CryoFIB milling large tissue samples for cryo-electron tomography

Cryo-electron tomography (cryo-ET) is a formidable technique to observe the inner workings of vitrified cells at a nanometric resolution in near-native conditions and in three-dimensions. One consequent drawback of this technique is the sample thickness, for two reasons: i) achieving proper vitrification of the sample gets increasingly difficult with sample thickness, and ii) cryo-ET relies on transmission electron microscopy (TEM), requiring thin samples for proper electron transmittance (<500 nm). For samples exceeding this thickness limit, thinning methods can be used to render the sample amenable for cryo-ET. Cryo-focused ion beam (cryo-FIB) milling is one of them and despite having hugely benefitted the fields of animal cell biology, virology, microbiology, and even crystallography, plant cells are still virtually unexplored by cryo-ET, in particular because they are generally orders of magnitude bigger than bacteria, viruses, or animal cells (at least 10 μm thick) and difficult to process by cryo-FIB milling. Here, we detail a preparation method where abaxial epidermal onion cell wall peels are separated from the epidermal cells and subsequently plunge frozen, cryo-FIB milled, and screened by cryo-ET in order to acquire high resolution tomographic data for analyzing the organization of the cell wall. This protocol was validated in: Curr Biol (2022), DOI: 10.1016/j.cub.2022.04.024.

Cryo-electron tomography (cryoET) has become a powerful technique at the interface of structural biology and cell biology, due to its unique ability for imaging cells in their native state and determining structures of macromolecular complexes in their cellular context. A limitation of cryoET is its restriction to relatively thin samples. Sample thinning by cryo-focused ion beam (cryoFIB) milling has significantly expanded the range of samples that can be analyzed by cryoET. Unfortunately, cryoFIB milling is low-throughput, time-consuming and manual. Here, we report a method for fully automated sequential cryoFIB preparation of high-quality lamellae, including rough milling and polishing. We reproducibly applied this method to eukaryotic and bacterial model organisms, and show that the resulting lamellae are suitable for cryoET imaging and subtomogram averaging. Since our method reduces the time required for lamella preparation and minimizes the need for user input, we envision the technique will render previously inaccessible projects feasible.

Cryo-electron tomography (cryo-ET) is a powerful method to elucidate subcellular architecture and to structurally analyze biomolecules in situ by subtomogram averaging, yet data quality critically depends on specimen thickness. Cells that are too thick for transmission imaging can be thinned into lamellae by cryo-focused ion beam (cryo-FIB) milling. Despite being a crucial parameter directly affecting attainable resolution, optimal lamella thickness has not been systematically investigated nor the extent of structural damage caused by gallium ions used for FIB milling. We thus systematically determined how resolution is affected by these parameters. We find that ion-induced damage does not affect regions more than 30 nanometers from either lamella surface and that up to ~180-nanometer lamella thickness does not negatively affect resolution. This shows that there is no need to generate very thin lamellae and lamella thickness can be chosen such that it captures cellular features of interest, thereby opening cryo-ET also for studies of large complexes.

Cryogenic-electron tomography (cryo-ET) has provided an unprecedented glimpse into the nanoscale architecture of cells by combining cryogenic preservation of biological structures with electron tomography. Micropatterning of extracellular matrix proteins is increasingly used as a method to prepare adherent cell types for cryo-ET as it promotes optimal positioning of cells and subcellular regions of interest for vitrification, cryo-focused ion beam (cryo-FIB) milling, and data acquisition. Here we demonstrate a micropatterning workflow for capturing minimally adherent cell types, human T-cells and Jurkat cells, for cryo-FIB and cryo-ET. Our affinity capture system facilitated the nanoscale imaging of Jurkat cells, revealing extracellular filamentous structures. It improved workflow efficiency by consistently producing grids with a sufficient number of well positioned cells for an entire cryo-FIB session. Affinity capture can be extended to facilitate high resolution imaging of other adherent and non-adherent cell types with cryo-ET.

Structural insights into functional regulation of the human CPEB3 prion by an amyloid-forming segment.

Cryo-Electron Tomography (cryo-ET) is a method that enables resolving the structure of macromolecular complexes directly in the cellular environment. 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. The PIE-scope can be retrofitted on existing microscopes, although the drawings we provide are meant to work on ThermoFisher DualBeams with small mechanical modifications those can be adapted on other brands.

