Interferometric scattering microscopy

Patent metadata

Field	Value
Publication number	US-12189105-B2
Application number	US-202318144372-A
Country	US
Kind code	B2
Filing date	May 8, 2023
Priority date	Jul 13, 2016
Publication date	Jan 7, 2025
Grant date	Jan 7, 2025

Abstract

An interferometric scattering microscope is adapted by performing spatial filtering of output light, which comprises both light scattered from a sample location and illuminating light reflected from the sample location, prior to detection of the output light. The spatial filtering passes the reflected illumination light but with a reduction in intensity that is greater within a predetermined numerical aperture than at larger numerical apertures. This enhances the imaging contrast for coherent illumination, particularly for objects that are weak scatterers.

First claim

The invention claimed is:

1. A method of characterizing interactions and/or assembly of individual biomolecules using an interferometric light scattering microscope, said interferometric light scattering microscope comprising:
a sample holder for holding a sample in a sample location;
an illumination source arranged to provide illuminating light;
a detector;
an optical system being arranged to direct illuminating light onto the sample location and being arranged to collect output light in reflection, the output light comprising both light scattered from the sample location and illuminating light reflected from the sample location, and to direct the output light to the detector; and
a spatial filter positioned to filter the output light, the spatial filter being arranged to pass output light but with a reduction in intensity that is greater within a predetermined numerical aperture than at larger numerical apertures.

2. The method according to claim 1 , wherein said method permits spatiotemporal characterization of the interactions of the biomolecules.

3. The method according to claim 1 , wherein said method permits the characterization of the interactions of the biomolecules in low-scattering in vitro conditions.

4. The method according to claim 1 , wherein said method permits the characterization of a distribution of complexes of biomolecules at high dynamic range.

5. The method according to claim 1 , wherein said method permits the characterization of the molecular dynamics of the interactions of biomolecules.

6. The method according to claim 1 , wherein the interactions of the biomolecule include glycoprotein cross-linking, amyloidogenic protein aggregation, and/or actin polymerization.

7. The method according to claim 1 , wherein the biomolecule is a single biomolecule.

8. The method according to claim 1 , wherein said biomolecule is a single protein.

9. The method according to claim 1 , wherein said biomolecule is a glycoprotein.

10. The method according to claim 1 , wherein the biomolecule assembles into an oligomeric species.

11. A method according to claim 1 , wherein the spatial filter is arranged to pass output light with a reduction in intensity within said predetermined numerical aperture to 10 −2 of the incident intensity or less.

12. A method according to claim 1 , wherein the spatial filter is arranged to pass output light with a reduction in intensity within said predetermined numerical aperture to 10 −4 of the incident intensity or more.

13. A method according to claim 1 , wherein the predetermined numerical aperture is less than 1.

14. A method according to claim 1 , wherein the illuminating light is spatially and temporally coherent.

15. A method according to claim 1 , wherein the optical system comprises a beam splitter arranged to split the optical paths for the illuminating light and the output light, the spatial filter being part of the beam splitter.

16. A method of identifying an oligomeric species in a sample by quantifying the mass of the species using an interferometric light scattering microscope, the interferometric light scattering microscope comprising:
a sample holder for holding the sample in a sample location;
an illumination source arranged to provide illuminating light;
a detector;
an optical system being arranged to direct illuminating light onto the sample location and being arranged to collect output light in reflection, the output light comprising both light scattered from the sample location and illuminating light reflected from the sample location, and to direct the output light to the detector; and
a spatial filter positioned to filter the output light, the spatial filter being arranged to pass output light but with a reduction in intensity that is greater within a predetermined numerical aperture than at larger numerical apertures.

17. The method according to claim 16 , wherein said oligomeric species is comprised of protein.

18. The method according to claim 16 wherein the oligomer is assembled from multiple components.

19. The method according to claim 16 wherein the oligomer is assembled from envelope or capsid proteins.

20. The method according to claim 16 wherein the oligomeric species is a membrane scaffold protein.

Assignees

• Univ Oxford Innovation Ltd

Inventors

• Kukura Philipp
• Weigel Alexander
• Benesch Justin

Classifications

• G02B21/36

  arranged for photographic purposes or projection purposes (G02B21/18 takes precedence){or digital imaging or video purposes including associated control and data processing arrangements (image data processing per se G06T)} · CPC title

• G02B21/26

  Stages; Adjusting means therefor · CPC title

• G02B21/008

  Details of detection or image processing, including general computer control · CPC title

• G01N15/1434

  Optical arrangements · CPC title

• G01G9/005

  using radiations, e.g. radioactive (analysing materials by the use of wave or particle radiation G01N23/00) · CPC title