Method and system for cross-sectioning a sample with a preset thickness or to a target site

Abstract

Linear fiducials including notches or chevrons with known angles relative to each other are formed such that each branch of a chevron appears in a cross-sectional face of the sample as a distinct structure. Therefore, when imaging the cross-section face during the cross-sectioning operation, the distance between the identified structures allows unique identification of the position of the cross-section plane along the Z axis. Then a direct measurement of the actual position of each slice can be calculated, allowing for dynamic repositioning to account for drift in the plane of the sample and also dynamic adjustment of the forward advancement rate of the FIB to account for variations in the sample, microscope, microscope environment, etc. that contributes to drift. An additional result of this approach is the ability to dynamically calculate the actual thickness of each acquired slice as it is acquired.Linear fiducials including notches or chevrons with known angles relative to each other are formed such that each branch of a chevron appears in a cross-sectional face of the sample as a distinct structure. Therefore, when imaging the cross-section face during the cross-sectioning operation, the distance between the identified structures allows unique identification of the position of the cross-section plane along the Z axis. Then a direct measurement of the actual position of each slice can be calculated, allowing for dynamic repositioning to account for drift in the plane of the sample and also dynamic adjustment of the forward advancement rate of the FIB to account for variations in the sample, microscope, microscope environment, etc. that contributes to drift. An additional result of this approach is the ability to dynamically calculate the actual thickness of each acquired slice as it is acquired.

What is claimed is: 
     
       1. A method for cross-sectioning a sample with a preset thickness, comprising:
 providing a sample having x, y and z dimensions with first and second linear fiducials each having ends electronically detectable on a first cross-section surface of the sample defined by the x-y plane and each extending from the first cross-section surface in a direction having the z-dimension component at known angles relative to the x-y surface; 
 exposing a second cross-section surface defined by the x-y plane with a material removal tool, where an x dimension distance between ends of the first and second linear fiducials exposed in each cross-sectioned surface changes along the z-dimension; 
 electronically calculating a first distance in the z-dimension between the first cross-section surface and the second cross-section surface based on a change in the x dimension distance and the angles; 
 automatically adjusting parameters to advance the material removal tool in the z dimension for exposing a third cross-section surface at a second distance in the z dimension from the second cross-section surface that is closer to the preset thickness than the first distance. 
 
     
     
       2. The method of  claim 1 , wherein the first and the second linear fiducials each extend from the first cross-section surface in the x-z plane. 
     
     
       3. The method of  claim 1 , wherein the at least first and second linear fiducials include grooves formed in the surface of the sample that is substantially perpendicular to the first cross-section surface. 
     
     
       4. The method of  claim 3 , wherein the at least first and second linear fiducials include protective layers formed over the grooves. 
     
     
       5. The method of  claim 1 , wherein the at least first and second linear fiducials include channels extending into the sample. 
     
     
       6. The method of  claim 1 , wherein the ends of the first and second linear fiducials have a predefined geometry electronically detectable on the first cross-section surface of the sample. 
     
     
       7. The method of  claim 1 , wherein the at least first and second linear fiducials are formed as a first chevron, and the angles of the first and second linear fiducials are the same. 
     
     
       8. The method of  claim 7 , wherein the at least first and second linear fiducials include a second chevron aligned with the first chevron in the x-dimension and formed behind the first chevron in the z-dimension. 
     
     
       9. The method of  claim 8 , wherein the at least first and second linear fiducials further includes a pair of parallel linear fiducials extending in the z-dimension substantially perpendicular to the first cross-section surface. 
     
     
       10. The method of  claim 1 , wherein exposing a second cross-section surface includes operating a focused ion beam to mill the sample from the first cross-section surface of the sample up to a distance in the z-dimension where the second cross-section surface is exposed. 
     
     
       11. The method of  claim 10 , wherein automatically adjusting parameters includes adjusting a milling rate of the focused ion beam. 
     
