A versatile cryo-transfer system, connecting cryogenic focused ion beam sample preparation to atom probe microscopy

Chandra Macauley; Martina Heller; Alexander Rausch; Frank Kümmel; Peter Felfer

doi:10.1371/journal.pone.0245555

. 2021 Jan 19;16(1):e0245555. doi: 10.1371/journal.pone.0245555

A versatile cryo-transfer system, connecting cryogenic focused ion beam sample preparation to atom probe microscopy

Chandra Macauley ^1,^2,^*, Martina Heller ^1,², Alexander Rausch ¹, Frank Kümmel ^1,^¤a, Peter Felfer ¹

Editor: Leigh T Stephenson³

PMCID: PMC7815152 PMID: 33465106

Abstract

Atom probe tomography (APT) is a powerful technique to obtain 3D chemical and structural information, however the ‘standard’ atom probe experimental workflow involves transfer of specimens at ambient conditions. The ability to transfer air- or thermally-sensitive samples between instruments while maintaining environmental control is critical to prevent chemical or morphological changes prior to analysis for a variety of interesting sample materials. In this article, we describe a versatile transfer system that enables cryogenic- or room-temperature transfer of specimens in vacuum or atmospheric conditions between sample preparation stations, a focused ion beam system (Zeiss Crossbeam 540) and a widely used commercial atom probe system (CAMECA LEAP 4000X HR). As an example for the use of this transfer system, we present atom probe data of gallium- (Ga)-free grain boundaries in an aluminum (Al) alloy specimen prepared with a Ga-based FIB.

1. Introduction

Cryogenic sample preparation and characterization has been developed and widely used in the biology community since the 1970s [1] and is only recently being applied more broadly to materials science. These techniques open up a wide range of materials, including soft materials and environmentally unstable materials, that can now be studied e.g. in electron microscopes such as transmission electron microscopes (TEMs) [2, 3] and scanning electron microscopes (SEMs) [4, 5]. Such cryogenic/environmental sample preparation techniques could also open up new research fields when applied to atom probe tomography (APT), an increasingly popular, high-resolution microscopy / mass spectrometry technique that is capable of determining the chemistry and structure of materials in 3-dimensions with single atom sensitivity [6]. Although conductive, non-site specific samples are relatively easily prepared with electropolishing [7], preparation of non-conductive or site-specific APT samples is almost always achieved using a combined focused ion beam/scanning electron microscope (FIB/SEM) to make samples with the required geometry [8–11]. Conventional transfer of samples from the FIB/SEM to the atom probe takes place at ambient temperature and pressure, causing chemical or morphological changes or even complete destruction of environmentally- or thermally-sensitive samples. Therefore, a system that enables cryogenic- and/or environmental-transfers is critical to broadening the research fields to which APT can be applied.

Some cryogenic- and environmental-transfer systems are commercially available and rely on a specimen shuttle transfer device or suitcase that docks with various instruments and maintains a user-defined environment during transfer [12–15]. These systems can be broadly separated based on if the transfer devices are a) actively cooled and pumped e.g. Ferrovac’s cryo suitcase [15, 16], b) only actively cooled e.g. Leica’s VCT500 [17, 18] and c) completely passive e.g. Quorum’s PP3006 CoolLok [14]. There are advantages and disadvantages to each of these systems. While the actively cooled and pumped Ferrovac Ultra High Vacuum (UHV) Cryo Transfer Module (CTM) provides the most controlled transfer conditions (vacuum below 10⁻⁹ mbar, temperature down to -180°C), the additional hardware required increases the size and weight of a liquid nitrogen-filled transfer device to ~1 m and 14 kg [16]. Furthermore, the Ferrovac suitcase requires a free CF40 port, which may not be available on well-equipped microscopes. On the other hand, the passive transfer systems are often more compact but require quick transfer times between instruments to minimize sample degradation. Despite the need for quick transfers, passive systems have successfully been used to observe hydrogen trapping in steels [19] and intact organic molecules [14]. A recent publication by McCarroll et al. provides an overview of some of the most common systems [20].

In this paper, we describe a custom-built versatile transfer system that enables transfer of samples that are sensitive to air or thermal exposure between sample preparation stations such as a FIB/SEM and a local electrode atom probe (LEAP). The paper is organized in the order of components/modifications used during the cryo-FIB preparation of an atom probe sample and subsequent transfer into the LEAP. Finally, to demonstrate the actively cooled cryogenic-, high vacuum-transfer capabilities of the system, an aluminum (Al) alloy was milled using a gallium (Ga) liquid-metal ion source FIB at cryogenic temperatures prior to being transferred using the described system to the LEAP. While Al alloys are not per-se environmentally sensitive, the exposure to Ga in general [21], and in the FIB in particular [22–24], leads to strong segregation of the Ga to the grain boundaries, in a process called liquid metal embrittlement. The altered chemical makeup of the grain boundary is then no longer representative of the original specimen [25]. However, the site-specific investigation of the grain boundaries in these Al alloys is of great interest, as they define the susceptibility to intergranular corrosion [26–28], a major issue in their use e.g. in the automotive industry [29]. As shown below, this problem can be circumvented by the use of low temperatures throughout the characterization and transfer process.

While the ability to quickly make environmentally controlled transfers between instruments has been shown by other groups, our system has unique advantages such as a small footprint and close shielding of the sample that prevents frost formation. Since it uses standard parts or components that can easily be milled, the system is highly adaptable to new experimental arrangements. Additionally, the system does not require a dedicated cryo port on the FIB/SEM, as it interlocks directly with the existing load lock door, a feature that is ideal for multi-user instruments that are not exclusively used for cryo experiments.

2. Materials and methods

In the course of this work, we have established a cryogenic route connecting a Zeiss Crossbeam 540 FIB/SEM (Carl Zeiss, Oberkochen, Germany), equipped with a Quorum cryogenic PP3005 SEMCool (Quorum Technologies, Bath, UK) cold stage, with a CAMECA 4000X HR local electrode atom probe (LEAP) system through a cryogenic transfer system designed by the authors. A general overview of the cryogenic/vacuum transfer system including relevant temperatures and pressures at the various steps of sample preparation, transfer and analysis is shown in Fig 1. Fig 1A, a flow chart of the preparation process is given, including approximate transfer times. All steps colored in blue indicate the use of new components designed and built in this work. Fig 1B gives a schematic overview of how the components interface with the FIB/SEM and LEAP. The sample is first transferred into the FIB/SEM analysis chamber, AC, using a custom transfer device (FIBTD) that attaches directly to the existing load lock, LL, Fig 1B, top. After FIB milling and/or SEM characterization, the sample is moved out of the FIB/SEM using the same, thermally-insulated, but not actively cooled FIBTD, in which the sample is handed off to an actively cooled transfer device (ACTD), with a standard KF16 vacuum flange (Fig 1B, center). The ACTD is used to transport the sample to the LEAP and attaches to a custom ‘cryo’ load lock, that allows for direct insertion of the sample into the LEAP’s buffer chamber, BC (Fig 1B, bottom). In the buffer chamber, a special specimen shuttle puck is waiting pre-cooled in a liquid nitrogen cooled insertable stage. The transfer rod in the LEAP is then used to move the sample onto the stage in the analysis chamber. In the cold chain, the sample can be kept at cryogenic temperatures at all times (< ca. - 160°C), so long as the hand-off times between systems, when the sample is not actively cooled, are kept low (some 10s of s, see S1 File). This was confirmed with temperature measurements shown in S1 and S2 Figs and S1 Table in S1 File. Here we use the phrase “cold chain” to describe the protocol of transferring samples from the FIB/SEM to the LEAP while maintaining cryogenic temperatures. The ACTD interfaces with other instrumentation through an industry standard KF16 flange, making it easily adaptable to multiple sample preparation and analysis stations. Although not discussed in detail in this paper, the ACTD connects to other platforms, shown in the dashed box in Fig 1A, such as a cryo electropolishing station and PVD coating/ion etching station. Photos of these systems are shown in S3 and S4 Figs in S1 File, respectively. Each component required for the extension of the commercial systems to cryo-capability was designed and built in-house, using standard vacuum parts whenever possible. Custom components were fabricated either by the Mechanics and Electronics Workshop of the Faculty of Engineering at Friedrich-Alexander-University (Erlangen, Germany) or using a miniature 5-axis CNC mill (Pocket NC, Bozeman, MT, USA).

