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A lamella (also known as a foil or membrane) prepared for Lift-Out. Top row SEM view, bottom FIB view. (a,b) Thinning of the lamella (c) The FIB 'undercut' is made prior to attaching the micromanipulator needle to the lamella (not shown). (d) The lamella is transferred to the support grid by the micromanipulator. (e,f) The lamella is mounted on the support grid in the so-called Vpost by depositing a platinum precursor gas with the FIB. (g,h) The sample is thinned to electron transparency prior to TEM. Scale bars (a,b,d,f,g) 5 µm (c) 10 µm (e) 30 µm (h) 3µm
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es A major challenge in electron microscopy is the production of suitable samples from hydrated biological and soft matter materials for sub‐nm resolution imaging in a cryo‐Transmission Electron Microscope (TEM). A well‐known solution for room temperature materials is called (in situ) Lift‐Out. It comprises of a fine needle that picks u...
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... but also a micromanipulator for removal of the lamella, either operated in situ in the vacuum of the FIB or ex situ as a standalone instrument 13 . The lamella is sometimes known as a foil or membrane. Once suitably thin, the lamella is mounted on a specialised support grid and can be transferred to the TEM for further imaging and analysis (Fig. ...
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... the lamella is attached to the LO-grid, the excess deposited ice / organometallic platinum which encases the lamella needs to be removed (Figs. 10a-d). The FIB mills away the excess material and continues thinning the lamella to electron transparency (200-300 nm). Milling currents starting at 500 pA and finishing at 50-30 pA provide careful removal of excess materials in a reasonable time frame. Care should be taken to avoid any cryo-condensed materials that attach the lamella to the ...
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... currents starting at 500 pA and finishing at 50-30 pA provide careful removal of excess materials in a reasonable time frame. Care should be taken to avoid any cryo-condensed materials that attach the lamella to the LO-grid post or the entire lamella could be lost (Fig 10c). The use of over and under tilting (+/-2 degrees or more) ensures parallel surfaces on either side of the lamella. ...
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... to the LO-grid post or the entire lamella could be lost (Fig 10c). The use of over and under tilting (+/-2 degrees or more) ensures parallel surfaces on either side of the lamella. As has been described at length, the entire Lift-Out procedure is complex. It can be summarised in the following workflow diagram (for an Omniprobe based procedure) (Fig. 11) which has been colour coded for easier ...
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... type, hardness and dimensions of the desired lamella), similar to a manual room temperature Lift-Out. The final examination of the lamella occurs once the lamella is safely transferred into the cryo-TEM. Once in the TEM, care must be taken to limit beam sample exposure and low-dose imaging regimes are advised. Images from cryo-FIB LO lamellae (Fig. 12) are shown and demonstrate that several ultrastructural features can be observed including cell membrane (Figs 12 a-c,f), nuclear membranes (Fig. 12a,b) and mitochondria (Fig. ...
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... in the TEM, care must be taken to limit beam sample exposure and low-dose imaging regimes are advised. Images from cryo-FIB LO lamellae (Fig. 12) are shown and demonstrate that several ultrastructural features can be observed including cell membrane (Figs 12 a-c,f), nuclear membranes (Fig. 12a,b) and mitochondria (Fig. 12c). ...
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... once the lamella is safely transferred into the cryo-TEM. Once in the TEM, care must be taken to limit beam sample exposure and low-dose imaging regimes are advised. Images from cryo-FIB LO lamellae (Fig. 12) are shown and demonstrate that several ultrastructural features can be observed including cell membrane (Figs 12 a-c,f), nuclear membranes (Fig. 12a,b) and mitochondria (Fig. ...
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... into the cryo-TEM. Once in the TEM, care must be taken to limit beam sample exposure and low-dose imaging regimes are advised. Images from cryo-FIB LO lamellae (Fig. 12) are shown and demonstrate that several ultrastructural features can be observed including cell membrane (Figs 12 a-c,f), nuclear membranes (Fig. 12a,b) and mitochondria (Fig. ...
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... lamella can be attached to the side of a post, on top of a post, or in a v-shaped post (Fig. 13a). Furthermore, it is possible to pre-mill a support grid prior to attaching of the lamella (Fig 14.). Part of the consideration involves the position of the hardware within the microscope, i.e. the micromanipulator and the ...
