Cryo-correlative Light Research Articles

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.

Correlative cryo super-resolution light and electron microscopy on mammalian cells using fluorescent proteins

Sample fixation by vitrification is critical for the optimal structural preservation of biomolecules and subsequent high-resolution imaging by cryo-correlative light and electron microscopy (cryoCLEM). There is a large resolution gap between cryo fluorescence microscopy (cryoFLM), ~400-nm, and the sub-nanometre resolution achievable with cryo-electron microscopy (cryoEM), which hinders interpretation of cryoCLEM data. Here, we present a general approach to increase the resolution of cryoFLM using cryo-super-resolution (cryoSR) microscopy that is compatible with successive cryoEM investigation in the same region. We determined imaging parameters to avoid devitrification of the cryosamples without the necessity for cryoprotectants. Next, we examined the applicability of various fluorescent proteins (FPs) for single-molecule localisation cryoSR microscopy and found that all investigated FPs display reversible photoswitchable behaviour, and demonstrated cryoSR on lipid nanotubes labelled with rsEGFP2 and rsFastLime. Finally, we performed SR-cryoCLEM on mammalian cells expressing microtubule-associated protein-2 fused to rsEGFP2 and performed 3D cryo-electron tomography on the localised areas. The method we describe exclusively uses commercially available equipment to achieve a localisation precision of 30-nm. Furthermore, all investigated FPs displayed behaviour compatible with cryoSR microscopy, making this technique broadly available without requiring specialised equipment and will improve the applicability of this emerging technique for cellular and structural biology.

Biological Applications at the Cutting Edge of Cryo-Electron Microscopy.

Cryo-electron microscopy (cryo-EM) is a powerful tool for macromolecular to near-atomic resolution structure determination in the biological sciences. The specimen is maintained in a near-native environment within a thin film of vitreous ice and imaged in a transmission electron microscope. The images can then be processed by a number of computational methods to produce three-dimensional information. Recent advances in sample preparation, imaging, and data processing have led to tremendous growth in the field of cryo-EM by providing higher resolution structures and the ability to investigate macromolecules within the context of the cell. Here, we review developments in sample preparation methods and substrates, detectors, phase plates, and cryo-correlative light and electron microscopy that have contributed to this expansion. We also have included specific biological applications.

Differentiation and Characterization of Excitatory and Inhibitory Synapses by Cryo-electron Tomography and Correlative Microscopy.

As key functional units in neural circuits, different types of neuronal synapses play distinct roles in brain information processing, learning, and memory. Synaptic abnormalities are believed to underlie various neurological and psychiatric disorders. Here, by combining cryo-electron tomography and cryo-correlative light and electron microscopy, we distinguished intact excitatory and inhibitory synapses of cultured hippocampal neurons, and visualized the in situ 3D organization of synaptic organelles and macromolecules in their native state. Quantitative analyses of >100 synaptic tomograms reveal that excitatory synapses contain a mesh-like postsynaptic density (PSD) with thickness ranging from 20 to 50 nm. In contrast, the PSD in inhibitory synapses assumes a thin sheet-like structure ∼12 nm from the postsynaptic membrane. On the presynaptic side, spherical synaptic vesicles (SVs) of 25–60 nm diameter and discus-shaped ellipsoidal SVs of various sizes coexist in both synaptic types, with more ellipsoidal ones in inhibitory synapses. High-resolution tomograms obtained using a Volta phase plate and electron filtering and counting reveal glutamate receptor-like and GABAA receptor-like structures that interact with putative scaffolding and adhesion molecules, reflecting details of receptor anchoring and PSD organization. These results provide an updated view of the ultrastructure of excitatory and inhibitory synapses, and demonstrate the potential of our approach to gain insight into the organizational principles of cellular architecture underlying distinct synaptic functions.SIGNIFICANCE STATEMENT To understand functional properties of neuronal synapses, it is desirable to analyze their structure at molecular resolution. We have developed an integrative approach combining cryo-electron tomography and correlative fluorescence microscopy to visualize 3D ultrastructural features of intact excitatory and inhibitory synapses in their native state. Our approach shows that inhibitory synapses contain uniform thin sheet-like postsynaptic densities (PSDs), while excitatory synapses contain previously known mesh-like PSDs. We discovered “discus-shaped” ellipsoidal synaptic vesicles, and their distributions along with regular spherical vesicles in synaptic types are characterized. High-resolution tomograms further allowed identification of putative neurotransmitter receptors and their heterogeneous interaction with synaptic scaffolding proteins. The specificity and resolution of our approach enables precise in situ analysis of ultrastructural organization underlying distinct synaptic functions.

