Fluorescence Lifetime Multiplexing for Particle Analysis
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Solution Overview
Problem
Current multiplexing technologies in particle and cell analysis face limitations due to spectral overlap, which restricts the number of concurrent spectral bands that can be detected, and existing methods for cell sorting are destructive, making nondestructive, highly multiplexed analysis and sorting challenging.
Innovation Solution
The use of fluorescence lifetime as an independent parameter, combined with spectral fluorescence labeling, to increase the number of distinguishable combinations for tagging cell characteristics, enabling reduced spectral crosstalk and expanded multiplexing capabilities in bead-based assays.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Adaptability or versatility
If multiple spectral bands are used for multiplexed detection, then the number of detectable markers increases, but spectral overlap and crosstalk increase
Solution Approach 1:
The patent introduces fluorescence lifetime as a new independent dimension for multiplexing, separate from spectral bands. By measuring both wavelength and lifetime, the system can distinguish between fluorophores that would otherwise be confusable through spectral overlap alone, enabling higher-order multiplexing with reduced crosstalk.
Solution Approach 2:
The patent employs composite fluorescent probes that combine multiple fluorophores with distinct lifetime characteristics. These composite probes allow simultaneous excitation at a single wavelength while maintaining distinguishable lifetime signatures, effectively separating signals that would overlap in spectral space.
2Ease of operation
If existing cell sorting methods are used, then cell separation is achieved, but the methods are destructive
Solution Approach 1:
The patent replaces destructive mechanical/electrical sorting methods (such as electrostatic deflection or physical barriers) with a nondestructive optical sorting approach. By using optical tweezers or similar non-contact manipulation techniques guided by fluorescence lifetime detection, cells can be sorted based on their fluorescent properties without physical damage.
3Adaptability or versatility
If spectral fluorescence labeling is used, then multiplexing capability is achieved, but the number of distinguishable combinations is limited
Solution Approach 1:
The patent adds fluorescence lifetime as an independent multiplexing dimension alongside spectral bands. This dimensional expansion allows the system to distinguish between fluorophores based on lifetime differences even when their emission spectra overlap, significantly increasing the number of simultaneously distinguishable combinations.
Solution Approach 2:
The patent segments the fluorescence signal analysis into separate spectral and temporal components. By analyzing the time-dependent decay characteristics of fluorescence emissions, the system can resolve individual fluorophore contributions within a mixed signal, enabling finer-grained multiplexing than spectral alone would allow.
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 enhances the number of possible combinations for identifying individual bead types, reducing costs and improving the efficiency of multiplexed assays by allowing for up to 10,000 or more combinations, thereby overcoming the limitations of spectral overlap and enabling nondestructive cell sorting.
Implementation Method 1
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.
Implementation Method 2
a detector including a number of spectral detection channels, the channels being sensitive to distinct wavelength sections of the electromagnetic spectrum and being configured to detect optical signals resulting from interactions between the beam and the sample
Data Source
AI summary
Described herein are apparatuses for analyzing an optical signal decay. In some embodiments, an apparatus includes: a source of a beam of pulsed optical energy; a sample holder configured to expose a sample to the beam; a detector comprising a number of spectral detection channels configured to convert the optical signals into respective electrical signals; and a signal processing module configured to perform a method. In some embodiments, the method includes: receiving the electrical signals from the detector; mathematically combining individual decay curves in the electrical signals into a decay supercurve, the supercurve comprising a number of components, each component having a time constant and a relative contribution to the supercurve; and numerically fitting a model to the supercurve.