WO2020160788A1 - Apparatus and method for preparing a sample for transmission microscopy investigations by ion beam initiated ablation - Google Patents

Apparatus and method for preparing a sample for transmission microscopy investigations by ion beam initiated ablation Download PDF

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Publication number
WO2020160788A1
WO2020160788A1 PCT/EP2019/053163 EP2019053163W WO2020160788A1 WO 2020160788 A1 WO2020160788 A1 WO 2020160788A1 EP 2019053163 W EP2019053163 W EP 2019053163W WO 2020160788 A1 WO2020160788 A1 WO 2020160788A1
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WIPO (PCT)
Prior art keywords
sample
ion beam
source device
beam source
sample preparation
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PCT/EP2019/053163
Other languages
French (fr)
Inventor
Hendrik SCHIKORA
Friedjof TELLKAMP
R.J. Dwayne Miller
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Max-Planck-Gesellschaft zur Förderung der Wissenschaften e. V.
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Priority to PCT/EP2019/053163 priority Critical patent/WO2020160788A1/en
Publication of WO2020160788A1 publication Critical patent/WO2020160788A1/en

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Classifications

    • H—ELECTRICITY
    • H01—ELECTRIC ELEMENTS
    • H01J—ELECTRIC DISCHARGE TUBES OR DISCHARGE LAMPS
    • H01J37/00—Discharge tubes with provision for introducing objects or material to be exposed to the discharge, e.g. for the purpose of examination or processing thereof
    • H01J37/30—Electron-beam or ion-beam tubes for localised treatment of objects
    • H01J37/305—Electron-beam or ion-beam tubes for localised treatment of objects for casting, melting, evaporating or etching
    • H01J37/3053—Electron-beam or ion-beam tubes for localised treatment of objects for casting, melting, evaporating or etching for evaporating or etching
    • H01J37/3056—Electron-beam or ion-beam tubes for localised treatment of objects for casting, melting, evaporating or etching for evaporating or etching for microworking, e.g. etching of gratings, trimming of electrical components
    • H—ELECTRICITY
    • H01—ELECTRIC ELEMENTS
    • H01J—ELECTRIC DISCHARGE TUBES OR DISCHARGE LAMPS
    • H01J37/00—Discharge tubes with provision for introducing objects or material to be exposed to the discharge, e.g. for the purpose of examination or processing thereof
    • H01J37/30—Electron-beam or ion-beam tubes for localised treatment of objects
    • H01J37/304—Controlling tubes by information coming from the objects or from the beam, e.g. correction signals
    • H—ELECTRICITY
    • H01—ELECTRIC ELEMENTS
    • H01J—ELECTRIC DISCHARGE TUBES OR DISCHARGE LAMPS
    • H01J2237/00—Discharge tubes exposing object to beam, e.g. for analysis treatment, etching, imaging
    • H01J2237/06—Sources
    • H01J2237/08—Ion sources
    • H01J2237/0812—Ionized cluster beam [ICB] sources
    • H—ELECTRICITY
    • H01—ELECTRIC ELEMENTS
    • H01J—ELECTRIC DISCHARGE TUBES OR DISCHARGE LAMPS
    • H01J2237/00—Discharge tubes exposing object to beam, e.g. for analysis treatment, etching, imaging
    • H01J2237/244—Detection characterized by the detecting means
    • H01J2237/2445—Photon detectors for X-rays, light, e.g. photomultipliers
    • H—ELECTRICITY
    • H01—ELECTRIC ELEMENTS
    • H01J—ELECTRIC DISCHARGE TUBES OR DISCHARGE LAMPS
    • H01J2237/00—Discharge tubes exposing object to beam, e.g. for analysis treatment, etching, imaging
    • H01J2237/244—Detection characterized by the detecting means
    • H01J2237/24455—Transmitted particle detectors
    • H—ELECTRICITY
    • H01—ELECTRIC ELEMENTS
    • H01J—ELECTRIC DISCHARGE TUBES OR DISCHARGE LAMPS
    • H01J2237/00—Discharge tubes exposing object to beam, e.g. for analysis treatment, etching, imaging
    • H01J2237/30—Electron or ion beam tubes for processing objects
    • H01J2237/304—Controlling tubes
    • H01J2237/30466—Detecting endpoint of process
    • H—ELECTRICITY
    • H01—ELECTRIC ELEMENTS
    • H01J—ELECTRIC DISCHARGE TUBES OR DISCHARGE LAMPS
    • H01J2237/00—Discharge tubes exposing object to beam, e.g. for analysis treatment, etching, imaging
    • H01J2237/30—Electron or ion beam tubes for processing objects
    • H01J2237/317—Processing objects on a microscale
    • H01J2237/3174—Etching microareas
    • H01J2237/31745—Etching microareas for preparing specimen to be viewed in microscopes or analyzed in microanalysers
    • H—ELECTRICITY
    • H01—ELECTRIC ELEMENTS
    • H01J—ELECTRIC DISCHARGE TUBES OR DISCHARGE LAMPS
    • H01J37/00—Discharge tubes with provision for introducing objects or material to be exposed to the discharge, e.g. for the purpose of examination or processing thereof
    • H01J37/02—Details
    • H01J37/22—Optical or photographic arrangements associated with the tube
    • H01J37/226—Optical arrangements for illuminating the object; optical arrangements for collecting light from the object

Definitions

  • Apparatus and method for preparing a sample for transmission microscopy investigations by ion beam initiated ablation Apparatus and method for preparing a sample for transmission microscopy investigations by ion beam initiated ablation
  • the present invention generally relates to a sample preparation apparatus and method for preparing a sample for transmission microscopy, in particular by employing ion beam initiated ablation.
  • a common method for determining the structure and/or dynamics of organic material is X-ray structure analysis using, e.g., synchrotron beams.
  • X-ray structure analysis of protein crystals protein crystallography
  • several methods for the production of macroscopic single crystals of different protein complexes are known. The principle here is that thicker crystals also produce a better diffraction image than thinner crystals, so that there are no efforts to produce thin crystal samples.
  • Electron microscopy investigations like transmission electron microscopy (TEM), scanning TEM (STEM) and/or electron diffraction microscopy, are important alternative methods to determine the structure and/or dynamics of materials. Since electrons interact much more strongly than, e.g., synchrotron beams with a material sample, the sample must be much thinner, ideally of the order of 10 nm to 100 nm. Samples of these dimensions are also denoted as ultra-thin samples.
  • An advantage of electron microscopy over, e.g., X-ray structure analysis with synchrotron beams is the relatively low price of the electron microscopy equipment.
  • the costs for the construction and operation of synchrotron sources are up to three orders of magnitude higher than for electron microscopes.
  • Microtoming involves cutting the sample material e. g. with a diamond blade.
  • the blade exerts mechanical stress on the sample which can destroy the crystalline structure of the sample material, leading to unwanted mosaic formation in diffraction images.
  • Samples that are not suitable for microtoming due to their softness can be embedded in a matrix. Shock freezing (vitrification) and subsequent cutting in the cryogenic state is another procedure for which commercial instruments are available.
  • Microtoming is in particular not suitable for the production of, e.g., ultra-thin protein crystal samples, as proteins have a sponge-like structure due to water retention and roll up during microtomy or assume a flake-like consistency, making them unsuitable for electron microscopy, e.g. TEM or diffraction
  • the ultra-thin sample should be transferrable under suitable conditions, comprising e.g. high vacuum and/or cryogenic temperatures, to an analytic instrument, like an electron microscope, which is arranged for measuring the structure and dynamics of the ultra-thin sample. Furthermore, for the production of several ultra-thin samples, the preparation method should be reproducible.
  • ion beam etching or ion beam thinning ion beam etching or ion beam thinning
  • ion beams from an ion beam source are shot onto the material sample surface and strike atoms from the sample upon impact, resulting in material removal.
  • Common sources are Gallium (Ga) and Argon (Ar) ion beam sources.
  • Ga-ion beam sources are easier to focus than Ar-ion beam sources, they are often used at Focused Ion Beam (FIB) workstations.
  • FIB Focused Ion Beam
  • Ar-ion beams also can be used for sample ablation, as shown with the low-energy Ar- ion milling apparatus Fischione Model 1040 NanoMill for thinning various kinds of inorganic samples in [1]
  • a major limitation of conventional ion beam thinning is the possible destruction and/or amorphization of the crystalline structure by high-energy ion bombardment. This problem is acceptable for thicker samples, e.g. in the micrometer (pm) range, since the crystalline structure is still present under the amorphous layer.
  • pm micrometer
  • precisely coordinated parameters such as angle of incidence, acceleration voltage or ion beam current must be used with increased effort for thinning inorganic samples.
  • ultra-thin organic samples can almost not be produced by conventional ion beam etching or ion beam thinning, since the structure of organic material, e.g.
  • a variant of the ion beam etching is the gas cluster ion beam (GCIB) technology, which allows more gentle material removal than the single ion beam technology in general and especially for organic samples.
  • GCIB gas cluster ion beam
  • clusters are generated, which can typically contain several hundred to about 4000 Ar-atoms.
  • On impact with the sample surface, a large fraction of the energy is distributed into the fragmentation of the cluster and not into the destruction of the sample structure.
