Method for inspecting a sample using an assembly comprising a scanning electron microscope and a light microscope

Abstract

The invention relates to a method for inspecting a sample with an assembly comprising a scanning electron microscope (SEM) and a light microscope (LM). The assembly comprises a sample holder for holding the sample. The sample holder is arranged for inspecting the sample with both the SEM and the LM, preferably at the same time. The method comprising the steps of: capturing a LM image of the sample in its position for imaging with the SEM; determining a position and dimensions of a region of interest in or on the sample using the LM image; determining values to which the SEM parameters need to be set to image the sample at a desired resolution; and capturing a SEM image of the region of interest, preferably using the first electron beam exposure of said region of interest.

The invention claimed is: 
     
       1. Method A method for inspecting a sample with an assembly comprising a scanning electron charged particle microscope and a light microscope, wherein the assembly comprises a sample holder for mounting a sample thereon, wherein the sample holder is arranged for inspecting the sample with both the scanning electron charged particle microscope and the light microscope, wherein the method comprising the steps of:
 capturing a light microscopy image of the sample in its position for imaging with the scanning electron charged particle microscope, the light microscopy image being captured using an optical microscope objective lens of the light microscope; 
 determining a position and physical dimensions of a region of interest in the sample based on the light microscopy image; 
 determining values to which the scanning electron charged particle microscope parameters need to be set to image the sample on the sample holder at a desired resolution, wherein said values to image the sample on the sample holder at the desired resolution are at least partially based on the light microscope image; and 
 capturing a scanning electron charged particle microscope image of the region of interest in the sample, wherein the scan settings for recording the scanning electron charged particle microscope image are determined from said values and said position and physical dimensions of said region of interest before the scanning electron charged particle microscope image is captured, 
 wherein the scanning electron charged particle microscope image of the region of interest is captured using a first electron charged particle beam exposure of said region of interest. 
 
     
     
       2. Method The method according to  claim 1 , wherein the values to which the scanning electron charged particle microscope parameters need to be set to image the sample at a desired resolution, is obtained from a focusing area on the sample or on the sample holder. 
     
     
       3. Method The method according to  claim 2 , wherein the position of said focusing area is determined using the light microscope. 
     
     
       4. Method The method according to  claim 2 , wherein the focusing area is located outside the region of interest, adjacent to the region of interest, or at an edge of said region of interest. 
     
     
       5. Method The method according to  claim 2 , wherein the sample or the sample holder is equipped with a designated focusing area. 
     
     
       6. Method The method according to  claim 2 , wherein the focusing area contains focusing markers, which focusing markers are arranged for retrieving the values needed to achieve a desired resolution. 
     
     
       7. Method The method according to  claim 1 , wherein said position and physical dimensions of said region of interest and/or the position of said focusing area, are determined by clicking pointers or drawing a rectangle on said light microscope image, in particular on the light microscope image as presented on a display screen of the assembly. 
     
     
       8. Method The method according to  claim 1 , wherein the sample or the sample holder comprises coordinate markers that are used to convert a light microscopy coordinate system associated with an image from the light microscopy to an electron a charge particle charge particle microscopy coordinate system associated with an image from the scanning electron charged particle microscope. 
     
     
       9. Method The method according to  claim 8 , wherein images of the coordinate markers are used to determine an equation to transform light microscopy image coordinates into electron charged particle microscopy image coordinates. 
     
     
       10. Method The method according to  claim 8 , wherein said coordinate markers are generated by exposing an area or multiple positions within an area on the sample or sample holder with the electron charged particle beam from the electron charged particle microscope so as to generate cathodoluminescence light that is detected by the light microscope. 
     
     
       11. Method The method according to  claim 1 , wherein the electron charged particle microscope image from the region of interest is captured by integrating multiple sequential scans of the region of interest and/or by interlaced scanning of scan lines in the region of interest. 
     
     
       12. Method The method according to  claim 1 , wherein the scanning of the region of interest for capturing of the electron charged particle microscope image is provided with wait time, which is arranged to provide sufficient time for non-damaging relaxation processes to take away energy from the sample which is induced by the irradiation of the sample by the electron charged particle beam of the electron charged particle microscope. 
     
     
       13. Method The method according to  claim 1 , wherein the integrated light microscope is used to monitor the sample during the capturing of the electron charged particle microscope image. 
     
     
       14. Method The method according to  claim 13 , wherein the monitoring comprises an evaluation of whether or not any damage is detected by the light microscope. 
     
     
       15. Method The method according to  claim 13 , wherein the electron charged particle beam exposure of the sample is monitored by observing the fluorescence light from indicators using said light microscope. 
     
     
       16. Method The method according to  claim 15 , wherein said indicators are arranged or are selected to stop emitting fluorescence light when they are exposed to a predetermined dose, or are exposed with a predetermined dose rate. 
     
     
       17. Method The method according to  claim 15 , wherein the observation of the indicators by the light microscope is used to establish a feedback to the electron charged particle microscope, wherein said feedback is arranged for adjusting the scanning procedure of the electron charged particle beam exposure to minimize further damage to the sample. 
     
     
       18. Method The method according to  claim 1 , wherein the electron charged particle microscope parameters are adjusted during the electron charged particle beam scanning of first pixels or lines of the image of the region of interest. 
     
     
       19. Method The method according to  claim 1 , wherein the light microscope is used to monitor the sample in time so as to follow dynamics in the sample, or to monitor the sample and watch for changes in light microscope image of the sample as a function of time. 
     