Fig 1 — In a) the workflow is shown with transfer times. Custom components designed and built as part of this work are blue while existing systems are black A schematic overview in b) shows the FIB transfer device connected directly to a Zeiss Crossbeam 540 FIB/SEM. Prepared samples can be transferred with an actively cooled transfer device (ACTD) that interfaces with existing instrument load locks (LL) into a CAMECA LEAP. AC: analysis chamber, BC: buffer chamber, RT: room temperature.

2.1 Cryogenic FIB/SEM stage and transfer device

The basis of the sample preparation in this work is the FIB/SEM. In our instrument, samples can be kept at any temperature from room temperature down to around -190°C, by using a Quorum PP3005 cryogenic stage. This stage is cooled by a stream of gaseous nitrogen, which is passed through liquid nitrogen in a heat exchanger and thus brought to near liquid nitrogen temperatures. Temperature regulation is achieved by adjusting the cold gas flowrate for coarse tuning in combination with a heating element for closed-loop temperature control. This thermally insulated cold stage attaches to the existing SEM stage, as shown in Fig 2A. This figure shows an APT sample mounted in a double threaded APT sample carrier (‘double nipple’) which is in turn mounted in a FIB cryo-shuttle.

Custom, pre-tilted (54°, which is the angle between the electron and ion beams) FIB cryo-shuttles with threads to accommodate the specimens were made to allow annular ion beam milling perpendicular to the sample surface without tilting the stage. This is useful since the cryo-stage is supplied with cooling gas through polymer tubes which get stiff at cold temperatures. While tilting at cryogenic temperatures is possible, it needs to be carried out carefully to not damage the gas lines or stage. By using pre-tilted samples, the ion beam can easily be used to cut into materials or sharpen tips into the required geometry for atom probe tomography. If perpendicular FIB milling is not required for a given experiment, but SEM imaging and cryo- or environmental-transfer to other systems is desired, non-pre-tilted FIB cryo-shuttles can be used as shown in Fig 2B.

To facilitate quick cryogenic- or environmental-transfers to/from the FIB/SEM, a transfer device was designed and built, as shown in Fig 2B. This FIB transfer device (FIBTD) interfaces with the existing Zeiss 80 mm Airlock without further modification, as shown in Fig 2C. It also allows for a hand-off to the actively cooled transfer device (ACTD), for FIB cryo-shuttles with pre-tilt (‘ACTD1’ in Fig 2B) and without pre-tilt (‘ACTD2’ in Fig 2B). The transfer device reduces the risk of chamber contamination and enables efficient characterization of multiple samples in a single FIB session, since without the means to transfer cold samples through the load-lock, the cold stage would need to be warmed to room temperature for sample exchanges and the entire analysis chamber vented. While commercial load-lock solutions exist that attach to the ports of the chamber, no free ports to attach such a solution were available in our current multi-user FIB/SEM instrument, which is not exclusively used for cryogenic characterization. To prevent unwanted vibrations in the FIB/SEM and to clear the view of the instrument operator, the FIBTD is easily removed after each transfer.

For a sample exchange, the existing Airlock roughing pump is used to pump down the airlock with the attached FIBTD prior to introducing the sample to the pre-cooled stage. This pumping operation takes ca. 30s until a vacuum sufficient for transfer is established. The generated vacuum of ~ 10⁻⁴ mbar thereby has proven to be sufficient to prevent frost built up on the specimen, as shown below in the analysis of the Al alloy. This is likely also a result of using dry nitrogen as a flushing gas for the load lock, keeping the H₂O partial pressure low. As the cryo-stage is ~18mm taller than the stock SEM stage, the stage has to be lowered to its minimum Z-height for sample exchange. A flexible bellows coupling (Figs 2B and 1) is used to allow the FIBTD to pivot slightly upwards, so the FIB cryo-shuttle on the FIBTD with the cold stage. A bayonet connection to the specimen shuttle (Fig 2D) is used to hold the shuttle at the end of the transfer rod securely, thus ensuring rapid, reliable transfers. In this FIBTD, no active cooling or pumping is available. The sample is, however, thermally insulated from the rest of the transfer device via a low thermal conductivity polyether ether ketone (PEEK) end-effector, 2 in Fig 2D, to minimize heat transfer. To measure the efficacy of this thermal isolation, specimen temperature was measured while a FIB cryo shuttle, pre-cooled by the cryo stage to -118°C, warmed up (since it was not actively cooled) during a simulated hand-off to the ACTD. As shown in S2 Fig in S1 File, the pre-cooled shuttle on the PEEK end-effector of the FIBTD warms up at 2.3°C/min at a pressure below 10⁻⁵ mbar. The importance of pressure on the warming rates of pre-cooled FIB cryo shuttles attached to the FIBTD transfer rod is demonstrated in S1 Table in S1 File.

2.2 Actively cooled transfer device (ACTD)

To facilitate transport of the samples between the FIB/SEM and the LEAP, an actively cooled transfer device (ACTD) was developed. The ACTD can be cooled using liquid nitrogen or potentially other cryogenic liquids via a cold finger, while temperature and pressure are monitored and logged to alert if all the liquid nitrogen is used up or a vacuum leak occurs. So long the reservoir does not run dry, the sample is can be as cold as -170° C in the ACTD. Active pumping through an ion getter pump is also possible but has so far not been used due to the short transfer times between instruments (< 10 minutes) within the facility. Active pumping would only be necessary for longer transfer times > 15 min, e.g. between institutions. The ACTD is shown in Fig 3A. Due to the small size of the atom probe samples, the ACTD can be small and compact, (41 cm long, 2 kg), and can easily be carried in one hand, while still allowing for bottom-out pressures in the range of 10⁻⁸ mbar through its almost entirely metal sealed construction with a magnetically-coupled transfer rod. These pressures are reached after a pump down of several hours to a day, depending on ambient humidity, if the ACTD was exposed to air. The main body of the ACTD is machined from a solid piece of 316 stainless steel, with welded-in CF 16 flanges and a KF 16 flange to the gate valve. As a result, the lowest achievable pressure is dictated by the gate valve, which is a KF 16 gate valve with aluminum body and rubber gasket sealed flanges (VAT Valves, Switzerland). Although these KF valves have the same leak tightness as their CF metal flanged, fluoroelastomer (FKM)-sealed counterparts, they cannot be heated beyond 80°C, limiting possible bake-outs. Such higher temperature bake-outs are only needed to reach ultra high vacuum (<10⁻⁹ mbar) conditions. Since the ACTD is being connected to the FIB/SEM, which has an ultimate pressure of 10⁻⁷ mbar, such bake-outs are not required.

Fig 3 — a) Photo of the ACTD showing the valve open and the copper end-component (labeled 1) extended. b) The rendered cross-section of the ACTD shows the threaded connection between the liquid nitrogen (LN₂) cold finger and the copper end-component (1). A low thermal conductivity PEEK component (2) thermally isolates the end-component from the rest of the transfer rod. Temperature is measured at the cold finger using a thermocouple (TC). c) A higher magnification inset of b) more clearly shows how the double nipple screws into the copper end-component during transfers, resulting in the atom probe tip being shielded. d) Transfers between the FIBTD and ACTD are enabled by the two-threaded double nipple that has a right-handed screw (RHS) thread and a left-handed screw (LHS) thread.

Not only does the small size of the ACTD aid in the ease of transfers, it also minimizes frost contamination through its comparably small volume. As seen in the photograph and computer aided design (CAD) cross-section drawing in Fig 3A and 3B, respectively, a 210 mL liquid nitrogen-filled steel vessel provides active cooling via a direct threaded connection between the copper end-component and the spout of the vessel, enabling temperatures of -160°C during transfer. A detailed inset is provided in Fig 3C to more clearly show how the atom probe sample is secured in the ACTD. To protect the user from cold metal parts, the steel vessel is surrounded by a 3D-printed plastic hull. The sample is held in a double-nipple, Fig 3D, that has a 5 mm diameter right-handed screw (RHS) thread on one side, and a left-handed screw (LHS) thread on the other. Such a design enables the double nipple to be transferred between the copper end-component shown in Fig 3A, and FIB cryo shuttle or LEAP shuttle pucks with one continuous twisting motion, as will be described in the next section. The sample itself can be a rough-electropolished tip crimped in a copper tube or a half-TEM grid that contains several tips. With a clear bore of a minimum 16mm, larger sample diameters could be accommodated if needed. During transfer, the sample is closely shrouded by the copper end-component (5 mm inner diameter), as shown in the higher magnification cross-section in Fig 3C, minimizing frost formation. In this way, as the sample is screwed into the transfer rod, any contaminant will condense on the outside of the copper sheath rather than on the sample. The copper end-component is thermally isolated from the rest of the aluminum transfer rod with a low thermal conductivity plastic (PEEK) component. Temperature is measured using a K-type thermocouple (TC in Fig 3B) in contact with the liquid nitrogen (LN₂) cold finger, since the rotating motion of the transfer rod does not allow for direct attachment to the sample or the end-effector. The pressure in the ACTD is monitored using a MEMS Pirani gauge (MKS instruments type 925), with a bottom-out pressure of 1x10^-5 mbar. A manually operated mini-gate valve (KF 16, VAT Group AG) isolates the ACTD and enables connection to the FIB transfer device, other sample preparation stations (see S1 File), and to the LEAP.