Exploring cryogenic focused ion beam milling as a Group III–V device fabrication tool

Cryogenic focused ion beam (Cryo-FIB) milling has become a standard step in the cryogenic electron tomography (Cryo-ET) workflow and is required to thin cells to electron-semitransparency. However, this destructive process removes the vast majority of the cellular material and raises a critical question: what thin section should be preserved for Cryo-ET analysis? Using a tri-coincident cryogenic FIB-SEM-LM system, we identify an interferometric optical response that can be used for targeting lamella production to fluorescently labeled structures with accuracy beyond the diffraction limit. Here we demonstrate this approach using synthetic samples of fluorescent beads embedded in micron-scale droplets of amorphous ice. We then apply the approach to capture virions inside host cells. Successful targeting is confirmed by Cryo-ET revealing clusters of virions in intracellular vesicles. The method does not require any fluorescent fiducials or axial registration and can be performed on any fluorescently labeled structure that is visible in widefield fluorescence microscopy.

Parasitology meets cryo-electron tomography – exciting prospects await

Beyond resolution: Cryo-electron tomography of microbial nanomachines in native host context.

Cryo-electron tomography (cryo-ET) has become a powerful approach to study the high-resolution structure of cellular macromolecular machines in situ. However, the current correlative cryo-fluorescence and electron microscopy lacks sufficient accuracy and efficiency to precisely prepare cryo-lamellae of target locations for subsequent cryo-ET. Here we describe a precise cryogenic fabrication system, ELI-TriScope, which sets electron (E), light (L) and ion (I) beams at the same focal point to achieve accurate and efficient preparation of a target cryo-lamella. ELI-TriScope uses a commercial dual-beam scanning electron microscope modified to incorporate a cryo-holder-based transfer system and embed an optical imaging system just underneath the vitrified specimen. Cryo-focused ion beam milling can be accurately navigated by monitoring the real-time fluorescence signal of the target molecule. Using ELI-TriScope, we prepared a batch of cryo-lamellae of HeLa cells targeting the centrosome with a success rate of ~91% and discovered new in situ structural features of the human centrosome by cryo-ET.

Human Immunodeficiency virus type 1 (HIV-1) is a lentivirus that infects non-dividing cells of the immune system. In non-dividing cells, nuclear import of the viral genome occurs through the nuclear pore complex (NPC), a large macromolecular assembly that forms a channel of ~40 nm across the nuclear envelope. This process requires interactions between NPC components known as nucleoporins and CA (capsid) proteins of HIV-1 post-fusion complexes. In mature HIV-1 virions, multiple copies of CA assemble into a lattice of hexamers and pentamers, forming a cone-shaped capsid core of ~60 nm in width and ~110 nm in length encasing the viral genome. Because of its large size, it was generally believed that the capsid core entirely or partially disassembled prior to its translocation through the NPC. However, nuclear entry and uncoating are rare events and challenging to characterize and therefore subject to a long-standing debate. Here, cryo-electron tomography (cryo-ET) on cryo-focused ion beam (FIB) milled T-cells was combined with subtomogram averaging (SA) to study the ultrastructure of HIV-1 capsids in the process of nuclear entry. Using a HIV-1 variant arrested at the nuclear pore due to a defect in binding of the CA lattice to the host cell protein CPSF6, snapshots of HIV-1 nuclear entry at multiple stages were captured. Reverse transcription-competent HIV-1 complexes were identified in the cytosol, docking to and within the NPC, and in the nucleoplasm of T cells. Surprisingly, in the cytosol and at the NPC, the viral complexes retained cone-shaped capsids highly resembling in size and geometry the mature capsid cores within intact virions. The density at their interior suggested that they were associated with the viral genome. The cone-shape capsids deeply enter into the NPC central channel with their narrow ends. The hexameric lattices of these capsids were intact or nearly intact. These findings argued against the current models of uncoating in the cytosol or at the NPC and rather supported translocation of intact HIV-1 capsids through the NPC. Instead, HIV-1 capsids uncoat in the nucleus of T cells. Inside the nucleus, tube-shape fragments still containing few lattice elements were observed. The lack of density at their inside suggested that the viral genome was released from these complexes. Uncoating may thus occur through the partial opening and remodeling of the hexameric lattices of HIV-1 capsids, rather than a step-wise disassembly. To address how an intact HIV-1 capsid can enter the central channel of NPC, the human NPC from T cells was structurally analyzed in cellulo by SA. The NPC overall structure was not altered upon infection but rather dilated in comparison to the structure previously obtained from purified envelopes. The diameter of the central channel of NPC in T cells corresponded to ~64 nm, suggesting that the translocation of intact HIV-1 capsid through the NPC is geometrically possible. The dilated conformation of NPCs in T cells is independent of HIV-1 infection, but represents a physiological condition of actively transporting NPCs.

