Method and apparatus for cryogenic and environmentally controlled sample handling - Patents.com

Abstract

A method for manipulating a cryogenic sample for subsequent examination includes placing a cryogenic sample on a working surface of a sample holder and placing the sample holder in a pumped cryostat chamber having a layer of cryogenic liquid therein. The cryostat chamber is vented and maintained at low humidity to create a cryogenic vapor layer above the cryogenic liquid, with the working surface of the sample holder within the cryogenic vapor layer and the low humidity and inert environment. Thereafter, while maintaining the cryogenic sample within the cryogenic vapor layer, the cryogenic sample is manipulated into a sample carrier housed in a different portion of the working surface.

Description

[Technical field]

[0001] 2. Background of the Invention 1. Field of the invention The present invention relates generally to the manipulation of samples prepared using a focused ion beam, and more particularly to detailed methods and systems for manipulating samples at the low temperatures required for examination using cryo-electron microscopy, or for spectroscopic or analytical examination via a wide range of methods. [Background technology]

[0002] 2. Description of the Prior Art Manipulating small specimens from larger bulk samples for examination with tools such as electron microscopes is extremely complex and has given rise to the development of several sophisticated processes. Unfortunately, there are currently no effective in situ (manipulation and examination inside the instrument that produced the specimen) or ex situ (manipulation and examination outside the instrument that produced the specimen) methods to adequately manipulate specimens under the cryogenic and environmentally controlled conditions for which such temperature-controlled conditions are required. For example, such cryogenic conditions may be required when glassy biological specimens or other beam-sensitive materials (e.g., polymers, organics, Li-based, Na-based, etc.) are used. Such materials must remain or be maintained at cryogenic temperatures or in a controlled environment during FIB milling and manipulation for subsequent cryo-EM (or ambient EM) or other characterization techniques and analysis.

[0003] Cryo-sample preparation must be performed at temperatures below -140°C to preserve the glassy phase of biological tissues and prevent the formation of ice crystals that can obscure the electron microscopy contrast and damage cells. Conventional methods of manipulating cryo-prepared focused ion beam (cryo-FIB) samples for subsequent examination by cryo-electron microscopy (cryo-EM) are performed under vacuum in a particle-optical instrument vacuum chamber (i.e., FIB) using a cryo-sample stage holder and a separate cryo-cooled probe, and are called cryo-in situ lift-out (cryo-INLO). These conventional cryo-INLO methods include (i) attaching the tip of a cryo-probe to the sample and then fixing it to a carrier grid with different deposition methods, and / or (ii) vibrating the tip of the cryo-probe relative to the sample, causing a local phase change of the material to attach the tip to the sample and fix it to a carrier grid, and / or (iii) gripping the sample with a cryogripper-type probe and fixing it to a carrier grid. In some cases, cryo-FIB milling may be required to grind the sample off the groove walls after the probe is attached, and / or to grind the tip of the cryo-probe off the sample after it has been attached to the specimen grid carrier (by deposition or phase change) so that the sample can be further cryo-FIB milled to thin it down for cryo-EM.

[0004] Cryo-FIB INLO requires the use of a probe tip (usually metal) that is maintained at low temperature while placed on the cryo-stage during FIB and that is configured as part of a cryostat independent of the cryo-sample holder so that the tip can be kept cold during sample extraction (i.e., "lift-out") from the bulk sample to keep the sample in the glassy phase.

[0005] Cryo-FIB INLO requires numerous complex steps that typically require 30-60 minutes to complete. Because the sample and probe tip are cooled independently, maintaining the cold sample and cold probe tip at the exact same low temperature during these steps can be difficult and can often result in thermal drift between the two when they come into contact. This thermal drift often results in the inability to form a strong bond, resulting in damage and failure at the deposition point, especially since this temperature stability must be maintained for several minutes during the deposition / attachment process. The cryo-INLO method requires cryogenic manipulation of FIB-prepared samples "inside" the FIB vacuum chamber along with numerous complex steps, which introduces the risk of sample destruction / loss, a low success rate of typically only one usable sample per day, and ultimately a significant increase in the cost per sample.

[0006] Cryo-ultrasectioning, an alternative to the cryo-FIB INLO method, uses knives to slice frozen thin samples for cryo-EM. The knives cause mechanical damage and the method is not ideal for localized cutting. The probes utilized for cryo-ultrasectioning are primarily used only to guide large sample slices (~0.5 mm or larger) onto the carbon-coated grid carrier surface and therefore have completely different geometry and size compared to samples prepared using FIB (~20 micrometers in length). Also, these probes cannot be used to "pluck", "pick", or "lift out" samples at localized locations from the cryo-contained bulk sample confined within the FIB-milled grooves and then subsequently transport / manipulate the thinned sample to the localized area of ​​the carrier grid. Thus, cryo-ultrasectioning applications and methods are not applicable to cryo-manipulation of thin layers or other small samples prepared using FIB.

