlative light and cryo-electron microscopy (cryo-CLEM) gives us a way to locate structures of interest in the electron microscope. In brief, the structures of interest are fluorescently tagged, and images from the cryo-fluorescent microscope (cryo-FM) maps are superimposed on those from the cryo-electron microscope (cryo-EM). By enhancing cryo-FM to include single-molecule localization microscopy (SMLM), we can achieve much better localization. The introduction of cryo-SMLM increased the yield of photons from fluorophores, which can benefit localization efforts. Dahlberg and Moerner (2021, Annual Review of Physical Chemistry, 72, 253-278) have a recent broad and elegant review of super-resolution cryo-CLEM. This paper focuses on cryo(F)PALM/STORM for the cryo-electron tomography community. I explore the current challenges to increase the accuracy of localization by SMLM and the mapping of those positions onto cryo-EM images and maps. There is much to consider: we need to know if the excitation of fluorophores damages the structures we seek to visualize. We need to determine if higher numerical aperture (NA) objectives, which add complexity to image analysis but increase resolution and the efficiency of photon collection, are better than lower NA objectives, which pose fewer problems. We need to figure out the best way to determine the axial position of fluorophores. We need to have better ways of aligning maps determined by FM with those determined by EM. We need to improve the instrumentation to be easier to use, more accurate, and ice-contamination free. The bottom line is that we have more work to do.

Development of a Cryo-correlative Light and Electron Microscopy Workflow at Simons Electron Microscopy Center

Development of a Cryo-correlative Light and Electron Microscopy Workflow at Simons Electron Microscopy Center

Advanced cryo-tomography workflow developments - correlative microscopy, milling automation and cryo-lift-out.

Cryo-electron tomography (cryo-ET) is a groundbreaking technology for 3D visualisation and analysis of biomolecules in the context of cellular structures. It allows structural investigations of single proteins as well as their spatial arrangements within the cell. Cryo-tomograms provide a snapshot of the complex, heterogeneous and transient subcellular environment. Due to the excellent structure preservation in amorphous ice, it is possible to study interactions and spatial relationships of proteins in their native state without interference caused by chemical fixatives or contrasting agents. With the introduction of focused ion beam (FIB) technology, the preparation of cellular samples for electron tomography has become much easier and faster. The latest generation of integrated FIB and scanning electron microscopy (SEM) instruments (dual beam microscopes), specifically designed for cryo-applications, provides advances in automation, imaging and the preparation of high-pressure frozen bulk samples using cryo-lift-out technology. In addition, correlative cryo-fluorescence microscopy provides cellular targeting information through integrated software and hardware interfaces. The rapid advances, based on the combination of correlative cryo-microscopy, cryo-FIB and cryo-ET, have already led to a wealth of new insights into cellular processes and provided new 3D image data of the cell. Here we introduce our recent developments within the cryo-tomography workflow, and we discuss the challenges that lie ahead. LAY DESCRIPTION: This article describes our recent developments for the cryo-electron tomography (cryo-ET) workflow. Cryo-ET offers superior structural preservation and provides 3D snapshots of the interior of vitrified cells at molecular resolution. Before a cellular sample can be imaged by cryo-ET, it must be made accessible for transmission electron microscopy. This is achieved by preparing a 200-300 nm thin cryo-lamella from the cellular sample using a cryo-focused ion beam (cryo-FIB) microscope. Cryo-correlative light and electron microscopy (cryo-CLEM) is used within the workflow to guide the cryo-lamella preparation to the cellular areas of interest. We cover a basic introduction of the cryo-ET workflow and show new developments for cryo-CLEM, which facilitate the connection between the cryo-light microscope and the cryo-FIB. Next, we present our progress in cryo-FIB software automation to streamline cryo-lamella preparation. In the final section we demonstrate how the cryo-FIB can be used for 3D imaging and how bulk-frozen cellular samples (obtained by high-pressure freezing) can be processed using the newly developed cryo-lift-out technology.

