Method for mapping the surface of a macromolecule
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Publication Number
US-12716829-B2
Publication Date
2026-08-25
Expiration Date
Abstract
In order to map the surface of a macromolecule, at least one fluorescent probe is introduced into a medium in which the macromolecule is embedded or will be embedded. Then, a plurality of spatial positions of the at least one fluorescent probe with regard to the macromolecule are determined via localization of the at least one singularized fluorescent probe with a simple standard deviation of no more than 2 nm. For this purpose, fluorescence light photons emitted by the singularized fluorescent probe are recorded. In addition, a bounding surface bounding the determined spatial positions with regard to the macromolecule is determined; and a three-dimensional map of at least a part of the surface of the macromolecule is generated from the bounding surface.
Core Innovation
The invention provides a method for mapping the surface of a macromolecule by introducing at least one fluorescent probe into a medium in which the macromolecule is embedded or will be embedded. The method determines a plurality of spatial positions of the fluorescent probe with regard to the macromolecule by localization of at least one singularized fluorescent probe, wherein fluorescence light photons emitted by the singularized fluorescent probe are registered. The localization is performed with single standard deviation not more than 2 nm, so that the probe positions are determined with high precision relative to the macromolecule.
From the localized positions, the method determines a bounding surface that bounds the determined spatial positions with regard to the macromolecule. A three-dimensional map of at least part of the surface of the macromolecule is generated from the bounding surface. The mapping is based on a probe-localized “negative image” that omits the macromolecule volume and instead captures the surface defined by the localized probe positions.
The approach further enables additional surface-chemistry information by relating probe-affinity groups to the bounding-surface map, such that probe affinities can be used to derive binding sites and binding constants or binding affinities associated with the macromolecule surface. Implementation options include localization using MinFlux or MinSTED, singularizing probes via diffusion and/or switching, activation, or selection mechanisms such as switchable, photoactivatable, or fluorogenic fluorescent probes, and using markers to track and fix macromolecule orientation and position.
Claims Coverage
Independent claim clm-00001 defines the core method of 3D surface mapping via singularized fluorescent probe localization and a bounding surface, with high-precision localization (single standard deviation not more than 2 nm) and generation of a 3D map from the bounding surface. Dependent claims refine the independent claim by adding quantitative constraints, singularization mechanisms, and specific localization platforms.
3D mapping of a macromolecule surface using singularized fluorescent probe localization and a bounding surface
introducing at least one fluorescent probe into a medium in which the macromolecule is embedded or will be embedded; determining a plurality of spatial positions of the at least one fluorescent probe with regard to the macromolecule by localization of the at least one singularized fluorescent probe at a single standard deviation of not more than 2 nm, wherein fluorescence light photons emitted by the singularized fluorescent probe are registered; determining a bounding surface bounding the determined spatial positions with regard to the macromolecule; and generating a three-dimensional map of at least a part of the surface of the macromolecule from the bounding surface.
High localization precision for determining singularized probe positions
determining spatial positions by localizing a singularized fluorescent probe at a single standard deviation of not more than 1 nm, and determining enough positions such that the average surface density is at least 100 positions per 100 square nanometers.
Minimum surface sampling density for the determined spatial positions
determining enough spatial positions of the fluorescent probe so that the average surface density of the determined positions on the surface of the macromolecule is at least 25 positions per 100 square nanometers.
Singularizing alternating subsets of equal fluorescent probes
achieve the spatial positions by singularizing alternating subsets of a total number of equal fluorescent probes included in the medium.
Singularizing probe subsets using switchable, photoactivatable, or fluorogenic fluorescent probes
singularize the alternating subsets by at least one of switching on/off switchable fluorescent probes, activating photoactivatable fluorescent probes, or selecting fluorogenic fluorescent probes.
Using MinFlux or MinSTED for localization of singularized probes
determine the spatial positions by localization of the at least one singularized fluorescent probe using MinFlux or MinSTED.
Across the independent claim and its refinements, the method’s central inventive concept is to map a macromolecule surface in three dimensions by localizing singularized fluorescent probes with high precision, bounding the localized positions with a bounding surface, and generating a 3D surface map; further refinements specify quantitative localization and surface-density constraints, singularization via alternating subsets and probe-control mechanisms, and localization using MinFlux or MinSTED.
Stated Advantages
Enables a three-dimensional map of at least part of the surface of the macromolecule from localized, singularized probe positions.
Enables additional surface-chemistry information based on probe affinity groups, binding sites, and derived binding constants or binding affinities.
Documented Applications
Mapping the surface of a macromolecule embedded in a medium.
Parallelization across multiple macromolecule copies and repeated measurements under changing conditions.
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