  • the GCIB technology has limited applications, e.g., it is mainly used in X-ray photoelectron spectroscopy (XPS) and in secondary ion mass spectrometry (SIMS), whereby in these applications not the material removal itself, but the analysis of the removed atoms is in the foreground.
  • XPS X-ray photoelectron spectroscopy
  • SIMS secondary ion mass spectrometry
  • GCIB surface smoothing of inorganic materials, such as sidewall smoothing of silicon (Si) described in [3], and etching of inorganic materials, such as semiconductor porous low-k injection laser diode (ILD) material in [4]
  • ILD semiconductor porous low-k injection laser diode
  • Objectives of the invention are to provide an improved apparatus and an improved method for preparing a material sample for electron microscopy, being capable of avoiding limitations of conventional techniques.
  • the apparatus and method for preparing a material sample for electron microscopy are to be capable of better avoiding sample damages and/or preserving the structure of the material sample.
  • the apparatus and method are to be capable of preparing ultra-thin, in particular organic, samples in a reproducible and/or automatable manner.
  • the above objective is solved by a sample preparation apparatus for preparing, in particular thinning, a sample for transmission electron microscopy investigations by ion beam initiated ablation.
  • the sample preparation apparatus comprises a sample holder device, which is arranged for accommodating a sample, and an ion beam source device, which is arranged for creating an ion beam and directing the ion beam toward the sample holder device.
  • the sample is arranged at the sample holder device for an irradiation with the ion beam.
  • the sample holder device and the ion beam source device are arranged in a vacuum chamber.
  • the ion beam source device is capable of ablating the sample by the effect of the ion beam.
  • the ion beam source device may comprise a source of Argon (Ar) or Gallium (Ga) ions.
  • the ion beam may comprise single atoms and/or clusters of atoms.
  • the ion beam source device preferably is arranged in a pivotable manner relative to the sample holder device, thus allowing an adjustment of the angle of incidence of the ion beam on the sample.
  • the sample preparation apparatus comprises a sample thickness detection device, which is arranged for monitoring a sample thickness, and a control unit, which is adapted for controlling the ion beam source device.
  • the sample thickness detection device can be completely or partially arranged in the vacuum chamber. Alternatively or additionally, it can be arranged outside the vacuum chamber, but optically coupled with the sample.
  • the control unit comprises e. g. a multi-purpose computer, like a personal computer (PC), being coupled with the ion beam source device.
  • PC personal computer
  • control unit is adapted for controlling the ion beam source device in dependency on an output signal of the sample thickness detection device.
  • this allows a controlled, damage free ion beam initiated ablation of the sample, in particular down to a thickness allowing electron microscopy with the sample.
  • the control unit is coupled with the sample thickness detection device for receiving at least one output signal therefrom.
  • the output signal is any electrical signal being derived from sample thickness detection and representing a current sample thickness.
  • the above objective is solved by a sample preparation method for preparing a sample for transmission electron microscopy investigations.
  • the sample preparation method comprises the steps of providing a sample, in particular on a sample holder device, creating an ion beam with an ion beam source device and directing the ion beam onto the sample.
  • the sample is thinned by the effect of the ion beam.
  • the ion beam source device is controlled by a control unit, and a sample thickness is monitored, in particular using a sample thickness detection device.
  • the ion beam source device is controlled by the control unit in dependency on a result of monitoring the sample thickness, in particular in dependency an output signal of the sample thickness detection device.
  • the ion beam source device preferably is controlled such that the sample is thinned down to a thickness adapted for the transmission electron microscopy investigation.
  • the method is performed using the sample preparation apparatus according the invention.
  • the ion beam source device is provided with a configuration (setting of operation conditions, in particular ion beam energy, ion beam flux and/or irradiation geometry) so that sample damages are avoided.
  • the use of an ion beam for ablating or thinning the sample may avoid damaging the sample.
  • the sample thickness provides a measure for a damage-free sample ablation and that the sample thickness can be monitored during the ablation process.
  • the ion beam source device configuration can be safely maintained and/or adjusted such that sample damages are avoided and thinning is stopped before the sample is destructed. Since the sample preparation method takes a certain amount of time, typically several hours, the method can be automated, i.e. the ablation of sample material can be monitored and stopped when the desired sample thickness is reached.
  • Damaging the sample may include at least one of a destruction of a, e.g. crystal, structure of the sample and a deposition of abrasive material within the sample material. Damage-free sample preparation is obtained if initial sample features, in particular topology and/or structure of the sample, are kept without changes or with negligible changes, in particular such that electron microscopy allows a characterization of the initial sample features.
  • the sample thickness detection device is arranged for monitoring the sample thickness in situ, i. e. the sample is arranged in the vacuum chamber, in particular in a condition without removing the sample from a position, where it is irradiated with the ion beam.
  • the sample thickness detection device is capable of monitoring the sample thickness, while operating the ion beam source device, particularly preferred during the ion beam initiated ablation or thinning procedure.
  • monitoring the sample thickness in situ during the ion beam initiated ablation or thinning procedure avoids the risk of damages from jolts and/or instabilities of the vacuum if repeated transfers among a sample preparation apparatus and a thickness measuring apparatus would be required.
  • sample thickness denotes a thickness, in particular a smallest thickness, of the sample during or after thinning, along a normal direction of the sample holder device.
  • Monitoring the sample thickness may comprise directly measuring the thickness of the sample.
  • monitoring the sample thickness may comprise directly determining at the sample as to whether the sample thickness is above, equal or below a predefined threshold value, e.g. when an electron transmission upon bombardment with electrons is observed.
  • sample refers to any organic or inorganic material sample to be investigated by electron microscopy, in particular transmission electron microscopy imaging or diffraction measurement.
  • the sample includes a protein crystal.
  • a protein crystal sample may comprise a protein crystal embedded in a casting compound.
  • the casting compound may comprise gum or glue, e.g. two-component glue.
  • the sample can be provided after an advance preparation of a thicker sample, e. g. by microtoming.
  • the sample preparation apparatus may be used for further thinning or ablating inorganic and/or organic macroscopic material samples that have been obtained by microtoming.
  • a typical size of a macroscopic protein crystal obtained by microtoming is of the order of 20 pm to 50 pm.
  • a typical size of an ultra-thin sample to be analyzed in a TEM is of the order of 10 nm to 100 nm.
  • the sample thickness detection device comprises an electron transmission detector unit.
  • the electron transmission detector unit includes an electron source being arranged for emitting, in particular focusing, a sensing electron beam towards the sample and an electron collector being arranged for receiving the electron beam transmitted through the sample.
  • the electron source is adapted for creating the sensing electron beam with an averaged power less that the electron beam power for an electron microscopy investigation.
  • the sensing electron beam is created such that a sample damage by the thickness detection is excluded.
  • the control unit preferably controls the ion beam source device in dependency on an output signal of the electron transmission detector unit.
  • the output signal is provided e. g. by an electron current transmitted through the sample.
  • the control unit preferably may stop an operation of the ion beam source device in dependency on the output signal of the electron transmission detector unit, e. g. an electron current sensed by the electron collector.
  • the operation of the ion beam source device can be continued for a predetermined period of time after detecting the threshold current for finally thinning the sample.
  • the sensing electron beam may be used to detect the thickness of a sample close to the ultra-thin thickness range required for, e.g., TEM.
  • the sensing electron beam may be used for detecting the thickness of a sample in a range of the order of 10 nm to 100 nm, i. e. one magnitude below the range of wavelengths of visible light.
  • the sensing electron beam may be generated at low cost by, e.g., the cathode of a commercially available cathode ray tube.
  • the ion beam source device may be operated as a gas cluster ion beam source (GCIB source).
  • GCIBs may perform a gentler thinning of a material sample than a low-energy beam composed of single atoms.
  • the use of a GCIB source represents an important inventive contribution of this embodiment, as GCIB based sample ablation or thinning for sample preparation purposes has not been described in the past.
  • GCIB sources rather have been used for surface smoothing or the etching of structures in thick samples.
  • the gas cluster ion beam source is an ion beam source being provided with a configuration such that ion clusters are emitted.
  • Parameters of the GCIB source may comprise e. g. a geometry of an ion beam creating nozzle, a nozzle pressure, an acceleration voltage and an acceleration path of the GCIB.
  • Parameters of the GCIB source in particular for creating an Ar-GCIB may comprise an Ar gas pressure, e. g. in a range from 8 to 20 bar, an acceleration voltage, e. g. in a range from 5kV to 10 kV, a Wien filter magnet current, e. g. in a range from 0 A to 3 A, an angle of incidence, e. g.
  • GCIB cluster parameters may determine GCIB cluster parameters, like a cluster size, an ion current and an ion energy.
  • GCIB clusters may have a size of typically a 100 up to 4000 atoms, e. g. Ar-atoms, e.g. of the order of 3000 atoms per cluster.
  • the GCIB cluster parameters may further comprise a diameter of the clusters, e. g. in a range from 100 pm to 500 pm.
  • the ion beam source device may create the ion beam in a first operation mode (single ion beam mode), such that the ion beam comprises single ions, and in a second operation mode (GCIB mode), such that the ion beam comprises ion clusters.
  • a macroscopic material sample may at first be ablated or thinned by using the single ion beam mode.
