Cryogenic Sample Vitrification for Electron Microscopy

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Solution Overview

Problem

Existing methods for preparing samples under cryogenic conditions for electron microscopy often result in the formation of crystalline ice, which damages biological structures and degrades image quality due to ice crystals scattering the electron beam.

Innovation Solution

A method involving a flat sample carrier with two considerable sides, where the sample material is applied and then partially submerged in a cryogenic liquid, with a stream of cryogenic liquid directed to each side to vitrify the sample quickly, followed by full submersion to cool below 136 K, effectively removing humid air and reducing crystalline ice formation.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Speed

If the sample carrier is partially submerged in cryogenic liquid and streams are directed to each side, then the vitrification speed is improved, but the device complexity increases

Engineering Contradiction:

Improvevitrification speedVSAvoidapparatus complexity

Core Design Contradiction:

SpeedVSDevice complexity

Solution Approach 1:

The cryogenic liquid delivery system is segmented into multiple independent streams, with each stream directed at a specific side of the sample carrier. This segmentation allows simultaneous cooling of both sides of the sample, dramatically increasing vitrification speed while keeping each individual stream simple and manageable

Inventive Principle:

Principle #1Segmentation

Solution Approach 2:

The cooling approach transitions from a single-direction or top-down method to a multi-dimensional approach where cryogenic liquid streams are directed at both sides of the sample carrier simultaneously. This dimensional change enables heat removal from multiple directions, accelerating vitrification without requiring excessively complex equipment

Inventive Principle:

Principle #17Another dimension (Dimensionality change)

2Temperature

If the sample carrier is fully submerged in cryogenic liquid, then the cooling efficiency is improved, but crystalline ice formation increases due to prolonged exposure time

Engineering Contradiction:

Improvecooling efficiencyVSAvoidice crystal formation control

Core Design Contradiction:

TemperatureVSManufacturing precision

Solution Approach 1:

The sample carrier is partially submerged in cryogenic liquid before the actual vitrification process begins. This preliminary submersion pre-cools the sample carrier and removes humid air from around the sample, creating optimal conditions for rapid vitrification when the full submersion occurs, thereby reducing total exposure time and preventing ice crystal formation

Inventive Principle:

Principle #10Preliminary action

Solution Approach 2:

The method rushes through the critical cooling phase by directing high-velocity streams of cryogenic liquid at both sides of the sample carrier simultaneously. This rapid cooling approach skips through the dangerous temperature zone where ice crystals could form, quickly transitioning the sample from liquid to vitrified state before crystallization can occur

Inventive Principle:

Principle #21Skipping (Rushing through)

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 reduces the time for vitrification and cooling, minimizing crystalline ice formation, resulting in better representation and quality of the sample material in cryo-electron microscope images.

Implementation Method 1

vitrifying the sample material by directing, from a level at or directly below the surface of the cryogenic liquid, at least one stream of cryogenic liquid to each considerable side of the sample material

Methodology Applied

Scientific EffectVitrification: Vitrification

Implementation Method 2

cooling the flat sample carrier to a temperature below 136 K

Methodology Applied

Scientific EffectRapid cooling: Cooling

Implementation Method 3

The stream of cryogenic liquid will 'push' the humid air away from the environment of the sample material and replace it by cryogenic liquid

Methodology Applied

Scientific EffectDisplacement:

Implementation Method 4

fully submerging the flat sample carrier with the vitrified sample material into the cryogenic liquid to cool the flat sample carrier to a temperature below 136 K

Methodology Applied

Scientific EffectCryogenic cooling: Cryogenics

Data Source

PatentUS20220381656A1Method and Apparatus for Preparing Samples Under Cryogenic Conditions for Imaging or Diffraction Experiments in an Electron Microscope

Publication Date: 2022.12.01 BORMANS BEHEER BV

AI summary

A method and apparatus for preparing samples for imaging under cryogenic conditions or diffraction experiments under cryogenic conditions in an electron microscope. One version of the method involves: partially submerging a flat sample carrier with sample material vertically into a reservoir containing a cryogenic liquid until all areas with sample material are positioned below the surface of the cryogenic liquid; vitrifying the sample material at least one stream of cryogenic liquid to each considerable side of the sample material on the flat sample carrier at or directly below the surface of the cryogenic liquid in the reservoir; and fully submerging the flat sample carrier with the vitrified sample material into the cryogenic liquid to cool the flat sample carrier to a temperature below about 136 K. Variations of the method and various features of the apparatus are described.