[0007] Ex situ lift-out (EXLO) is a well-known "ambient" (room temperature and conditions) probing technique that is typically used to manipulate samples for subsequent transmission electron microscopy (EM) or other analytical characterization after a localized focused ion beam (FIB) milling routine has been performed. In addition to EM, lift-outs prepared with FIB have been analyzed by a number of techniques, including but not limited to scanning electron microscopy, confocal microscopy, Auger microscopy, scanning probe microscopy, secondary ion mass spectrometry, Raman spectroscopy, synchrotron, X-ray analysis, and other similar spectroscopy techniques.

[0008] With EXLO, samples are FIB milled inside a charged particle vacuum environment and the sample is manipulated "outside" the FIB into a thin film (e.g. carbon, holey carbon, Formvar, etc.) coated or slotted EM grid guided by an optical microscope and micromanipulator system on a motorized sample stage at ambient temperature conditions. The advantages of the EXLO method for FIB samples are: (i) it is localized; (ii) it has a high success rate; (iii) it is fast and simple and therefore more cost-effective; and (iv) if necessary, the sample can be further thinned by the FIB. Currently, the main drawback of EXLO is that the method is limited only to manipulation in air at room temperature (e.g. ambient) conditions. EXLO manipulation of FIB samples can be performed primarily with van der Waals forces attaching the probe tip to the sample, or via vacuum-assisted manipulation, or via a gripper-type probe.

[0009] INLO techniques exist that allow for sample manipulation in cryogenic environments, but these have drawbacks and complications that reduce throughput and increase the possibility of sample destruction, and EXLO techniques exist that have advantages over existing INLO techniques, but these techniques are limited to operation in air at room temperature (e.g., ambient) conditions only and therefore are not yet adequately adapted for sample manipulation at cryogenic temperatures.

[0010] There is therefore a need to provide a more effective method for manipulating small cryogenic samples which avoids the problems of the prior art. Summary of the Invention

[0011] Summary of the Invention A method for manipulating a cryo-sample for subsequent inspection includes first placing a cryo-sample on a working surface of a sample holder, which is then placed in a cryostat chamber having a layer of cryo-liquid above a cryo-vapor layer with the working surface of the sample holder located within the cryo-vapor layer, and then manipulating the cryo-sample into a sample carrier contained in a different portion of the working surface while maintaining the cryo-sample within the cryo-vapor layer.

[0012] An apparatus for manipulating a cryo-sample includes a cryostat chamber having a layer of cryogenic liquid and a cryogenic vapor layer above the cryogenic liquid. A cryogenic sample holder is located within the cryostat chamber and configured to maintain a cryogenic sample on a working surface located only within the cryogenic vapor layer. The apparatus may further include a transfer chamber maintained under vacuum and having a layer of cryogenic liquid therein, and a cryo-jacket located within the cryogenic liquid of the transfer chamber. The cryo-jacket has a bottom wall in contact with but elevated above an upper surface of the cryogenic liquid to form an interior compartment configured to receive the cryogenic sample holder such that the holder does not contact the cryogenic liquid.

[0013] The present invention develops a fast and easy reproducible EXLO method that can be applied to cryogenically and environmentally prepared FIB samples. This is novel since EXLO has only been performed at ambient conditions so far. The present invention is applicable to the manipulation of FIB milled or other small samples (e.g., fiber, particle, or laser cut samples) outside of the instrument that produces such samples. The present invention may utilize many different probe designs, consisting of, but not limited to, glass or metal probes that are raised to a tiny needle tip, metallic glass probes, hollow glass or metal probes with aperture geometries that match the geometry of the sample being manipulated, gripper or end effector probes, and probes that utilize other attachment mechanisms such as glue. The present method and apparatus are designed to keep the sample vitreous during all transfers and manipulations, and are performed in a very time-efficient manner. In addition, samples prepared using cryogenic FIB may not require FIB milling entirely or may leave small tabs of material remaining on either side of the sample. The manipulation is performed on a working surface in the cryogenic vapor phase such that the FIB sample, sample carrier, and probe are all at the same cryogenic temperature as described below. [Brief description of the drawings]

[0014] BRIEF DESCRIPTION OF THE DRAWINGS The above and other objects, features and advantages of the present invention will become more readily apparent from the following detailed description of preferred embodiments of the present invention, which proceeds with reference to the accompanying drawings.