In the present study we review our experience with 900 consecutive percutaneous muscle biopsies over the period 1993 to 2007. We examined the advantages and limitations of the procedure, biopsy site preferences, diagnostic range, frequency of diagnoses and quality of histopathology. Demographics, referral patterns and patients' perceptions of the procedure were also assessed. Cases were identified through the London Health Sciences Centre Department of Pathology database. Standard biopsy procedures were followed using a manual trocar style instrument. With a neuropathology technologist in attendance at all biopsies, biopsies were oriented in the fresh state and snap frozen. Most referrals for muscle biopsy were from neuromuscular neurologists. The procedure was found to be efficient, well-tolerated and produced high quality specimens in all diagnostic categories. No major complications occurred. Failure to obtain an adequate tissue sample, although uncommon (< 2%), was usually due to marked obesity, edema or muscle wasting. Bleeding at the site was rarely problematic and no wound infections were reported. Needle muscle biopsies represent an efficient alternative to open biopsies when peripheral nerve sampling is not required and when large tissue samples are not needed for extensive biochemical analyses.

Qualitative and quantitative analysis of cellular DNA content may be clinically useful in the prognostic evaluation of certain types of malignant tumors, including breast carcinoma. Flow cytometric (FCM) analysis has been the most frequently used procedure for DNA analysis, but it requires a reasonably large tissue sample. Computer-based image analysis (IA) now allows imprint, cytospin, and needle aspiration smear preparations and other small tissue samples to be used. To resolve concern about the diagnostic efficacy of small tissue samples in the use of IA, the authors performed a comparative study of FCM analysis and IA using 115 fresh-frozen breast carcinomas. Feulgen-stained imprint preparations for IA and single-cell suspensions from the same fresh-frozen tissue for FCM analysis were used, and the respective histograms were compared. The results were concordant in 90.4% (104 of 115) of the cases, but 11 specimens yielded discordant data. IA provided histograms with a somewhat lower resolution and a relatively high coefficient of variation for the G0/G1 peak, thus rendering occasional tumors, which were near-diploid aneuploid by FCM analysis (four cases), not amenable to diagnosis by aneuploid characterization. In three additional cases, FCM analysis showed aneuploid hyperdiploid (two cases) and multiploid (one case) histograms, but IA only demonstrated a diploid peak. Conversely, in four other cases, aneuploid peaks were recognized only by IA. Computerized IA has significant advantages over FCM analysis, including lower cost, the ability to analyze very small specimens, the capability of detecting rare high ploidy cells, the capacity to classify cellular populations according to specific morphologic type, and the fact that no destructive enzyme or chemical digestion is required for specimen preparation, thereby preserving the integrity of fragile cells.

Large fat-biopsy samples are necessary for extensive simultaneous assays. Subcutaneous adipose tissue is generally obtained either by needle-biopsy aspiration (approximately 1 g) or by open surgical biopsy for larger samples. We propose a reliable technique that permits the removal of large adipose tissue samples percutaneously. A vertical 5-mm slit was made in the abdominal skin of 15 obese patients, and a liposuction mini-cannula (10 cm x 3 mm) attached to a 20-mL sterile plastic syringe was inserted. A vacuum was created by withdrawing the syringe plunger and fixing it with a specially adapted clamp. We obtained 3- to 15-g samples of adipose tissue without conspicuous scarring or visible contour changes. A significant correlation between the body mass index and the amount of adipose tissue was observed (r = .50, p < .05). Isolated adipocytes remained highly viable, as evidenced by glucose transport under both basal and maximal insulin-stimulated conditions. The mini-liposuction procedure was fast, and once the mini-cannula was removed from the wound, patients could immediately move about freely. The new technique described here for fat biopsy using a mini-cannula is less traumatic than open surgical biopsy, and it permits the removal of larger adipose tissue samples than do the usual biopsy techniques. Furthermore, adipose cells are removed intact, and the simplicity of the method facilitates repeat biopsies.