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... lamella can be attached to the side of a post, on top of a post, or in a v-shaped post (Fig. 13a). Furthermore, it is possible to pre-mill a support grid prior to attaching of the lamella (Fig 14.). Part of the consideration involves the position of the hardware within the microscope, i.e. the micromanipulator and the GIS. ...
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... relevance of the configuration of the hardware is illustrated in Figure 13. Fig. 13b) shows the water GIS and the micromanipulator located on the same side of the chamber. ...
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... relevance of the configuration of the hardware is illustrated in Figure 13. Fig. 13b) shows the water GIS and the micromanipulator located on the same side of the chamber. Testing proved this configuration less routinely successful in comparison to having the water GIS on the opposite site to the manipulator (Fig. 13c&d). Mounting a GIS from the opposite side to the manipulator takes advantage that the port is at a ...
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... relevance of the configuration of the hardware is illustrated in Figure 13. Fig. 13b) shows the water GIS and the micromanipulator located on the same side of the chamber. Testing proved this configuration less routinely successful in comparison to having the water GIS on the opposite site to the manipulator (Fig. 13c&d). Mounting a GIS from the opposite side to the manipulator takes advantage that the port is at a lower angle, thus deposition is more from the side of the ...
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... configuration depends on the specific LO-grids being used. Ideally, a clear line of sight should be present between the nozzle of the GIS and the lamella being attached. In the case, where the manipulator and the GIS approach the LO-grid from opposite sides, shadowing of the lamella by surrounding posts rendered the water deposition ineffective (Fig. ...
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... a GIS from one port to another may not be a practical solution. A simple solution avoiding shadowing is attaching the lamella to the left side of far-left post (Fig. 13d). This straightforward solution is proven reproducible and simple (Fig. ...
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... a GIS from one port to another may not be a practical solution. A simple solution avoiding shadowing is attaching the lamella to the left side of far-left post (Fig. 13d). This straightforward solution is proven reproducible and simple (Fig. ...
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... alternative 'pocket' approach was originally published by Mahamid 21 and recently refined by Schaffer 22 demonstrated using the Kleindiek cryo-gripper solution. Figure 14 illustrates the steps involved in preparation and its use with the Omniprobe solution. The process is performed at room temperature and consists of pre-fabrication (Fig. 14a) and the addition of platinum 'shoulders' to the top of the LO-grid post (Fig. 14c) by ion beam induced deposition (FIB deposition). ...
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... alternative 'pocket' approach was originally published by Mahamid 21 and recently refined by Schaffer 22 demonstrated using the Kleindiek cryo-gripper solution. Figure 14 illustrates the steps involved in preparation and its use with the Omniprobe solution. The process is performed at room temperature and consists of pre-fabrication (Fig. 14a) and the addition of platinum 'shoulders' to the top of the LO-grid post (Fig. 14c) by ion beam induced deposition (FIB deposition). This is followed by FIB-milling of 'pockets' into top of the shoulders and the LO-grid to give a slot into which the lamella can me inserted (Fig 14. d-f). This allows for a lamella to be firmly attached ...
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... and recently refined by Schaffer 22 demonstrated using the Kleindiek cryo-gripper solution. Figure 14 illustrates the steps involved in preparation and its use with the Omniprobe solution. The process is performed at room temperature and consists of pre-fabrication (Fig. 14a) and the addition of platinum 'shoulders' to the top of the LO-grid post (Fig. 14c) by ion beam induced deposition (FIB deposition). This is followed by FIB-milling of 'pockets' into top of the shoulders and the LO-grid to give a slot into which the lamella can me inserted (Fig 14. d-f). This allows for a lamella to be firmly attached while still being visible in the cryo-TEM at high tilt angles. The pocket approach ...
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... process is performed at room temperature and consists of pre-fabrication (Fig. 14a) and the addition of platinum 'shoulders' to the top of the LO-grid post (Fig. 14c) by ion beam induced deposition (FIB deposition). This is followed by FIB-milling of 'pockets' into top of the shoulders and the LO-grid to give a slot into which the lamella can me inserted (Fig 14. d-f). This allows for a lamella to be firmly attached while still being visible in the cryo-TEM at high tilt angles. ...