[0015] [Figure 1] FIG. 1 is a perspective view showing ex situ lift-out (EXLO) using a solid conical glass probe tip as known in the prior art. [Diagram 2] FIG. 2 is a plan view showing EXLO operation on a small area of ​​a carbon coated grid as known in the prior art. [Diagram 3]FIG. 3 is a side view showing back-orientation scanning of a sample prepared using an asymmetric FIB as known in the prior art onto a slotted grid surface. [Figure 4] FIG. 4 is a perspective view of a sample and lift-out geometry using a beveled fixed probe tip with an elliptical probe flat surface as known in the prior art. [Diagram 5] FIG. 5 is a top view showing manipulation of a sample prepared using FIB onto a small portion of a carbon coated EM grid carrier using vacuum / suction forces through a hollow glass probe tip as known in the prior art. [Figure 6] 6A and 6B are perspective and side cross-sectional views, respectively, of a sample holder held within the cryojacket and forming a preferred feature of the present invention. [Figure 7] 7A and 7B are perspective and side cross-sectional views, respectively, of a sample holder held on a moat support structure in accordance with the teachings of the present invention. [Figure 8] FIG. 8 is a schematic side view illustrating the movement of a sample holder through various stages from sample formation to manipulation of the sample into a carrier in accordance with the teachings of the present invention. [Figure 9] FIG. 9 is a flow diagram of a low temperature EXLO step performed in accordance with the teachings of the present invention. [Figure 10] FIG. 10 is a flow diagram listing alternative probing method steps that can be used in conjunction with the low temperature EXLO steps of FIG. [Figure 11] FIG. 11 lists a possible workflow following an operation on a selected carrier grid according to the teachings of the present invention. [Figure 12A] FIG. 12A is an optical microscope image of a cryo-sample on a sample grid carrier. [Figure 12B] FIG. 12B is a cryo-EM image of the cryo-sample on the sample grid carrier. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0016] Detailed Description In the following, novel methods are described for EXLO manipulation of samples, performed under cryogenic and environmentally controlled conditions. These cryo-EXLO methods can be used for manipulation of vitreous biological and / or frozen samples, for environmentally or beam-sensitive materials (e.g., polymers, organics, Li-based, Na-based, etc.) that need to remain cold or be kept cold in a controlled environment during FIB milling, and for manipulation for subsequent cryo-EM (or ambient EM) or other characterization techniques and analysis. Vitreous samples are initially prepared at low temperatures by cooling the sample very quickly (e.g., plunge freezing) or by high pressure freezing.

[0017] While the present invention is directed to novel EXLO manipulation of samples under cryogenic conditions, it is first important to understand how EXLO is currently used at ambient conditions.

[0018] EXLO manipulation of samples to a grid at ambient conditions can be performed by different probe methods. One conventional method utilizes van der Waals attractive forces by touching the outer surface of a glass (or metal-coated glass) probe 104 to a sample 103 in a FIB-milled groove wall 102 below the sample surface 101 of a bulk sample 100, as shown in FIG. 1. As shown in a small portion 201 of a grid of 3 mm overall diameter in FIG. 2, the sample 103 attached to the probe 104 can be transferred or manipulated towards a sample carrier such as a carbon, perforated carbon, or Formvar coating 202 supported by a metal grid bar 203. Van der Waals attractive forces are orders of magnitude larger than electrostatic or gravitational forces acting on small samples (Giannuzzi et al., 2015). With EXLO, the tip of a conical hollow or solid glass probe is heated and drawn to a small point (e.g., ∼1 micrometer) and the curved outer surface of the probe is used to pick up or lift out a sample prepared with the FIB (Figure 1) and then manipulated towards a carbon coated EM grid carrier, a holey carbon coated EM grid carrier or a similar surface (Figure 2). After lift-out has been performed, the probe can be rotated to optimize the position of the sample for manipulation onto the selected sample carrier, or a second probe can be used to assist the sample manipulation process with respect to the first probe.

[0019] Previous patents by the present owner (disclosing the EXLO method (referred to as the "EXpressLO method"), for example as described in U.S. Pat. Nos. 8,740,209, 8,789,826 and 10,801,926) describe methods of EXLO by manipulation of a prepared sample using a sample carrier and an asymmetric focused ion beam (FIB) into a slotted sample grid carrier (FIGS. 3 and 4). Advantages of these EXpressLO methods are: (i) no carbon or thin film support is required to support the sample, allowing advanced microscopy without interference from carbon films; (ii) the sample can be post-processed after manipulation into the slotted carrier; (iii) rear manipulation for additional FIB milling eliminates FIB curtaining artifacts in the area of ​​interest; and (iv) beveled surfaces optimize the EXLO geometry and increase van der Waals attraction.

[0020] The EXpressLO lift-out method, shown in FIG. 3, uses a conical glass probe tip 104, as described above, to pick up or lift out an asymmetrically hollowed out sample 103, drawn to a fine point (e.g., 1 micrometer) and prepared using a FIB, using the outer surface of the probe for subsequent manipulation onto an EXpressLO Open Half Grid Slotted Sample Carrier Grid 302. The EXpressLO carrier grid 302 supports the sample via a recessed slotted surface 301 defined by the top and bottom edges 303 and 304 of the grid. The top and bottom edges 303 and 304 of the grid may be separated by a height of 5 micrometers or more. Prior to manipulation, the probe may be rotated so that the sample 103 remains on the grid surface 301, or the sample may be poked or rotated with a second probe or grid edge 303 to optimize the orientation of the sample 103 on top of the probe 104 before manipulating the probe through the slot. The surface of a sample can be analyzed by EM by passing an electron beam through the sample across an open slot, or its internal structure can be analyzed.