High-vacuum optical platform for cryo-CLEM (HOPE): A new solution for non-integrated multiscale correlative light and electron microscopy.

Cryo-correlative light and electron microscopy (cryo-CLEM) offers a unique way to analyze the high-resolution structural information of cryo-vitrified specimen by cryo-electron microscopy (cryo-EM) with the guide of the search for unique events by cryo-fluorescence microscopy (cryo-FM). To achieve cryo-FM, a trade-off must be made between the temperature and performance of objective lens. The temperature of specimen should be kept below devitrification while the distance between the objective lens and specimen should be short enough for high resolution imaging. Although special objective lens was designed in many current cryo-FM approaches, the unavoided frosting and ice contamination are still affecting the efficiency of cryo-CLEM. In addition, the correlation accuracy between cryo-FM and cryo-EM would be reduced during the current specimen transfer procedure. Here, we report an improved cryo-CLEM technique (high-vacuum optical platform for cryo-CLEM, HOPE) based on a high-vacuum optical stage and a commercial cryo-EM holder. The HOPE stage comprises of a special adapter to suit the cryo-EM holder and a high-vacuum chamber with an anti-contamination system. It provides a clean and enduring environment for cryo specimen, while the normal dry objective lens in room temperature can be used via the optical windows. The 'touch-free' specimen transfer via cryo-EM holder allows least specimen deformation and thus maximizes the correlation accuracy between cryo-FM and cryo-EM. Besides, we developed a software to perform semi-automatic cryo-EM acquisition of the target region localized by cryo-FM. Our work provides a new solution for cryo-CLEM and can be adapted for different commercial fluorescence microscope and electron microscope.

Shrinkage of freeze-dried cryosections of cells: Investigations by EFTEM and cryo-CLEM

Freeze-drying of cryosections of cells or tissues is considered to be the most efficient preparation for microanalysis purpose related to transmission electron microscopy. It allows the measurements of ions and water contents at the ultrastructural level. However an important drawback is associated to freeze-drying: the shrinkage of the cryosections. The aim of this paper is the investigation of this phenomenon by means of three different methods applied to both hydrated and dehydrated cryosections: direct distance measurements on fiducial points, thickness measurements by energy filtered transmission microscopy (EFTEM) and cryo-correlative light electron microscopy (cryo-CLEM). Measurements in our experimental conditions reveal a lateral shrinkage around 10% but the most important result concerns the lack of differential shrinkage between most of the cellular compartments.

Actin Organization in Cells Responding to a Perforated Surface, Revealed by Live Imaging and Cryo-Electron Tomography

In a 3D environment, motile cells accommodate their protruding and retracting activities to geometrical cues. Dictyostelium cells migrating on a perforated film explored its holes by forming actin rings around their border and extending protrusions through the free space. The response was initiated when an actin wave passed a hole, and the rings persisted only in the PIP3-rich territories surrounded by a wave. To reconstruct actin structures from cryo-electron tomograms, actin rings were identified by cryo-correlative light and electron microscopy, and thin wedges of relevant regions were obtained by cryo-focused ion-beam milling. Retracting stages were distinguished from protruding ones by the accumulation of myosin-II. Early actin rings consisted of filaments pointing upright from the membrane, entangled with a meshwork of filaments close to the membrane.Branches identified at later stages suggested that formin-based nucleation of filaments was followed by Arp2/3-mediated network stabilization, which prevented buckling of the force-generating filaments.

Cross-membranes orchestrate compartmentalization and morphogenesis in Streptomyces.

Far from being simple unicellular entities, bacteria have complex social behaviour and organization, living in large populations, and some even as coherent, multicellular entities. The filamentous streptomycetes epitomize such multicellularity, growing as a syncytial mycelium with physiologically distinct hyphal compartments separated by infrequent cross-walls. The viability of mutants devoid of cell division, which can be propagated from fragments, suggests the presence of a different form of compartmentalization in the mycelium. Here we show that complex membranes, visualized by cryo-correlative light microscopy and electron tomography, fulfil this role. Membranes form small assemblies between the cell wall and cytoplasmic membrane, or, as evidenced by FRAP experiments, large protein-impermeable cross-membrane structures, which compartmentalize the multinucleoid mycelium. All areas containing cross-membrane structures are nucleoid-restricted zones, suggesting that the membrane assemblies may also act to protect nucleoids from cell-wall restructuring events. Our work reveals a novel mechanism of controlling compartmentalization and development in multicellular bacteria.

Targeted nano analysis of water and ions in the nucleus using cryo-correlative microscopy.

