Focused Ion Beam Milling for MicroED Sample Preparation
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Solution Overview
Problem
Microcrystal electron diffraction (MicroED) methods face challenges in sample preparation due to the strong interaction between matter and electrons, which limits the thickness of crystals that can be investigated, often resulting in mechanical destruction, inefficient blotting, and ice contamination, affecting the hydration and lattice structure of macromolecular crystals.
Innovation Solution
The use of a focused ion beam to generate lamellae of macromolecular protein crystals for continuous rotation MicroED, where the crystals are coated with an electrically conductive material and milled and polished to ideal thickness, eliminating the need for blotting optimization and allowing for crystals of various sizes to be processed efficiently.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Ease of manufacture
If traditional blotting method is used to prepare microcrystal samples, then sample preparation can be completed, but mechanical destruction of crystals occurs and ice contamination affects crystal structure
Solution Approach 1:
The patent replaces the mechanical blotting system with an ion beam milling system. Instead of using mechanical force to remove excess ice and crystals, a focused ion beam is used to precisely mill the ice layer to the desired thickness, eliminating mechanical contact with the crystals and preventing their destruction
Solution Approach 2:
The patent introduces an intermediate process step of ion beam milling between sample application and data collection. This intermediate step allows precise control of ice thickness and removal of contamination without directly contacting the crystals, serving as a mediator that protects crystal integrity while achieving the desired sample preparation
2Productivity
If crystal thickness is increased to accommodate larger crystals, then more crystals can be processed, but electron absorption increases and limits investigation capability
Solution Approach 1:
The patent changes the thickness parameter of the ice layer containing the crystals to an optimal range (50-500 nm) that balances crystal size accommodation with electron beam penetration. This parameter optimization allows larger crystals to be processed while maintaining sufficient electron transmission for diffraction
3Ease of manufacture
If conventional sample preparation is used, then standard procedures can be followed, but extensive time is spent optimizing blotting and screening crystals
Solution Approach 1:
The patent replaces the trial-and-error mechanical blotting optimization process with a controlled ion beam milling process. The ion beam parameters (current, duration, angle) can be precisely controlled and reproduced, eliminating the need for extensive empirical optimization and reducing preparation time
Solution Approach 2:
The patent performs preliminary milling of the ice layer to a standard thickness range before crystal selection and data collection. This preliminary preparation creates a standardized sample format that facilitates faster crystal screening and reduces the time needed for optimization during the actual experiment
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 approach streamlines sample preparation, increases the scope of crystals suitable for MicroED, reduces the time spent screening for crystals, and achieves higher signal-to-noise ratio, higher resolution, and better refinement statistics by maintaining the structural integrity of the crystals.
Implementation Method 1
milling the coated microcrystal with a first ion beam to yield a milled microcrystal
Implementation Method 2
polishing the milled microcrystal with a second ion beam to yield a polished microcrystal
Implementation Method 3
diffracting the electrons from the polished microcrystal to yield a multiplicity of diffraction patterns
Data Source
AI summary
A sample preparation method includes disposing a microcrystal on an electrically conductive grid, coating the microcrystal with an electrically conductive material to yield a coated microcrystal, milling the coated microcrystal with a first ion beam to yield a milled microcrystal, and polishing the milled microcrystal with a second ion beam to yield a polished microcrystal. A length of a side of the milled microcrystal is between about 250 nm and about 500 nm, and a length of the corresponding side of the polished microcrystal is between about 150 nm and about 250 nm. Assessing the crystal structure of the polished microcrystal includes rotating the polished microcrystal while accelerating electrons toward the polished microcrystal, diffracting the electrons from the polished microcrystal to yield a multiplicity of diffraction patterns, and assessing, from the multiplicity of diffraction patterns, the crystal structure of the polished microcrystal.