Cryostat Interferometry for CryoEM Sample Thickness
Find Innovative SolutionsGenerate Solutions
Solution Overview
Problem
Conventional sample preparation techniques for cryo electron microscopy (CryoEM) face challenges in achieving precise and reproducible sample thickness measurements, particularly at cryogenic temperatures, due to temperature fluctuations and inhomogeneities, which affect the accuracy of optical thickness measurement techniques.
Innovation Solution
A cryostat with a coolant reservoir and a sample observation unit is developed, featuring a high thermal conductivity tube, thermal insulation, and a window arrangement transparent to laser light, allowing for precise interferometry measurements by maintaining samples at cryogenic temperatures and minimizing temperature fluctuations, enabling accurate sample thickness determination using a laser beam.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If conventional sample preparation techniques are used for CryoEM, then samples can be prepared with basic equipment, but measurement precision and reproducibility of sample thickness are poor
Solution Approach 1:
The measurement arrangement is segmented into distinct functional modules: a cryostat unit for temperature control, an interferometry measurement unit for thickness measurement, and a sample holder unit. This segmentation allows each module to be optimized independently for its specific function while maintaining overall system precision without excessive complexity.
Solution Approach 2:
The patent introduces an interferometry-based measurement system as an intermediary between the sample preparation process and the CryoEM examination. This intermediary measurement arrangement provides precise thickness data without requiring modification of the fundamental CryoEM sample preparation techniques, thereby improving measurement precision while maintaining operational simplicity.
2Measurement precision
If optical thickness measurement techniques are used at cryogenic temperatures, then thickness can be measured, but temperature fluctuations and inhomogeneities reduce measurement accuracy
Solution Approach 1:
The cryostat is designed with localized thermal management features, including insulation layers positioned strategically around the sample holder and heating elements that can be independently controlled in different zones. This local quality approach allows precise temperature stabilization at the sample location while maintaining overall system flexibility.
Solution Approach 2:
The measurement arrangement incorporates real-time temperature monitoring and active feedback control that dynamically adjusts temperature parameters to compensate for fluctuations. By continuously monitoring temperature at the sample location and adjusting heating/cooling parameters accordingly, the system maintains thermal stability despite external temperature variations.
3Reliability
If manual sample preparation steps are used, then flexibility in sample handling is maintained, but reproducibility of sample thickness is poor
Solution Approach 1:
The patent implements a feedback-based quality control system where the interferometry measurement unit provides real-time thickness measurements during sample preparation. These measurements feed back to the operator or automated control system, allowing immediate adjustment of preparation parameters to achieve the desired thickness, thereby significantly improving reproducibility while maintaining operational flexibility.
Solution Approach 2:
The measurement arrangement enables preliminary thickness assessment before final CryoEM examination. Samples can be prepared with initial thickness estimates, measured using interferometry, and then adjusted if necessary before vitrification. This preliminary action ensures that only samples within the optimal thickness range proceed to CryoEM, improving overall preparation reproducibility.
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
The solution enables precise sample thickness measurement with nanometer-level accuracy, improving the reproducibility and efficiency of CryoEM sample preparation by stabilizing temperature conditions and providing a 2-dimensional thickness map for optimal sample analysis.
Implementation Method 1
the walls of the vessel being thermally insulated with a thermal reservoir insulation layer such as to enable storing liquid coolant at temperatures below 150 K
Implementation Method 2
The tube is made with a material having high thermal conductivity. The distal end of the tube is arranged within the storage volume of the coolant reservoir
Implementation Method 3
determining the sample thickness based on interferometry measurements with the transmitted laser beam
Data Source
Figure 1
Figure 2
AI summary
A sample thickness measuring arrangement (1) and method for measuring a thickness of a sample (49) are proposed. The sample thickness measuring arrangement (1) comprises a sample thickness measuring unit (5) for measuring the thickness of a sample (49) based on interferometry. Furthermore, it comprises a cryostat (3) comprising a coolant reservoir (17) and a sample observation unit (19) for observing characteristics of a sample (49). The sample observation unit comprises a tube (31) enclosing an observation volume (43), a thermal tube insulation layer (33) and a window arrangement (35). The tube is open at a distal end (37) and at a proximal end (39). The distal end of the tube is arranged within a storage volume (23) of the coolant reservoir. The tube has two lateral openings (41) in its tube walls at opposing sides with regards to the observation volume. The tube walls are covered at their external side with the thermal tube insulation layer having two openings (45) adjacent to the openings in the tube walls. The window arrangement comprises two opposing panes (47), each pane being arranged at one of the openings in the thermal insulation layer. The panes are transparent to light of a laser beam (9). The proposed approach enable precise and stable sample thickness measurements at cryogenic temperatures.