Granted Patent B2

US 12,510,462 · App. 17/058,445 · Granted Dec 30, 2025

Inventors: George G. Daaboul (Watertown, MA); David S. Freedman (Newton Highlands, MA); Amit Deliwala (Andover, MA)

Assignee: Unchained Labs

G01N15/1456G01N15/0211G01N33/54366G02B5/285G01N2015/0238G01N2015/025

Abstract

Claims (29)

1 . A method for detecting particles located substantially in a target plane above and in proximity to a top surface of a reflective substrate via simultaneous detection of fluorescence and single-particle scattering, the method comprising:

(a) directing illumination light from an illumination source towards the top surface of the reflective substrate, thereby illuminating the particles along with the reflective substrate;

(b) detecting, with one or more detectors, a portion of the illumination light that is scattered by the particles and reflected by the reflected substrate;

(c) directing excitation light from an excitation source towards the top surface of the reflective substrate, wherein the excitation source is separate from the illumination source, and wherein the excitation light has a wavelength corresponding to an excitation band of a fluorescent species within or on a surface of the particles, thereby exciting the fluorescent species;

(d) detecting, with one or more detectors, fluorescent light emitted from the fluorescent species as a result of excitation by the excitation light; and

(e) processing, (i) data corresponding to the detected portion of the illumination light that is scattered by the particles and reflected by the reflective substrate and (ii) data corresponding to the detected fluorescent light and to locate at least a portion of the particles,

wherein the reflective substrate comprises a multi-layered optical interference coating comprising a stack of three or more layers on top of a base layer, wherein a thickness of each of the one or more layers in the stack is less than or equal to about 200 nm.

2 . The method of claim 1 , wherein the stack comprises a series of alternating high and low refractive index layers, each high refractive index layer having a refractive index greater than a refractive index of a preceding layer and each low refractive index layer having a refractive index less than a refractive index of a preceding layer.

3 . The method of claim 2 , wherein each high refractive index layer has a refractive index ranging from 2.3 to 4.

4 . The method of claim 2 , wherein each low refractive index layer has a refractive index ranging from 1.1 to 1.7.

5 . The method of claim 2 , wherein the series of alternating high and low refractive index layers comprises 3 layers.

6 . The method of claim 1 , wherein:

step (b) comprises imaging, with a first imaging detector a first portion of the reflective substrate, thereby obtaining a scattering image representing illumination light that is reflected by the reflective substrate and scattered by the particles, and detected by the first imaging detector;

step (d) comprises imaging, with a second imaging detector a second portion of the reflective substrate, thereby obtaining a fluorescence image representing fluorescent light emitted from the fluorescent species as a result of excitation by the excitation light and detected by the second imaging detector; and

in step (e):

(i) the data corresponding to the detected portion of the illumination light that is scattered by the particles and reflected by the reflective substrate comprises the scattering image and;

(ii) the data corresponding to the detected fluorescent light comprises the fluorescence image.

7 . The method of claim 6 , wherein step (b) and/or step (d) comprises using an objective having a numerical aperture (NA) sufficiently high to allow for single particle imaging.

8 . The method of claim 6 , wherein step (e) comprises:

detecting, within the scattering image, one or more first particle image features, each corresponding to a particular particle; and

detecting, within the fluorescence image, one or more second particle image features, each corresponding to a particular particle.

9 . The method of claim 8 , comprising, for each of at least a portion of the one or more first particle image features, using an intensity of the first particle image feature to determine a size of the particular particle to which it corresponds.

10 . The method of claim 8 , comprising, for each of at least a portion of the one or more second particle image features, using an intensity of the second particle image feature to determine a size of the particular particle to which it corresponds.

11 . The method of claim 8 , comprising for each of at least a portion of the one or more second particle image features, determining presence of a molecular target within and/or on the particular particle to which it corresponds.

12 . The method of claim 8 , comprising, for each first particle image feature, matching the first particle image feature to a corresponding second particle image feature determined to correspond to a same particular particle, thereby co-locating the particle in the scattering image and the fluorescence image.

13 . The method of claim 1 , comprising: incubating the particles with a complex comprising the fluorescent species; and contacting the particles with the reflective substrate.

14 . The method of claim 1 , wherein at least a portion of the particles have a diameter ranging from about 10 nm to about 1000 nm.

15 . The method of claim 1 , wherein at least a portion of the particles have a diameter ranging from about 1.0 μm to about 10 μm.

16 . The method of claim 1 , wherein the particles comprise an extracellular vesicle, an exosome, or a liposome.

Assignments (3)

Continuity (3)

References Cited (157)