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US-12620552-B2 - Method for micromachining a biological sample for creating a lamella for analysis in a cryo-charged particle microscope

US12620552B2US 12620552 B2US12620552 B2US 12620552B2US-12620552-B2

Abstract

The disclosure relates to a method for micromachining a biological sample for creating a lamella for analysis in a Cryo-Charged Particle Microscope (Cryo-CPM). The method comprising the steps of providing a biological sample on a sample carrier; Locating a sample area on the sample carrier, said sample area comprising a region of interest having biological material from which a lamella can be created; and Micromachining at least part of the biological sample so as to remove material in a part of the sample area surrounding the region of interest, in order to increase a visual contrast between the biological material in the region of interest and its surroundings. With the increased visual contrast a location for a potential lamella can be identified.

Inventors

  • Matej Dolník
  • Veronika Vrbovská
  • Radim Kříž
  • Jakub Kuba
  • Tilman Franke

Assignees

  • FEI COMPANY

Dates

Publication Date
20260505
Application Date
20231129
Priority Date
20221130

Claims (16)

  1. 1 . A method for micromachining a biological sample for creating a lamella for analysis in a Cryo-Charged Particle Microscope (Cryo-CPM), the method comprising: providing a biological sample on a sample carrier; locating a sample area on the sample carrier, said sample area surrounding a region of interest having biological material from which a lamella can be created; micromachining at least part of the sample carrier so as to remove material in a part of the sample area adjacent to the region of interest, in order to increase a visual contrast between the biological material in the region of interest and a portion of the sample area surrounding the biological material; identifying, by a processing unit using the increased visual contrast between the biological material in the region of interest and its surroundings, a location within the biological material in the region of interest from which a lamella can be created; and micromachining the sample to produce said lamella from the identified location within the biological material.
  2. 2 . The method according to claim 1 , further comprising using a micromachining tool in at least one of micromachining at least part of the sample carrier and micromachining the sample to produce said lamella.
  3. 3 . The method according to claim 1 , further comprising imaging the sample using a charged particle microscope.
  4. 4 . The method according to claim 3 , wherein identifying the location within the biological material in the region of interest from which the lamella can be created is performed on an image obtained by the charged particle microscope.
  5. 5 . The method according to claim 1 , wherein identifying the location within the biological material in the region of interest includes identifying, by the processing unit, a separation edge between the region of interest and the portion of the sample area surrounding the biological material.
  6. 6 . The method according to claim 1 , further comprising changing the orientation of the sample carrier after micromachining the at least part of the sample carrier.
  7. 7 . The method according to claim 1 , further comprising providing a focused ion beam (FIB) for at least one of micromachining at least part of the sample carrier and micromachining the sample to produce said lamella.
  8. 8 . The method according to claim 7 , wherein micromachining the sample to produce said lamella includes positioning said beam at an angle with respect to a plane defined by said sample carrier.
  9. 9 . The method according to claim 7 , wherein micromachining at least part of the sample carrier includes positioning said beam substantially orthogonal to a plane defined by said sample carrier.
  10. 10 . The method according to claim 1 , further comprising: providing a focused ion beam (FIB); positioning said sample carrier substantially orthogonal relative to the beam prior to micromachining at least part of the sample carrier; and changing, after micromachining the at least part of the sample carrier, the position of the sample carrier with respect to the beam prior to micromachining the sample to produce said lamella.
  11. 11 . The method according to claim 10 , further comprising obtaining, using charged particle microscopy, an image of said region of interest and identifying, by the processing unit, said location.
  12. 12 . The method according to claim 11 , wherein identifying the location within the biological material in the region of interest includes identifying, using said processing unit, a separation edge between the region of interest and the portion of the sample area surrounding the biological material.
  13. 13 . The method according to claim 1 , wherein ice particle contamination on the sample area is evaluated and accounted for in selecting the lamella.
  14. 14 . The method according to claim 1 , further comprising calculating a milling angle based on the identified location within the biological material in the region of interest from which the lamella can be created.
  15. 15 . The method according to claim 1 , wherein identifying the location within the biological material in the region of interest from which the lamella can be created includes measuring a height of the biological material in the region of interest.
  16. 16 . The method according to claim 1 , wherein micromachining the at least part of the sample carrier so as to remove material in the part of the sample area surrounding the region of interest includes removing material of the sample area in front of, in a rear of, to a right of, and to a left of the biological material.

