Nanoparticle Enumeration and Sizing by 3D Fluorescence Tracking
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Solution Overview
Problem
Existing methods for nanoparticle enumeration and characterization, such as electron microscopy, dynamic light scattering, flow cytometry, and nanoparticle tracking analysis, face challenges in resolving size and concentration of polydisperse nanoparticle suspensions, particularly for extracellular vesicles, due to limitations in resolution, throughput, and sensitivity to sample heterogeneity.
Innovation Solution
A method using 3D imaging-based techniques with epifluorescent microscopy to track nanoparticles in suspension, enabling accurate quantification and size distribution by identifying unique nanoparticles through spatial tracking and excluding overlapping tracks, and utilizing charged surfaces for selective attachment.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If electron microscopy is used for nanoparticle imaging, then size and ultrastructural information resolution is improved, but throughput and enumeration capability deteriorates
Solution Approach 1:
The patent replaces electron microscopy (a complex mechanical/electrical system requiring vacuum and specialized equipment) with light-based optical microscopy combined with fluorescence labeling. This substitution maintains sufficient measurement precision for nanoparticle size while dramatically improving throughput and enabling routine enumeration without specialized equipment
Solution Approach 2:
The patent changes the detection parameter from direct electron imaging to fluorescence signal detection. By labeling nanoparticles with fluorescent markers and detecting their optical signals, the system achieves adequate size resolution through optical methods while enabling high-throughput imaging and counting that electron microscopy cannot provide
2Productivity
If light scattering-based methods are used for nanoparticle detection, then throughput is improved, but measurement precision and reliability for polydisperse samples deteriorates
Solution Approach 1:
The patent uses fluorescence labeling to change the detection approach from measuring light scattering intensity (which varies with particle composition and size in complex ways) to detecting fluorescent signal intensity. This allows for more reliable size characterization in polydisperse samples because fluorescence intensity can be calibrated to particle size independently of composition variations
Solution Approach 2:
The patent introduces fluorescent labels as an intermediary between the nanoparticle and the detection system. These labels provide a consistent optical signal that can be reliably correlated with particle size, overcoming the limitations of direct light scattering methods where the signal depends on both size and composition in an unpredictable manner
3Productivity
If flow cytometry is used for nanoparticle enumeration, then throughput is improved, but measurement precision for particles below 500 nm deteriorates due to swarming effect
Solution Approach 1:
The patent segments the detection process into individual particle imaging events rather than bulk flow measurement. By capturing images of particles as they pass through the field of view and analyzing them individually, the system avoids the swarming effect where multiple particles are detected as a single event, thereby improving counting accuracy for small particles while maintaining throughput
Applied Scientific Principles
This section explains which scientific principles are used to turn an abstract innovation direction into a practical engineering solution.
Function Achieved in This Case
Enables accurate quantification and characterization of heterologous nanoparticles in suspension, overcoming limitations of existing methods by providing high resolution and throughput, even in polydisperse samples, with improved sensitivity and reliability.
Implementation Method 1
Fluorescent imaging has allowed for impressive EV tracking
Implementation Method 2
Due to the constraints of physics, including the diffraction limit of light microscopy (~200 nm) and Brownian motion of objects, direct imaging of nanoscale particles in suspension is challenging
Data Source
AI summary
The present invention provides a method for quantifying moving heterologous nanoparticles in a suspension by imaging. The quantifying method comprises acquiring at least one z-stack of images within the suspension; tracking the nanoparticles in the images to identify unique nanoparticles; and enumerating the unique nanoparticles. Also provided is a method for characterizing size distribution of moving heterologous nanoparticles in a suspension by imaging. The characterization method comprises acquiring time lapse images; tracking the nanoparticles in the images to identify unique nanoparticles; determining the locations of each of the unique particles; determining the size of each of the unique nanoparticles; and aggregating the sizes of the unique nanoparticles.