Particle characterization using optical microscopy

Patent metadata

Field	Value
Publication number	US-2022012456-A1
Application number	US-201917296208-A
Country	US
Kind code	A1
Filing date	Nov 22, 2019
Priority date	Nov 22, 2018
Publication date	Jan 13, 2022
Grant date	—

Abstract

A method of optically characterizing individual molecules/molecular complexes, or other particles, in solution. The method comprises flowing a solution comprising the molecules/molecular complexes into an imaging region of a microfluidic channel, wherein the imaging region of the microfluidic channel has a first lateral dimension of greater than 1 μm in an x-direction wherein the x-direction is perpendicular to a direction of the flow; capturing a succession of images of the individual molecules/molecular complexes in the imaging region; tracking movement of the individual molecules/molecular complexes in at least the x-direction in the imaging region using the succession of images; and characterizing the individual molecules/molecular complexes from the tracked movement. In some implementations the characterizing comprises determining a diffusion coefficient of the molecules/molecular complexes from the tracked movement.

First claim

1 . A method of optically characterizing individual molecules/molecular complexes in solution, the method comprising:
flowing a solution comprising the molecules/molecular complexes into an imaging region of a microfluidic channel, wherein the imaging region of the microfluidic channel has a first lateral dimension of greater than 1 μm in an x-direction wherein the x-direction is perpendicular to a direction of the flow; capturing a succession of images of the individual molecules/molecular complexes in the imaging region; tracking movement of the individual molecules/molecular complexes in at least the x-direction in the imaging region using the succession of images; characterizing the individual molecules/molecular complexes from the tracked movement.

2 . A method as claimed in claim 1 wherein the characterizing comprises determining a diffusion coefficient of the molecules/molecular complexes from the tracked movement.

3 . A method as claimed in claim 1 wherein the tracking further comprises tracking movement of the molecules/molecular complexes in a y-direction along the direction of the flow, and generating an approximately flat flow profile for the flowing solution in the x-direction.

4 . A method as claimed in claim 3 wherein generating the approximately flat flow profile comprises flowing the solution using electro-osmosis.

5 . A method as claimed in claim 1 comprising flowing the solution into a manifold, and flowing the solution from the manifold into a plurality of the imaging regions in a respective plurality of the microfluidic channels coupled to the manifold.

6 . A method as claimed in claim 3 wherein the microfluidic channels coupled to the manifold are blind microfluidic channels.

7 . A method as claimed in claim 1 wherein the microfluidic channel has an outlet, and wherein the tracking of movement of the molecules/molecular complexes is performed whilst the solution is flowing through the microfluidic channel.

8 . A method as claimed in claim 1 wherein capturing the succession of images comprises capturing the images using interferometric scattering optical microscopy.

9 . A method as claimed in claim 8 wherein the imaging region has a light-reflecting interface, and wherein capturing the images using interferometric scattering optical microscopy comprises, for each image, illuminating a molecule/molecular complex in the imaging region with coherent light using an objective lens such that the light is reflected from the interface and scattered by the molecule/molecular complex; capturing the reflected light and the scattered light using the objective lens; and providing the captured reflected and scattered light to an imaging device to image interference between the reflected light and the scattered light.

10 . A method as claimed in claim 8 further comprising restricting movement of the molecules/molecular complexes in a z-direction in the imaging region, wherein the z-direction is a lateral direction perpendicular to the x-direction, wherein the restricting comprises limiting movement of the molecules/molecular complexes in the z-direction to a distance of less than 2λ, 3/2λ, λ, or

λ
2

,

where λ is the wavelength of coherent light used for the interferometric scattering optical microscopy.

11 . A method as claimed in claim 8 wherein capturing the succession of images comprises, for each of the images of the succession of images, capturing a sequence of interference images, wherein an interference images comprises an image of interference generated by light scattered by the molecules/molecular complexes, and processing the sequence of interference images to provide the image of the succession of images.

12 . A method as claimed in claim 11 wherein processing the sequence of interference images comprises determining a location map image representing a map of one or both of intensity maxima and intensity minima across the sequence of interference images.

13 . A method as claimed in claim 12 wherein processing the sequence of interference images further comprises low-pass filtering the location map image.

14 . A method as claimed in claim 8 further comprising determining a mass of an individual molecule/molecular complex from one or more images captured using the interferometric scattering optical microscopy.

15 . A method as claimed in claim 14 wherein the characterizing comprises determining a ratio of the mass to a size of the individual molecule/molecular complex.

16 . A method as claimed in claim 1 wherein capturing the succession of images comprises capturing the images using an interferometric scattering optical microscope, and wherein a focus of the interferometric scattering optical microscope is set away from an edge of the microfluidic channel in a z-direction perpendicular to the x-direction.

17 . A method as claimed in claim 1 wherein the molecules/molecular complexes comprises a biological molecules/complexes, and wherein the solution comprises an aqueous solution.

18 . A system for optically characterizing individual molecules/molecular complexes in solution, the system comprising:
a microfluidic channel having an imaging region, wherein the imaging region of the microfluidic channel has a first lateral dimension of greater than 1 μm in an x-direction, wherein the x-direction is perpendicular to a direction of flow in the imaging region of the microfluidic channel; a drive system to flow a solution comprising the molecules/molecular complexes into the imaging region; an optical image capture system to capture a succession of images of the individual molecules/molecular complexes in the imaging region; a processor to track movement of the individual molecules/molecular complexes in at least the x-direction in the imaging region using the succession of images and to determine data characterizing the individual molecules/molecular complexes from the tracked movement.

19 . A system as claimed in claim 18 comprising a microfluidic manifold and plurality of the microfluidic channels coupled to the microfluidic manifold each with a respective imaging region; and wherein the drive system comprises an electro-osmotic drive system.

20 . A system as claimed in claim 18 wherein the imaging region has a light-reflecting interface, and wherein the optical image capture system comprises an interferometric scattering optical microscope, the microscope comprising: a source of coherent light; an objective lens to direct the coherent light to illuminate the imaging region such that the light is reflected from the interface and scattered by a molecule/molecular complex in the imaging region, wherein the objective lens is configured to capture the reflected light and the scattered light; and an imaging device configured to image interference between the reflected light and the scattered light.

21 .- 22 . (canceled)

Assignees

• Cambridge Entpr Ltd

Inventors

• Knowles Tuomas Pertti Jonathan
• Challa Pavan Kumar
• Saar Kadi Liis
• Peter Quentin Alexis
• Kaminski Clemens
• Vanderpoorten Oliver

Classifications

• G02B21/0016

  Technical microscopes, e.g. for inspection or measuring in industrial production processes · CPC title

• G06V20/695 Primary

  Preprocessing, e.g. image segmentation · CPC title

• G01N15/0227 Primary

  using imaging; using holography · CPC title

• G02B21/008

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

• G01N2015/0238

  Single particle scatter · CPC title