2.3 Hand-off between the FIBTD and the ACTD

The FIBTD must connect with the ACTD to move cryogenically-prepared, or otherwise environmentally-sensitive samples to the LEAP. The ACTD can connect with the 54°-angled flange, as shown in the CAD drawing in Fig 4A, or if the sample is not mounted on a pre-tilted cryo-shuttle it can connect to the perpendicular flange (ACTD 2 in Fig 4A). Fig 4B shows both the FIBTD and the ACTD connected to the FIB/SEM load-lock. In this configuration, a (usually) pre-pumped ACTD is connected to the FIB transfer arm that is at ambient pressure. The FIB transfer device is then pumped down with the FIB/SEM load-lock, and the gate valve of the ACTD is opened. At this point, the vacuum is maintained by the turbo pump of the FIB/SEM system, resulting in a pressure of < 10⁻⁵ to 10⁻⁶ mbar, as measured by the wide-range gauge of the FIB/SEM, which is significantly lower compared to moving the specimen into the FIB/SEM, where initially only the FIB/SEM load lock roughing pump maintains the vacuum. When the FIB cryo-shuttle is first removed from the cryo stage, Fig 4C, it must be rotated 90° along the axis of the FIB transfer rod, so that the pre-tilted double nipple and atom probe sample are parallel to the axis of the ACTD, Fig 4D. The threaded copper sheath of the ACTD is then screwed on to the double nipple, enshrouding the sample, Fig 4E. Thanks to the two-threaded design of the double nipple, the same twisting direction is used to first screw the copper sheath onto the double nipple before the double nipple is unscrewed from the FIB cryo shuttle, as shown in the movie provided in the S1 File. The ACTD transfer rod is then retracted, the gate valve is closed and the ACTD is transported to the LEAP while maintaining vacuum conditions below the measurement limit of the attached Pirani gauge (10⁻⁵ mbar), simply by cryo-sorption for the relatively short time (ca. 5 min.) it takes to be transported from the FIB/SEM to the LEAP in our facility. This efficacy of this method has not yet been tested at another facility that requires much longer transfer times. During the exchange process, the sample is always connected to cold parts of either the FIBTD or the ACTD, so no temperature rise occurs (for temperature / pressure data from the hand-off see S1 File).

Fig 4 — a) CAD drawing depicting the 54° connection between the FIBTD and the ACTD. The perpendicular port for the ACTD that is labeled ACTD 2 can be used with the FIB cryo shuttles that are not pre-tilted. b) The connected transfer devices interface directly to the Zeiss load lock. When the FIB cryo shuttle is first removed from the FIB, c), it must be rotated 90° along the axis of the FIBTD transfer rod to line-up with the ACTD as shown in d). The copper end-component of the ACTD then surrounds the sample, e) as the double nipple is passed from the FIBTD to the ACTD.

2.4 Modifications to the LEAP

In order to allow the samples to be inserted into the atom probe without breaking the vacuum / cold chain, modifications to our atom probe were also needed. To this end, we implemented a solution that enables direct transfer of the samples onto the LEAP transfer rod that moves samples into the main analysis chamber. The sample transfer takes place in what is commonly referred to as the buffer chamber of the LEAP, after which the sample can be quickly transferred into the analysis chamber. This solution consists of a 70mm long cryo-transfer chamber, labeled 1 in the CAD drawing in Fig 5A and 5D, which was installed to the right of the existing buffer chamber. This necessitated the installation of a slightly longer LEAP transfer rod (609 mm) in order to accommodate the additional length. The flange also contains a viewport for specimen alignment (2) as well as two other flanges (3) that accommodate: 1) a movable stage, highlighted with the square in Fig 5A and shown in detail in 5B and 5C, capable of in-situ heating and cryogenic cooling, and 2) the stage’s electrical connections. The stage can be cooled with liquid nitrogen via a vessel (4 in Fig 5) that is also connected to the linear drive of the stage. When not in use, the stage can be retracted (Fig 5C, top), preventing interference with the normal use of the LEAP transfer rod. Prior to a cryo transfer, a custom LEAP shuttle puck is attached to the LEAP transfer rod, which is retracted fully before inserting the cryo stage, Fig 5C, bottom. The LEAP puck can then be placed on the cryo-stage to pre-cool before a cryo transfer is initiated. The ACTD connects directly to a flexible bellows coupling welded onto the cryo LL (5 in Fig 5). A 80 l/s turbo pump, as part of a pumping station configuration (Pfeiffer HiCube 80 Eco, 6) pumps down the volume of pipe between the ACTD and a VAT gate valve (7) while the pressure is measured by a combined Pirani and cold cathode vacuum gauge (8). When the pressure inside the pipe reaches a desired value, usually 10⁻⁶ mbar, the mini gate valve of the ACTD is opened. Once the pressure inside the ACTD is better than 5 x 10⁻⁶ mbar, the gate valve to the LEAP buffer chamber is opened and the sample, which was cooled throughout the process with liquid nitrogen, can be screwed into the pre-cooled, custom LEAP puck, Fig 5B. During this hand-off, the LEAP puck is held in place by the actively cooled cryo stage, Fig 5C, bottom. A video of the complete cryo-transfer into the LEAP is provided in the S1 File. Once the hand-off is complete, the ACTD transfer rod is retracted and the cryo load lock VAT gate valve is closed. The LEAP transfer rod is used to remove the custom LEAP puck from the cryo stage, which is then retracted, and the puck can be moved into the LEAP analysis chamber. Since all of the additional equipment is mounted outside the regular buffer chamber of the LEAP instrument and the ACTD is easily removed when not in use, normal operation by regular users is unimpeded.

Fig 5 — a) The rendered CAD drawing indicates the modifications to the LEAP, described in the text. A detailed view of the cryogenic-stage, is shown in b) with a custom LEAP shuttle in place as it would be during the hand-off of the sample and double nipple from the ACTD. The region highlighted with an orange box in a) is shown in c) to illustrate the movement of the cryo-stage from the perspective of the analysis chamber. d) A photo shows in detail the connection of the ACTD to the LEAP.

2.5 Utilization of the above cryo-transfer system to study Al alloys

To demonstrate the efficacy of the cryogenic-, environmental-transfer capabilities of the system, we chose to show the prevention of FIB-induced Ga segregation to the grain boundaries in the Al–Ga system. Normally, if Al is exposed to Ga, severe segregation of Ga to the grain boundaries of the Al is observed, as was discussed in the introduction. At low temperatures, we hypothesized that this phenomenon could be suppressed by inhibiting Ga diffusion.

To provide a sufficient chance of encountering one or more grain boundaries in the experiment, ultrafine-grained Al samples made of the solution hardened Al alloy, AA5754, were first rough electropolished in air at room temperature prior to transferring to the pre-cooled cryogenic FIB/SEM stage. The Al samples had been processed by accumulative roll bonding (ARB), which was first introduced by Tsuji et al. [30]. The microstructure and mechanical properties of the alloy used in this work were published by Hausöl et al. [31]. After 8 ARB-passes, a grain size in the normal rolling direction of 50–100 nm is achieved [31]. The small grain size was chosen to increase the likelihood that a grain boundary was present within the final atom probe tip. The sample was sharpened using a Ga-ion FIB/SEM at -140°C. Rather high energy ion beam parameters of 30 kV and 300pA were used during the final ion milling to cause the largest amount of Ga-implantation, which is usually undesirable [10] when preparing atom probe samples and often avoided by using lower voltages [32, 33]. However, knock on damage by the Ga ions was not a concern, since the main objective was to introduce a large amount of Ga ions as a “worst case scenario” for liquid metal embrittlement.

