Dark-field microscopy for 3D nanoparticle localization
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Solution Overview
Problem
Conventional fluorescent microscopy methods fail to provide three-dimensional imaging of cells without altering the function of nano-scale drug carriers, as they require fluorescent labels that can interfere with drug delivery and increase complexity.
Innovation Solution
The development of wide-field microscopy methods using dark-field illumination and multiple point spread functions to acquire three-dimensional images of unstained and fluorescent cells, allowing for the determination of nano-particle locations without fluorescent labeling, and employing computational deconvolution to correct image focus across a wide spectral range.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If conventional fluorescent microscopy methods are used to image cells in three dimensions, then cell structure can be visualized, but fluorescent labels must be introduced which alter the function of nano-particles and significantly increase preparation complexity
Solution Approach 1:
The invention extracts and eliminates the fluorescent labeling step from the imaging process. By using dark-field microscopy to detect scattered light from nano-particles directly, the method removes the need for fluorophore attachment, thereby preserving nano-particle function while reducing preparation complexity
Solution Approach 2:
The invention replaces the optical fluorescence detection system with a dark-field scattering detection system. Instead of using fluorophores that emit light at specific wavelengths, the method detects light scattered by nano-particles, substituting a mechanical/optical detection approach that does not require chemical labeling
2Loss of information
If fluorescent labels are attached to nano-particles for imaging, then cell uptake and intracellular processing can be visualized, but the intended function for drug delivery is altered
Solution Approach 1:
The invention extracts the detection capability from the nano-particle itself by detecting scattered light in dark-field microscopy, rather than requiring the nano-particle to emit fluorescence. This extraction of the labeling requirement preserves the nano-particle's drug delivery function while still enabling visualization of cell uptake and intracellular processing
Solution Approach 2:
The invention creates an optical copy or representation of the nano-particle's location and movement by detecting scattered light patterns. Instead of modifying the nano-particle with fluorophores, the method captures light scattering information that replicates the nano-particle's spatial information without altering its physical or chemical properties
3Volume of stationary object
If confocal fluorescence microscopy is used to image thin sections over a volume, then three-dimensional cell structure can be viewed, but the method requires fluorescent labeling and increases difficulty of cell preparation
Solution Approach 1:
The invention extracts the three-dimensional imaging capability from fluorescence-based methods and transfers it to dark-field microscopy by detecting scattered light from nano-particles across multiple focal planes. This eliminates the need for fluorescent labeling while maintaining volumetric imaging capability
Solution Approach 2:
The dark-field microscopy system is designed to perform multiple functions: it can image both the cell structure and the nano-particles simultaneously without requiring different labeling strategies. The single dark-field setup provides universal imaging capability for both biological and nanomaterial components
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 precise three-dimensional localization of nano-particles within cells without altering their function, reducing complexity and allowing for diverse nano-particle configurations to be observed across a broad wavelength range, thus advancing nanomedicine research.
Implementation Method 1
acquisition of image data from functionalized subject particles within unstained and fluorescent cell preparations... using broadband illumination scattered from the cell volume... Such an image can be acquired with a dark-field illumination method
Implementation Method 2
The new methods employ multiple point spread functions (PSF) to correct the image focus across a wide spectral range... The new deconvolution methods automatically detect cell structure and subject particles in images
Implementation Method 3
Such an image can be acquired with a dark-field illumination method and the use of image sectioning techniques rather than by conventional fluorescence methods
Data Source
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AI summary
Disclosed are various embodiments for methods and systems for three-dimensional imaging of subject particles in media through use of dark-field microscopy. Some examples, among others, include a method for obtaining a three-dimensional (3D) volume image of a sample, a method for determining a 3D location of at least one subject particle within a sample, a method for determining at least one spatial correlation between a location of at least one subject particle and a location of at least one cell structure within a cell and/or other similar biological or nonbiological structure, a method of displaying a location of at least one subject particle, method for increasing the dynamic range of a 3D image acquired from samples containing weak and strong sources of light, and method for sharpening a 3D image in a vertical direction.