     
       20. Method The method according to  claim 1 , wherein the method comprises the additional steps of:
 recording fluorescence images during the scanning of the electron charged particle beam of the scanning electron charged particle microscope over the region of interest in the sample for capturing the scanning electron charged particle microscope image, to monitor for damage or to monitor for changes in the fluorescent light of the indicators, and 
 adjusting the scanning electron charged particle microscope parameters during said scanning of the electron charged particle beam based on information from the recorded fluorescence images. 
 
     
     
       21. Method The method according to  claim 1 , wherein the sample is arranged in a vacuum-tight capsule which comprises one or more thin membranes that are substantially transparent for electrons charged particles and through which the scanning electron charged particle imaging can be done. 
     
     
       22. Method The method according to  claim 21 , wherein the vacuum-tight capsule comprises a focusing area, and wherein the focusing area contains focusing markers, which focusing markers are arranged for retrieving the values needed to achieve a desired resolution. 
     
     
       23. Method The method according to  claim 21 , wherein said one or more thin membranes comprises a focusing membrane, wherein said focusing membrane is used for finding the focusing settings. 
     
     
       24. Method The method according to  claim 21 , wherein the light microscope is used to obtain an image of the sample under the membrane areas and to select the regions of interest within the membrane areas. 
     
     
       25. Method The method according to  claim 21 , wherein the vacuum- tight capsule is connected to one or more reservoirs for liquid, wherein said capsule and reservoirs are arranged to establish a flow of liquid at the location of the sample. 
     
     
       26. Method The method according to  claim 25 , wherein at least one of said one or more reservoir holds a liquid containing scavenger species, in particular scavenger molecules, such as or scavenger molecules that include vitamin molecules. 
     
     
       27. Method The method according to  claim 26 , wherein said scavenger species are introduced into the vacuum-tight capsule or are present in the sample only when the sample is scanned by the electron charged particle beam. 
     
     
       28. Method The method according to  claim 25 , wherein the flow of liquid is synchronized with the electron charged particle beam scanning for capturing the scanning electron charged particle microscope image of the region of interest, or wherein the flow of liquid is substantially stopped when the sample is not being scanned by the electron charged particle beam. 
     
     
       29. Method The method according to  claim 1 , wherein the light microscope is used to identify the starting and/or stop time for capturing of the scanning electron charged particle microscope image of the region of interest. 
     
     
       30. Method The method according to  claim 1 , wherein the sample is monitored with the light microscope during the capture of the scanning electron charged particle microscope image of the region of interest, wherein information obtained with the light microscope is used to adjust the scan settings for capturing the scanning electron charged particle microscope image of the region of interest during the scan and/or to control the flow from the reservoirs. 
     
     
       31. Method The method according to  claim 1 , wherein the sample comprises markers that can be observed with the light microscope, wherein changes in the observed markers with said light microscope are used to adjust said scan settings. 
     
     
       32. Method The method according to  claim 31 , wherein said markers are arranged to emit fluorescence light, and wherein the intensity of said fluorescence light is dependent on the dose and/or the dose rate deposited by the scanning electron charged particle microscope. 
     
     
       33. Assembly An assembly comprising a light microscope and an electron a charged particle microscope, wherein the assembly comprises a processing unit, wherein the processing unit is provided with a computer program having instructions adapted to perform a method for inspecting a sample with the assembly, wherein the assembly comprises a sample holder for mounting a sample thereon, wherein the sample holder is arranged for inspecting the sample with both the scanning electron charged particle microscope and the light microscope, wherein the method comprises comprising the steps of:
 capturing a light microscopy image of the sample in its position for imaging with the scanning electron charged particle microscope, the light microscopy image being captured using an optical microscope objective lens of the light microscope; 
 determining a position and physical dimensions of a region of interest in the sample based on the light microscopy image; 
 determining values to which the scanning electron charged particle microscope parameters need to be set to image the sample on the sample holder at a desired resolution, wherein said values are at least partially based on the light microscope image; and 
 capturing a scanning electron charged particle microscope image of the region of interest in the sample, wherein the scan settings for recording the scanning electron charged particle microscope image are determined from said values and said position and physical dimensions of said region of interest before the scanning electron charged particle microscope image is captured. 
 
     
     
       34. A non-transitory computer readable medium, having recorded thereon a computer program which, when provided on a processing unit of an assembly comprising a light microscope and an electron a charged particle microscope, said computer program has instructions to perform a method for inspecting a sample with the assembly, wherein the assembly comprises a sample holder for mounting a sample thereon, wherein the sample holder is arranged for inspecting the sample with both the scanning electron charged particle microscope and the light microscope, wherein the method comprises comprising the steps of:
 capturing a light microscopy image of the sample in its position for imaging with the scanning electron charged particle microscope, the light microscopy image being captured using an optical microscope objective lens of the light microscope; 
 determining a position and physical dimensions of a region of interest in the sample based on the light microscopy image; 
 determining values to which the scanning electron charged particle microscope parameters need to be set to image the sample on the sample holder at a desired resolution, wherein said values are at least partially based on the light microscope image; and 
 capturing a scanning electron charged particle microscope image of the region of interest in the sample, wherein the scan settings for recording the scanning electron charged particle microscope image are determined from said values and said position and physical dimensions of said region of interest before the scanning electron charged particle microscope image is captured. 
 
     
     
       35. The method according to  claim 1 , wherein the charged particle microscope comprises a scanning electron microscope, and wherein the charged particle beam comprises an electron beam. 
     
     
       36. The assembly according to  claim 33 , wherein the charged particle microscope comprises a scanning electron microscope, and wherein the charged particle beam comprises an electron beam. 
     
     
       37. The non-transitory computer readable medium according to  claim 34 , wherein the charged particle microscope comprises a scanning electron microscope, and wherein the charged particle beam comprises an electron beam.