Particle Analysis Using Fluorescence Lifetime Multiplexing
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Solution Overview
Problem
Current multiplexing techniques in flow cytometry and cell sorting face challenges due to spectral overlap, which limits the number of concurrent spectral bands that can be employed, and existing methods like mass cytometry are destructive and not suitable for selection and sorting of cells.
Innovation Solution
The use of fluorescence lifetime as an independent parameter, combined with spectral labeling, to generate a highly multiplexed set of combinations for uniquely tagging cell characteristics, reducing or eliminating spectral crosstalk, and enabling nondestructive analysis and sorting.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Adaptability or versatility
If multiple fluorescent tags are used simultaneously for multiplexed analysis, then the number of detectable cell characteristics increases, but spectral overlap occurs between adjacent fluorescence spectra
Solution Approach 1:
The patent transitions from spectral-domain multiplexing to time-domain multiplexing by measuring fluorescence lifetime. Instead of detecting multiple colors simultaneously in the spectral domain, the system uses time-resolved detection to distinguish fluorophores based on their decay time constants, adding a temporal dimension to the detection process and eliminating spectral overlap issues
Solution Approach 2:
The patent changes the detection parameter from spectral wavelength to fluorescence lifetime. By measuring the time constant of fluorescence decay rather than the wavelength of emitted light, the system can distinguish between different fluorophores without the spectral overlap that plagues conventional multiplexed fluorescence detection
2Loss of information
If spectral coverage is maximized to increase information extraction, then more fluorescence bands are detected, but spectral overlap between adjacent bands increases
Solution Approach 1:
The patent adds a temporal dimension to fluorescence detection by measuring lifetime in addition to intensity. This allows the system to extract information from fluorophores based on their decay characteristics rather than relying solely on spectral separation, enabling dense spectral packing without overlap
3Adaptability or versatility
If mass cytometry is used for highly multiplexed analysis, then the number of detectable tags increases, but the analysis becomes destructive and cell sorting is not possible
Solution Approach 1:
The patent replaces the physical destruction mechanism of mass cytometry with optical detection. Instead of vaporizing cells for mass spectrometry analysis, the system uses nondestructive fluorescence lifetime measurement, allowing cells to remain intact for subsequent sorting and analysis
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 significantly increases the number of combinations available for labeling and identifying cell types, reducing spectral overlap and allowing for nondestructive analysis and sorting, thereby enhancing the multiplexing capacity and efficiency of particle and cell analysis.
Implementation Method 1
The particle analysis apparatus employs pulsed optical excitation and time-resolved detection of fluorescence decay
Implementation Method 2
a detector configured to convert said optical signals into respective electrical signals
Data Source
AI summary
A particle analyzer, comprising a source of a beam of pulsed optical energy; a detector comprising a number of spectral detection channels to detect optical signals resulting from interactions between the beam and particles in a sample (such as, e.g., fluorescence signals), and to convert the optical signals into respective electrical signals; optical paths from the source to the sample and from the sample to the detector; a flowcell connected with the optical paths and with a flow path for a suspension of particles; a signal processing module capable of: receiving the electrical signals from the detector; mathematically combining individual decay curves in the signals into a decay supercurve; allocating individual components of the supercurve to discrete bins of predetermined time constants; and quantifying the relative contribution of individual components to the supercurve; a particle sorting actuator; an actuator driver; and at least one particle collection receptacle.