3. Results and discussion

After preparing the sharpened atom probe sample in the FIB at -140°C (133 K), the cryogenic- and environmental-transfer system was used to transfer the sample to the LEAP while maintaining a measured temperature of -160°C in the ACTD. For comparison, we have also carried out experiments where we have cryo-FIB milled specimens at -140°C and transferred them into the atom probe at room temperature. These samples yielded only very small datasets without a grain boundary, or no data, as opposed to the cryo-transferred samples, which yielded good data in all cases. We attribute this to fractures caused by the well-known effect of Ga embrittling the grain boundaries, as an atom probe experiment puts a large mechanical stress onto the sample. The temperature above which this embrittlement effect comes into play has not yet been determined.

The results from an experiment with cryo-milling and cryo-transfer are presented in Fig 6. In Fig 6A, the microstructure of the nanocrystalline-Al is shown with the analysis direction of the APT reconstruction indicated. In this figure, a camera image of the sample in the analysis chamber of the atom probe clearly demonstrates that the sample was transferred without any discernible frost formation. The small volume design and in particular the copper-sheath surrounding the sample, effectively mitigate frost formation with this transfer system. Water molecules were also not encountered in the mass spectrum (see S5 and S6 Figs in S1 File). This is interesting as H₂O is often found in APT data as a contaminant after conventional electrochemical or FIB preparation. A SEM image of the FIB milled sample is also shown, demonstrating the thin and highly elongated sample shape that is the result of the high primary Ga⁺ ion energy (30keV) beam used to mill the sample. This shape is also evident in the shape of the 3D atom map of 100k randomly picked Al atoms in the dataset, presented in Fig 6B. The dataset was collected using voltage pulsing at a pulse frequency of 200kHz, a temperature of 40K, a detection rate of 0.5%, and a pulse fraction of 20%. From this 3D atom map, we have chosen a section containing a grain boundary for detailed analysis (Fig 6C). The presence of a grain boundary can clearly be observed through the change of the location of the crystallographic poles visible in the field desorption patterns [34]. Three field desorption patterns corresponding to locations above, in the middle and below the grain boundary are displayed together with the 3D distribution of the all detected Ga atoms in Fig 6C. A movie of the evolution of the field desorption patterns during the experiment and a movie of the 3D distribution of Ga as well as the APT data are included in the S1 File. In the 3D Ga distribution, there is already some structure visible, indicating the presence of a grain boundary. Most importantly, it is clear that Ga has not segregated at the grain boundary, in stark contrast to previous TEM [21, 22] and APT [23–25] investigations of aluminum alloys prepared with a Ga-FIB at room temperature. To quantify this claim, we have created a 1D concentration profile through the grain boundary as indicated in Fig 6C, shown in Fig 6D. This concentration profile confirms that no segregation is present at the grain boundary, but a rise in Ga beyond the boundary along the direction of the ion penetration is observed. This is typical for ion implantation into a polycrystal, if the second crystal has an orientation towards the ion path with higher stopping power as compared to the first one. In fact, for the entire sample, a concentration profile typical for ion implantation is observed (Fig 6E). This concentration profile was taken along the analysis direction of the atom probe sample. Since Al is a relatively light element with low stopping power, the maximum concentration of the implantation is found at around 100 nm for 30keV Ga⁺ ions. We do expect the implantation profile to significantly deviate from that of a flat sample though, since forward sputtering (escape of energetic ions through the side of the sample) would play a significant role in such a slim sample. In this sample, the overall implantation profile (Ga_tot) had to be estimated from the observed Ga⁺⁺ implantation profile, by assuming a constant charge state ratio between Ga⁺ and Ga⁺⁺. This is owing to the fact that for the pulse frequency of 200kHz used in the experiment, the flight times of the Ga⁺ ions were too long to be registered for about the first half of the data collected.

The cryogenic-temperatures during FIB-milling and transfer therefore sufficiently inhibit Ga diffusion to the grain boundaries, which is in-line with the recent results of Lilensten et al. [35]. Despite using a significantly higher final FIB voltage (i.e. implanting Ga deeper in the sample) than was used in the work of Lilensten, Ga diffusion to the grain boundary was still inhibited in part due to the cryogenic transfer of the specimens to the LEAP in this work. Taken together, these results have tremendous implications for the study of Al alloys, for example in the research areas of intergranular corrosion and strengthening mechanisms, which are of major importance in the automotive industry [29]. The ability to accurately characterize grain boundaries in Al alloys, will undoubtedly enable the elucidation of intergranular corrosion mechanisms. While additional investigations are required to identify the exact influence of FIB-milling and transfer temperatures on Ga diffusivity in Al alloys, these preliminary results show that maintaining cryogenic temperatures is a way to obtain Ga-free grain boundaries in such materials without resorting to alternative plasma-based FIB systems [36–38].

4. Conclusions and outlook

In this study, a custom-built versatile transfer system that enables quick transfer of samples that are sensitive to air or thermal exposure between sample preparation stations such as a Zeiss Crossbeam FIB/SEM and a local electrode atom probe (LEAP 4000X HR) was presented. The transfer system can easily be adapted to interface with any other instrumentation possessing a standard KF 16 port. In our lab, this includes a cryogenic electropolishing station, a field ion microscope and a coating system. A FIB transfer device (FIBTD) attaches directly to the existing FIB load lock, obviating the need for a specific cryo port on the microscope. Modifications to the LEAP enable quick, environmental transfer of samples directly into the buffer chamber using an actively cooled transfer device (ACTD) and a cryogenically cooled transfer stage in the LEAP. The ACTD maintains temperatures below -150° C throughout the transfer process. Using the transfer system, it was possible to observe Ga-free grain boundaries in an Al alloy, despite the samples being prepared with a Ga-based FIB.

Compared to other systems presented in literature, the ACTD is significantly smaller and incorporates close cryo-shielding of the sample to prevent frost formation on the sample during transfer. Additionally, the system can be easily modified to suit various experimental or analysis conditions and sample geometries since it is based on standard or easily milled components. This flexibility will be key as we study new materials systems such as those that are normally liquid at room temperature.

Supporting information

S1 File

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S1 Video

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S2 Video

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S3 Video

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Data Availability

All relevant data are within the manuscript and its Supporting Information files. The APT file is available on Figshare: https://figshare.com/articles/dataset/R56_02188-Cryo_Al_pos/12895898.

Funding Statement

C.M., M.H. and P.F. acknowledge financial support by the Bavarian Ministry of Economic Affairs and Media, Energy and Technology for the joint projects in the framework of the Helmholtz Institute Erlangen-Nürnberg for Renewable Energy (IEK-11) of Forschungszentrum Jülich. The authors would also like to acknowledge funding by the Deutsche Forschungsgemeinschaft (DFG) via the Cluster of Excellence ‘Engineering of Advanced Materials’ (project EXC 315). The funders provided support in the form of salaries for authors CM and MH, but did not have any role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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PLoS One. doi: 10.1371/journal.pone.0245555.r001

Decision Letter 0

Leigh T Stephenson

9 Nov 2020

PONE-D-20-27616

A versatile cryo-transfer system, connecting cryogenic focused ion beam sample preparation to atom probe microscopy

PLOS ONE

Dear Dr. Macauley,

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Reviewer #1: The manuscript titled “A versatile cryo-transfer system, connecting cryogenic focused ion beam sample preparation to atom probe microscopy” has been reviewed. The manuscript describes the development of unique hardware that enables the protected transfer of specimens between a cryogenic FIB/SEM and a LEAP for atom probe (AP) analysis. Specifically, specimen transfer to either the FIB/SEM or AP is performed using separate custom specimen shuttle suitcase devices (called “transfer arms” in the manuscript). The transfer device for FIB/SEM transfer maintains the specimen at passive cryogenic temperatures and passively at high vacuum conditions. The transfer device for the AP is capable of maintaining a specimen actively at cryogenic temperatures (-160 °C), while passively holding high vacuum conditions. Details of how the two different transfer devices are docked to the FIB/SEM and AP instruments are also discussed. In addition, a unique custom specimen carrier (called a double nipple) was developed to allow transfer between the FIB/SEM transfer device and the AP transfer device. For the latter, the design of the transfer device enables the specimen to be fully isolated within an actively cooled sub-volume so as to eliminate frost contamination during transfer. The efficacy of these devices, chambers, and described protocols are demonstrated via the analysis of an Al alloy intentionally exposed to relatively high Ga ion flux while at cryogenic temperatures, to show that Ga does not segregate to GBs. Appropriately referenced past reports by others show that Ga does indeed segregate to GBs in Al, when exposed to high Ga ion flux at room temperature.

