Volume scanning electron microscopy of serial thick tissue sections with gas cluster milling
地域：
VA
VA Ashburn
摘要
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.
說明書
This application is a non-provisional of, and claims priority under 35 U.S.C § 119 to, U.S. Provisional Patent Application No. 62/627,000, filed on Feb. 6, 2018, entitled “VOLUME SCANNING ELECTRON MICROSCOPY OF SERIAL THICK TISSUE SECTIONS WITH GAS CLUSTER ION MILLING,” and to U.S. Provisional Patent Application No. 62/629,501, filed on Feb. 12, 2018, entitled “VOLUME SCANNING ELECTRON MICROSCOPY OF SERIAL THICK TISSUE SECTIONS WITH GAS CLUSTER MILLING,” and to U.S. Provisional Patent Application No. 62/801,509, filed on Feb. 5, 2019, entitled “SERIAL THICK-SECTION GAS CLUSTER ION BEAM SCANNING ELECTRON MICROSCOPY,” the disclosures of which are incorporated herein in their entireties.
Many modalities of electron microscopy (EM) can probe cellular structure at the nanometer scale. However, despite considerable progress over the past decade in developing high-resolution three-dimensional (3D) imaging, there remain important limitations reflecting an inherent trade-off between resolution and the size of the 3D volume. For demanding applications such as tracing neuronal processes, high resolution in the z axis, parallel to the direction of the electron beam of the electron microscope, in addition to the xy plane, is critical. Focused Ion Beam Scanning Electron Microscopy (FIB-SEM) offers this capability, with xy and z resolution all <10 nm. FIB-SEM can generate 3D images with superior z-axis resolution, yielding data with isotropic voxels that is therefore more readily interpretable than available with other techniques.
權(quán)利要求
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 angle of greater than 10 degrees and less than 80 degrees to the surface of the sample. 29. The system of claim 27 , wherein the beam of particles is directed at the sample at an angle of greater than 20 degree and less than 45 degrees to the surface of the sample. 30. The system of claim 27 , 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. 31. The system of claim 27 , 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. 32. The system of claim 27 , wherein the sample is cut into a plurality of sections that are loaded onto a substrate. 33. The system of claim 32 , wherein the beam milling system is configured for generating a beam of particles directed at the plurality of sections to mill away successive surface layers from the sections simultaneously. 34. The system of claim 32 , wherein the beam milling system is configured for scanning the beam of particles over the sections. 35. The system of claim 32 , 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. 36. The system of claim 32 , wherein each section is less than 50 microns thick. 37. The system of claim 32 , wherein the processor configured for generating a three dimensional image of the sample based on the imaging of the successive layers of the sample. 38. The system of claim 27 ,wherein the sample is cut into a plurality of sections that each are milled into a plurality of surface layers by the beam of particles 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. 39. The system of claim 27 , further comprising a sample cooking system configured for irradiating the sample to increase the bulk conductivity of the sample. 40. The system of claim 39 , wherein the sample cooking system is configured for irradiating the sample with electrons. 41. The system of claim 40 , 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. 42. The system of claim 39 , wherein the scanning electron microscope system includes the sample cooking system. 43. The system of claim 27 , wherein the scanning electron beam microscope system includes a multibeam SEM system providing a plurality of electron beams to a surface of the sample to image the surface in parallel. 44. The system of claim 27 , 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.
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