  • a relatively large fraction of the initial thickness of the macroscopic sample may be removed in a relatively short period of time by means of the single ion beam mode.
  • the ion beam source may be operated in the single ion mode for initial thinning of the, e.g. microtomed, macroscopic sample from a micro-meter range thickness down to 1 pm.
  • the upper layer of the intermediate sample obtained in this way after completing the first operation mode may be amorphous.
  • the upper layer of the intermediate sample may contain impurities of ion beam material.
  • the intermediate sample is further ablated or thinned in the second operation mode by using the GCIB.
  • the operation in GCIB mode may remove any possible amorphous and/or impure layer of the intermediate sample.
  • the ion beam source may be operated in the GCIB mode for ablating the sample down to a range of the order of 10 nm to several hundred nanometers.
  • the first and second operation modes may be refined by adjusting the ion beam parameters.
  • the control unit may control the ion beam source device such that it is operated in the first operation mode as long as the sample thickness is above a first threshold thickness.
  • the control unit may switch the ion beam source device to the second operation mode when the sample thickness is equal to or below the first threshold thickness.
  • the point in time of switching from a first operation mode to a second operation mode, or refinements thereof, may depend on a predefined minimum or threshold thickness of the intermediate sample.
  • the control unit may automatically switch the operation modes, so that an ultra-thin sample advantageously can be prepared from a macroscopic sample without, e.g. continuous, human supervision, e.g. overnight.
  • a manual switching between the first and second operations modes can be provided. Manual switching can be provided by switching the ion beam source device directly or via the control unit.
  • the sample thickness detection device may further comprise an optical measuring unit, which is adapted for measuring the sample thickness in situ.
  • the optical measuring unit includes a light source device and an optical detector device, being arranged for optically measuring the sample thickness.
  • the control unit of the sample preparation apparatus is arranged for controlling the light source device of the optical measuring unit.
  • the control unit may control the intensity of a light beam created by the light source device.
  • the control unit may control the wavelength of the light beam.
  • the control unit controls the ion beam source device in dependency on an output signal of the optical measuring unit.
  • the optical measuring unit allows a thickness detection in a thickness range above a sensitivity range of the electron transmission detector unit. This facilitates a fast ablation of the sample in a thickness range where the sample is less sensitive.
  • the optical detector device may include e. g. an optical microscope with a camera sensor being arranged for measuring the sample thickness by at least one of a transmission measurement and an interferometric measurement.
  • the optical measuring unit may comprise at least one of a transmitted light microscope, a Fizeau interferometer, a Shear interferometer, a phase-contrast microscope and a differential interference contrast microscope.
  • other types of optical detector devices can be provided, using e. g. a single photodiode for sensing sample transmission.
  • the optical measuring unit may measure the thickness of the sample, in a range equal to or above the wavelength of the light used for the detection. As an example, if the light source device comprises a green laser, thicknesses below about 550 nm can be measured. Thus, the optical measuring unit may be used to monitor the ablation of a macroscopic sample from above 1 pm down to about 400 nm or less, e. g. 100 nm of sample thickness.
  • the optical measuring unit and the electron transmission detector unit can be jointly provided, and the can be simultaneously used for monitoring the sample thickness.
  • the control unit reads out the sample thicknesses from both components and slows down the ablation or thinning procedure or stops it when the desired sample thickness is reached.
  • the optical measuring unit can be used for detecting as to whether the threshold thickness for switching to the second operation mode has been reached.
  • the optical measuring unit may indicate to the control unit that a predefined minimum or threshold thickness of the sample is reached in the first operation mode for fast milling.
  • the control unit may initiate that the ion beam source switches to the second operation mode for gentle ablation upon indication from the optical unit that the threshold thickness has been reached.
  • the sample holder device may be coupled with a cooling unit so that the sample can be cooled to cryogenic temperatures during the preparation thereof.
  • a cooling unit so that the sample can be cooled to cryogenic temperatures during the preparation thereof.
  • this facilitates preparing samples which require vitrification and subsequent ablation at cryogenic temperatures to preserve their structure, e.g. the structure of a protein crystal.
  • protein crystals contain water which is necessary for the crystalline structure. Placing these crystals in a vacuum would lead at least to a damage of the crystal structure or even to destruction.
  • the use of the cooling unit facilitates the procedure of ion ablating/thinning for vitrified protein crystals possible.
  • the sample holder device is adapted for accommodating multiple samples, advantages for preparing more than one ultra-thin sample in series are obtained. Multiple samples can be arranged side by side on a support surface of the sample holder device. With a preferred example, more than one ultra-thin sample of identical sample material may be used to reproduce and/or verify experimental results. Alternatively or in addition, accommodating several samples of identical and/or distinct sample material on the sample holder allows for automated ultra-thin sample preparation without human supervision, e.g. overnight or during weekends.
  • the sample holder device preferably may comprise an arrangement of grids, like TEM grids, e.g. six outer grids arranged around a central grid. A grid may have a diameter of 3 mm.
  • the invention allows to automatically thin several samples one after the other over a longer period of time.
  • the sample holder device may be mounted on a displacement device.
  • the displacement device may be arranged for at least one of stepwise scanning, translating and rotating the sample holder device. Displacements of the sample relative to the ion beam may be advantageous for a uniform ablation of the sample material.
  • the displacement device scans the sample so that the ion beam hits several locations and the sample material is removed uniformly.
  • each sample within a multiple sample arrangement may be ablated consecutively, e.g. after the output signal of the sample thickness detection device drops below a predefined target value for the given sample.
  • the displacement device may initiate or perform a transfer of a sample from the vacuum chamber of the sample preparation apparatus to an analyzer, e.g. an electron microscope.
  • the optical measuring unit can be provided for monitoring the uniformity of material removal, e. g. using the optical microscope.
  • the control unit can be arranged for adjusting the sample position relative to the ion beam with the displacement device.
  • the sample holder device may carry a scintillator arranged for emitting light in response to irradiation with the ion beam.
  • the scintillator facilitates an adjustment of the ion beam relative to the sample(s) and/or a correction of the ion beam adjustment.
  • the scintillator has a predetermined position on the sample holder device relative to the sample(s) thereon.
  • the control unit may control a translation and/or rotation of the sample holder device and/or a direction of the ion beam until the scintillator emits light in response to irradiation with the ion beam.
  • control unit may control the translation and/or rotation of the sample holder device and/or the direction of the ion beam for irradiating the sample to be thinned.
  • the scintillator preferably comprises cerium-doped or cerium-based scintillator material.
  • the vacuum chamber may comprise a loading unit.
  • the loading unit may couple a mobile transfer chamber to the vacuum chamber.
  • the loading unit may be used to transfer a sample or multiple samples on a sample holder to an analyzer, e.g. an electron microscope, under vacuum conditions.
  • Figure 1 a sample preparation apparatus according to embodiments of the invention.
  • sample preparation apparatus for preparing an ultra-thin sample from a thick macroscopic material sample, in particular an organic sample such as a protein crystal, by ion beam initiated ablation is described. It is emphasized that the application of the invention is not restricted to thinning of protein crystals, but rather possible also with other organic or inorganic material samples.
  • sample preparation apparatus of Figure 1 can be used for implementing different embodiments of the invention. Details of the
  • a sample preparation apparatus 100 comprises a sample holder device 10 and an ion beam source device 20 within a vacuum chamber 30, which is equipped with a pumping system (not shown). Furthermore, a sample thickness detection device 40 is provided, which comprises an electron transmission detector unit 41 and an optical measuring unit 44. Both components of the sample thickness detection device 40 are coupled with a control unit 50 controlling the ion beam source device 20.
  • a cooling unit 60 for cooling a sample 1 at the sample holder device 10 and/or a loading unit 70 are provided as shown in Figure 1.
  • the sample holder device 10 comprises a platform 11 providing a support surface for accommodating at least one sample 1 and being mounted on a displacement device 12.
  • the platform 11 has a structure, like a grid or lattice structure, allowing a transmission of a sensing electron beam, and it can be separable from the displacement device, e. g. for loading the platform 11 with the sample 1 outside the vacuum chamber 30 and introducing the platform 11 with the sample 1 into the vacuum chamber for performing the thinning process.
  • the displacement device 12 preferably comprises an XYZ manipulator. Alternatively or in addition, the displacement device 12 may allow translations and/or rotations of the sample relative to the ion beam source device 20.
  • the platform 11 may comprise one or more TEM grids directly connected to the displacement device 12, or the platform 11 may be connected to the displacement device 12 and carry one or more TEM grids, each with one or more samples.
  • the control unit 50 can be coupled with the displacement device 12 for controlling the position of the sample relative to the ion beam source device 20.
  • the ion beam source device 20 is arranged of producing an ion beam 21, e.g. an Ar-ion beam with an energy in a range from 5 keV to 10 keV, in particular an Ar-GCIB with a cluster energy of 10 keV, and directing the ion beam 21 towards the sample holder device 10.
  • the ion beam source device 20 comprises at least one of a single ion beam source, a GCIB source and a combination source being capable of switching from a single ion beam mode to a GCIB mode.
  • the ion beam source device 20 comprises a commercially available system, like the GCIB 1 OS manufactured by IONOPTIKA.
