Method of processing ion beam based on optical microscopy imaging

1 . A method of ion beam processing based on optical microscopy imaging, comprising:

marking a surface of a sample using an ion beam, so as to obtain a registration reference pattern;

performing a three-dimensional optical imaging on the sample to obtain a first three-dimensional light microscopy image;

projecting the first three-dimensional light microscopy image to a cutting angle of the ion beam, and determining a first position of a to-be-researched target in the first three-dimensional light microscopy image based on the registration reference pattern; and

performing an ion beam imaging on the sample to obtain an image excited by an ion beam, and determining, according to the first position, a second position of the to-be-researched target in the image excited by the ion beam,

wherein the method further comprises:

thinning the sample according to the second position to obtain a second slice containing the to-be-researched target;

performing a three-dimensional optical imaging on the second slice to obtain a second three-dimensional light microscopy image, wherein a magnification of the three-dimensional optical imaging on the second slice is greater than a magnification of the three-dimensional optical imaging on the sample;

projecting the second three-dimensional light microscopy image to the cutting angle of the ion beam to obtain a two-dimensional projection image;

determining a third position of the to-be-researched target in the second slice based on the two-dimensional projection image; and

thinning the sample according to the third position to obtain a third slice containing the to-be-researched target.

2 . The method according to claim 1 , further comprising:

thinning the sample according to the second position to obtain a first slice containing the to-be-researched target.

3 . The method according to claim 1 , wherein the performing a three-dimensional optical imaging on the sample or the performing a three-dimensional optical imaging on the second slice comprises:

loading the sample or the second slice onto a light microscopy imaging position in an electron microscopy vacuum chamber, so as to keep the sample horizontal; and

performing, in a direction perpendicular to the sample, a multi-channel three-dimensional imaging on the sample or the second slice by using a three-dimensional optical imaging technology.

4 . The method according to claim 3 , wherein the three-dimensional optical imaging technology comprises a confocal imaging method, a structured light illumination imaging method, a three-dimensional single molecule localization imaging method, or a light sheet imaging method.

5 . The method according to claim 3 , wherein an image of the registration reference pattern or a slice surface morphology information is obtained using a bright field imaging, and an image of the to-be-researched target is obtained using a fluorescence imaging.

6 . The method according to claim 1 , wherein the projecting the first three-dimensional light microscopy image to a cutting angle of the ion beam or the projecting the second three-dimensional light microscopy image to the cutting angle of the ion beam to obtain a two-dimensional projection image comprises:

projecting the first three-dimensional light microscopy image and the second three-dimensional light microscopy image to the two-dimensional projection image with a same viewing angle as the ion beam.

7 . The method according to claim 6 , wherein the determining a first position of a to-be-researched target in the first three-dimensional light microscopy image based on the registration reference pattern comprises:

measuring a two-dimensional pixel distance from the to-be-researched target in the two-dimensional projection image to a center of the registration reference pattern;

converting the two-dimensional pixel distance to a true distance based on a pre-calibrated optical imaging pixel point size; and

determining the first position based on the true distance.

8 . The method according to claim 2 , wherein a thickness of the first slice is at a level of one hundred nanometers, and the first slice is suitable for a transmission electron imaging or an electron tomography imaging.

9 . The method according to claim 1 , wherein a thickness of the third slice is at a level of one hundred nanometers, the third slice is suitable for a transmission electron imaging or an electron tomography imaging, and a thickness of the second slice is at a level of micrometers.

10 . The method according to claim 2 , further comprising:

performing the three-dimensional optical imaging on the first slice to obtain a third three-dimensional light microscopy image;

analyzing the third three-dimensional light microscopy image and extracting a type information and a distribution information of the to-be-researched target in the first slice; and

guiding and assisting in a collection and an analysis of cryo-electron tomography imaging data based on the type information and the distribution information of the to-be-researched target.

11 . The method according to claim 10 , wherein guiding and assisting in a collection and an analysis of cryo-electron tomography imaging data based on the type information and the distribution information of the to-be-researched target comprises:

performing a transmission electron imaging on the first slice to obtain a transmission electron microscopy image;

locating a fourth position of the to-be-researched target in the transmission electron microscopy image based on the type information and the distribution information of the to-be-researched target;

delineating a collection range of the cryo-electron tomography imaging data in the transmission electron microscopy image based on the fourth position; and

associating a three-dimensional reconstructed cryo-electron tomography image with a corresponding third three-dimensional light microscopy image of the first slice, and distinguishing a type and a position of the to-be-researched target using a fluorescence information, so as to assist in an analysis of a cryo-electron tomography image.

12 . The method according to claim 1 , further comprising:

performing the three-dimensional optical imaging on the third slice to obtain a third three-dimensional light microscopy image;

analyzing the third three-dimensional light microscopy image and extracting a type information and a distribution information of the to-be-researched target in the third slice; and

guiding and assisting in a collection and an analysis of cryo-electron tomography imaging data based on the type information and the distribution information of the to-be-researched target.

13 . The method according to claim 12 , wherein the guiding and assisting in a collection and an analysis of cryo-electron tomography imaging data based on the type information and the distribution information of the to-be-researched target comprises:

performing a transmission electron imaging on the third slice to obtain a transmission electron microscopy image;

locating a fourth position of the to-be-researched target in the transmission electron microscopy image based on the type information and the distribution information of the to-be-researched target;

delineating a collection range of the cryo-electron tomography imaging data in the transmission electron microscopy image based on the fourth position; and

associating a three-dimensional reconstructed cryo-electron tomography image with a corresponding third three-dimensional light microscopy image of the third slice, and distinguishing a type and a position of the to-be-researched target using a fluorescence information, so as to assist in an analysis of a cryo-electron tomography image.