[0021] The orientation of the sample is shown by milling asymmetric hollows using a Focused Ion Beam (FIB) instrument. This sample asymmetry 103 can be easily visualized using an optical microscope used for the lift-out and micro-manipulation process to position the sample in the desired orientation. During the manipulation process, the probe can be rotated and / or the sample can be poked with a fixed second probe, or via a second micromanipulator, or with the protruding surface of the EXpressLO grid, so that the sample can be manipulated to remain on the EXpressLO grid surface in order to orient the sample to its desired position before manipulating the probe through the EXpressLO grid slot. Figure 4 shows an example of a bevel ellipsoid 401 relative to the probe axis 402 optimized for van der Waals lift-out. Manipulation to the sample grid carrier is performed with or without probe rotation.

[0022] Other methods of manipulating the sample under ambient conditions use vacuum suction as a holding force (FIG. 5) or use various grippers or end effectors or by dipping the probe into the glue to attach the sample to the probe for transfer to the glue-containing grid. In one example, vacuum can be applied through a beveled hollow glass tube 501 to lift out the approached FIB-milled free sample from the top of the sample (Wang, U.S. Pat. No. 7,041,985) or to lift out the FIB-milled sample from its surface if the sample is still attached to the bulk FIB-milled groove wall (Arjavac, U.S. Pat. No. 8,455,821; Arjavac, U.S. Pat. No. 8,890,064). For either of these prior art vacuum methods, the sample 502 is finally manipulated into a carbon-coated (202) EM grid carrier 201 using vacuum or suction forces through a hollow glass probe to aid in the lift-out and manipulation process shown in FIG. 5. Depending on the particular vacuum probing method used, the FIB sample must not be completely FIB milled or a small amount of material remains on both sides of the FIB sample and is released during the operation. All of these prior art probing methods mentioned above describe operation under ambient conditions.

[0023] Turning now to the EXLO procedures and apparatus specific to samples requiring low temperatures and / or highly controlled environmental conditions, FIG. 6A shows a perspective view of a cryojacket 600 used to hold, isolate and protect a sample, such as sample 103 (FIG. 1), during a portion of the process described below. The cryojacket 600 is preferably formed of a unitary body 602 of gold plated copper material. It is shaped with a lower dovetail end 604 and has a rectangular top that is used to facilitate insertion and removal of the cryojacket from the cryogenic liquid using tweezers or forceps. A slot 606 is formed through the top surface 608 of the jacket body 602 which serves as an entrance to an interior compartment 610 of the jacket body. A sample holder, also referred to as a cryoholder 612, is shown held within the interior compartment 610 of the cryojacket 600, the various parts of which are described further below. The upward facing surface of the holder 612 includes attachment points, such as bayonet-type receptors or threaded holes, shown here as threaded holes 613, so that a rod or other transfer device (see FIG. 8) can be used to move the cryo-holder 612 in / out of the FIB 800 (FIG. 8) through the FIB airlock 814 and into the cryo-jacket inner compartment 610.

[0024] FIG. 6B shows a side view of the cryojacket 600 along section line 6B-6B of FIG. 6A, with the cryojacket 600 partially immersed within a layer of cryogenic fluid 614 held within a cryostat chamber 616. The cryostat chamber 616 is preferably formed with a base 618 and sidewalls 620 configured to hold the cryogenic fluid 614 therein. A slot 609 formed in the base 618 is shaped to receive the cryojacket dovetail 604 and prevent the cryojacket 600 from tilting as the cryoholder 612 is moved in and out of the slot 609. The cryogenic fluid 614 is disposed within the cryostat chamber 616 to a layer thickness 622 sufficient to contact the sides and bottom of the cryojacket 600. However, the cryojacket sidewalls 624 are of sufficient height to prevent a lop layer 626 of the cryogenic liquid from spilling beyond the upper height of the sidewalls 624 and into the interior compartment 610. Thus, although the inner compartment 610 and the retained cryo-holder 612 remain out of contact with the cryo-fluid 614, they are still cooled due to their proximity to the cryo-fluid 614 and the cryo-jacket's outer surfaces 604, 624 which contact it.

[0025] The cryo-holder 612 includes a frost-protection lid 628 that is rotatably attached to the main body 630 via screws or pins 632. FIG. 6B shows the cryo-holder lid 628 in a closed position such that the cryo-holder lid 628 completely covers the working surface 634 (FIG. 7A) while the holder 612 is held within the cryo-jacket 600. The lid 628 protects the working surface 634 from contamination and limits the growth of ice crystals on a sample placed thereon. The cryo-jacket 600 is located within the cryostat 616 such that a cryogenic liquid 614 (e.g., liquid nitrogen [LN2], liquid helium, liquid ethane, liquid argon, or other similar cryogenic liquid) surrounds and cools the jacket, which in turn cools the cryo-holder 612.

