Focus-Stacked Sample Preparation for Precise 3D Milling
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Solution Overview
Problem
Current imaging techniques, such as focused ion beam (FIB) and fluorescence microscopy, face challenges in accurately identifying features at different depths due to limited depth of focus, leading to inefficiencies in sample preparation for transmission electron microscopy (TEM) lamellae, especially on surfaces with topographic variations.
Innovation Solution
The implementation of focus stacking to combine images at various focus depths into a single composite image, allowing for accurate selection of sample locations and generation of depth maps, which enables precise three-dimensional positioning for subsequent operations like milling.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If fluorescence imaging is used to locate features in the interior of a sample, then features at different depths can be identified, but the process requires sifting through multiple images at different focus depths which is tedious and time-consuming
Solution Approach 1:
The patent applies focus stacking to combine multiple 2D images taken at different focal depths into a single composite image that effectively represents a 3D view of the sample. This allows features at different depths to be simultaneously visualized in one image, eliminating the need to manually search through multiple focal planes and significantly reducing the time required to locate and identify features of interest.
2Area of stationary object
If SEM imaging is used for surface imaging with large depth of focus, then surface features can be visualized, but correlating SEM views with FIB views of the same surface is difficult and error-prone particularly for surfaces with significant topographic variations
Solution Approach 1:
The patent merges the imaging capability with the milling capability by using the same FIB instrument for both functions. The focus-stacked composite image generated by the FIB imager provides both the visual information needed for feature identification and the precise spatial coordinates needed for accurate feature location and subsequent milling operations, eliminating correlation errors between separate SEM and FIB systems.
3Manufacturing precision
If FIB milling is used to prepare lamellae, then thin samples for TEM can be produced, but the limited depth of focus of FIB makes it challenging to accurately identify features at different depths
Solution Approach 1:
The patent implements a feedback loop where the FIB imager continuously captures images at different focal depths, applies focus stacking to generate composite images and depth maps, and uses this information to guide and adjust the milling process. The depth map provides real-time feedback on the topography and depth of features, allowing for precise control of lamella thickness and accurate identification of features at different depths throughout the milling process.
Data Source
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AI summary
Methods and apparatus apply focus stacking to sample preparation, improving accuracy of analytic tasks, facilitating automation, and improving throughput. Focus stacking is applied to a set of sample images having different focus depths, to produce a composite image in which features at different depths are in focus and, optionally, a depth map. A sample location is selected from the composite image and a localized material removal, measurement, or imaging operation is performed based on the sample location. A depth value from the depth map is used to set a working depth of a tool for performing the localized operation. Applications include lamella preparation for cryogenic TEM analysis of biological samples by focused ion beam milling operation. Other applications, techniques, and variations are disclosed.