Particle Sorting via Fluorescence Lifetime Multiplexing
Find Innovative SolutionsGenerate Solutions
Solution Overview
Problem
Current multiplexing techniques in flow cytometry and cell sorting face challenges due to spectral overlap, limiting the number of concurrent spectral bands that can be detected and leading to inefficient use of the electromagnetic spectrum and contamination of signals, while existing methods like mass cytometry are destructive and not suited for selection and sorting.
Innovation Solution
The use of fluorescence lifetime as an independent parameter, combined with spectral fluorescence labeling, to generate highly multiplexed combinations for 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 spectral bands are used simultaneously for multiplexed detection, then the number of detectable cell characteristics increases, but spectral overlap causes signal contamination and reduces detection accuracy
Solution Approach 1:
The patent transitions from single-parameter spectral detection to multi-parameter detection by adding fluorescence lifetime as a second independent dimension. Each fluorophore is characterized by both its emission wavelength and its lifetime, creating a two-dimensional identification space that resolves spectral overlaps. This allows simultaneous detection of more than 10 fluorophores without signal contamination, as each has a unique combination of wavelength and lifetime even when their spectra overlap in the traditional one-dimensional approach.
2Adaptability or versatility
If mass cytometry is used to increase multiplexing capability, then the number of detectable characteristics increases, but the method is destructive and cannot be used for selection and sorting
Solution Approach 1:
The patent changes the detection parameter from mass-to-charge ratio (as in mass cytometry) to fluorescence lifetime in nanoseconds. This parameter change enables non-destructive detection, as fluorescence lifetime measurement does not require ionization or physical disruption of the sample. The method maintains sample integrity for subsequent sorting operations while achieving high multiplexing capability through the combination of spectral and temporal information.
3Area of stationary object
If spectral coverage is maximized by using adjacent fluorescence bands, then the range of detectable wavelengths increases, but spectral overlap increases causing crosstalk between channels
Solution Approach 1:
The patent resolves spectral crosstalk by adding the time dimension through fluorescence lifetime measurement. Even when fluorophores have overlapping emission spectra, their distinct lifetime characteristics allow complete separation of signals. The system measures both wavelength and lifetime for each fluorophore, creating unique spectral-temporal fingerprints that eliminate crosstalk entirely, enabling full utilization of the electromagnetic spectrum without signal interference.
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 highly multiplexed analysis and sorting without damaging the samples, thereby enhancing the accuracy and efficiency of particle analysis.
Implementation Method 1
a detector, the detector comprising a number of spectral detection channels, said channels being sensitive to distinct wavelength sections of the electromagnetic spectrum and being configured to detect optical signals resulting from interactions between said beam and said sample, said channels being further configured to convert said optical signals into respective electrical signals
Implementation Method 2
The presence or absence of the fluorophore (and therefore of the antigen the fluorophore-conjugated antibody is intended to specifically bind to) can then be established by excitation of the cells in the sample by optical means and the detection (if present) of the fluorescence emission from the fluorophore
Data Source
Figure 1~3
Figure 4(a)~4(d)
Figure 5
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.