[0026] FIG. 7A shows a perspective view of the cryo-holder 612 placed on top of the trench holder 700. The trench holder 700 is located within a layer 702 (shown here in outline by a shadowed line) of cryo-fluid 704 during operation. As will be understood below, the cryo-fluid 704 may be the same as that used with the cryo-jacket 600 in FIGS. 6A and 6B so that the cryo-jacket 600 and trench holder 700 are located in the same pool of cryo-fluid within the cryostat 616, thereby allowing them to be cooled. Furthermore, the cryo-layer 702 may be the same as that shown as layer 622 (FIG. 6B). However, the top surface 705 of layer 702 may have a reduced height compared to layer 622 due to boiling off of the cryo-fluid 614 over time during the maneuvering process, for example, when the sample holder 612 is moved to the cryo-jacket 600 and then to the trench holder 700, and when the sample is being moved from a resting surface on the work surface to a grid carrier as described in more detail below.

[0027] The side walls 706 of the moat holder 700 extend upwardly on all sides, preferably but not necessarily above the cryogenic layer 702, to form a peripheral ridge 708 around the perimeter of the upper surface. The cryogenic holder 612 is thus set on this upper surface and surrounded by this peripheral ridge 708 such that the holder 612 is held in place and prevented from slipping into the cryogenic fluid 704. The upper surface of the moat holder 700 on which the cryogenic holder 612 is located is preferably high enough so that the working surface 634 is within a cryogenic vapor layer 710 that forms above the layer 702 of cryogenic fluid 704 due to the slow evaporation of the cryogenic fluid 704 over time. The resulting cryogenic vapor layer 710 will typically be a combination of cryogenic gas from the evaporating substantially inert cryogenic liquid 704 and an additional inert environment within the chamber.

[0028] To accurately measure the operating temperature at which the sample is operated within the vapor layer 710, a temperature sensor (not shown) can be suspended above the cryogenic fluid layer 702 at a height substantially equal to the height of the working surface 634. The temperature of liquid nitrogen (LN2) is most typically around -196°C or lower, while the temperature at which vitreous samples are operated is preferably between -195°C and -160°C. The height above the top surface 705 of the cryogenic fluid is typically related to temperature. For example, for LN2, the closer the operating temperature is to the -196°C temperature of the cryogenic fluid, the closer the working surface is to the top surface 705. For example, 17 cm above the cryogenic fluid surface 705 is approximately -161°C, 12 cm above is -167°C, 7 cm above is -168°C, etc. However, because a microscope is typically used as part of an EXLO system to facilitate manipulation of the microscope sample from mount 712 to carrier 714 within the working surface 634 of the sample holder 612, manipulation is limited to a very narrow range of heights above the upper surface 705 of the cryogenic fluid, typically between 18-35 mm above the working surface 634, which matches the focal length of the microscope.

[0029] The work surface 634 of the holder 612 preferably includes a pair of spaced apart mounting surfaces upon which a sample is manipulated. A sample, such as a sample 103 formed in a bulk substrate 100 (FIG. 1) or a vitreous sample of a frozen grid of yeast cells, is mounted in the sample mount 712 and manipulated (i.e., moved) toward the grid or other sample carrier 714 via a probe 716 using various methods described herein. The sample may be held in the sample mount location 712 using releasable clips or other means (not shown).

[0030] The specimen grid carrier 714 is held within the recessed area via a clip (not shown) with slit sides 715 so that tweezers / forceps can be inserted into the grid carrier 714 to grasp the grid carrier 714 and move the mounted specimen for manipulation to the equipment for inspection.

[0031] 7B is a side view of the assembly along section line 7B-7B of FIG. 7A. A layer 702 of cryogenic liquid (e.g., LN2) is contained within the cryostat 616. Above the surface 705 of the layer 702 of cryogenic liquid (e.g., LN2) is a layer 720 of cryogenic vapor 710 maintained at a low humidity (e.g., less than 10% relative humidity) and a substantially inert environment (e.g., nitrogen gas). The cryostat 616 is filled with the cryogenic liquid 702 such that the surface 705 of the cryogenic liquid is at a level below the working surface 634 to cool the sample holder trench support 700 including the sample holder 612. The working surface 634 may be customized to accommodate defined positions for different bulk samples and one or more sample grid carriers for cryo-FIB milling. A frost cover 628 is shown in an open position (see FIG. 7A ) that is used to cover the working surface 634 on the holder 612 during the cryo-transfer step so that a probe 716 having the desired tip geometry can be used for cryo-EXLO operations. Note that the geometry of the working surface 634 and probe operations in the vapor phase 720 above the cryo-liquid level 705 allows the FIB sample on the mounting surface 712, the grid carrier 714, and the probe 716 to all operate at the same cryogenic temperature.

