Volume scanning electron microscopy of serial thick tissue sections with gas cluster milling

Patent metadata

Field	Value
Publication number	US-11177110-B2
Application number	US-201916269256-A
Country	US
Kind code	B2
Filing date	Feb 6, 2019
Priority date	Feb 6, 2018
Publication date	Nov 16, 2021
Grant date	Nov 16, 2021

Abstract

A microscopy system includes a gas cluster beam system configured for generating a beam of gas clusters directed toward a sample to irradiate a sample and mill away successive surface layers from the sample, a scanning electron microscope system configured for irradiating the successive surface layers of the sample with an electron beam and for imaging the successive surface layers of the sample in response to the irradiation of the surface layer, and a processor configured for generating a three dimensional image of the sample based on the imaging of the successive layers of the sample.

First claim

What is claimed is:

1. A microscopy system comprising:
a gas cluster beam system configured for generating a beam of gas clusters directed toward a sample to irradiate the sample and mill away successive surface layers from the sample, wherein the gas cluster beam system is configured to mill away successive surface layers from the sample until an entire depth of the sample has been milled away at a position on the sample;
a scanning electron microscope system configured for irradiating the successive surface layers of the sample with an electron beam and for imaging the successive surface layers of the sample in response to the irradiation of the surface layer; and
a processor configured for generating a three dimensional image of the sample based on the imaging of the successive layers of the sample, wherein the processor is configured for assigning lengths of a milling step for different positions on the sample perpendicular to a normal direction to the surface of the sample based on the number of milling steps required to mill away all of the sample at the different positions.

2. The system of claim 1 , wherein gas clusters in the beam include more than 100 atoms per cluster.

3. The system of claim 1 , wherein gas clusters in the beam include more than 1000 atoms per cluster.

4. The system of claim 1 , wherein the beam of gas clusters is directed toward the sample at an angle of greater than 10 degrees to the surface of the sample.

5. The system of claim 1 , wherein the beam of gas clusters is directed toward the sample at an angle of greater than 10 degrees and less than 80 degrees to the surface of the sample.

6. The system of claim 1 , wherein the beam of gas clusters is directed toward the sample at an angle of greater than 20 degree and less than 45 degrees to the surface of the sample.

7. The system of claim 1 , wherein the gas cluster beam system includes a vacuum system through which the beam of gas clusters is directed toward the sample, the vacuum system including residual gas molecules that, during a collision with a gas cluster of the beam of gas clusters, break the gas cluster into two or more subclusters.

8. The system of claim 1 , wherein the energy of gas clusters in the beam of gas clusters is such that the average energy of an atom in a gas cluster in the beam is greater than the energy required to remove an atom from a surface layer of the sample.

9. The system of claim 1 , wherein the energy of individual atoms in the gas clusters in the beam of gas clusters is between 0.2 and 20 eV.

10. The system of claim 1 , wherein the energy of gas clusters in the beam of gas clusters is such that the average energy of an atom in a gas cluster in the beam is between one to five times the energy required to remove an atom from a surface layer of the sample.

11. The system of claim 1 , wherein the scanning electron microscope system is configured for irradiating the successive surface layers of the sample with a plurality of electron beams and for imaging the successive surface layers of the sample in response to the irradiation of the surface layer by the multiple electron beams.

12. The system of claim 1 , further comprising:
a stage supporting the sample, wherein the stage is configured to rotate the sample about an axis different from an axis of the gas cluster ion beam.

13. The system of claim 1 , wherein the sample is cut into a plurality of sections that are loaded onto a substrate.

14. The system of claim 13 , wherein the gas cluster ion beam system is configured for generating a beam of gas clusters for irradiating the plurality of sections to mill away successive surface layers from the sections simultaneously.

15. The system of claim 13 , wherein the gas cluster ion beam system is configured for scanning the beam of gas clusters over the sections.

16. The system of claim 13 , wherein the scanning electron microscope system is configured for irradiating the successive surface layers of the sample with a plurality of electron beams and for imaging the successive surface layers of the sample in response to the irradiation of the surface layer by the multiple electron beams.

17. The system of claim 13 , wherein each section is less than 50 microns thick.

18. The system of claim 13 , wherein the processor is configured for generating a three dimensional image of the sample based on the imaging of the successive layers of the sample.

19. The system of claim 13 , wherein the beam of gas clusters includes neutral gas clusters.

20. The system of claim 13 ,
wherein the sample is cut into a plurality of sections that each are milled into a plurality of surface layers by the beam of gas clusters and whose plurality of surface layers are imaged in response to the irradiation by the electron beam, and
wherein the processor is further configured for generating the three dimensional image of the sample based on the imaging of the plurality of surface layers of the plurality of sections.

21. The system of claim 20 , wherein the processor is configured for generating the image of the sample based on stitching together 3D images of each of the sections.

22. The system of claim 20 , further comprising a sample cooking system configured for irradiating the sample to increase the bulk conductivity of the sample.

23. The system of claim 22 , wherein the sample cooking system is configured for irradiating the sample with electrons.

24. The system of claim 23 , wherein the electrons of the sample cooking system have energies greater than 10 keV and sufficient to penetrate and irradiate the whole thickness of the sample.

25. The system of claim 22 , wherein the scanning electron microscope system includes the sample cooking system.

26. The system of claim 1 , further comprising a substrate having a surface configured to support the sample, the surface being coated with a high contrast material to indicate in an image generated by the scanning electron microscope system when the sample is entirely milled away at a particular x-y position on the sample.

27. A microscopy system comprising:
a beam milling system configured for generating a beam of particles, wherein the beam of particles includes neutral gas clusters, directed at a sample to irradiate the sample and to mill away successive surface layers from the sample wherein the beam of particles is directed at the sample at an angle of greater than 10 degrees to the successive surface layers of the sample, wherein the beam milling system is configured to mill away successive surface layers from the sample until an entire depth of the sample has been milled away;
a scanning electron microscope system configured for irradiating the successive surface layers of the sample with an electron beam and for imaging the successive surface layers of the sample in response to the irradiation of the surface layer; and
a processor configured for generating a three dimensional image of the sample based on the imaging of the successive layers of the sample, wherein the processor is configured for assigning lengths of a milling step for different positions on the sample perpendicular to a normal direction to the surface of the sample based on the number of milling steps required to mill away all of the sample at the different positions.

28. The system of claim 27 , wherein the beam of particles is directed at the sample at an ang

Assignees

• Hughes Howard Med Inst

Inventors

• Hayworth Kenneth J
• Hess Harald F
• Xu C Shan
• Peale David

Classifications

• H01J2237/2067

  Surface alteration · CPC title

• H01J37/3056

  for microworking, e. g. etching of gratings or trimming of electrical components · CPC title

• H01J37/222

  Image processing arrangements associated with the tube · CPC title

• H01J2237/0812

  Ionized cluster beam [ICB] sources · CPC title

• G01N23/2251

  using incident electron beams, e.g. scanning electron microscopy [SEM] · CPC title