Overall, the description of all the custom hardware and development of a unique approach is impressive. Despite other groups reporting similar developments for the FIB-based cryogenic preparation and environmentally protected transfer of specimens for APT analysis, the approach by the authors is unique and offers advantages (e.g. specimen shielding and small foot print) and is worthy of publication.

However, before this manuscript should be considered for publication, I would argue that the authors address the following comments and concerns outlined below. The order in which these concerns are described follow the chronological order encounter in the manuscript.

1. Sentence beginning with “Preparation of non-conductive or site-specific APT samples…” is awkward and makes too broad of assumption. As written, it implies that one cannot make site-specific samples from non-conductive, conductive, or semiconductive specimens for that matter. Additionally, the use of “exclusively” when referring to use of a FIB/SEM as being most easily or even exclusivity achieved is too broad. I agree that for site specific analysis, this is the true, but there are other material specimens worthy of APT analysis, such as pure metals, which do not require site-specific targeting, where electropolishing is much easier.

2. References 7-10: These references should be placed at the end of the sentence since they all apply to all subjects in this sentence.

3. Regarding sentence: “Some cryogenic- and environmental-transfer systems are commercially available and rely on a shuttle or suitcase that docks with various instruments and maintains a user-defined environment during transfer [11–14].” This is a good opportunity to help perpetuate a correct (logical) description with explicit nomenclature. I would argue that "Shuttle" refers to something that is used to secure a specimen (e.g. APT specimen pucks, can be called APT specimen shuttle pucks, where the word "puck" really describes a generic shape (disc-shaped), where "shuttle" describes the action (to transport). Suitcase describes a device which could hold other objects within it (i.e. isolated from the environment). So I would strongly suggest using the phrase "specimen shuttle suitcase device" to describe it. My understanding is that other publications describe such a device using various phrases, but if you were to use this phrase, maybe the community can adopt it in an effort to somewhat standardize terminology. This concept will be reiterated below.

4. Regarding the paragraph and discussion within that starts with “Some cryogenic- and environmental-transfer systems are…”: Since you go into superficially describing some of these systems in detail, please provide a more detailed comparison of the various transfer systems as suitcase devices that are: a) completely passive (e.g quorum; Perea et al.); b) actively cooled only (Leica, Gerstl et al.); c) actively cooled and pumped (Ferrovac; Stephenson et al.). Additionally, describing pros and cons will provide the context and clear distinction of the approach described here.

5. Typo: the word “Northwestern” is wrong. It should be Northwest, so that it reads “Pacific Northwest National Laboratory of the US Department of Energy…”

6. Regarding the sentence starting with “In this paper, we describe a custom-built, robust, and versatile transfer system…”, the word “robust” as used here doesn’t seem relevant as a descriptor. Please provide evidence of the system being ‘robust’.

7. Regarding sentence starting with “To demonstrate the cryogenic-, high vacuum-transfer capabilities of the system,”: It would be good to define "cryogenic" in this context. I suspect you mean that your device has active cooling. This would be more precise, otherwise given your implied definition here, even the Quorum transfer system is 'cryogenic' even though it is NOT actively cooled. This is an opportunity to highlight positive differences your system brings.

8. Regarding paragraph starting with “The ability to quickly make such environmentally-controlled transfers between instruments enables the application of atom probe tomography to previously inaccessible but increasingly important research fields beyond materials science, such as chemistry and biology.” While this is true, several other groups have now shown this. BUT what is it about your contribution here that is different? How does it improve or better enable new science? Again, use this as an opportunity to highlight positive differences your system brings.

9. Regarding the sentence beginning with “The cold-chain consists of optional initial cooling of the sample to the desired temperature, e.g. by plunge freezing.” It is confusing to use the phrase “cold-chain” as a descriptor of a protocol, without being explicit about what that means. Please consider rewriting this to say something like Here we use the phrase “cold-chain” to describe the protocol of XXXX. If this not the first instance of using this phrase as I describe here, then consider defining it in the location where it first appears. Additionally, other instances of this phrase are unhyphenated. Please be consistent.

10. Regarding the general organization: The organization of the manuscript is somewhat confusing, making it challenging for readers to understand the design and implementation of all the parts. To aid, please create separate sections describing: 1) modifications to the FIB/SEM; 2) modification to the APT system; 3) design of ancillary equipment such as sputter coater and electropolishing system; 4)design and utilization of the specimen shuttle suitcase device for transfer of specimens between the various tools described above.

11. Regarding sentence “Custom, pre-tilted (54°, which is the angle between the electron and ion beams) and non-pre-tilted cryo-shuttles with threads to accommodate the specimens were made to allow annular ion beam milling perpendicular to the sample surface without tilting the stage.” Please provide a picture description of this as a separate figure for both types of specimen shuttles; consider amending such images/drawings in existing figures. Additionally, nomenclature is confusing when describing 'shuttles'. This was brought up already above.

12. General comments: You describe your transfer devices as “transfer arms”. The phrase transfer arms, I believe, sells the design and function short. What I mean is that what you describe is more of a device, in that it serves much more purpose than just an arm that transfers specimens. This again, this is where you have the opportunity to be more precise and provide descriptive nomenclature. Strongly consider using the phrase “specimen shuttle suitcase transfer device” to define your “transfer arms”, and you can then shorten this description as “transfer device”.

13. Regarding the sentence “The generated vacuum of ~ 10-4 mbar thereby has proven to be sufficient to prevent frost built up on the specimen.” Please provide a statement that evidence of this is shown below during the exemplary analysis of an Al alloy. Otherwise, the reader is left wondering why such a conjectured statement was made.

14. Be explicit in referring to which “transfer arm” you are referring to in the sentence “In this first transfer arm, no active cooling or pumping is available”.

15. Regarding the sentence “Measured heating rates of the shuttle while uncooled at the end of the PEEK end-effector at a vacuum of around 10-2 mbar are in the range of 2.5°C/min.” Considering conductive thermal heat transfer, the rate in a change of temperature depends on the temperature of the specimen relative to the temperature of the object it is in contact with. This change in temperature per unit time is not linear as you imply. Also, please revise this sentence to be more clear. I am to understand that your specimen is starting off as cold, so it has been 'cooled'; it is not 'uncooled' as you say. Be more precise by saying your specimen "is not actively cooled".

16. Awkward sentence structure. Please revise: “Switzerland). While these valves have the same leak tightness as their metal flanged, fluoroelastomer (FKM)-sealed counterparts, they cannot be heated beyond 80°C, limiting possible bake-outs. However, moving samples from the FIB/SEM with ultimate pressures not below 10-7 mbar, this is not of any concern.

17. Regarding Fig. 3. Please provide an inset image that is a zoomed in region of where the double nipple screws into your transfer device, and how it is enclosed (shielded). It is hard to decern without any arrows or labels in the figure. This will help the reader to make more sense of the respective description in the paragraphs that follow.

18. Regarding the sentences “The sample is held in a double-nipple, Fig 3C, that has a 5 mm diameter right-handed screw thread on one side, and a left-handed screw thread on the other. Such a design enables the double nipple to be transferred between the copper end-component shown in Fig 3A, and FIB or LEAP sample holders with one continuous twisting motion.” Please provide a reference in the figures to how this is done. Consider adding additional figure panels to help explain this visually.

19. Regarding the sentence “This will hold true so long as sufficient vacuum is maintained that leads to molecular flow of the residual gas atoms.” Wouldn't this be the case until the pressure is well above atm pressure (i.e. turbulent or laminar flow)? Maybe I’m mistaken, but as such, this statement does not make sense as used here.

20. Regarding the sentence that ends with “…the roughing pump maintains the vacuum.” What explicitly are you referring to?...FIB/SEM load lock?

21. Regarding the sentence: “During transfer, the FIB cryo-shuttle is rotated 90��along the axis of the FIB transfer arm, so that the pre-tilted double nipple and atom probe sample are parallel to the axis of the LEAP transfer arm, Fig 4C. The threaded copper sheath of the LEAP transfer”. Please add labels (with arrows) that indicate the orientation and direction of the LEAP transfer arm, the SEM transfer arm, and the location of the SEM load lock/SEM chamber.

