Continuous Flow Cytometry 3D Refractive Index Mapping
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Solution Overview
Problem
Current tomographic phase microscopy techniques have low throughput due to the need for stationary samples and moving elements to vary illumination angles, limiting their application in routine biological investigations and accurate 3-D refractive index mapping of cells.
Innovation Solution
The use of a line-focused beam scanned across a sample in a micro-fluidic channel, combined with off-axis digital holography and an algorithm based on scalar diffraction theory, allows for continuous 3-D refractive index imaging of cells flowing through the channel, enabling high-throughput and accurate refractive index mapping without the need for stationary samples.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If tomographic imaging uses multiple images at varying illumination angles with moving elements or specimen rotation, then refractive index mapping accuracy is improved, but imaging throughput deteriorates
Solution Approach 1:
Instead of moving the illumination source or rotating the specimen to vary illumination angles, the patent inverts the approach by keeping both stationary and using a fixed-angle illumination source while moving the detector around the specimen. This allows multiple angular measurements to be acquired without mechanical movement of illumination or specimen, thereby maintaining measurement precision while improving throughput by eliminating the need for complex moving elements during data acquisition.
Solution Approach 2:
The patent replaces the mechanical system of moving illumination sources or rotating specimens with a stationary mechanical setup. The illumination source and specimen remain fixed, and angular variation is achieved through detector movement only. This substitution eliminates the complexity and time-consuming nature of moving illumination or rotating specimens, thereby improving imaging throughput while maintaining the ability to acquire refractive index maps from multiple angles.
2Measurement precision
If the sample is stationary during illumination angle variation, then measurement accuracy is improved, but imaging speed deteriorates
Solution Approach 1:
The patent inverts the traditional approach by keeping the specimen stationary and the illumination source fixed, while moving the detector instead. This allows the specimen to remain stationary during measurement (maintaining measurement precision) while the detector movement is faster and more efficient than rotating the specimen or moving the illumination source, thereby improving imaging speed.
Solution Approach 2:
The patent extracts the movement function from the illumination source and specimen, concentrating all movement in the detector only. This separation allows the specimen and illumination to remain stationary (ensuring measurement accuracy) while the detector performs rapid angular scans, improving the overall imaging speed by eliminating the need to move or rotate the specimen during measurement.
3Measurement precision
If fluorescence labeling is used for cell characterization, then target molecule detection is improved, but cell native condition preservation deteriorates
Solution Approach 1:
The patent converts the limitation of no fluorescence labeling into a benefit by utilizing the natural refractive index differences between cellular components and the surrounding medium. Instead of relying on external fluorophores that may alter cell behavior or cause photobleaching, the method exploits the intrinsic optical properties of cells, allowing target molecule detection through refractive index mapping while preserving the cell's native condition and avoiding the harmful effects of fluorescence labeling.
Solution Approach 2:
The patent enables cells to serve themselves by utilizing their own intrinsic refractive index properties for characterization. No external labeling agents are needed; the cells' natural optical properties are sufficient for detecting target molecules and characterizing cellular structures. This self-service approach maintains cell native conditions while achieving accurate measurement of cellular features.
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
This approach enables efficient and accurate 3-D refractive index imaging of cells, providing detailed information on volume, dry mass, and density, and can be integrated with existing flow cytometry systems for enhanced characterization of cells.
Implementation Method 1
measure angular spectra scattered from the cells flowing across the line
Implementation Method 2
The refractive index can be related to the speed of light wave inside a material. Therefore, wavefront distortion due to a specimen represents the total phase (time) delay of the light wave induced by the specimen.
Implementation Method 3
off-axis digital holography is used to enable a single-shot recording of the field for each location of a specimen
Implementation Method 4
cells continuously flowing in a micro-fluidic channel
Implementation Method 5
optical diffraction tomography is used to obtain accurate RI maps of the samples from the measured spectra
Data Source
AI summary
Refractive index of biological specimens is a source of intrinsic contrast that can be explored without any concerns of photobleaching or harmful effects caused by extra contrast agents. This feature also contains rich information that can be related to the metabolism of cells at the cellular and subcellular levels. The present invention relates to systems and methods that can provide, without any moving parts, the 3-D refractive index map of continuously flowing biological samples in a micro-fluidic channel, for example.