Optical configuration methods for spectral scatter flow cytometry
Inventors
Welsh, Joshua Aden • Jones, Jennifer C. • Telford, William G. • Berzofsky, Jay A. • Rosner, Ari
Assignees
US Department of Health and Human Services
Publication Number
US-10876955-B2
Publication Date
2020-12-29
Expiration Date
2038-10-23
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Abstract
Apparatus include an illumination source configured to produce and direct a multi-wavelength illumination beam to a microfluidic target that can include nanotags, a detector configured to receive a multi-wavelength detection beam from the microfluidic target and to produce a detection signal, wherein the multi-wavelength detection beam comprises light that is elastically side-scattered by an interaction between the multi-wavelength illumination beam and the nanotags in the microfluidic target, and a processor configured to receive the detection signal and to determine the presence of the nanotags in the microfluidic target by comparing multiple wavelength side-scatter intensity characteristics of the detection signal with predetermined multi-wavelength elastic side-scatter intensity profiles of one or more nanotag types. Methods are also disclosed that determine the presence of different nanotags responsive to a multi-wavelength detection beam based on a detected signal and predetermined multi-wavelength elastic side-scatter intensity profiles for different nanotag types.
Core Innovation
The invention relates to apparatus and methods for flow cytometry using a multi-wavelength illumination source configured to produce a multi-wavelength illumination beam directed to a microfluidic target that can include nanotags. The apparatus includes a detector configured to receive a multi-wavelength detection beam comprising elastically side-scattered light from interactions between the illumination beam and nanotags in the microfluidic target, and a processor configured to determine the presence of nanotags by comparing multiple wavelength side-scatter intensity characteristics of the detection signal with predetermined multi-wavelength elastic side-scatter intensity profiles of nanotag types.
The background identifies a problem in the detection, resolution, and sorting of nanoscale particles such as extracellular vesicles (EVs) and other nanoparticles smaller than approximately 500 nm using conventional flow cytometry methods like fluorescent activated cell sorting (FACS). Existing tools lack the capability to characterize such small particles or detect single molecules on the surface of EVs due to electronic noise and signal loss. A need exists for improved reagents, methods, and apparatus that can detect individual nanoparticles and molecular components with high sensitivity and specificity.
The invention addresses this problem by providing a spectral scatter flow cytometry system that uses multi-wavelength illumination and detection to exploit distinctive scattering spectral characteristics of nanotags composed of different nanomaterials, such as gold and silver nanoparticles. The system employs advanced optical configurations including wavelength separation, chromatic focusing compensation, and multi-channel detectors to acquire side-scatter intensity data across multiple wavelengths. By comparing detected signals with predetermined spectral profiles and performing deconvolution, the system can identify and enumerate different nanotags, including single nanotags attached to EVs or other nanoparticles, overcoming prior detection limitations.
Claims Coverage
The claims cover both apparatus and method aspects comprising multiple inventive features focused on multi-wavelength illumination, detection, and analysis for detection of nanotags in a microfluidic target.
Multi-wavelength illumination and detection for nanotag identification
An apparatus including an illumination source producing a multi-wavelength beam directed to a microfluidic target with nanotags, and a detector that receives elastically side-scattered multi-wavelength light and produces a detection signal. A processor compares multiple wavelength side-scatter intensity characteristics with predetermined profiles to determine the presence of nanotags.
Deconvolution-based determination of nanotags and extracellular vesicles
Determining the presence of nanotags, including multiple types attached to the same extracellular vesicle, by deconvolving the detection signal using predetermined multi-wavelength elastic side-scatter intensity profiles.
Broadband and supercontinuum illumination with wavelength separation and chromatic focus correction
Using a broadband illumination source, such as a supercontinuum laser, with wavelength separation into multiple subbeams that are directed and focused at the microfluidic target accounting for chromatic focusing distance to reduce focus error.
Multi-wavelength side-scatter detection with prism optics and microlens arrays
A detection system that collects elastically side-scattered light, separates it spatially into detection subbeams using prism optics, focuses them with a microlens array, and detects them with detector channels such as avalanche photodiodes or photomultiplier tubes.
Monochromatic laser source illumination with collinear beam alignment and chromatic focus optimization
An illumination source comprising multiple monochromatic lasers at different wavelengths combined along a collinear optical path and focused at a common location at the microfluidic target, using beam focusing optics and dichroic elements that optimize chromatic focusing.
Dual collection optics with differing parameters for improved Mie resonance exploitation
First and second collection optics arranged perpendicularly on opposite sides of the microfluidic target, with different collection parameters such as collection angle and aperture geometry to shift Mie resonances and increase dynamic range and size/refractive index resolution of detected particles.
Chromatic delta focus compensation and spatial arrangement of detection subbeams
Detection systems with dichroic optical elements arranged to separate multi-wavelength detection beams into subbeams detected by spaced optical detectors arranged based on focusing distances and optical path length differences induced by chromatic aberration and delta-focus profiles.
Optical fiber assemblies with configurable apertures for chromatic focus correction
Optical fiber assemblies having apertures at fiber ends spaced apart along propagation direction to accommodate chromatic delta-induced focusing differences, including translation stages for lateral and longitudinal adjustment, slit or shaped aperture geometries, and optical blocks with absorptive areas to reduce stray light.
Methods for multi-wavelength spectral scatter detection and analysis
Methods include directing a multi-wavelength illumination beam to a microfluidic target, elastically side-scattering the beam, detecting multiple detection subbeams separated by wavelength to produce a detection signal, and determining nanotag presence by comparison with predetermined spectral profiles using deconvolution.
The claims collectively define an apparatus and method for spectral scatter flow cytometry employing multi-wavelength illumination and spectral deconvolution of multi-wavelength side-scatter signals to detect and characterize nanotags and extracellular vesicles with enhanced sensitivity and specificity using sophisticated optical configurations and data processing.
Stated Advantages
Improved detection and resolution of single nanoparticles and molecular components smaller than 500 nm, overcoming signal loss and noise issues in conventional flow cytometry.
Ability to identify and differentiate multiple nanotag types simultaneously using distinctive multi-wavelength spectral side-scatter profiles.
Enhanced sensitivity allowing single particle and single molecule detection, including on extracellular vesicles.
Optical configurations that compensate for chromatic aberration and utilize Mie resonance to improve accuracy in size and refractive index determination.
Flexibility in illumination sources, including broadband and monochromatic lasers, enabling optimization for various nanoparticle compositions and sizes.
Modular detection system designs that increase dynamic range and detection capability via dual collection optics and chromatic focus compensation.
Documented Applications
Detection, analysis, and sorting of extracellular vesicles (EVs) and other nanoscale particles released by cells, based on molecular nanotags attached to these particles.
Single molecule detection and enumeration on the surface of EVs or nanoscale particles, enabling phenotyping by labeling multiple epitopes with distinct nanotags.
Use in clinical and research settings for improved nanoparticle detection, resolution, and sorting.
Instrumentation including microfluidic flow cytometers equipped with multi-wavelength illumination and detection for biomedical research, tumor marker detection in blood samples, and particle phenotyping.
Flow cytometry apparatus retrofitting or modification to enable detection and spectral deconvolution of nanoscale particle labels non-fluorescently by elastic side scattering across multiple wavelengths.
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