22. Regarding the sentence: “The sample transfer takes place in the buffer chamber,…”. Please explain the what the “buffer chamber is. This phrase is jargon for the specific instrument and is familiar to the APT community explicitly as you use it. Consider stating, "The sample transfer takes place in what is commonly referred to as the buffer chamber of the Local Electrode Atom Probe,"

23. Regarding the sentence: “This is the fastest conceivable route.” This is not explicitly true. Direct transfer into the analysis chamber would be the quickest route, but is not practical given LEAP design. Also, this route would create potential contamination of the AC. For these reasons, BC connection makes the most sense, as going through the LL would create extra steps of having to use the LEAP-specific specimen puck carousels. .

24. Regarding the sentence: “The stage can be cooled with liquid nitrogen via a vessel (4 in Fig 5) that is also connected to the linear drive of the stage.” Please show schematic details of this to provide details that any reader would as about how the stage is "moved".

25. Regarding the sentence: “During the hand-over, the LEAP puck is held in place…”. Please provide additional details describing how this done. Is it done while holding the stage on the LEAP transfer arm? or does the LEAP transfer arm then grab the sample from the "movable" stage after hand off, from which it can then be loaded into the AC? Again, showing schematically is necessary for clarification.

26. Regarding the sentence: “To demonstrate the efficacy of the cryogenic-, environmental-transfer capabilities of the system, we chose to show the prevention of liquid metal embrittlement in the Al – Ga system.”. I’m having trouble understanding how APT analysis shows “the prevention of liquid metal embrittlement”. Such a determination would require some mechanical testing analysis to show this. Instead APT is able to confirm the distribution of Ga relative to GBs, and an inference would then be made that the lack of GB enrichment of Ga leads to less mechanical embrittlement. Maybe you are referring to the observations/statements implying a relatively high APT analysis yield compared to other studies which showed low yield at relatively low applied biases (i.e. fractured at lower stresses). Please be explicit in explaining such phenomena.

27. General statements regarding Figures:

a. Fig. 1b. label the FIB/SEM transfer device connected to the LL of the FIB/SEM

b. Provide image of the 90deg type of FIB/SEM specimen pucks; Figure 2 only shows the 54 degree version

c. Add error bars (consider shaded line bands) to the plots in Fig. 6D and E.

Reviewer #2: 1. There is some analysis regarding the distribution of Ga++ and the presence of MgOH complex ions within the sample that I believe needs to be conducted again in relation to the suggestions I have made in the uploaded comments.

4. Although well written, it is difficult to follow the train of thought due to the use of multiple and overlapping names for key components. This needs to be fixed prior to publication.

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Reviewer #1: Yes: Daniel Perea

Reviewer #2: Yes: Ingrid E. McCarroll

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PLoS One. 2021 Jan 19;16(1):e0245555. doi: 10.1371/journal.pone.0245555.r002

Author response to Decision Letter 0

24 Dec 2020

Reviewer 1:

The manuscript titled “A versatile cryo-transfer system, connecting cryogenic focused ion beam sample preparation to atom probe microscopy” has been reviewed. The manuscript describes the development of unique hardware that enables the protected transfer of specimens between a cryogenic FIB/SEM and a LEAP for atom probe (AP) analysis. Specifically, specimen transfer to either the FIB/SEM or AP is performed using separate custom specimen shuttle suitcase devices (called “transfer arms” in the manuscript). The transfer device for FIB/SEM transfer maintains the specimen at passive cryogenic temperatures and passively at high vacuum conditions. The transfer device for the AP is capable of maintaining a specimen actively at cryogenic temperatures (-160 °C), while passively holding high vacuum conditions. Details of how the two different transfer devices are docked to the FIB/SEM and AP instruments are also discussed. In addition, a unique custom specimen carrier (called a double nipple) was developed to allow transfer between the FIB/SEM transfer device and the AP transfer device. For the latter, the design of the transfer device enables the specimen to be fully isolated within an actively cooled sub-volume so as to eliminate frost contamination during transfer. The efficacy of these devices, chambers, and described protocols are demonstrated via the analysis of an Al alloy intentionally exposed to relatively high Ga ion flux while at cryogenic temperatures, to show that Ga does not segregate to GBs. Appropriately referenced past reports by others show that Ga does indeed segregate to GBs in Al, when exposed to high Ga ion flux at room temperature.

We have modified the sentence to make it clear that electropolishing is used for conductive, non-site specific samples but that the FIB/SEM is often used to make APT samples from non-conductive or site-specific samples.

2. References 7-10: These references should be placed at the end of the sentence since they all apply to all subjects in this sentence.

Fixed.

The terminology used in the paper was indeed confusing and we agree that more standardized terminology would benefit the community. We have made changes so that the term “shuttle” is now only used when describing components in which specimens are secured (eg. FIB cryo-shuttle, APT specimen shuttle pucks). Any device used to transport specimen shuttles between instruments is now referred to as a “specimen shuttle transfer device” or “transfer device” for short. To differentiate between the two transfer devices, they are now referred to as the ‘FIB transfer device’ and the ‘actively cooled transfer device (ACTD)’ throughout the paper.

5. Typo: the word “Northwestern” is wrong. It should be Northwest, so that it reads “Pacific Northwest National Laboratory of the US Department of Energy…”

Apologies for the typo. It is corrected.

The sentence was removed and replaced with the following to highlight the advantages of our system:

The following sentence was added to the introduction: “The paper is organized in the order of components/modifications used during a cryo-FIB preparation of an atom probe sample and subsequent transfer into the LEAP.”

The figure has been modified accordingly.

See response to point 3).

The following was added to the end of the aforementioned sentence: “…, as shown below in the analysis of the Al alloy.”

14. Be explicit in referring to which “transfer arm” you are referring to in the sentence “In this first transfer arm, no active cooling or pumping is available”.

The sentence was changed as follows: “In this FIB transfer device, no active cooling or pumping is available.” Additionally the second transfer arm is now only referred to as the ‘actively cooled transfer device, ACTD’, as clarified in point 3).

“To measure the efficacy of this thermal isolation, specimen temperature was measured while a FIB cryo shuttle, pre-cooled by the cryo stage to -118°C, warmed up (since it was not actively cooled) during a simulated hand-off to the ACTD. As shown in Fig S2, the pre-cooled shuttle on the PEEK end-effector of the FIBTD warms up at 2.3°C/min at a pressure below 10-5 mbar. “

The sentences have been revised and are now: ‘Although these KF valves have the same leak tightness as their CF metal flanged, fluoroelastomer (FKM)-sealed counterparts, they cannot be heated beyond 80°C, limiting possible bake-outs. Such higher temperature bake-outs are only needed to reach ultra high vacuum (<10-9 mbar) conditions. Since the ACTD is being connected to the FIB/SEM, which has an ultimate pressure of 10-7 mbar, such bake-outs are not required.’

An inset is now shown in Fig. 3C including labels and arrows.

While we have added annotations and additional panels to Fig 3 and Fig 4, we think the best way to understand the mechanism of sample hand-off is with a movie. This movie is now provided in the supplementary information for the paper.

The sentence in question was removed.

20. Regarding the sentence that ends with “…the roughing pump maintains the vacuum.” What explicitly are you referring to?...FIB/SEM load lock?

“…FIB/SEM load lock…” was added to clarify which pump provides the vacuum when moving samples INTO the FIB/SEM.

Additional figure panels, labels, and arrows have been added to Fig 4 to better explain the orientation.

The sentence has been changed to: “The sample transfer takes place in what is commonly referred to as the buffer chamber of the LEAP, after which…”. The LEAP acronym was defined in the introduction and methods section of the paper.

We agree with the reviewer and have deleted “this is the fastest conceivable route”.

The sentence has been changed to “…FIB-induced Ga segregation to the grain boundaries in the Al – Ga system.”. While it has been well documented that Ga-segregation causes embrittlement of Al alloys, the review is correct that we did not explicitly study the mechanical properties. It is true that our analysis yield was substantially better for cryo-prepared and transferred samples compared to samples either prepared with room T Ga-based FIB or transferred at room temperature.

27. General statements regarding Figures:

a. Fig. 1b. label the FIB/SEM transfer device connected to the LL of the FIB/SEM Revised.

b. Provide image of the 90deg type of FIB/SEM specimen pucks; Figure 2 only shows the 54 degree version Both FIB cryo shuttles are now shown

c. Add error bars (consider shaded line bands) to the plots in Fig. 6D and E. Shaded line bands have been added.