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... the transferred is successfully made, throw out the liquid nitrogen and use a hairdryer to warm up the cup and other components in the cup to drive away the condensed water. Images in Figure 15 demonstrate the progressive build-up of contamination on a Lift-Out support grid over 25 minutes during multiple transfers. Following the second transfer (Fig. 15d,e) both posts show significant contamination clearly demonstrating that retaining nitrogen for more than 15-20 minutes is inadvisable. ...
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... the transferred is successfully made, throw out the liquid nitrogen and use a hairdryer to warm up the cup and other components in the cup to drive away the condensed water. Images in Figure 15 demonstrate the progressive build-up of contamination on a Lift-Out support grid over 25 minutes during multiple transfers. Following the second transfer (Fig. 15d,e) both posts show significant contamination clearly demonstrating that retaining nitrogen for more than 15-20 minutes is inadvisable. This type of liquid nitrogen-born contamination can easily block the site of interest on the lamella, rendering hours of work ...
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... minimise handling the Lift-Out grid, the slot and half grids can be pre-aligned and glued together in advance of the LO-procedure (Fig. 16). This 'one piece' solution not only solves the risk of falling through but adds a robust rim for handling the grid away from the posts in the centre of the half grid. An opening is cut in the slot grid, allowing the micromanipulator to reach the half-grid to mount the lamella (Fig. 16b). The solution with the combined grids solves the ...
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... pre-aligned and glued together in advance of the LO-procedure (Fig. 16). This 'one piece' solution not only solves the risk of falling through but adds a robust rim for handling the grid away from the posts in the centre of the half grid. An opening is cut in the slot grid, allowing the micromanipulator to reach the half-grid to mount the lamella (Fig. 16b). The solution with the combined grids solves the main issues ...
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... minimisation of handling LO-grids was done by gluing the grid to the retaining ring (clip ring) of a modified Gatan 626 side loading rod (Fig. 16c). This solution required the design of a cryo-sledge that accepts this modified clip ring in a vertical orientation (Fig. 16d) (for deposition and milling) and allows quick transfer of the clip-ring to the cooled cryo-TEM holder. The result was a noticeable lack of frost on the LO-grid posts (Figs. 16e,f) which is attributed to the ...
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... minimisation of handling LO-grids was done by gluing the grid to the retaining ring (clip ring) of a modified Gatan 626 side loading rod (Fig. 16c). This solution required the design of a cryo-sledge that accepts this modified clip ring in a vertical orientation (Fig. 16d) (for deposition and milling) and allows quick transfer of the clip-ring to the cooled cryo-TEM holder. The result was a noticeable lack of frost on the LO-grid posts (Figs. 16e,f) which is attributed to the rapid transfer of the grid from This article is protected by copyright. All rights ...
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... of a modified Gatan 626 side loading rod (Fig. 16c). This solution required the design of a cryo-sledge that accepts this modified clip ring in a vertical orientation (Fig. 16d) (for deposition and milling) and allows quick transfer of the clip-ring to the cooled cryo-TEM holder. The result was a noticeable lack of frost on the LO-grid posts (Figs. 16e,f) which is attributed to the rapid transfer of the grid from This article is protected by copyright. All rights ...
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... or water) that can rapidly build up on the surface of the sample. This could obscure the area of interest or cause complications when extracting the second (or third) lamella. There will also be a build-up of the condensation layer on the micromanipulator, however, this layer can be removed by exposing the 'needle' to the FIB for a few minutes (Fig. 17). Additional time for this step should be factored into the overall time demands of the ...
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... coating takes place in a preparation chamber if using Quorum system, if using other cryo-stages the location of sputtering and order of steps may vary. Figure 12. TEM/Scanning Transmission Electron Microscope (STEM) micrographs resulting from cryo-LO work demonstrated using yeast (S. cerevisiae). ...