  • Operational parameters of the ion beam source device 20 are selected by the user in dependency on the particular application conditions, e. g. using reference data or preliminary tests with reference samples.
  • the ion beam 21 has a beam convergence below 2 pm, and it has a spot size in a range from 100 pm to 500 pm at the support surface of the sample holder device 10.
  • the electron transmission detector unit 41 comprises an electron source 42 and an electron collector 43.
  • the electron source 42 produces a sensing electron beam directed through the sample 1 to the electron collector 43.
  • the electron source 42 comprises a cathode with a focusing optic, as available from conventional cathode ray display tubes (e. g. television tubes).
  • the cathode may be operated in pulsed mode for reducing the averaged power of the sensing electron beam.
  • the sensing electron beam may comprise pulses with a frequency of 0,1 Hz and an energy of 1 kV.
  • the electron collector 43 comprises a Faraday cup, which is arranged for detecting electrons passing through the sample 1 accommodated on the sample holder device 10.
  • a transmission of the electron beam through the sample 1 is typically detected with sample thicknesses in the nanometer range, e.g. below 100 nm.
  • the sample thickness detection device 40 may comprise only the electron transmission detector unit 41 for detecting a completed preparing condition wherein the sample is sufficiently thin for an electron microscopy investigation.
  • the sample thickness detection device 40 also includes the optical measuring unit 44.
  • the optical measuring unit 24 comprises at least one, e.g. coherent, light source 45 being arranged for directing a light beam 46 towards the sample holder device 10.
  • the optical measuring unit 44 further comprises an optical microscope 47, with, e.g., a long working distance microscope lens 48.
  • the optical measuring unit 44 is capable of measuring the thickness of the sample 1 in a range of the wavelength of the light emitted by the light source 45, e.g. around 500 nm for a green laser source, by evaluating absorption and/or interference patterns.
  • the at least one light source 45 of the optical measuring unit 44 may be controlled by the control unit 50.
  • the optical microscope 47 may be adapted for a transmission (or absorption) measurement at the sample.
  • a sample thickness can be measured by sensing the light power transmitted through the sample and comparing it with prestored and/or measured calibration data.
  • the optical microscope 47 may be configured as a phase contrast microscope and/or a differential interference contrast microscope. For interference measurements of the sample thickness, the optical microscope 47 may provide at least one of a transmitted light
  • the cooling unit 60 comprises e. g. a cryogenic storage vessel 61, e.g. an LN2 Dewar vessel, a cooling braid 62 and a cooling finger 63.
  • the cooling finger 63 e. g. made of Cu, is directly connected directly with the platform 11 of the sample holder device 10. Furthermore, the cooling finger 63 is connected via the cooling braid 62, e. g.
  • the cooling unit 60 can be provided with a temperature sensor (not shown), e. g. at the platform 11.
  • the cooling unit 60 can be omitted e. g. if sample cooling is not necessary, e. g. if the sample is sufficiently stable in the vacuum chamber 30 even at room temperature (around 20 °C).
  • the vacuum chamber 30 can be equipped with a loading unit 70 as shown in Figure 1.
  • the loading unit 70 comprises a load lock 71, a transfer chamber 72, a transfer rod 73 and a cryogenic device 74 (optionally provided).
  • the loading unit 70 allows to transfer a sample under vacuum conditions to a TEM (not shown).
  • a protein crystal sample obtained directly from a crystallization process or after a preliminary shaping by microtoming, is placed on the sample holder device 10.
  • the sample is placed under vacuum and optionally cooled on the platform 11 of outside the vacuum chamber 30 and subsequently inserted by means of the loading unit 70 into the vacuum chamber 10.
  • the sample 1 is placed with an auxiliary tool (not shown) on the platform 11 within the vacuum chamber 30.
  • the sample 1 is cooled and/or kept at cryogenic temperatures by means of the cooling unit 60.
  • the sample 1 is arranged e. g. on a carrier element, like an electron microscopy grid. If multiple samples are to be arranged on the platformll, each sample preferably is positioned on a separate carrier element, so that multiple carrier elements each with one sample are arranged on the platform 11, and/or multiple samples are positioned on one carrier element.
  • the ion beam source device 20 is operated for creating the ion beam 21.
  • a scintillator (not shown) being provided with a predetermined position relative to the sample 1 on the sample holder device 10, the ion beam 21 can be adjusted.
  • the scintillator can be monitored visually or with the optical microscope 47, and the displacement device 12 is moved until the ion beam 21 hits the scintillator. Subsequently, the displacement device 12 is displaced by translations and/or rotations until the sample 1 is placed in the ion beam 21.
  • the ion beam source device 20 is operated in a first operation mode for creating the ion beam 21 with single ions.
  • the sample thickness is continuously or repeatedly measured during the ablation with the ion beam 21, using the optical measuring unit 44.
  • a threshold thickness of e. g. 450 nm is reached, the ion beam source device 20 is switched to a second operation mode for creating the ion beam 21 with ion clusters.
  • the electron transmission detector unit 41 is switched on for further monitoring the sample thickness.
  • the sample thickness is continuously or repeatedly tested by focusing a sensing electron beam to the sample and detecting, whether the sensing electron beam is transmitted through the sample.
  • the ablation with the ion beam 21 comprising the ion clusters can be continued for a predetermined period of time, or the ion beam source device 20 is switched off immediately.
  • Continued ablation can be provided in dependency on the application conditions, e. g. if the sample is to be further thinned using reference data which provide the allowable period of time for further ablation without sample destruction.
  • the sample preparation is completed.
  • the sample can be transported to an analyzer, e.g. an electron microscope, e. g. on the platform 11 through the loading unit 70.
  • an analyzer e.g. an electron microscope
  • a further sample can be provided for ion beam irradiation and the ablation or thinning procedure takes place again.
  • removing samples from the vacuum chamber preferably is conducted only after thinning all samples on the sample holder device 10.

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Abstract

A sample preparation apparatus (100) for ion beam ablation based preparing a sample (1), like a protein crystal, for a transmission electron microscopy investigation comprises a sample holder device (10) being arranged for accommodating a sample (1), an ion beam source device (20) being arranged for creating an ion beam (21) and directing the ion beam (21) toward the sample holder device (20), wherein the sample holder device (10) and the ion beam source device (20) are arranged in a vacuum chamber (30) and the ion beam source device (20) is capable of ablating the sample (1) by the effect of the ion beam (21), a sample thickness detection device (40) being arranged for monitoring a sample thickness, and a control unit (50) being adapted for controlling the ion beam source device (20), wherein the control unit (50) is adapted for controlling the ion beam source device (20) in dependency on an output signal of the sample thickness detection device (40). Furthermore, a sample preparation method for preparing a sample for a transmission electron microscopy investigation is described.

Description

Apparatus and method for preparing a sample for transmission microscopy investigations by ion beam initiated ablation
Technical Field
The present invention generally relates to a sample preparation apparatus and method for preparing a sample for transmission microscopy, in particular by employing ion beam initiated ablation.
Technical Background
In the present specification, reference is made to the following prior art illustrating the technical background of the invention:
[1] M. Mitome in "Journal of Electron Microscopy" volume 62, issue 2, 1 April 2013, pages 321 - 326;
[2] A. C. DCirr et al. in "Ultramicroscopy" volume 98, issue 1, December 2003, pages 51 - 55;
[3] N. Toyoda et al. in "Nuclear Instruments and Methods in Physics Research B" volume 232, 2005, pages 195-199;
[4] R. MacCrimmon et al. in "Nuclear Instruments and Methods in Physics Research Section B:
Beam Interactions with Materials and Atoms" volume 242, issues 1-2, January 2006, pages 427-430; and
[5] P. J. Cumpson et al. in "Surface and Interface Analysis" volume 45, issue 13, December 2013, pages 1859-1868.
The understanding of structure and/or dynamic processes within organic material forms the basis e. g. for future pharmaceutical and medical applications. A common method for determining the structure and/or dynamics of organic material, e.g. protein crystals, is X-ray structure analysis using, e.g., synchrotron beams. For the X-ray structure analysis of protein crystals (protein crystallography), several methods for the production of macroscopic single crystals of different protein complexes are known. The principle here is that thicker crystals also produce a better diffraction image than thinner crystals, so that there are no efforts to produce thin crystal samples. Electron microscopy investigations, like transmission electron microscopy (TEM), scanning TEM (STEM) and/or electron diffraction microscopy, are important alternative methods to determine the structure and/or dynamics of materials. Since electrons interact much more strongly than, e.g., synchrotron beams with a material sample, the sample must be much thinner, ideally of the order of 10 nm to 100 nm. Samples of these dimensions are also denoted as ultra-thin samples.
An advantage of electron microscopy over, e.g., X-ray structure analysis with synchrotron beams is the relatively low price of the electron microscopy equipment. The costs for the construction and operation of synchrotron sources are up to three orders of magnitude higher than for electron microscopes.
For producing material samples for electron microscopy, the technique of microtoming is conventionally used. Microtoming involves cutting the sample material e. g. with a diamond blade. As a disadvantage, the blade exerts mechanical stress on the sample which can destroy the crystalline structure of the sample material, leading to unwanted mosaic formation in diffraction images. Samples that are not suitable for microtoming due to their softness can be embedded in a matrix. Shock freezing (vitrification) and subsequent cutting in the cryogenic state is another procedure for which commercial instruments are available.