The cell nucleus is a crowded volume in which the concentration of macromolecules is high. These macromolecules sequester most of the water molecules and ions which, together, are very important for stabilization and folding of proteins and nucleic acids. To better understand how the localization and quantity of water and ions vary with nuclear activity, it is necessary to study them simultaneously by using newly developed cell imaging approaches. Some years ago, we showed that dark-field cryo-Scanning Transmission Electron Microscopy (cryo-STEM) allows quantification of the mass percentages of water, dry matter, and elements (among which are ions) in freeze-dried ultrathin sections. To overcome the difficulty of clearly identifying nuclear subcompartments imaged by STEM in ultrathin cryo-sections, we developed a new cryo correlative light and STEM imaging procedure. This combines fluorescence imaging of nuclear GFP-tagged proteins to identify, within cryo ultrathin sections, regions of interest which are then analyzed by STEM for quantification of water and identification and quantification of ions. In this chapter we describe the new setup we have developed to perform this cryo-correlative light and STEM imaging approach, which allows a targeted nano analysis of water and ions in nuclear compartments.

Chapter 8 - Correlative Cryo-Fluorescence and Cryo-Soft X-Ray Tomography of Adherent Cells at European Synchrotrons

Cryo-soft X-ray tomography (cryo-SXT) is a synchrotron-hosted imaging technique used to analyze the ultrastructure of intact, cryo-prepared cells. Correlation of cryo-fluorescence microscopy and cryo-SXT can be used to localize fluorescent proteins to organelles preserved close to native state. Cryo-correlative light and X-ray microscopy (cryo-CLXM) is particularly useful for the study of organelles that are susceptible to chemical fixation artifacts during sample preparation for electron microscopy. In our recent work, we used cryo-CLXM to characterize GFP-LC3-positive early autophagosomes in nutrient-starved HEK293A cells (Duke et al., 2013). Cup-shaped omegasomes were found to form at "hot-spots" on the endoplasmic reticulum. Furthermore, cryo-SXT image stacks revealed the presence of large complex networks of tubulated mitochondria in the starved cells, which would be challenging to model at this scale and resolution using light or electron microscopy. In this chapter, we detail the cryo-CLXM workflow that we developed and optimized for studying adherent mammalian cells. We show examples of data collected at the three European synchrotrons that currently host cryo-SXT microscopes, and describe how raw cryo-SXT datasets are processed into tomoX stacks, modeled, and correlated with cryo-fluorescence data to identify structures of interest.

Chapter 10 - Correlative Cryo-Fluorescence Light Microscopy and Cryo-Electron Tomography of Streptomyces

Light microscopy and electron microscopy are complementary techniques that in a correlative approach enable identification and targeting of fluorescently labeled structures in situ for three-dimensional imaging at nanometer resolution. Correlative imaging allows electron microscopic images to be positioned in a broader temporal and spatial context. We employed cryo-correlative light and electron microscopy (cryo-CLEM), combining cryo-fluorescence light microscopy and cryo-electron tomography, on vitrified Streptomyces bacteria to study cell division. Streptomycetes are mycelial bacteria that grow as long hyphae and reproduce via sporulation. On solid media, Streptomyces subsequently form distinct aerial mycelia where cell division leads to the formation of unigenomic spores which separate and disperse to form new colonies. In liquid media, only vegetative hyphae are present divided by noncell separating crosswalls. Their multicellular life style makes them exciting model systems for the study of bacterial development and cell division. Complex intracellular structures have been visualized with transmission electron microscopy. Here, we describe the methods for cryo-CLEM that we applied for studying Streptomyces. These methods include cell growth, fluorescent labeling, cryo-fixation by vitrification, cryo-light microscopy using a Linkam cryo-stage, image overlay and relocation, cryo-electron tomography using a Titan Krios, and tomographic reconstruction. Additionally, methods for segmentation, volume rendering, and visualization of the correlative data are described.

Targeted nano analysis of water and ions using cryocorrelative light and scanning transmission electron microscopy

Cryo fluorescence imaging coupled with the cryo-EM technique (cryo-CLEM) avoids chemical fixation and embedding in plastic, and is the gold standard for correlated imaging in a close to native state. This multi-modal approach has not previously included elementary nano analysis or evaluation of water content. We developed a new approach allowing analysis of targeted in situ intracellular ions and water measurements at the nanoscale (EDXS and STEM dark field imaging) within domains identified by examination of specific GFP-tagged proteins. This method allows both water and ions- fundamental to cell biology- to be located and quantified at the subcellular level. We illustrate the potential of this approach by investigating changes in water and ion content in nuclear domains identified by GFP-tagged proteins in cells stressed by Actinomycin D treatment and controls. The resolution of our approach was sufficient to distinguish clumps of condensed chromatin from surrounding nucleoplasm by fluorescence imaging and to perform nano analysis in this targeted compartment.