[0032] 8 and 9 show a schematic and flow diagram, respectively, of a process for moving a specimen from formation through manipulation in a cryogenic environment. In step 901, a specimen holder 612 is placed on a cryogenically cooled stage 804 in a focused ion beam (FIB) chamber 802, and a frost lid 628 is rotated open to expose a bulk sample 806 to a FIB milling instrument 808, which may also include a scanning electron microscope column 809. If such a sample is glassy or otherwise has properties that must be maintained in a cryogenic environment, the bulk sample 806 is cryogenically placed in the FIB and maintained in a cryogenic environment, preferably at or below -140°C. The FIB 808 focuses a beam 810 on the bulk sample 806 to mill a thin specimen 812 suitable for subsequent examination by an instrument such as an electron microscope. Once the specimen 812 has been milled, the frost lid 628 is closed and the working surface 624 of the sample and specimen holder 612 is covered to minimize the formation of ice contamination. It will be understood that FIG. 8 is schematic only, and the illustrated dimensions of the bulk sample 806 and specimen 812 are exaggerated relative to the size of the holder 612 as they are made visible in the figure. In reality, the bulk sample and specimen will be many times smaller than the specimen holder 612 and will be barely visible in the actual dimensional representation of FIG. 8. In step 902, after cryo-milling the specimen 812, the specimen holder 612 and milled specimen 812 are transferred through the airlock 814 to the vacuum chamber 816 with the lid 628 closed. The specimen holder 612 is positioned so that it sits vertically within the interior cavity 610 (FIG. 6B) of the cryo-jacket 600. As mentioned above, the cryo-jacket sits within a bath of cryo-fluid 614, which is itself held within the cryostat 616, where the top height 626 of the cryo-fluid does not exceed the height of the sidewall 620 of the cryostat 616. The chamber 816 and assembly are evacuated via pump and transferred through airlock 820 to a controlled, inert environment.

[0033] In step 903, the entire cryostat 616 is transferred through the airlock 820 into the glovebox chamber 822. The environmental conditions of the glovebox chamber 822 are then controlled via valve 824 to maintain less than 10% relative humidity and an inert gas environment within the chamber 822. Once inside the glovebox 822, the chamber 816 is vented and the cryostat 616 is removed from the chamber 816. Over time, the upper height of the cryogenic fluid 614 boils off and decreases in height from a height 626 just below the sidewall height of the cryojacket 600 to a height 705 just below the top height of the moat support 700. Additionally, additional LN2 can be added as needed to the interior of the glovebox 822 to bring the cryogenic fluid height 705 to the desired depth. Venting the assembly creates the conditions for a cryogenic vapor layer 710 (shown in FIGS. 7A and 7B) to collect just above the upper fluid level 705. A gripper such as forceps 826 is used to grasp the closed sample holder 612 from within the cryojacket 600 and move the sample holder onto the support surface of the trench support 700. The entire cryostat 616 is positioned on the EXLO station so that a user can manually manipulate the FIB sample, the cryostat, and other features of the cryostat through a sealed glove hole opening 828 that penetrates the side wall of the glove box 822.

[0034] In step 904, the lid 628 is removed to expose the sample 812 in a cryogenic vapor layer environment for manipulation. The EXLO method as described above is then used to lift the sample 812 out of the bulk sample 806 and manipulate it into a selected sample carrier, such as the grid carrier 714 (FIG. 7A) or the slotted grid shown in FIG. 3. The frost cover 628 may be closed and the cryostat 618 can be moved outside the glove box 822. The grid carrier 714 containing the sample 812 can then be transferred to an inspection tool (not shown) for analysis of the cryogenic sample using a variety of methods, such as electron microscopy, Raman spectroscopy, Auger spectroscopy, x-ray fluorescence spectroscopy, secondary ion mass spectrometry, or other suitable characterization or analysis techniques. All steps within the glove box 822, including transferring the cryostat from the airlock 820 to the glove box 822, venting the glove box 822, opening the sample holder lid 628, and manipulating the sample from the sample placement surface 712 to the holder 714, can be performed manually through the sealed glove hole opening 828.

[0035] Thus, the described method and associated apparatus allows for sample manipulation to occur in a low temperature, environmentally controlled environment, such as the glove box 822, that is located outside the instrument where the sample was generated, such as the FIB chamber 802. The described method and apparatus also allows for the transfer of the cryo-holder from the instrument where the sample was generated to a cryo-jacket within the cryostat, where the cryo-holder is maintained at a low temperature without immersing the cryo-holder directly in a cryogen. Thus, the described method and apparatus allows for the sample to be maintained in a vitreous state throughout the entire transfer and manipulation process. This has been found to reduce the total time required for sample manipulation to less than 10 minutes.

[0036] It is preferred that the environment in which the manipulation of the sample occurs, e.g., the glove box chamber (PVC / plexi, etc.), is substantially inert and maintained at a relatively low humidity percentage, preferably less than 10% (i.e., ambient water vapor within the glove box). This low humidity reduces the possibility of ice crystal formation / contamination on the sample, thus keeping the FIB milled sample surface "clean" for accurate inspection with the inspection tool. The humidity and inert conditions can be controlled using a pump and desiccant system and gas supply 824.