Citations
... Cryogenic in situ lift out is performed inside the FIB vacuum chamber and can be challenging due to its lengthy and complex steps. Potential temperature gradients between the micromanipulator and the specimen surface requiring multiple nontrivial probe attachments may lead to possible sample preparation failures (Parmenter & Nizamudeen, 2021). We recently applied cryogenic ex situ lift out (cryo-EXLO) for a successful manipulation of FIB-milled yeast, while maintaining specimen vitrification (Giannuzzi et al., 2023). ...
In this study, a conjugate radiation/conduction multimode heat transfer analysis of cryogenic focused ion beam (FIB) milling steps necessary for producing ex situ lift out specimens under cryogenic conditions (cryo-EXLO) is performed. Using finite volume for transient heat conduction and enclosure theory for radiation heat transfer, the analysis shows that as long as the specimen is attached or touching the FIB side wall trenches, the specimen will remain vitreous indefinitely, while actively cooled at liquid nitrogen (LN2) temperatures. To simulate the time needed to perform a transfer step to move the bulk sample containing the FIB-thinned specimen from the cryo-FIB to the cryo-EXLO cryostat, the LN2 temperature active cooling is turned off after steady-state conditions are reached and the specimen is monitored over time until the critical devitrification temperature is reached. Under these conditions, the sample will remain vitreous for >3 min, which is more than enough time needed to perform the cryo-transfer step from the FIB to the cryostat, which takes only ∼10 s. Cryo-transmission electron microscopy images of a manipulated cryo-EXLO yeast specimen prepared with cryo-FIB corroborates the heat transfer analysis.
... The remaining connection to the bulk material is removed, the micromanipulator is used to transfer the extracted volume, and redeposition milling is used to attach the volume to the receiver grid 31 . Finally, an electron-transparent lamella is prepared 32 . ...
... The study of multicellular organisms and tissues by cryo-ET, however, holds enormous potential for biological discovery, and technical advances are thus needed. We anticipate the combination of recent hardware and workflow improvements [30][31][32] with the increased throughput of Serial Lift-Out to make cryo-ET data acquisition from high-pressure-frozen material more attainable. In addition, plasma FIB technology, while available for some years, was recently introduced to the cryo-FIB community 13,35,36 and may further improve cryo-lift-out throughput by increased ablation rates. ...
Cryo-focused ion beam milling of frozen-hydrated cells and subsequent cryo-electron tomography (cryo-ET) has enabled the structural elucidation of macromolecular complexes directly inside cells. Application of the technique to multicellular organisms and tissues, however, is still limited by sample preparation. While high-pressure freezing enables the vitrification of thicker samples, it prolongs subsequent preparation due to increased thinning times and the need for extraction procedures. Additionally, thinning removes large portions of the specimen, restricting the imageable volume to the thickness of the final lamella, typically <300 nm. Here we introduce Serial Lift-Out, an enhanced lift-out technique that increases throughput and obtainable contextual information by preparing multiple sections from single transfers. We apply Serial Lift-Out to Caenorhabditis elegans L1 larvae, yielding a cryo-ET dataset sampling the worm’s anterior–posterior axis, and resolve its ribosome structure to 7 Å and a subregion of the 11-protofilament microtubule to 13 Å, illustrating how Serial Lift-Out enables the study of multicellular molecular anatomy.
... However, cryo-TEM has historically been limited by sample preparation techniques (Doerr, 2016, Cheng et al., 2015. New developments in cryo-sample preparation methods (Klumpe et al., 2022, Parmenter & Nizamudeen, 2021, Long et al., 2022, Hayles & DAM, 2021, Li et al., 2023 and the development of cryogenic shuttle transfer systems have significantly expanded the capacity of cryo-TEM (Wagner et al., 2020, Huang et al., 2022, Schaffer et al., 2019. ...
