Single Molecule Imaging via Soft-Landing Electrospray and Low-Energy Electron Transmission
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Solution Overview
Problem
Current methods for imaging single molecules, such as X-ray crystallography and cryo-electron microscopy, require averaging over many molecules, which masks biologically relevant structural details and is not feasible for membrane proteins that do not crystallize, and existing technologies like XFELs still necessitate averaging over millions of molecules.
Innovation Solution
A method involving soft-landing electrospray ion deposition of single molecules onto a transparent receiving layer in a vacuum chamber, followed by low-energy electron transmission imaging and reconstruction to obtain detailed images of individual molecules without destruction, using an apparatus with a carrier substrate and means for depositing and imaging single molecules under vacuum conditions.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If averaging over many molecules is used in X-ray crystallography or cryo-electron microscopy, then structural data can be obtained, but biologically relevant conformational details are lost and membrane proteins cannot be studied
Solution Approach 1:
The invention extracts and images individual single molecules separately rather than averaging them in ensembles. By isolating single molecules on a graphene substrate and imaging them individually with low-energy electrons, the method retrieves conformational information that would otherwise be lost in averaging processes
Solution Approach 2:
The invention changes the electron energy parameter to low-energy range (5-1000 eV), which fundamentally alters the interaction between electrons and molecules. This parameter change enables elastic scattering to dominate over inelastic scattering, allowing sufficient signal accumulation from single molecules without radiation damage that plagues high-energy methods
2Illumination intensity
If high-energy electrons are used in state-of-the-art TEMs, then imaging capability is achieved, but the strong inelastic scattering cross-section prevents accumulation of sufficient elastic scattering events for high-resolution reconstruction
Solution Approach 1:
The invention fundamentally changes the electron beam energy parameter from high-energy (conventional TEM) to low-energy (5-1000 eV) range. This parameter change reverses the scattering cross-section relationship, making elastic scattering dominant and enabling sufficient signal accumulation from single molecules
Solution Approach 2:
The invention introduces graphene as an intermediary substrate that supports single molecules while being transparent to low-energy electrons. The graphene layer enables stable positioning of molecules and provides a reference for imaging while minimizing interference with the electron-molecule interaction
3Power
If X-ray Free Electron Lasers with enhanced brightness are used, then single molecule imaging might be achieved, but current and foreseeable XFEL performance still requires averaging over at least 1 million molecules
Solution Approach 1:
The invention changes the imaging modality from X-ray to low-energy electron imaging, exploiting the fundamentally different scattering characteristics of low-energy electrons. This parameter change enables single molecule imaging with much lower signal requirements compared to XFEL methods
Solution Approach 2:
The invention replaces the X-ray imaging mechanism with low-energy electron imaging. The substitution exploits elastic scattering of low-energy electrons which has a much higher cross-section for single molecules, eliminating the need for million-molecule averaging required by XFELs
4Quantity of substance
If soft-landing electrospray ion deposition is used to deposit single molecules, then single molecules can be isolated on the receiving layer, but the deposition process must be conducted under vacuum conditions
Solution Approach 1:
The invention uses electrospray ion deposition as an intermediary process to transfer single molecules from solution phase to vacuum-compatible substrate. The electrospray source generates charged molecular ions that can be guided through vacuum and deposited gently on the graphene layer without requiring high vacuum throughout the entire system
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
Enables high-resolution imaging of single molecules, including proteins, without averaging, allowing for the capture of structural details and dynamic processes, and is applicable to a wide range of molecules, including large biomolecules and protein complexes.
Implementation Method 1
the receiving layer being substantially transparent for low-energy electrons with a kinetic energy of 5 to 1,000 eV
Implementation Method 2
depositing single molecules onto said receiving layer by means of softlanding electrospray ion deposition
Implementation Method 3
the strong inelastic scattering cross-section for both, X-rays and high-energy electrons as employed in the state-of-the-art aberration corrected TEMs, inhibits accumulation of sufficient elastic scattering events required in order to reveal high-resolution reconstruction of just one molecule
Data Source
AI summary
A method of imaging single molecules, comprises the steps of: a) providing an assembly comprising a carrier substrate having a substrate face with an aperture, the aperture being covered with a receiving layer attached to the substrate face, the receiving layer being substantially transparent for low-energy electrons with a kinetic energy of 5 to 1,000 eV; b) depositing single molecules onto said receiving layer by means of soft-landing electrospray ion deposition, whereby a single molecule loaded receiving layer is formed; c) acquiring an in-line low-energy electron transmission pattern of said single molecule loaded receiving layer; and d) applying a reconstruction procedure to said electron transmission pattern to obtain at least one image of a single molecule on said single molecule loaded receiving layer. The above steps a) to c) are conducted under vacuum conditions.