Description

CROSS REFERENCE TO RELATED APPLICATIONS This application claims priority to European Application No. EP22210709.6, filed Nov. 30, 2022, the entire contents of which is herein incorporated by reference. The invention relates to a method and system for micromachining a biological sample for creating a lamella for analysis in a Cryo-Charged Particle Microscope. Charged-particle microscopy is a well-known and increasingly important technique for imaging microscopic objects, particularly in the form of electron microscopy. Historically, the basic genus of electron microscope has undergone evolution into a number of well-known apparatus species, such as the Transmission Electron Microscope (TEM), Scanning Electron Microscope (SEM), and Scanning Transmission Electron Microscope (STEM), and also into various sub-species, such as so-called “dual-beam” apparatus (e.g. a FIB-SEM), which additionally employ a “machining” Focused Ion Beam (FIB), allowing supportive activities such as ion-beam milling or Ion-Beam-Induced Deposition (IBID), for example. More specifically: In a SEM, irradiation of a sample by a scanning electron beam precipitates emanation of “auxiliary” radiation from the sample, in the form of secondary electrons, backscattered electrons, X-rays and photoluminescence (infrared, visible and/or ultraviolet photons), for example; one or more components of this flux of emanating radiation is/are then detected and used for image accumulation purposes.In a TEM, the electron beam used to irradiate the sample is chosen to be of a high-enough energy to penetrate the sample (which, to this end, will generally be thinner than in the case of an SEM sample); the flux of transmitted electrons emanating from the sample can then be used to create an image. When such a TEM is operated in scanning mode (thus becoming a STEM), the image in question will be accumulated during a scanning motion of the irradiating electron beam. As an alternative to the use of electrons as irradiating beam, charged particle microscopy can also be performed using other species of charged particles. In this respect, the phrase “charged particle” should be broadly interpreted as encompassing electrons, positive ions (e.g. Ga or He ions), negative ions, protons and positrons, for instance. It should be noted that, in addition to imaging and performing (localized) surface modification (e.g. milling, etching, deposition, etc.), a charged particle microscope may also have other functionalities, such as performing spectroscopy, examining diffractograms, etc. In what follows, the invention will—by way of example—often be set forth in the specific context of electron microscopy (EM). However, such simplification is intended solely for clarity/illustrative purposes and should not be interpreted as limiting. Samples for electron microscope imaging require certain preparation for observation under transmitted light or electron radiation. For example, thin slices (or sections) of a sample are typically cut or milled from a bulk sample in a grid or tube. The cutting or milling can be performed by a focused ion beam (FIB) system, or within a dual beam system that includes both a FIB and an electron microscope. Examples of such dual beam systems include the Quanta 3D DualBeam systems from FEI Corporation (Hillsboro, OR, USA). After the thin slices are prepared using the FIB, the sample must then be transferred to a platform suitable for imaging. Sample preparation is in particular a challenge for biological samples (such as cells, cell components, single-cellular organisms, etc.). As these biological samples need to be stored and studied in a body of aqueous liquid (such as water, electrolyte, cell fluid, blood plasma, etc.), they present significant challenges vis-à-vis their examination in a charged particle microscope (CPM), since: An aqueous liquid introduced into a (quasi-)vacuum environment of a CPM will start to outgas/boil, thus tending to degrade the sample;To prevent this, the sample can be provided on a sample carrier, after which it can be frozen, using a cryogenic liquid, before being introduced into said vacuum. Such freezing must generally be performed very rapidly, with the aim of achieving sample vitrification (solidification into an amorphous, glass-like phase) without significant ice crystallization. After this, the sample needs to be kept at cryogenic temperatures, i.e. at or below −150° C. Once the biological bulk sample is created, thinning using a cryo-focused ion beam (cryo-FIB) can be used to create one or more thin lamellae, that can then be studied in a TEM or SEM, for example. The thinning by cryo-FIB has proved an optimal, artifact-free preparation method. Sample preparation for cellular cryo-ET, whether performed directly on thin cellular peripheries or following FIB micromachining, routinely involves seeding of adherent cells directly on EM grids. Standard EM grids are 3 mm diameter metal meshes overlaid with a delicate perforat