[0037] It is understood that the described manipulation methods can be performed with one or more probes, any of which may use primarily van der Waals forces to lift out the sample with a solid surface probe, vacuum or suction assisted forces with a hollow probe, probes with grippers or end effectors, and probes with glue assisted forces, as discussed in the prior art. In addition, one or more sample carriers can be used in the process for manipulation of samples prepared with one or more cryo-FIBs. Also, multiple working surfaces can be located in the cryostat such that the working surfaces are in the cryo-vapor. As an example, one working surface can contain the bulk sample with the cryo-FIB sample, and another surface can be used for the sample grid carrier. Some cryo-EMs use conventional grids that are clipped to larger support structures and can be handled by an automatic grid loader. For such autoloaders, the grids can be pre-clipped to these support structures for the cryo-EXLO process. Alternatively, the working surface can consist of a sample grid carrier or a pre-clipped sample grid carrier that is pre-mounted in the EM holder, which reduces additional cryo-transfer steps. In addition, one or more probe types can be used to optimize sample manipulation. All or part of the EXLO system may be contained within an enclosure that provides environmental control. The EXLO system may be accessed through wired or wireless / Bluetooth control of airlocks, glove ports, or direct computerized functions.

[0038] FIG. 9 shows a basic workflow diagram of the cryo-EXLO process steps. In step 901, a cryo-holder 612 (FIG. 7A) with a frost-lid that can be opened or closed via gravity or by using tweezers or other tools is used to place a bulk sample on the working surface that will be FIB milled to produce a thin sample for subsequent cryo-EM. The holder working surface may be customized to include separate areas that grip, hold, or support a sample carrier grid. The holder is cryo-transferred into the FIB using conventional methods. An intermediate step in the vacuum load lock before positioning on the cryo-FIB stage may include metal sputter coating with its lid open. Protective FIB deposition and FIB milling are performed using traditional cryo-FIB milling methods combined with conventional EXLO FIB milling protocols. After FIB milling, in step 902, the frost-cover is closed and the cryo-holder is vacuum-transferred to a cryo-jacket located inside the cryostat containing a cryogen (e.g., liquid nitrogen, LN2). The cryo-jacket keeps the cryo-holder cold during transfer without immersing the holder in the cryogen (e.g., LN2). After the holder is transferred to the cryostat, the holder is ejected and the assembly is transferred through an airlock to a controlled low humidity (less than 10% relative humidity) and substantially inert environment (e.g., nitrogen gas). During step 903, the assembly is vented and the holder is cryo-transferred from the cryo-jacket onto a moat support in the cryostat such that the holder working surface is located in the cryogen vapor above the cryogen (e.g., LN2) surface, and the entire cryostat is placed on the EXLO station housed in a humidity-controlled, inert environment. The frost cover is opened and a cold specimen grid carrier or a cold pre-clipped specimen grid carrier is placed on a defined area of ​​the holder working surface. Alternatively, a cryo-EM holder rod with a suitable specimen grid carrier can be placed in the cryostat in the cryogen vapor. Thereafter, in step 904, EXLO is performed using any number of the probing techniques described above in FIG. 10 to manipulate one or more FIB samples into the selected sample grid carrier.

[0039] FIG. 10 shows a work flow diagram illustrating another selection of probing methods applicable with the cryo-EXLO 904. It is understood that multiple probes and more than one probe type can be used for the cryo-EXLO operation process steps. During step 1001, a cone or bevel probe utilizing mainly van der Waals forces can be used to manipulate the sample prepared with the cryo-FIB and cryo-transfer it to the selected grid carrier. During another step 1002, a hollow probe utilizing vacuum or suction assistance can be used to manipulate the sample prepared with the cryo-FIB and cryo-transfer it to the selected grid carrier. During another step 1003, a gripper or probe with an end effector can be used to manipulate the sample prepared with the cryo-FIB and cryo-transfer it to the selected grid carrier. During another step 1004, a glued probe can be used to manipulate the sample prepared with the cryo-FIB and cryo-transfer it to the selected grid carrier.

[0040] FIG. 11 illustrates the steps following manipulation of the specimen into the selected grid carrier. During step 1110, the frost cover is closed and the cryostat housing the holder is removed from the environmental chamber. The frost cover is opened and one or more grid carriers are cryo-stored or transferred to the cryo-EM. During step 1120, if the specimen grid carrier is pre-loaded in a cryo-EM holder, the holder can be cryo-transferred directly to the cryo-EM or the specimen grid carrier can be cryo-stored. During step 1130, if a thick cryo-FIB specimen is manipulated into the slotted grid carrier, the slotted grid carrier can be cryo-transferred back to the cryo-FIB for additional FIB thinning before cryo-storage or cryo-transfer to the cryo-EM.

[0041] FIG. 12A is an optical microscope image acquired at the EXLO station immediately after a low-temperature EXLO run, showing a vitreous yeast sample 915 grafted onto a carbon film membrane 916 supported by a copper grid frame 917.

[0042] FIG. 12B is a bright field energy filtered cryo-EM image of the vitreous yeast sample 915, the carbon film 916, and the copper grid 917.

[0043] Having described and illustrated the principles of the invention in its preferred embodiments, it will be apparent that the invention may be modified in arrangement and detail without departing from such principles. Other materials and processes may be used, and samples may be formed from beam-sensitive materials that must remain cool, though not necessarily at temperatures below -140°C, during formation, manipulation, and inspection. We therefore claim all such modifications and variations as may come within the spirit and scope of the following claims.