Reliable and consistent preparation of atom probe tomography (APT) specimens from aqueous and hydrated biological specimens remains a significant challenge. One particularly difficult process step is the use of a focused ion beam (FIB) instrument for preparing the required needle-shaped specimen, typically involving a "lift-out" procedure of a small sample of material. Here, two alternative substrate designs are introduced that enable using FIB only for sharpening, along with example APT datasets. The first design is a laser-cut FIB-style half-grid close to those used for transmission-electron microscopy, that can be used in a grid holder compatible with APT pucks. The second design is a larger, standalone self-supporting substrate called a "crown", with several specimen positions that self-aligns in APT pucks, prepared by electrical discharge machining (EDM). Both designs are made nanoporous, to provide strength to the liquid-substrate interface, using chemical and vacuum dealloying. We select alpha brass a simple, widely available, lower-cost alternative to previously proposed substrates. We present the resulting designs, APT data, and provide suggestions to help drive wider community adoption.
... The 81 remaining connection to the bulk material is removed, the micromanipulator is used to transfer the 82 extracted volume and redeposition milling (Schreiber et al 2018) is used to attach the volume to the 83 receiver grid. Finally, an electron transparent lamella is prepared (Parmenter & Nizamudeen 2021). 84 ...
Cryo-focused ion beam milling of frozen-hydrated cells and subsequent cryo-electron tomography (cryo-ET) has enabled the structural elucidation of macromolecular complexes directly inside cells. Application of the technique to multicellular organisms and tissues, however, is still limited by sample preparation. While high-pressure freezing enables the vitrification of thicker samples, it prolongs subsequent preparation due to increased thinning times and the need for extraction procedures. Additionally, thinning removes large portions of the specimen, restricting the imageable volume to the thickness of the final lamella, typically < 300 nm. Here, we introduce Serial Lift-Out, an enhanced lift-out technique that increases throughput and obtainable contextual information by preparing multiple sections from single transfers. We apply Serial Lift-Out to C. elegans L1 larvae yielding a cryo-ET dataset sampling the worm's anterior-posterior axis and resolve its ribosome structure to 7 A, illustrating how Serial Lift-Out enables the study of multicellular molecular anatomy.
... As for the VHUT-cryoFIB method 8 requiring additional cryo-ultramicrotomy operations, our method is much more efficient when counting the total time of sample preparation. Importantly, different from other published methods 8,9,[30][31][32] , our method is just based on the fundamental functions and operations of the cryoFIB and HPF instruments, and doesn't need customized instruments and additional tools (Supplementary Table 4). The major problem currently affecting the success rate is ice contamination, which occurs during the transfer process after milling and can significantly reduce the area suitable for cryoET data collection. ...
Cryo-electron tomography (cryoET) is a powerful tool for exploring the molecular structure of large organisms. However, technical challenges still limit cryoET applications on large samples. In particular, localization and cutting out objects of interest from a large tissue sample are still difficult steps. In this study, we report a sample thinning strategy and workflow for tissue samples based on cryo-focused ion beam (cryoFIB) milling. This workflow provides a full solution for isolating objects of interest by starting from a millimeter-sized tissue sample and ending with hundred-nanometer-thin lamellae. The workflow involves sample fixation, pre-sectioning, a two-step milling strategy, and localization of the object of interest using cellular secondary electron imaging (CSEI). The milling strategy consists of two steps, a coarse milling step to improve the milling efficiency, followed by a fine milling step. The two-step milling creates a furrow–ridge structure with an additional conductive Pt layer to reduce the beam-induced charging issue. CSEI is highlighted in the workflow, which provides on-the-fly localization during cryoFIB milling. Tests of the complete workflow were conducted to demonstrate the high efficiency and high feasibility of the proposed method.
... Lamellae of larger samples can be created by extracting biological material with a FIB lift-out approach. With this method, a 3-to 5-µm-thick lamella is created perpendicular to the surface of a high-pressure frozen sample and transferred to a slot on another grid with a cryo-micromanipulator [74][75][76][77] or cryo-gripper 78,79 , followed by fine FIB fabrication of lamellae. The thick ice layer can be removed with a cryo-microtome or freeze fracturing before the lift-out procedure 80,81 . ...