However, many organic samples cannot be produced with this method because the mechanical stress caused by the blade destroys the organic material structure. Microtoming is in particular not suitable for the production of, e.g., ultra-thin protein crystal samples, as proteins have a sponge-like structure due to water retention and roll up during microtomy or assume a flake-like consistency, making them unsuitable for electron microscopy, e.g. TEM or diffraction
experiments. Microtoming is also challenging as the ultra-thin sample should be transferrable under suitable conditions, comprising e.g. high vacuum and/or cryogenic temperatures, to an analytic instrument, like an electron microscope, which is arranged for measuring the structure and dynamics of the ultra-thin sample. Furthermore, for the production of several ultra-thin samples, the preparation method should be reproducible.
Currently, there is no established and easy-to-use alternative to microtoming for the preparation of ultra-thin organic material samples, like protein crystals, for the determination of their structure and/or dynamics. Another approach for general sample ablation or thinning consists in ion beam etching or ion beam thinning [1] Herein, ion beams from an ion beam source are shot onto the material sample surface and strike atoms from the sample upon impact, resulting in material removal. Common sources are Gallium (Ga) and Argon (Ar) ion beam sources. As Ga-ion beam sources are easier to focus than Ar-ion beam sources, they are often used at Focused Ion Beam (FIB) workstations. However, Ar-ion beams also can be used for sample ablation, as shown with the low-energy Ar- ion milling apparatus Fischione Model 1040 NanoMill for thinning various kinds of inorganic samples in [1]
A major limitation of conventional ion beam thinning is the possible destruction and/or amorphization of the crystalline structure by high-energy ion bombardment. This problem is acceptable for thicker samples, e.g. in the micrometer (pm) range, since the crystalline structure is still present under the amorphous layer. For sample thicknesses in the range of a few nanometers as required for, e.g., TEM, precisely coordinated parameters such as angle of incidence, acceleration voltage or ion beam current must be used with increased effort for thinning inorganic samples. In contrast to inorganic materials, ultra-thin organic samples can almost not be produced by conventional ion beam etching or ion beam thinning, since the structure of organic material, e.g. protein crystals, is much more sensitive and is even more easily destroyed by ion bombardment. In [2], e.g. a method of thinning a sample of the organic semiconductor diindenoperylene (DIP) is described using an Ar-ion mill operated at 4 keV, which, while using specific carrier substances and grinding and polishing techniques to prepare the material sample for the Ar-ion milling, only gave a fraction of 30-50% of the sample specimen delivering reasonable TEM images.
A variant of the ion beam etching is the gas cluster ion beam (GCIB) technology, which allows more gentle material removal than the single ion beam technology in general and especially for organic samples. Instead of bombarding the sample with individual Ar-ions, so-called clusters are generated, which can typically contain several hundred to about 4000 Ar-atoms. On impact with the sample surface, a large fraction of the energy is distributed into the fragmentation of the cluster and not into the destruction of the sample structure. However, up to now, the GCIB technology has limited applications, e.g., it is mainly used in X-ray photoelectron spectroscopy (XPS) and in secondary ion mass spectrometry (SIMS), whereby in these applications not the material removal itself, but the analysis of the removed atoms is in the foreground. Other applications of the GCIB technology involve surface smoothing of inorganic materials, such as sidewall smoothing of silicon (Si) described in [3], and etching of inorganic materials, such as semiconductor porous low-k injection laser diode (ILD) material in [4] Within the context of organic, in particular protein, structures, as a first application in [5] the sputter yield of bovine collagen was estimated upon Ar-GCIB depth profiling on an organic/inorganic material interface.
Generally, there does not exist a method of preparing ultra-thin organic samples in a controlled way without substantial damaging the structure of the sample material. This problem does not relate only to thinning of protein crystals, but also to other organic sample, like thins films of organic semiconductors or pigments.
Objective of the invention
Objectives of the invention are to provide an improved apparatus and an improved method for preparing a material sample for electron microscopy, being capable of avoiding limitations of conventional techniques. In particular, the apparatus and method for preparing a material sample for electron microscopy are to be capable of better avoiding sample damages and/or preserving the structure of the material sample. Alternatively or in addition, the apparatus and method are to be capable of preparing ultra-thin, in particular organic, samples in a reproducible and/or automatable manner.
Summary of the invention
These objectives are solved by an apparatus and a method comprising the features of the independent claims, resp.. Advantageous embodiments of the invention are defined in the dependent claims.
According to a first general aspect of the invention, the above objective is solved by a sample preparation apparatus for preparing, in particular thinning, a sample for transmission electron microscopy investigations by ion beam initiated ablation. The sample preparation apparatus comprises a sample holder device, which is arranged for accommodating a sample, and an ion beam source device, which is arranged for creating an ion beam and directing the ion beam toward the sample holder device. The sample is arranged at the sample holder device for an irradiation with the ion beam. The sample holder device and the ion beam source device are arranged in a vacuum chamber. The ion beam source device is capable of ablating the sample by the effect of the ion beam. The ion beam source device may comprise a source of Argon (Ar) or Gallium (Ga) ions. The ion beam may comprise single atoms and/or clusters of atoms. The ion beam source device preferably is arranged in a pivotable manner relative to the sample holder device, thus allowing an adjustment of the angle of incidence of the ion beam on the sample.
Furthermore, the sample preparation apparatus comprises a sample thickness detection device, which is arranged for monitoring a sample thickness, and a control unit, which is adapted for controlling the ion beam source device. The sample thickness detection device can be completely or partially arranged in the vacuum chamber. Alternatively or additionally, it can be arranged outside the vacuum chamber, but optically coupled with the sample. The control unit comprises e. g. a multi-purpose computer, like a personal computer (PC), being coupled with the ion beam source device.
According to the invention, the control unit is adapted for controlling the ion beam source device in dependency on an output signal of the sample thickness detection device. Advantageously, this allows a controlled, damage free ion beam initiated ablation of the sample, in particular down to a thickness allowing electron microscopy with the sample. The control unit is coupled with the sample thickness detection device for receiving at least one output signal therefrom. The output signal is any electrical signal being derived from sample thickness detection and representing a current sample thickness.
According to a second general aspect of the invention, the above objective is solved by a sample preparation method for preparing a sample for transmission electron microscopy investigations. The sample preparation method comprises the steps of providing a sample, in particular on a sample holder device, creating an ion beam with an ion beam source device and directing the ion beam onto the sample. The sample is thinned by the effect of the ion beam. The ion beam source device is controlled by a control unit, and a sample thickness is monitored, in particular using a sample thickness detection device.
According to the invention, the ion beam source device is controlled by the control unit in dependency on a result of monitoring the sample thickness, in particular in dependency an output signal of the sample thickness detection device. The ion beam source device preferably is controlled such that the sample is thinned down to a thickness adapted for the transmission electron microscopy investigation. Preferably, the method is performed using the sample preparation apparatus according the invention. The ion beam source device is provided with a configuration (setting of operation conditions, in particular ion beam energy, ion beam flux and/or irradiation geometry) so that sample damages are avoided. Advantageously, the use of an ion beam for ablating or thinning the sample may avoid damaging the sample. The inventors have found, that the sample thickness provides a measure for a damage-free sample ablation and that the sample thickness can be monitored during the ablation process. By controlling the ion beam source device with the control unit, the ion beam source device configuration can be safely maintained and/or adjusted such that sample damages are avoided and thinning is stopped before the sample is destructed. Since the sample preparation method takes a certain amount of time, typically several hours, the method can be automated, i.e. the ablation of sample material can be monitored and stopped when the desired sample thickness is reached.
Damaging the sample may include at least one of a destruction of a, e.g. crystal, structure of the sample and a deposition of abrasive material within the sample material. Damage-free sample preparation is obtained if initial sample features, in particular topology and/or structure of the sample, are kept without changes or with negligible changes, in particular such that electron microscopy allows a characterization of the initial sample features.
The sample thickness detection device is arranged for monitoring the sample thickness in situ, i. e. the sample is arranged in the vacuum chamber, in particular in a condition without removing the sample from a position, where it is irradiated with the ion beam. Preferably, the sample thickness detection device is capable of monitoring the sample thickness, while operating the ion beam source device, particularly preferred during the ion beam initiated ablation or thinning procedure. As an additional advantage, monitoring the sample thickness in situ during the ion beam initiated ablation or thinning procedure avoids the risk of damages from jolts and/or instabilities of the vacuum if repeated transfers among a sample preparation apparatus and a thickness measuring apparatus would be required.
The term "sample thickness" denotes a thickness, in particular a smallest thickness, of the sample during or after thinning, along a normal direction of the sample holder device. Monitoring the sample thickness may comprise directly measuring the thickness of the sample. Alternatively or in addition, monitoring the sample thickness may comprise directly determining at the sample as to whether the sample thickness is above, equal or below a predefined threshold value, e.g. when an electron transmission upon bombardment with electrons is observed. The term "sample" refers to any organic or inorganic material sample to be investigated by electron microscopy, in particular transmission electron microscopy imaging or diffraction measurement. Preferably, the sample includes a protein crystal. A protein crystal sample may comprise a protein crystal embedded in a casting compound. The casting compound may comprise gum or glue, e.g. two-component glue. The sample can be provided after an advance preparation of a thicker sample, e. g. by microtoming. Thus, the sample preparation apparatus may be used for further thinning or ablating inorganic and/or organic macroscopic material samples that have been obtained by microtoming. For example, a typical size of a macroscopic protein crystal obtained by microtoming is of the order of 20 pm to 50 pm. A typical size of an ultra-thin sample to be analyzed in a TEM is of the order of 10 nm to 100 nm.