Claims

1. 1. A method for manipulating a cryogenic sample for subsequent examination, comprising: A cryo-sample is placed on the working surface of the sample holder; placing the sample holder in a cryostat chamber having a layer of cryogenic liquid, with a layer of cryogenic vapor above the cryogenic liquid, with the working surface of the sample holder located within the layer of cryogenic vapor; manipulating the cryogenic sample into a sample carrier contained in a different portion of the working surface while maintaining the cryogenic sample within the cryogenic vapor layer; method.

2. the step of manipulating the cryogenic sample ex situ includes at least one of attaching the cryogenic sample to a solid surface probe tip using primarily van der Waals forces, attaching the cryogenic sample to a hollow surface probe tip using vacuum or suction assisted forces, attaching the cryogenic sample to a probe tip using a gripper or end effector, and attaching the cryogenic sample to a probe tip using glue adhesion forces; The method of claim 1.

3. The method of claim 1 , wherein the low temperature vapor layer is substantially inert.

4. 4. The method of claim 3, wherein the cryogenic liquid is liquid nitrogen (LN2), liquid helium, liquid ethane, or liquid argon.

5. 10. The method of claim 1, further comprising the step of maintaining the environment in which said manipulating step occurs at a relative humidity of less than 10%.

6. placing a moat support structure within the cryogenic liquid; placing the sample holder on the moat support structure such that the working surface of the sample holder is elevated into the low temperature vapor layer; The method of claim 1 further comprising:

7. 7. The method of claim 6, wherein the moat support structure includes an upper surface elevated above an upper surface of the cryogenic liquid, and the step of placing the sample holder on the moat support includes supporting the sample holder on the upper surface of the moat support.

8. The method of claim 1 , wherein the step of manipulating the cryo-sample to a different portion of the work surface comprises moving the cryo-sample to a sample grid carrier.

9. The method of claim 8 , wherein the sample grid carriers are located on the different portions of the working surface.

10. 10. The method of claim 9, further comprising the step of analyzing the cryogenic sample after said manipulating step using electron microscopy, Raman spectroscopy, Auger spectroscopy, X-ray fluorescence spectroscopy, secondary ion mass spectrometry or other suitable characterization or analysis technique.

11. placing a bulk sample on the sample holder; moving the sample holder containing the cryosample from the instrument into a cryo-jacket; maintaining the sample holder and the cryojacket under vacuum; The method of claim 1 further comprising:

12. After the step of generating the cryo-sample with the instrument and prior to the step of moving the sample holder into the cryo-jacket, covering the working surface of the sample holder with a frost cover. The method of claim 11 further comprising:

13. venting the cryostat chamber such that the cryostat chamber is maintained in a primarily inert environment and at a relatively low humidity; transferring the sample holder from the cold jacket to a moat support structure within the cryostat chamber; The method of claim 12 further comprising:

14. 14. The method of claim 13, further comprising generating the cryosample from the bulk sample in an instrument at a temperature below (-140°C).

15. 14. The method of claim 13, wherein the sample is a biological sample, the method further comprising maintaining the sample in a vitreous state throughout transport and manipulation steps.

16. 1. An apparatus for manipulating cryogenic samples, comprising: a cryostat chamber having a layer of cryogenic liquid and a layer of cryogenic vapor above the cryogenic liquid; a cryosample holder located within the cryostat chamber and configured to maintain a cryosample on a working surface located solely within the cryovapor layer of relatively low humidity (less than 10%) under substantially inert conditions; An apparatus comprising:

17. 17. The apparatus of claim 16, further comprising a sample grid carrier mounted on the working surface of the cryo-sample holder, the carrier configured to receive the cryo-sample for subsequent analysis using a method selected from the group consisting of electron microscopy, Raman spectroscopy, Auger spectroscopy, X-ray fluorescence spectroscopy, secondary ion mass spectrometry or other suitable characterization or analysis technique.

18. 17. The apparatus of claim 16, further comprising a moat support structure positioned within the cryogenic liquid, the sample holder positioned on the moat support, the sample holder working surface not contacting the cryogenic liquid, and the working surface of the sample holder elevated into the cryogenic vapor layer.

19. 20. The apparatus of claim 18, wherein the moat support structure includes an upper surface elevated above a lower surface of the cryogenic liquid, the sample holder being positioned on the upper surface of the moat support.

20. a cryojacket positioned within the cryostat's cryogenic liquid and having a bottom wall and a side wall contacting but elevated above an upper surface of the cryogenic liquid to form an interior compartment configured to receive the cryogenic sample holder such that the holder does not contact the cryogenic liquid; 20. The apparatus of claim 16, further comprising:

21. 17. The apparatus of claim 16, wherein the cryogenic sample holder includes a frost lid configured to move between an open position exposing the work surface and a closed position covering the work surface.

22. 17. The apparatus of claim 16, wherein the cryogenic liquid is one taken from the group consisting of liquid nitrogen (LN2), liquid helium, liquid ethane, and liquid argon.

Images