Cryogenic electron microscopy and data processing enable the determination of structures of isolated macromolecules to near-atomic resolution. However, these data do not provide structural information in the cellular environment where macromolecules perform their native functions, and vital molecular interactions can be lost during the isolation process. Cryogenic focused ion beam (FIB) fabrication generates thin lamellae of cellular samples and tissues, enabling structural studies on the near-native cellular interior and its surroundings by cryogenic electron tomography (cryo-ET). Cellular cryo-ET benefits from the technological developments in electron microscopes, detectors and data processing, and more in situ structures are being obtained and at increasingly higher resolution. In this Review, we discuss recent studies employing cryo-ET on FIB-generated lamellae and the technological developments in ultrarapid sample freezing, FIB fabrication of lamellae, tomography, data processing and correlative light and electron microscopy that have enabled these studies. Finally, we explore the future of cryo-ET in terms of both methods development and biological application. This Review describes advances in cryogenic electron tomography on focused ion beam lamellae, highlighting the key benefits of this technology for in situ structural biology and discussing important future directions.
... This occupies the FIB for the better part of a day which bottlenecks the process of performing cryo-TEM and reduces access to the FIB for other tasks. A recent publication detailed specific times for all cryo-FIB milling and cryo-INLO manipulation steps (Parmenter & Nizamudeen, 2021). The cryo-INLO steps denoted can take up to 60 min to accomplish, with the entire cryo-FIB milling and cryo-INLO process consuming the majority of a workday. ...
... Together with the rapid cryo-EXLO process, the preparation of four complete EXLO-type specimens was therefore significantly faster compared to the typical cryo-INLO approach which requires final thinning steps after the lift out is completed (Parmenter & Nizamudeen, 2021). The yeast cells were cryotransferred to the lift out station inside the glove box as described above. ...
This work describes cryogenic ex situ lift out (cryo-EXLO) of cryogenic focused ion beam (cryo-FIB) thinned specimens for analysis by cryogenic transmission electron microscopy (cryo-TEM). Steps and apparatus necessary for cryo-EXLO are described. Methods designed to limit ice contamination include use of an anti-frost lid, a vacuum transfer assembly, and a cryostat. Cryo-EXLO is performed in a cryostat with the cryo-shuttle holder positioned in the cryogenic vapor phase above the surface of liquid N2 (LN2) using an EXLO manipulation station installed inside a glove box maintained at < 10% relative humidity and inert (e.g., N2 gas) conditions. Thermal modeling shows that a cryo-EXLO specimen will remain vitreous within its FIB trench indefinitely while LN2 is continuously supplied. Once the LN2 is cut off, modeling shows that the EXLO specimen will remain vitreous for over 4 min, allowing sufficient time for the cryo-transfer steps which take only seconds to perform. Cryo-EXLO was applied successfully to cryo-FIB-milled specimen preparation of a polymer sample and plunge-frozen yeast cells. Cryo-TEM of both the polymer and the yeast shows minimal ice contamination with the yeast specimen maintaining its vitreous phase, illustrating the potential of cryo-EXLO for cryo-FIB-EM of beam-sensitive, liquid, or biological materials.
... A major challenge with a full cryogenic workflow is that the precursor gas deposits rapidly onto any exposed surface cooled below its condensation temperature (Perea et al., 2017), leading to uncontrolled uniformity and thickness. Although this condensed material can be locally 'cured' to a conducting solid material through the application of an electron beam or ion beam, this requires care to ensure the material is 'cured' for the full thickness and does not contain any residual condensed gas that may expand upon warming (PARMENTER & NIZAMUDEEN, 2020). This larger scale deposition is extremely quick and has been proposed to reduce time for such areas as electron beam lithography (Salvador-Porroche et al., 2020) but for the purposes of APT sample preparation (Córdoba et al., 2019;Orús et al., 2021), it has limited application due to the lack of site specificity and reduction in thickness control. ...
... Si-coupon) to weld the parts together. Similar approaches have been used for cryo-specimen preparation by FIB in the biological sciences (Parmenter & Nizamudeen, 2020). Redeposition is caused by the incoming ion beam and causes a mixing of the sputtered materials with the incoming beam (Cairney & Munroe, 2003) and had already been used to create welds for lift-outs (Montoya et al., 2007;Kuba et al., 2020) or fill pores to facilitate further specimen preparation (Zhong et al., 2020). ...