According to a preferred embodiment of the invention, the sample thickness detection device comprises an electron transmission detector unit. The electron transmission detector unit includes an electron source being arranged for emitting, in particular focusing, a sensing electron beam towards the sample and an electron collector being arranged for receiving the electron beam transmitted through the sample. The electron source is adapted for creating the sensing electron beam with an averaged power less that the electron beam power for an electron microscopy investigation. The sensing electron beam is created such that a sample damage by the thickness detection is excluded. With this embodiment, the control unit preferably controls the ion beam source device in dependency on an output signal of the electron transmission detector unit. The output signal is provided e. g. by an electron current transmitted through the sample. Advantageously, if the electron collector detects an electron transmission through the sample, in particular an electron current above a predetermined threshold current, the sample is considered to be thin enough for the electron microscopy investigation. Thus, the control unit preferably may stop an operation of the ion beam source device in dependency on the output signal of the electron transmission detector unit, e. g. an electron current sensed by the electron collector. Alternatively, the operation of the ion beam source device can be continued for a predetermined period of time after detecting the threshold current for finally thinning the sample.
The sensing electron beam may be used to detect the thickness of a sample close to the ultra-thin thickness range required for, e.g., TEM. Alternatively or in addition, the sensing electron beam may be used for detecting the thickness of a sample in a range of the order of 10 nm to 100 nm, i. e. one magnitude below the range of wavelengths of visible light. Advantageously, the sensing electron beam may be generated at low cost by, e.g., the cathode of a commercially available cathode ray tube.
According to a further advantageous embodiment of the invention, the ion beam source device may be operated as a gas cluster ion beam source (GCIB source). Advantageously, GCIBs may perform a gentler thinning of a material sample than a low-energy beam composed of single atoms. The use of a GCIB source represents an important inventive contribution of this embodiment, as GCIB based sample ablation or thinning for sample preparation purposes has not been described in the past. GCIB sources rather have been used for surface smoothing or the etching of structures in thick samples.
There is no sample thickness detection in situ for conventional single ion beam sources since, due to the high energy of a single ion in the ion beam, the angle of incident is usually very flat, e.g. smaller than 10° from the sample surface, and the sample is rotated around the surface normal for uniform sample removal. The evaluation of rotating samples is not as easy as with a rigid sample. By using a GCIB, the energy per atom within an ion cluster is much lower than in a single ion beam. The angle of incident of the GCIB can be chosen much larger, e.g. between 30° and 90° relative to the sample surface. In this case, steady rotation is not required, and a sample thickness detection can be performed in situ, preferably during the sample ablation or thinning procedure.
The gas cluster ion beam source is an ion beam source being provided with a configuration such that ion clusters are emitted. Parameters of the GCIB source may comprise e. g. a geometry of an ion beam creating nozzle, a nozzle pressure, an acceleration voltage and an acceleration path of the GCIB. Parameters of the GCIB source in particular for creating an Ar-GCIB may comprise an Ar gas pressure, e. g. in a range from 8 to 20 bar, an acceleration voltage, e. g. in a range from 5kV to 10 kV, a Wien filter magnet current, e. g. in a range from 0 A to 3 A, an angle of incidence, e. g. in a range from 10° to 90°, typ. 45°, an extractor voltage, e. g. in a range from-100 V to -2 kV, and a voltage of a lens for focusing, e. g. in a range from 4 kV to 7 kV. These parameters may determine GCIB cluster parameters, like a cluster size, an ion current and an ion energy. GCIB clusters may have a size of typically a 100 up to 4000 atoms, e. g. Ar-atoms, e.g. of the order of 3000 atoms per cluster. The GCIB cluster parameters may further comprise a diameter of the clusters, e. g. in a range from 100 pm to 500 pm. At impact on the sample, a large fraction of the cluster energy is distributed into fragments of the cluster without damaging the inorganic and/or organic, e.g. protein crystal, structure of the sample. With a further, particularly preferred embodiment of the invention, the ion beam source device may create the ion beam in a first operation mode (single ion beam mode), such that the ion beam comprises single ions, and in a second operation mode (GCIB mode), such that the ion beam comprises ion clusters. A macroscopic material sample may at first be ablated or thinned by using the single ion beam mode. A relatively large fraction of the initial thickness of the macroscopic sample may be removed in a relatively short period of time by means of the single ion beam mode. For example, the ion beam source may be operated in the single ion mode for initial thinning of the, e.g. microtomed, macroscopic sample from a micro-meter range thickness down to 1 pm. The upper layer of the intermediate sample obtained in this way after completing the first operation mode may be amorphous. Alternatively or in addition, the upper layer of the intermediate sample may contain impurities of ion beam material.
Subsequently, the intermediate sample is further ablated or thinned in the second operation mode by using the GCIB. The operation in GCIB mode may remove any possible amorphous and/or impure layer of the intermediate sample. As an example, the ion beam source may be operated in the GCIB mode for ablating the sample down to a range of the order of 10 nm to several hundred nanometers. Alternatively or in addition, the first and second operation modes may be refined by adjusting the ion beam parameters.
The control unit may control the ion beam source device such that it is operated in the first operation mode as long as the sample thickness is above a first threshold thickness. The control unit may switch the ion beam source device to the second operation mode when the sample thickness is equal to or below the first threshold thickness. The point in time of switching from a first operation mode to a second operation mode, or refinements thereof, may depend on a predefined minimum or threshold thickness of the intermediate sample. The control unit may automatically switch the operation modes, so that an ultra-thin sample advantageously can be prepared from a macroscopic sample without, e.g. continuous, human supervision, e.g. overnight. Alternatively, a manual switching between the first and second operations modes can be provided. Manual switching can be provided by switching the ion beam source device directly or via the control unit.
According to a further preferred embodiment, the sample thickness detection device may further comprise an optical measuring unit, which is adapted for measuring the sample thickness in situ. The optical measuring unit includes a light source device and an optical detector device, being arranged for optically measuring the sample thickness. Preferably, the control unit of the sample preparation apparatus is arranged for controlling the light source device of the optical measuring unit. The control unit may control the intensity of a light beam created by the light source device. Alternatively or in addition, the control unit may control the wavelength of the light beam. With this embodiment, the control unit controls the ion beam source device in dependency on an output signal of the optical measuring unit.
Advantageously, the optical measuring unit allows a thickness detection in a thickness range above a sensitivity range of the electron transmission detector unit. This facilitates a fast ablation of the sample in a thickness range where the sample is less sensitive.
The optical detector device may include e. g. an optical microscope with a camera sensor being arranged for measuring the sample thickness by at least one of a transmission measurement and an interferometric measurement. According to preferred variants, the optical measuring unit may comprise at least one of a transmitted light microscope, a Fizeau interferometer, a Shear interferometer, a phase-contrast microscope and a differential interference contrast microscope. Alternatively, other types of optical detector devices can be provided, using e. g. a single photodiode for sensing sample transmission.
The optical measuring unit, in particular using the optical microscope, may measure the thickness of the sample, in a range equal to or above the wavelength of the light used for the detection. As an example, if the light source device comprises a green laser, thicknesses below about 550 nm can be measured. Thus, the optical measuring unit may be used to monitor the ablation of a macroscopic sample from above 1 pm down to about 400 nm or less, e. g. 100 nm of sample thickness.
The optical measuring unit and the electron transmission detector unit can be jointly provided, and the can be simultaneously used for monitoring the sample thickness. The control unit reads out the sample thicknesses from both components and slows down the ablation or thinning procedure or stops it when the desired sample thickness is reached.
According to a particularly advantageous variant of the invention, the optical measuring unit can be used for detecting as to whether the threshold thickness for switching to the second operation mode has been reached. The optical measuring unit may indicate to the control unit that a predefined minimum or threshold thickness of the sample is reached in the first operation mode for fast milling. The control unit may initiate that the ion beam source switches to the second operation mode for gentle ablation upon indication from the optical unit that the threshold thickness has been reached.
According to a further preferred embodiment, the sample holder device may be coupled with a cooling unit so that the sample can be cooled to cryogenic temperatures during the preparation thereof. Advantageously, this facilitates preparing samples which require vitrification and subsequent ablation at cryogenic temperatures to preserve their structure, e.g. the structure of a protein crystal. For example, protein crystals contain water which is necessary for the crystalline structure. Placing these crystals in a vacuum would lead at least to a damage of the crystal structure or even to destruction. The use of the cooling unit facilitates the procedure of ion ablating/thinning for vitrified protein crystals possible.