Cryo-ET Workflow for Understanding Ion Channels Localization on the Nodes of Ranvier

The localization of ion channels on myelinated axon is closely related with the saltatory conduction of action potential (AP). Abnormal changes in these channels contribute to multiple mental diseases. The development of cryo-Electron Tomography (cryo-ET) has provided a promising prospect for peering into ion channels in their native environment at high resolution. Previous achievements are reviewed here on cryo-ET. Accordingly, a cryo-ET workflow is designed for understanding ion channels localization in myelinated axon, especially nodes of Ranvier, which are significant for the saltatory conduction involved in the propagation of high-speed AP. The workflow is divided into six parts: the preparation of neural cultures with myelin, antibodies and immunofluorescence staining, frozen-hydrated sample preparation, cryo-ET imaging, cryo-correlative light and electron microscopy (cryo-CLEM) imaging, three-dimensional (3D) reconstruction and refinement. The purpose is to conceive a possible solution for the problems related to ion channel compounds including localization, conformation dynamics, accessory structures of ion channel and transient regulatory factors, and thus provide insights into treating neurological diseases caused by abnormal ion channels activity.

Advances in Cryo-Correlative Light and Electron Microscopy: Applications for Studying Molecular and Cellular Events.

Cryo-correlative light and electron microscopy (Cryo-CLEM) is materializing as a widespread approach amalgamating the advantages of both fluorescence light microscopy (FLM) as well as three dimensional (3D) cryo-electron tomography (cryo-ET) to reveal the ultrastructure of significant target molecules with specific cellular functions. Cryo-CLEM allows imaging of cells by means of fluorescence microscopy exhibiting the location of the destined molecule at high temporal and spatial resolution while cryo-ET is employed to analyze the 3D structure at a molecular resolution in close-to-physiological condition. Present review focuses upon the practical strategies for Cryo-CLEM and recent technical developments that will assist the broad implementation of this technique to investigate and answer questions pertaining to various biological events occurring in the cell.

PIE-scope, integrated cryo-correlative light and FIB/SEM microscopy.

Cryo-electron tomography (cryo-ET) is emerging as a revolutionary method for resolving the structure of macromolecular complexes in situ. However, sample preparation for in situ Cryo-ET is labour-intensive and can require both cryo-lamella preparation through cryo-focused ion beam (FIB) milling and correlative light microscopy to ensure that the event of interest is present in the lamella. Here, we present an integrated cryo-FIB and light microscope setup called the Photon Ion Electron microscope (PIE-scope) that enables direct and rapid isolation of cellular regions containing protein complexes of interest. Specifically, we demonstrate the versatility of PIE-scope by preparing targeted cryo-lamellae from subcellular compartments of neurons from transgenic Caenorhabditis elegans and Drosophila melanogaster expressing fluorescent proteins. We designed PIE-scope to enable retrofitting of existing microscopes, which will increase the throughput and accuracy on projects requiring correlative microscopy to target protein complexes. This new approach will make cryo-correlative workflow safer and more accessible.

PIE-scope, integrated cryo-correlative light and FIB/SEM microscopy

PIE-scope, integrated cryo-correlative light and FIB/SEM microscopy

An efficient protocol of cryo-correlative light and electron microscopy for the study of neuronal synapses

Cryo-electron tomography is an emerging electron microscopy technique for determining three-dimensional structures of cellular architectures near their native state at nanometer resolution, with a shortcoming of lack of specific labels. Fluorescence light microscopy, on the other hand, specifically visualizes target cellular and molecular components with fluorescent labels, but is limited to a resolution of tens to hundreds nanometers. Combining the advantages of the two techniques, we have developed a cryo-correlative light and electron microscopy system. Our system consists a custom-designed cryo-chamber that allows for fluorescence imaging of frozen-hydrated samples, and an algorithm to achieve accurate correlation. With this system and our optimized protocol, high-quality tomograms of neuronal synapses labelled by specific fluorescent tags in cultured hippocampal neurons are obtained at high efficiency.