Workflows have been developed in the past decade to enable atom probe tomography analysis at cryogenic temperatures. The inability to control the local deposition of the metallic precursor from the gas-injection system (GIS) at cryogenic temperature makes the preparation of site-specific specimens by using lift-out extremely challenging in the focused-ion beam. Schreiber et al. exploited redeposition to weld the lifted-out sample to a support. Here we build on their approach to attach the region-of-interest and additionally strengthen the interface with locally sputtered metal from the micromanipulator. Following standard focused-ion beam annular milling, we demonstrate atom probe analysis of Si in both laser pulsing and voltage mode, with comparable analytical performance as a pre-sharpened microtip coupon. Our welding approach is versatile, as various metals could be used for sputtering, and allows similar flexibility as the GIS in principle.
... These each pose their own unique challenges. Much of the fundamentals and process development of room temperature FIB lift-out can be found in the literature, along with some of the first methods papers for cryo-FIB [8,13,20,22,25,[35][36][37][38][39][40][41][42]. ...
... In the first approach the needle is brought into contact with the lamella and then the GIS chemistry, either ice or Pt precursor is allowed to flow for a brief period of time. The GIS chemistry will create the material connection by coating the entire working area, including the ROI/needle interface [38,41]. Once connected to the needle the remaining connection to the substrate is cut away and the lamella can be lifted out. ...
Primarily driven by structural biology, the rapid advances in cryogenic electron microscopy (cryo-EM) techniques are now being adopted and applied by materials scientists. Samples that inherently have electron transparency can be rapidly frozen (vitrified) in amorphous ice and imaged directly on a cryogenic transmission electron microscopy (cryo-TEM), however this is not the case for many important materials systems, which can consist of layered structures, embedded architectures, or be contained within a device. Cryogenic focused ion beam (cryo-FIB) lift-out procedures have recently been developed to extract intact regions and interfaces of interest, that can then be thinned to electron transparency and transferred to the cryo-TEM for characterization. Several detailed studies have been reported demonstrating the cryo-FIB lift-out procedure, however due to its relative infancy in materials science improvements are still required to ensure the technique becomes more accessible and routinely successful. Here, we review recent results on the preparation of cryo-TEM lamellae using cryo-FIB and show that the technique is broadly applicable to a range of soft matter and beam sensitive energy materials. We then present a tutorial that can guide the materials scientist through the cryo-FIB lift-out process, highlighting recent methodological advances that address the most common failure points of the technique, such as needle attachment, lift-out and transfer, and final thinning.
... Several approaches to generate thin sections of cells in their hydrated state were developed (Al-Amoudi et al., 2011;Bokstad et al., 2012;Dubochet et al., 2007), however, the introduction of cryo-FIB milling for biological samples (Marko et al., 2007) paved the way to reproducibly and reliably produce sections of cells in their unperturbed, native state without cutting artifacts (Engel et al., 2015;Harapin et al., 2015;Medeiros et al., 2018;Rigort et al., 2012). Recently developed lift-out techniques even allow to generate thin sections of multi-cellular organisms for cryo-ET analysis (Parmenter and Nizamudeen, 2021;Schaffer et al., 2019), although technical difficulty and low throughput make the investigation of organisms still challenging. Impressive insights into the cellular interior are more routinely described using single-cellular model organisms like S. cerevisiae (Allegretti et al., 2020;Delarue et al., 2018;Kralt et al., 2022), C. reinhardtii Bykov et al., 2017;van den Hoek et al., 2022), bacteria (Khanna et al., 2021;Rapisarda et al., 2019;Weiss et al., 2019) or eukaryotic cells which can be directly grown on EM grids (Harapin et al., 2015;Kronenberg-Tenga et al., 2021;Mahamid et al., 2016;Mosalaganti et al., 2022;Schuller et al., 2021). ...
Cryo-electron tomography (cryo-ET) combines a close-to-life preservation of the cell with high-resolution three-dimensional (3D) imaging. This allows to study the molecular architecture of the cellular landscape and provides unprecedented views on biological processes and structures. In this review we mainly focus on the application of cryo-ET to visualize and structurally characterize eukaryotic cells – from the periphery to the cellular interior. We discuss strategies that can be employed to investigate the structure of challenging targets in their cellular environment as well as the application of complimentary approaches in conjunction with cryo-ET.