If, according to another modification of the invention, the sample holder device is adapted for accommodating multiple samples, advantages for preparing more than one ultra-thin sample in series are obtained. Multiple samples can be arranged side by side on a support surface of the sample holder device. With a preferred example, more than one ultra-thin sample of identical sample material may be used to reproduce and/or verify experimental results. Alternatively or in addition, accommodating several samples of identical and/or distinct sample material on the sample holder allows for automated ultra-thin sample preparation without human supervision, e.g. overnight or during weekends. The sample holder device preferably may comprise an arrangement of grids, like TEM grids, e.g. six outer grids arranged around a central grid. A grid may have a diameter of 3 mm. Advantageously, the invention allows to automatically thin several samples one after the other over a longer period of time.
According to a further advantageous embodiment, the sample holder device may be mounted on a displacement device. The displacement device may be arranged for at least one of stepwise scanning, translating and rotating the sample holder device. Displacements of the sample relative to the ion beam may be advantageous for a uniform ablation of the sample material. As an example, the displacement device scans the sample so that the ion beam hits several locations and the sample material is removed uniformly. Alternatively or in addition, by displacing the sample holder, each sample within a multiple sample arrangement may be ablated consecutively, e.g. after the output signal of the sample thickness detection device drops below a predefined target value for the given sample. Alternatively or in addition, the displacement device may initiate or perform a transfer of a sample from the vacuum chamber of the sample preparation apparatus to an analyzer, e.g. an electron microscope. Advantageously, the optical measuring unit can be provided for monitoring the uniformity of material removal, e. g. using the optical microscope. With this embodiment, the control unit can be arranged for adjusting the sample position relative to the ion beam with the displacement device.
Optionally, the sample holder device may carry a scintillator arranged for emitting light in response to irradiation with the ion beam. Advantageously, the scintillator facilitates an adjustment of the ion beam relative to the sample(s) and/or a correction of the ion beam adjustment. Preferably, the scintillator has a predetermined position on the sample holder device relative to the sample(s) thereon. The control unit may control a translation and/or rotation of the sample holder device and/or a direction of the ion beam until the scintillator emits light in response to irradiation with the ion beam. Subsequently, the control unit may control the translation and/or rotation of the sample holder device and/or the direction of the ion beam for irradiating the sample to be thinned. The scintillator preferably comprises cerium-doped or cerium-based scintillator material.
According to another advantageous embodiment, the vacuum chamber may comprise a loading unit. The loading unit may couple a mobile transfer chamber to the vacuum chamber. The loading unit may be used to transfer a sample or multiple samples on a sample holder to an analyzer, e.g. an electron microscope, under vacuum conditions.
Brief description of drawing
Further details of embodiments of the sample preparation apparatus and method are described with reference to the attached drawing, which show in:
Figure 1 a sample preparation apparatus according to embodiments of the invention.
Detailed description of preferred embodiments
In the following, a sample preparation apparatus and method for preparing an ultra-thin sample from a thick macroscopic material sample, in particular an organic sample such as a protein crystal, by ion beam initiated ablation is described. It is emphasized that the application of the invention is not restricted to thinning of protein crystals, but rather possible also with other organic or inorganic material samples. Features of the sample preparation apparatus of Figure 1 can be used for implementing different embodiments of the invention. Details of the
configuration and operation of an ion beam source device are not described in the following, as far as they are known from prior art.
According to Figure 1, a sample preparation apparatus 100 comprises a sample holder device 10 and an ion beam source device 20 within a vacuum chamber 30, which is equipped with a pumping system (not shown). Furthermore, a sample thickness detection device 40 is provided, which comprises an electron transmission detector unit 41 and an optical measuring unit 44. Both components of the sample thickness detection device 40 are coupled with a control unit 50 controlling the ion beam source device 20. Optionally, a cooling unit 60 for cooling a sample 1 at the sample holder device 10 and/or a loading unit 70 are provided as shown in Figure 1.
The sample holder device 10 comprises a platform 11 providing a support surface for accommodating at least one sample 1 and being mounted on a displacement device 12. The platform 11 has a structure, like a grid or lattice structure, allowing a transmission of a sensing electron beam, and it can be separable from the displacement device, e. g. for loading the platform 11 with the sample 1 outside the vacuum chamber 30 and introducing the platform 11 with the sample 1 into the vacuum chamber for performing the thinning process. The displacement device 12 preferably comprises an XYZ manipulator. Alternatively or in addition, the displacement device 12 may allow translations and/or rotations of the sample relative to the ion beam source device 20. With examples, the platform 11 may comprise one or more TEM grids directly connected to the displacement device 12, or the platform 11 may be connected to the displacement device 12 and carry one or more TEM grids, each with one or more samples. The control unit 50 can be coupled with the displacement device 12 for controlling the position of the sample relative to the ion beam source device 20.
The ion beam source device 20 is arranged of producing an ion beam 21, e.g. an Ar-ion beam with an energy in a range from 5 keV to 10 keV, in particular an Ar-GCIB with a cluster energy of 10 keV, and directing the ion beam 21 towards the sample holder device 10. The ion beam source device 20 comprises at least one of a single ion beam source, a GCIB source and a combination source being capable of switching from a single ion beam mode to a GCIB mode. For example, the ion beam source device 20 comprises a commercially available system, like the GCIB 1 OS manufactured by IONOPTIKA. Operational parameters of the ion beam source device 20 are selected by the user in dependency on the particular application conditions, e. g. using reference data or preliminary tests with reference samples. Preferably, the ion beam 21 has a beam convergence below 2 pm, and it has a spot size in a range from 100 pm to 500 pm at the support surface of the sample holder device 10.
The electron transmission detector unit 41 comprises an electron source 42 and an electron collector 43. The electron source 42 produces a sensing electron beam directed through the sample 1 to the electron collector 43. Preferably, the electron source 42 comprises a cathode with a focusing optic, as available from conventional cathode ray display tubes (e. g. television tubes). The cathode may be operated in pulsed mode for reducing the averaged power of the sensing electron beam. As an example, the sensing electron beam may comprise pulses with a frequency of 0,1 Hz and an energy of 1 kV. The electron collector 43 comprises a Faraday cup, which is arranged for detecting electrons passing through the sample 1 accommodated on the sample holder device 10. A transmission of the electron beam through the sample 1 is typically detected with sample thicknesses in the nanometer range, e.g. below 100 nm.
The sample thickness detection device 40 may comprise only the electron transmission detector unit 41 for detecting a completed preparing condition wherein the sample is sufficiently thin for an electron microscopy investigation. However, with a preferred embodiment of the invention, the sample thickness detection device 40 also includes the optical measuring unit 44. The optical measuring unit 24 comprises at least one, e.g. coherent, light source 45 being arranged for directing a light beam 46 towards the sample holder device 10. Furthermore, the optical measuring unit 44 further comprises an optical microscope 47, with, e.g., a long working distance microscope lens 48. The optical measuring unit 44 is capable of measuring the thickness of the sample 1 in a range of the wavelength of the light emitted by the light source 45, e.g. around 500 nm for a green laser source, by evaluating absorption and/or interference patterns. The at least one light source 45 of the optical measuring unit 44 may be controlled by the control unit 50.
The optical microscope 47 may be adapted for a transmission (or absorption) measurement at the sample. A sample thickness can be measured by sensing the light power transmitted through the sample and comparing it with prestored and/or measured calibration data. Additionally or alternatively, the optical microscope 47 may be configured as a phase contrast microscope and/or a differential interference contrast microscope. For interference measurements of the sample thickness, the optical microscope 47 may provide at least one of a transmitted light
interferometer, a Fizeau interferometer, and a Shear interferometer. The cooling unit 60 comprises e. g. a cryogenic storage vessel 61, e.g. an LN2 Dewar vessel, a cooling braid 62 and a cooling finger 63. The cooling finger 63, e. g. made of Cu, is directly connected directly with the platform 11 of the sample holder device 10. Furthermore, the cooling finger 63 is connected via the cooling braid 62, e. g. made of Cu, with the cryogenic storage vessel 61, so that the platform 11 and the sample carried thereon can be cooled down to a temperature approaching the temperature of liquid nitrogen or the temperature of vapor of liquid nitrogen in the cryogenic storage vessel 61, in particular in a range from - 140 °C to - 180 °C. For monitoring the temperature, the cooling unit 60 can be provided with a temperature sensor (not shown), e. g. at the platform 11. The cooling unit 60 can be omitted e. g. if sample cooling is not necessary, e. g. if the sample is sufficiently stable in the vacuum chamber 30 even at room temperature (around 20 °C).
The vacuum chamber 30 can be equipped with a loading unit 70 as shown in Figure 1. The loading unit 70 comprises a load lock 71, a transfer chamber 72, a transfer rod 73 and a cryogenic device 74 (optionally provided). The loading unit 70 allows to transfer a sample under vacuum conditions to a TEM (not shown).
In the following, an exemplary embodiment of the sample preparation method is described with reference to thinning a protein crystal. Firstly, a protein crystal sample, obtained directly from a crystallization process or after a preliminary shaping by microtoming, is placed on the sample holder device 10. For example, the sample is placed under vacuum and optionally cooled on the platform 11 of outside the vacuum chamber 30 and subsequently inserted by means of the loading unit 70 into the vacuum chamber 10. Alternatively, the sample 1 is placed with an auxiliary tool (not shown) on the platform 11 within the vacuum chamber 30. The sample 1 is cooled and/or kept at cryogenic temperatures by means of the cooling unit 60. The sample 1 is arranged e. g. on a carrier element, like an electron microscopy grid. If multiple samples are to be arranged on the platformll, each sample preferably is positioned on a separate carrier element, so that multiple carrier elements each with one sample are arranged on the platform 11, and/or multiple samples are positioned on one carrier element.
