Holographic characterization of protein aggregates

Patent metadata

Field	Value
Publication number	US-11747258-B2
Application number	US-202217826848-A
Country	US
Kind code	B2
Filing date	May 27, 2022
Priority date	Feb 8, 2016
Publication date	Sep 5, 2023
Grant date	Sep 5, 2023

Abstract

Systems and methods for holographic characterization of protein aggregates. Size and refractive index of individual aggregates in a solution can be determined. Information regarding morphology and porosity can be extracted from holographic data.

First claim

What is claimed is:

1. A method of characterizing a sample of plurality of particles, comprising:
flowing the sample in a medium through an observation volume of a holographic microscope;
generating a first set of holograms based upon holographic video microscopy of a particle P n of a first cluster of the plurality of particles within the observation volume at a first time;
characterizing each of the particle of the first cluster of particles as a sphere;
determining the refractive index and the radius for characterized sphere of the first cluster of particles
generating a second set of holograms based upon holographic video microscopy of a second cluster of particles within the observation volume at a second time;
characterizing each of the particles of the second cluster of particle as a sphere; and
determining the refractive index and the radius for each of the particles of the second cluster of particles by characterizing of each of the particles of the first cluster and each of the particles of the second cluster as a fractal cluster of fractal dimension D where:

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with Ø being a volume fraction of particle, a 0 being the particle radius, and a p being the particles radius, n 0 being a refractive index of a particle, n m being a refractive index of the medium, and n p being an apparent refractive index of the respective particle cluster.

2. The method of claim 1 , further comprising monitoring synthesis of the plurality of particles based upon comparison of the first particle refractive index and radius and the second particle refractive index and radius.

3. The method of claim 1 , further comprising, wherein flowing the sample is at a rate of up to 100 μm s −1 .

4. The method of claim 1 , further comprising prior to generating the first set of holograms, adding salt.

5. The method of claim 1 , wherein determining the refractive index and the radius comprises application of Lorenz-Mie theory.

6. The method of claim 2 , further comprising determining whether synthesis of the plurality of particles has concluded.

7. The method of claim 1 , wherein determining the refractive index and the radius of the particles of the first cluster of particles and determining the refractive index and the radius of the particles of the second cluster of particles comprise determining a probability density for refractive index and radius of the particles of the first cluster of particles and determining a probability density for refractive index and radius of the particles of the second cluster of particles, respectively.

8. The method of claim 1 , wherein at least one protein aggregate is present in both the first cluster of particles and the second cluster of particles and further wherein the trajectory of the at least one particle is determined.

9. A computer-implemented machine for characterizing a plurality of particles, comprising:
a processor;
a holographic microscope comprising a coherent light, a specimen stage having an observation volume, an objective lens, and an image collection device, the holographic microscope in communication with the processor; and
a tangible computer-readable medium operatively connected to the processor and including computer code configured to:
flow the sample in a medium through an observation volume of a holographic microscope;
generate a first of holograms based upon holographic video microscopy of a particle P n of a first cluster of the plurality of particles within the observation volume at a first time;
characterize each of the particles of the first cluster of the plurality of particles as a sphere;
determine the refractive index and the radius for characterized sphere of the first cluster of particles
generate a second of holograms based upon holographic video microscopy of a second cluster of the plurality particles within the observation volume at a second time;
characterize each of the particles of the second cluster of particle as a sphere; and
determine the refractive index and the radius for each of the particles of the second cluster by the characterizing of each of the particles of the first cluster and of the second cluster as a fractal cluster of fractal dimension D where:

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⁡
(

f
⁡
(

n
p

)

-

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⁡
(

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m

)

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(

n
0

)

-

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(

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m

)

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=

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D

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Assignees

• Univ New York

Inventors

• Grier David G
• Ward Michael D
• Zhong Xiao
• Wang Chen
• Philips Laura A
• Ruffner David B
• Cheong Fook Chiong

Classifications

• G01N15/0227 Primary

  using imaging; using holography · CPC title

• G01N15/0211

  Investigating a scatter or diffraction pattern · CPC title

• G01N15/147 Primary

  the analysis being performed on a sample stream · CPC title

• G01N2015/0038

  Investigating nanoparticles · CPC title

• G01N2015/0053

  in liquids, e.g. trouble · CPC title