     
       12. The method of  claim 1 , wherein exposing a second cross-section surface includes cutting the sample with an in-microscope ultramicrotome at a distance in the z-dimension where the second cross-section surface is exposed. 
     
     
       13. The method of  claim 12 , wherein automatically adjusting parameters includes advancing a position of the in-microscope ultramicrotome in the z-dimension. 
     
     
       14. The method of  claim 1 , further including acquiring and displaying a first image of the first cross-section surface at a first resolution on a display. 
     
     
       15. The method of  claim 14 , further including scanning at least one exact region of interest in the first image defined by an arbitrary outline positioned on the first image. 
     
     
       16. The method of  claim 15 , further including acquiring second image of the at least one exact region of interest at a second resolution greater than the first resolution, and,
 further including overlaying the second image of the at least one exact region of interest at the second resolution, over the arbitrary outline positioned on the first image. 
 
     
     
       17. The method of  claim 16 , further including
 scanning at the second cross-section surface, the same at least one exact region of interest defined by the arbitrary outline from the image, 
 acquiring a third image of the at least one exact region of interest at the second resolution, and 
 displaying the third image in the absence of the first image, on the display. 
 
     
     
       18. The method of  claim 14 , further including
 scanning a first exact region of interest in the first image that includes at least one of the ends defined by an arbitrary outline positioned on the first image, 
 acquiring a second image of the first exact region of interest at a second resolution greater than the first resolution, 
 moving a stage supporting the sample by a predetermined amount in a predetermined direction, 
 scanning a second exact region of interest defined by the arbitrary outline at a position shifted by the predetermined amount in the predetermined direction, 
 acquiring a third image of the second exact region of interest at the second resolution, 
 computing a positional offset between the ends in the second image and the third image, and 
 applying the positional offset to shift a beam that executes the scanning. 
 
     
     
       19. An apparatus for cross-sectioning a sample with a preset thickness, comprising:
 a stage for supporting the sample, the sample having x, y and z dimensions with first and second linear fiducials each having ends electronically detectable on a first cross-section surface of the sample defined by the x-y plane, and each extending from the first cross-section surface in a direction having the z-dimension component at known angles relative to the x-y surface; 
 a material removal tool configured to expose a second cross-section surface defined by the x-y plane where an x dimension distance between ends of the at least first and second linear fiducials exposed in each cross-sectioned surface changes along the z dimension; and,
 a computer workstation configured to
 calculate a first distance in the z-dimension between the first cross-section surface and the second cross-section surface based on a change in the x dimension distance and the angles, and 
 automatically adjust parameters to advance the material removal tool in a z-dimension for exposing a third cross-section surface at a second distance in the z dimension from the second cross-section surface that is closer to the preset thickness than the first distance. 
 
 
 
     
     
       20. The apparatus of  claim 19 , wherein the material removal tool includes a focused ion beam controlled to mill the sample from the first cross-section surface of the sample up to a distance in the z-dimension where the second cross-section surface is exposed, and the computer work-station is configured to adjust a milling rate of the focused ion beam. 
     
     
       21. The apparatus of  claim 20 , further including a scanning electron microscope (SEM) configured to provide imaging data for the computer workstation, the computer workstation being configured to control the SEM to
 scan the first cross-section surface for display as a first image at a first resolution on the computer workstation, and 
 scan at least one exact region of interest in the first image defined by an arbitrary outline positioned on the first image at a second resolution greater than the first resolution. 
 
     
     
       22. The apparatus of  claim 20 , wherein the computer workstation is further configured to overlay the second image of the at least one exact region of interest at the second resolution, over the arbitrary outline positioned on the first image. 
     
     
       23. The method of claim 1, wherein the first and second linear fiducials are formed as linear structures. 
     
     
       24. The method of claim 5, wherein the channels are formed as holes extending into the sample. 
     
     
       25. The method of claim 19, wherein the first and second linear fiducials are formed as linear structures. 
     
     
       26. The method of claim 19, wherein the first and second linear fiducials are formed as holes extending into the sample.