Crenellated Sputter Ring for In Situ Cryo-EM Charge Mitigation
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Solution Overview
Problem
Sample charging in charged-particle beam microscopy, such as SEM and FIB processing, is challenging for nonconductive samples, particularly those with thick ice, making it difficult to locate and image areas of interest and prepare lamella for TEM imaging.
Innovation Solution
A sample holder with a conductive material and a sputterable surrounding member allows for in situ application of a conductive coating by directing a charged particle beam at a glancing angle, enabling milling and sputtering without removing the sample, thereby reducing charging artifacts.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If a nonconductive sample with thick ice is imaged in SEM or processed with FIB, then the sample can be examined, but sample charging occurs making it difficult to locate and image areas of interest and impede FIB processing
Solution Approach 1:
A conductive coating is applied to the sample surface before SEM imaging or FIB processing to prevent charging artifacts. The coating is deposited in advance using a sputter coating device with a conductive target positioned near the sample stage, ensuring the sample is pre-conditioned for successful imaging and processing without charging issues.
Solution Approach 2:
A conductive coating layer serves as an intermediary between the nonconductive sample and the charged particle beam. This intermediate conductive layer dissipates accumulated charge, preventing charging artifacts while allowing the underlying nonconductive sample to be imaged and processed.
2Object-affected harmful factors
If a conductive coating is applied to a nonconductive sample, then charging artifacts are reduced, but the sample must be removed from the microscope for external coating application
Solution Approach 1:
The SEM microscope is equipped with both imaging capabilities and an integrated sputter coating device, allowing it to perform multiple functions. The conductive coating can be applied within the microscope chamber using the integrated coating device, eliminating the need to remove the sample for external coating and streamlining the workflow.
Solution Approach 2:
The sputter coating device is merged with the SEM microscope system. The coating device includes a conductive target positioned near the sample stage, allowing simultaneous or sequential coating and imaging operations within the same instrument chamber, improving ease of operation.
3Adaptability or versatility
If FIB milling is performed on a nonconductive sample, then additional sample areas can be revealed, but charging artifacts impede the milling process
Solution Approach 1:
A conductive coating is applied to the sample surface before FIB milling to prevent charging artifacts during the milling process. The coating is deposited in advance using the integrated sputter coating device, ensuring the sample is pre-conditioned for successful FIB processing.
Solution Approach 2:
A conductive coating layer serves as an intermediary during FIB milling of nonconductive samples. This intermediate layer dissipates accumulated charge from the ion beam, preventing charging artifacts that would otherwise impede the milling process and allow versatile sample processing.
4Ease of operation
If the sample holder includes a sputterable surrounding member, then in situ conductive coating can be applied, but the device complexity increases
Solution Approach 1:
The sample holder is designed with a surrounding member that serves multiple functions: it provides mechanical support for the sample, acts as a sputterable target for conductive coating deposition, and can be positioned to control the coating geometry. This multi-functional design enables in situ coating while managing complexity through integration.
Solution Approach 2:
The sample holder surrounding member is segmented or configured with specific regions designated for sputtering. The member may include openings or be positioned at angles to allow the ion beam to access specific areas of the sample for coating, while maintaining structural integrity. This segmentation allows controlled in situ coating without excessive complexity.
Applied Scientific Principles
This section explains which scientific principles are used to turn an abstract innovation direction into a practical engineering solution.
Function Achieved in This Case
This method enables effective imaging and milling of nonconductive samples by applying a conductive coating, allowing for reduced charging artifacts and facilitating 3D visualization of sample features.
Implementation Method 1
A sputterable material is situated opposite the at least one window to define a CPB sputtering path through the at least one window to the sputterable material
Implementation Method 2
Sample processing by, for example, CPB milling can be accomplished with the CPB at a glancing angle with respect to the sample surface
Data Source
AI summary
To reduce charging artifacts in electron microscopy, a notched ring of sputterable material can be situated about a sample surface. An ion beam can be directed through a notch at to sputter the sputterable material onto the sample surface. Sputtering can be performed after low-angle focused ion beam (FIB) milling at the same sample tilts. The sample can be rotated about an axis and sputtering performed at multiple rotation angles. Upon sputtering of the conductive coating, the sample can be reoriented and imaged. These steps can be repeated to produce a 2D image stack for 3D image reconstruction.