Reviewer 2

Overall Statement

This study presents results of an in-house design of a unique cryogenic transfer system, primarily connecting a Ga-FIB/SEM to an atom probe. The work also showcases the elimination of Ga segregation to grain boundaries, commonly observed in room temperature ion beam milling and specimen transfer. Although the work overall is very promising and relevant to the scientific community, I do have some concerns related to the content. My primary concerns are: 1) there are some results that I believe need further analysis and clarification before they are ready for publication, 2) the overall presentation of the work is confusing and requires clarification, specifically in the area of naming conventions and overall workflow, and 3) the conclusions seem rather broad and do not directly reflect the findings in the paper.

Detailed Comments

1. The acronym TEM is not introduced in the document. This could be introduced in the 1st paragraph of the introduction.

The acronym has been included.

2. 2nd paragraph of the introduction it sounds as though MPIE has a commercial interest in the Ferrovac system: “In collaboration with CAMECA and the Max Planck Institute for Iron Research in Dusseldorf, Ferrovac offers a large…”. It might be better to say that in collaboration with these companies/institutes that “Ferrovac has developed a …”. (Unless of course MPIE has a commercial interest that I am not aware of…?)

The suggested changes have been made.

3. The last sentence of the 2nd paragraph in the introduction is a bit vague: “Pacific Northwestern National Laboratory of the US Department of Energy that offers even more possibilities”. Please be more specific about the further possibilities offered by this system.

4. In the sentence ending “through a cryogenic shuttle device designed by the authors, which is introduced below”, remove “which is introduced below” as the first object introduced is the transfer arm, which may then be mistaken for the shuttle device.

The suggested changes have been made.

5. In the sentence “A general overview of the system including relevant temperatures and pressures at the various steps of sample preparation …”, please add ‘cryogenic/vacuum transfer’ before ‘system’.

The suggested changes have been made.

6. It is unclear how you would actually plunge freeze the sample prior to transfer to the FIB. Would this be accomplished in some kind of controlled environment?

As plunge freezing was not used in the specific experiments presented in this paper, we removed it from the paper for clarity.

7. Fig. 1 is very difficult to comprehend and would benefit from rearranging the information. This image is critical to the reader’s capacity to understand the overall interconnectedness of the system and the overall workflow. Care should be taken to make sure it is as clear as possible.

a. Fig. 1a should be removed and this information incorporated into a single workflow diagram. (Note that temperatures are already incorporated into Figures b,c, and d resulting in double-up of information)

b. I would suggest placing the FIB/SEM at the top of the image, with operating properties beside it. Separate the transfer arm from the FIB/SEM and place this below the FIB/SEM with the transfer shuttle beside it providing appropriate information for both. Place the LEAP at the bottom (‘CAMECA’ is not really necessary and just takes up space) with appropriate information. Join each component with arrows (as in current Fig. 1a, however make the arrows long enough that the required information can be provided adjacent to them. It would also be clearer if you separated the cryo LL from the LEAP for the purposes of clearly articulating the workflow and isolating the newly designed components from existing infrastructure.

c. Where you have written -196 dC - RT, please change this to -196 dC to RT, otherwise it looks like an equation

d. Where and how does plunge freezing fit into this workflow?

We appreciate the constructive feedback and have significantly changed the figure based on your suggestions. A workflow is shown in part a) of the figure, clearly indicating what components have been designed and built as part of this work. A schematic workflow is shown in part b), using the same color scheme, to indicate the temperatures and pressures at critical points. We have left the FIB transfer device connected to the FIB because one advantage of our system is that the FIB load lock does not need to be changed to accommodate the transfer device. As mentioned previously, plunge freezing has been removed from the diagram since it was not used in this study.

8. In the sentence “After FIB milling and/or SEM characterization, the sample is moved out of the FIB/SEM using the same, thermally-insulated, but not actively-cooled transfer arm, in which the sample is handed off to another, actively-cooled transfer arm, with a standard KF16 vacuum flange (Fig 1C)”, the name transfer arm is used to describe two distinct parts of the system. Please be consistent with notation and refer to each component using a distinct naming convention. Alternatively, if you mean that the ‘sample is handed off to another, actively-cooled transfer arm that delivers the sample to the transfer shuttle’ then please be more specific in the detail.

The terminology used in the paper was indeed confusing. We have made changes so that the term “shuttle” is now only used when describing components in which specimens are secured (eg. FIB cryo-shuttle, APT specimen shuttle pucks). Any device used to transport specimen shuttles between instruments is now referred to as a “specimen shuttle transfer device” or “transfer device” for short. To differentiate between the two transfer devices, they are now referred to as the ‘FIB transfer device’ and the ‘actively cooled transfer device (ACTD)’ throughout the paper.

9. In reference to point 8, a number of other items have different names in the text and in the image, please check through the text carefully and be consistent with all naming conventions throughout. The constant changing of names and similarity of names made following the thread of the article extremely difficult.

See response to previous point.

10. Assuming that the cryo electropolishing station is the station used for initial plunge freezing (discussed in the main text), the image of the cryo electropolishing unit should be included in Figure 1.

As plunge freezing was not used in the described experiments, mention of it has been removed from the paper.

11. Fig 2:

a. Add annotations to a)

b. Change port names to TS1 and TS2 to represent transfer shuttle 1 and 2. APT 1 and 2 suggest that it will be connected to the atom probe directly.

c. A better name could be chosen for the cryo-shuttle, at the moment it is too similar to transfer shuttle, which also operates at cryo temperatures.

Annotations have been added and APT 1 and 2 have been changed to ACTD1 and ACTD2, in agreement with the new name for this transfer device. See response to point 8.

12. It is not clear how the transfer arm enables the analysis of multiple samples, as indicated in the following sentence “the transfer arm reduces the risk of chamber contamination and enables efficient characterization of multiple samples.” It appears to me that only one sample can be transferred at any one time.

The sentence has been changed to more clearly indicate why having a FIB transfer device is essential to analyzing multiple samples in a single FIB session. “The transfer device reduces the risk of chamber contamination and enables efficient characterization of multiple samples in a single FIB session, since without the means to transfer cold samples through the load-lock, the cold stage would need to be warmed to room temperature for sample exchanges and the entire analysis chamber vented.”

13. “This transfer arm can be used to plunge freeze liquid containing samples prior to characterization (see supplementary materials) and interfaces with the existing Zeiss 80 mm Airlock without further modification”. It is unclear to me how the transfer arm can be used to plunge freeze liquid containing samples without exposure to air. If air exposure is necessary in the process, then ice will build-up on the sample making the method redundant. Please explain further the initial experimental transfer step of samples into the transfer arm under a controlled environment.

See response to points 6 and 10 above.

14. “This pumping operation takes ca. 40s until a vacuum sufficient for transfer is established.” This is longer than the 30s, stated in the supplementary section, as the maximum time in order not to exceed the crystallization temperature of ice.

References to plunge freezing have been removed from the manuscript and supplementary information since it is not relevant for the experimental results shown in this paper.

15. The supplementary section states the crystallization temperature of water ice to be -160 dC. Please provide a reference, as my understanding was that cryo EM scientists aim to keep the temperature below -150 dC, and that this is sufficient to maintain vitreous ice. Perhaps pressures should be considered in the statement of transition temperatures?

See previous response. Pressures are absolutely important. A new table showing the heating rate as a function of pressure is provided in the supplementary information.

16. 2.2 Atom probe transfer shuttle. This shuttle can be attached to multiple instruments, perhaps a more generic name would be more appropriate.

We have changed the name to ‘actively cooled transfer device’.

17. Section 2.2: “So long the reservoir does not run dry, the sample is near liquid nitrogen temperature in the transfer shuttle.” The supplementary information indicates that the minimum temperature of the sample is approx. -170 dC, which is not so near liquid nitrogen temperature. Please add the specific temperature to the text. The specific temperature is now used.

18. “However, moving samples from the FIB/SEM with ultimate pressures not below 10-7 mbar, this is not of any concern.” This sentence is confusing, please reword.

The sentence has been changed to: “Although these KF valves have the same leak tightness as their CF metal flanged, fluoroelastomer (FKM)-sealed counterparts, they cannot be heated beyond 80°C, limiting possible bake-outs. Such higher temperature bake-outs are only needed to reach ultra high vacuum (<10-9 mbar) conditions. Since the ACTD is being connected to the FIB/SEM, which has an ultimate pressure of 10-7 mbar, such bake-outs are not required.”