The ion beam source device 20 is operated for creating the ion beam 21. By means of a scintillator (not shown) being provided with a predetermined position relative to the sample 1 on the sample holder device 10, the ion beam 21 can be adjusted. The scintillator can be monitored visually or with the optical microscope 47, and the displacement device 12 is moved until the ion beam 21 hits the scintillator. Subsequently, the displacement device 12 is displaced by translations and/or rotations until the sample 1 is placed in the ion beam 21.
In a first processing step, the ion beam source device 20 is operated in a first operation mode for creating the ion beam 21 with single ions. The sample thickness is continuously or repeatedly measured during the ablation with the ion beam 21, using the optical measuring unit 44. When a threshold thickness of e. g. 450 nm is reached, the ion beam source device 20 is switched to a second operation mode for creating the ion beam 21 with ion clusters. Simultaneously, the electron transmission detector unit 41 is switched on for further monitoring the sample thickness. The sample thickness is continuously or repeatedly tested by focusing a sensing electron beam to the sample and detecting, whether the sensing electron beam is transmitted through the sample. When transmitted electrons of the sensing electron beam are detected by the electron collector 43, the ablation with the ion beam 21 comprising the ion clusters can be continued for a predetermined period of time, or the ion beam source device 20 is switched off immediately. Continued ablation can be provided in dependency on the application conditions, e. g. if the sample is to be further thinned using reference data which provide the allowable period of time for further ablation without sample destruction.
Subsequently, the sample preparation is completed. After the threshold sample thickness is reached, the sample can be transported to an analyzer, e.g. an electron microscope, e. g. on the platform 11 through the loading unit 70. Alternatively, a further sample can be provided for ion beam irradiation and the ablation or thinning procedure takes place again. In this case, removing samples from the vacuum chamber preferably is conducted only after thinning all samples on the sample holder device 10.
Many advantages of the present invention will be fully understood from the foregoing description, and it will be apparent that various changes may be made in the form, construction and arrangement of the units and devices without departing from the scope of the invention and/or without sacrificing all of its advantages. The features of the invention disclosed in the above description, the drawings and the claims can be of significance individually, in combination or sub-combination for the implementation of the invention in its different embodiments. Since the invention can be varied in many ways, it will be recognized that the invention should be limited only by the scope of the claims.

Claims

Claims
1. Sample preparation apparatus (100), being adapted for ion beam ablation based prepar ing a sample (1) for a transmission electron microscopy investigation, comprising:
a sample holder device (10) being arranged for accommodating a sample (1), an ion beam source device (20) being arranged for creating an ion beam (21) and directing the ion beam (21) toward the sample holder device (20), wherein the sample holder device (10) and the ion beam source device (20) are arranged in a vacuum chamber (30) and the ion beam source device (20) is capable of ablating the sample (1) by the effect of the ion beam (21),
a sample thickness detection device (40) being arranged for monitoring a sample thick ness, and
a control unit (50) being adapted for controlling the ion beam source device (20), characterized in that
the control unit (50) is adapted for controlling the ion beam source device (20) in depend ency on an output signal of the sample thickness detection device (40).
2. Sample preparation apparatus according to claim 1, wherein
the sample thickness detection device (40) comprises an electron transmission detector unit (41), and
the control unit (50) is adapted for controlling the ion beam source device (20) in depend ency on an output signal of the electron transmission detector unit (41).
3. Sample preparation apparatus according to claim 2, wherein
the control unit (50) is adapted for stopping an operation of the ion beam source device (20) in dependency on the output signal of the electron transmission detector unit (41).
4. Sample preparation apparatus according to one of the foregoing claims, wherein
the ion beam source device (20) is adapted to be operated as a gas cluster ion beam source (GCIB source).
5. Sample preparation apparatus according to one of the foregoing claims, wherein the ion beam source device (20) is adapted for creating the ion beam (21) in a first opera tion mode, such that the ion beam comprises single ions, and in a second operation mode, such that the ion beam (21) comprises ion clusters.
6. Sample preparation apparatus according to claim 5, wherein
the control unit (50) is adapted for controlling the ion beam source device (20) such that it is operated in the first operation mode as long as the sample thickness is above a threshold thick ness, and
the control unit (50) is adapted for switching the ion beam source device (20) to the sec ond operation mode when the sample thickness is equal to or below the threshold thickness.
7. Sample preparation apparatus according to one of the foregoing claims, wherein
the sample thickness detection device (40) further comprises an optical measuring unit (44) being adapted for measuring the sample thickness, and
the control unit (50) is adapted for controlling the ion beam source device (20) in depend ency on an output signal of the optical measuring unit (44).
8. Sample preparation apparatus according to claim 7, wherein
the optical measuring unit (44) includes an optical microscope being arranged for measur ing the sample thickness by at least one of a transmission measurement and an interferometric measurement.
9. Sample preparation apparatus according to claims 6 and 7, wherein
the optical measuring unit (44) is adapted for measuring the threshold thickness.
10. Sample preparation apparatus according to one of the foregoing claims, wherein
the sample holder device (10) is coupled with a cooling unit (60) so that the sample can be cooled to cryogenic temperatures.
11. Sample preparation apparatus according to one of the foregoing claims, wherein
the sample holder device (10) is adapted for accommodating multiple samples.
12. Sample preparation apparatus according to one of the foregoing claims, wherein the sample holder device (10) is coupled with a displacement device (11), being arranged for at least one of stepwise scanning, translating and rotating the sample holder device (10).
13. Sample preparation apparatus according to one of the foregoing claims, wherein
the sample holder device (10) has a scintillator being arranged for emitting light in re sponse to irradiation with the ion beam (21).
14. Sample preparation apparatus according to one of the foregoing claims, wherein
the vacuum chamber (10) comprises a loading unit (70) being arranged for coupling a mo bile transfer chamber (71) to the vacuum chamber (10).
15. Sample preparation method, including preparing a sample for a transmission electron mi croscopy investigation, the method comprising the steps of:
providing a sample (1),
creating an ion beam (21) with an ion beam source device (20) and directing the ion beam (21) onto the sample (1), so that the sample (1) is ablated by the effect of the ion beam (21), wherein the ion beam source device (20) is controlled by a control unit (50), and
monitoring a sample thickness with a sample thickness detection device (40),
characterized in that
the ion beam source device (20) is controlled by the control unit (50) in dependency on an output signal of the sample thickness detection device (40).
16. Sample preparation method according to claim 15, wherein
the sample thickness detection device (40) comprises an electron transmission detector unit (41), and
the ion beam source device (20) is controlled by the control unit (50) in dependency on an output signal of the electron transmission detector unit (41).
17. Sample preparation method according to claim 16, wherein
an operation of the ion beam source device (20) is stopped after a detection of electron transmission above a threshold current through the sample with the electron transmission detec tor unit (41).
18. Sample preparation method according to one of the claims 15 to 17, wherein
the ion beam source device (20) is operated as a gas cluster ion beam source (GCIB source), wherein the ion beam (21) comprises ion clusters.
19. Sample preparation method according to one of the claims 15 to 18, wherein
the ion beam source device (20) creates the ion beam (21) in a first operation mode, such that the ion beam (21) comprises single ions, and in a second operation mode, such that the ion beam (21) comprises ion clusters.
20. Sample preparation method according to claim 19, wherein
the ion beam source device (20) is operated in the first operation mode as long as the sample thickness is above a threshold thickness, and
the ion beam source device (20) is switched to the second operation mode when the sam ple thickness is equal to or below the threshold thickness.
21. Sample preparation method according to one of the claims 15 to 20, wherein
the sample thickness detection device (40) further comprises an optical measuring unit (44) being adapted for measuring the sample thickness, and
the ion beam source device (20) is controlled by the control unit (50) in dependency on an output signal of the optical measuring unit (44).
22. Sample preparation method according to claims 20 and 21, wherein
the threshold thickness is measured with the optical measuring unit (44).
23. Sample preparation method according to one of the claims 15 to 22, including the step of cooling the sample to cryogenic temperatures.
24. Sample preparation method according to one of the claims 15 to 23, including the step of at least one of stepwise scanning, translating and rotating sample (1) during ion beam ir radiation.
25. Sample preparation method according to one of the claims 14 to 24, including the step of monitoring a position of the ion beam (21) relative to the sample (1) by using a scintillator being arranged for emitting light in response to irradiation with the ion beam (21).
26. Sample preparation method according to one of the claims 15 to 25, including the step of transferring the sample after thinning with a mobile transfer chamber (71) into a trans mission electron microscopy apparatus.
27. Sample preparation method according to one of the claims 15 to 26, wherein
the sample (1) comprises a protein crystal.
PCT/EP2019/053163 2019-02-08 2019-02-08 Apparatus and method for preparing a sample for transmission microscopy investigations by ion beam initiated ablation WO2020160788A1 (en)

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