19. “enabling temperatures of -160 °C during transfer.” This sentence is now in contrast to the statement, “So long the reservoir does not run dry, the sample is near liquid nitrogen temperature in the transfer shuttle.”. See response to point 16.

20. The choice of colour for the tip in Fig 3b makes it difficult to see the tip against a similar background colour. Please change.

A zoomed-in inset has been added, as well as labels, to make it easier to see the critical components.

21. Fig. 3, consider indicating the location of the sample with an arrow and label for those not familiar with the appearance of APT samples.

Labels, arrows and additional panels have been added to Fig 3.

22. In section 2.4 you refer to the existing atom probe transfer arm as “LEAP transfer arm”, this becomes particularly confusing now given that the transfer shuttle is often referred to as “LEAP transfer arm”. Please use distinct names. The name of the transfer device has now been changed to actively cooled transfer device (ACTD).

23. “capable of in-situ heating and cryogenic cooling, and its electrical connections”, ‘and its electrical connections’ doesn’t make sense in this sentence. Please clarify. The sentence has been modified to emphasize that the additional flanges on the cryo transfer chamber include a viewport, a movable cryogenic/heating stage and the electrical connections required for the stage.

24. “can be transferred to a pre-cooled, custom LEAP puck”, it would be better here if you said “can be screwed into a pre-cooled…” to remind readers that the sample is not transferred on a puck but via a double-nipple. The suggested change has been made.

25. “During this hand-over, the LEAP puck is held in place by the actively-cooled cryogenic stage, to make the alignment of the double nipple and the puck easier.” It is not clear from this sentence what is making the alignment of the double nipple and puck easier. The comment about it making alignment easier was removed. Prior to the design and construction of the cryo stage, pucks were first pre-cooled in the AC and the handoff between the ACTD and the custom puck happened with the puck attached to the LEAP transfer rod. The puck moved slightly on the transfer rod, making the hand-off challenging. The cryo-stage therefore makes the hand-off easier by holding the puck in place.

26. “with the long axis of the sample indicated” Please indicate in this sentence that you mean the long axis of the atom probe tip. The sentence has been modified to include analysis direction instead of long axis.

27. “This is interesting as H2O is often found in APT data as a contaminant after conventional electrochemical or FIB preparation”. Note that you have Mg2O peaks and what looks like a large Mg2H2O peak between the Ga2+ peaks and again MgOHx between 40-50 Da (double check). It is common in Mg containing data not to see H or O peaks, because these elements bond to and evaporate with the Mg in the sample. If you are inclined to continue to mention the lack of H2O in the dataset, you must also mention the presence of the Mg-O complex ions in the dataset.

Based on the following mass spectra, the peaks in question do not fit those of Mg2O, Mg2H2O or MgOHx.

28. Please list the detection rate as a part of the atom probe parameters. Included.

29. Figure 6:

a. This image is overly compact. Please separate the images or place c and d below a and b.

b. Make sure the scale bars do not overlap with the images.

c. Please redo the scale bar for c, I don’t believe this scale bar represents all three axes accurately.

The image has been modified slightly. The scale bar in c does accurately represent all three axes as it is an orthographic projection.

30. Something odd is happening in the video of the desorption map. The edges of the map change significantly throughout the acquisition and poles seem to come and go in relation to the moving of the edges. A few things may cause this: 1) significant vibration of the tip, 2) significant fractures to the tip and 3) slow drifting of the tip. All of which will affect the data. Please address this in the text and discuss implications on the data.

The field desorption maps change because the custom cryo-shuttle pucks used in the LEAP experiments had not yet been optimized to fit perfectly. During the course of an atom probe experiment, the sample shuttle puck would move slightly due to being slightly too small for the puck holder in the LEAP. This issue has since been fixed with precisely sized custom LEAP shuttle pucks.

31. I am concerned with the use of Ga++ as an indicator for Ga+, due to the overlap between Mg2O and Ga++. This analysis needs to be redone considering this overlap and its implications on Ga concentration. This is particularly of concern given that the peak for the heavier Ga isotope is greater than the peak of the lighter Ga isotope. Furthermore, Ga++ does not necessarily always evaporate after FIB preparation, so it could be that these peaks are entirely related to the Mg-O-H peaks.

See response to point 27.

32. The second paragraph of the conclusion reads more like an introduction than a conclusion. I think that it is necessary to rewrite this paragraph to focus on the specific outcomes of this particular study, such as the observation of Ga free grain boundaries, the rapid transfer times, the shielding of the tip during transfer and the low temperatures maintained during transfer.

The second paragraph has been removed and replaced with a new paragraph that focuses specifically on the advantages of our system and the Ga-free grain boundary result.

Supplementary section

1. The figures are not directly referenced in the text. This is particularly confusing when it is stated that “shown below are the temperature and pressure logs” when the below image is of the experimental set-up. Please be specific in referring to the images in the text.

Revised.

2. Again further names are given to the shuttle ‘atom probe shuttle’ and ‘LEAP transfer shuttle’, a consistent name is required for the transfer shuttle.

FIBTD, ACTD and FIB cryo shuttle are now used consistently throughout the paper and supplementary information.

Attachment

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PLoS One. doi: 10.1371/journal.pone.0245555.r003

Decision Letter 1

Leigh T Stephenson

4 Jan 2021

A versatile cryo-transfer system, connecting cryogenic focused ion beam sample preparation to atom probe microscopy

PONE-D-20-27616R1

Dear Dr. Macauley,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org.

If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org.

Kind regards,

Leigh T. Stephenson

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Dear Dr Macauley,

Thanks for your patience w.r.t. the reviewing process. As you noted in your letter, the reviewers did take their time but I think you and they can be pleased with the result. Thank you for taking the time to respond to each of the reviewers' points in turn. The method employed by your group certainly has advantages and I hope that the technical solutions to the problems encountered by the initially ill-fitting puck have successfully remedied the observed shifting in the field desorption maps. I look forward to seeing this "in print".

Kind regards,

Leigh Stephenson (MPIE)

Reviewers' comments:

PLoS One. doi: 10.1371/journal.pone.0245555.r004

Acceptance letter

Leigh T Stephenson

8 Jan 2021

PONE-D-20-27616R1

A versatile cryo-transfer system, connecting cryogenic focused ion beam sample preparation to atom probe microscopy

Dear Dr. Macauley:

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org.

If we can help with anything else, please email us at plosone@plos.org.

Thank you for submitting your work to PLOS ONE and supporting open access.

Kind regards,

PLOS ONE Editorial Office Staff

on behalf of

Dr. Leigh T. Stephenson

Academic Editor

PLOS ONE

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

S1 File

(DOCX)

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S1 Video

(MOV)

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S2 Video

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S3 Video

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Attachment

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Attachment

Submitted filename: Response to reviewers.docx

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Data Availability Statement

All relevant data are within the manuscript and its Supporting Information files. The APT file is available on Figshare: https://figshare.com/articles/dataset/R56_02188-Cryo_Al_pos/12895898.

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PERMALINK

A versatile cryo-transfer system, connecting cryogenic focused ion beam sample preparation to atom probe microscopy

Chandra Macauley

Martina Heller

Alexander Rausch

Frank Kümmel

Peter Felfer

Roles

Abstract

1. Introduction

2. Materials and methods

Fig 1. An experimental overview of the transfer system.

2.1 Cryogenic FIB/SEM stage and transfer device

Fig 2. The cryo stage and FIB transfer device (FIBTD) enable characterization, sample preparation and transfer at cryogenic conditions.

2.2 Actively cooled transfer device (ACTD)

Fig 3. The actively cooled transfer device (ACTD) and the double nipple sample shuttle enable transfers to the atom probe.

2.3 Hand-off between the FIBTD and the ACTD

Fig 4. Samples are handed-off between the FIBTD and the ACTD while the FIBTD is connected to the FIB load lock (LL).

2.4 Modifications to the LEAP

Fig 5. Modifications were made to a LEAP to enable cryo/environmental transfers.

2.5 Utilization of the above cryo-transfer system to study Al alloys

3. Results and discussion

Fig 6. Results of cryo-preparation of nanocrystalline-Al.

4. Conclusions and outlook

Supporting information

Data Availability

Funding Statement

References

Decision Letter 0

Leigh T Stephenson

Roles

Author response to Decision Letter 0

Decision Letter 1

Leigh T Stephenson

Roles

Acceptance letter

Leigh T Stephenson

Roles

Associated Data

Supplementary Materials

Data Availability Statement

ACTIONS

PERMALINK

RESOURCES

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