Phasor FLIM for Label-Free Metabolic State Discrimination
Find Innovative SolutionsGenerate Solutions
Solution Overview
Problem
Current methods for detecting tissue components in living tissues using multi-photon microscopy are limited by the inability to accurately assign auto-fluorescence signals to specific intrinsic molecular sources due to overlapping emission spectra and complex fluorescence intensity decays, which complicates the discrimination between different fluorescent species.
Innovation Solution
The use of the phasor approach in fluorescence lifetime microscopy allows for straightforward interpretation of intrinsic fluorescence signals by generating images based on decay properties rather than resolving molecular species' lifetimes, enabling the separation of tissue components through cluster analysis of phasor distributions and identification of specific fluorophores like GFP, collagen, FAD, NADH, retinol, and retinoic acid.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If multi-photon microscopy is used to detect tissue components, then high resolution and long term imaging of living tissues is achieved, but the ability to accurately assign auto-fluorescence signals to specific intrinsic molecular sources is limited due to overlapping emission spectra
Solution Approach 1:
The patent transforms the fluorescence decay analysis from time-domain resolution into frequency-domain representation using phasors. Each pixel's fluorescence decay is converted to a phasor point in a 2D complex plane, where the position encodes decay characteristics. This dimensional transformation allows differentiation of fluorophores with overlapping spectra by their unique phasor positions, resolving the contradiction between measurement precision and analysis complexity.
Solution Approach 2:
The patent replaces the traditional mechanical/mathematical fitting procedure with a geometric/phaser-based approach. Instead of iteratively fitting exponential decay models to resolve fluorophore contributions, the method uses cluster analysis of phasor positions to automatically identify and separate tissue components. This substitution eliminates the complexity of fitting procedures while improving the accuracy of molecular source assignment.
2Measurement precision
If multi-exponential fitting is used to resolve fluorescence intensity decays, then attempts are made to separate tissue components, but the overlapping of emission spectra and complex lifetime distribution make it difficult to assign specific tissue components to exponential decays
Solution Approach 1:
The patent replaces the information-intensive fitting procedure with an information-preserving phasor transformation. The phasor representation maintains all decay characteristics in a compact geometric form, avoiding the information loss inherent in fitting procedures that must assume specific decay models. Cluster analysis of phasor positions then naturally separates tissue components based on their unique decay signatures without requiring prior assumptions about the number or types of fluorophores present.
3Measurement precision
If emission wavelength discrimination is used to identify fluorophores, then some separation is achieved, but the overlapping of emission spectra of different fluorescent species limits the discrimination capability
Solution Approach 1:
The patent moves the discrimination problem from the spectral dimension to the temporal dimension by analyzing fluorescence decay characteristics. The phasor transformation encodes temporal decay information into a 2D representation where fluorophores with different decay profiles occupy distinct positions. This allows discrimination of fluorescent species based on their unique decay signatures rather than relying on spectral separation, reducing the need for complex spectral resolution while improving discrimination accuracy.
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 method effectively discriminates multiple tissue components, maps metabolic states, and predicts stem cell differentiation pathways without requiring fitting procedures or prior assumptions, providing a non-invasive optical tool for monitoring metabolic changes and cell sorting.
Implementation Method 1
Auto-fluorescence in live tissues arises from endogenous proteins and physiologically relevant fluorophores such as collagen, elastin, porphyrin, retinoids, flavins, nicotinamide adenine dinucleotide, hemoglobin and serotonin
Implementation Method 2
Two-photon excited fluorescence alone cannot assign auto-fluorescence signal to specific intrinsic molecular sources
Data Source
AI summary
A label-free imaging method to monitor stem cell metabolism discriminates different states of stem cell as they differentiate in a living tissues. We use intrinsic fluorescence biomarkers and the phasor approach to Fluorescence Lifetime Imaging Microscopy (FLIM). We identify and map intrinsic fluorophores such as collagen, retinol, retinoic acid, flavins, nicotinamide adenine dinucleotide (NADH) and porphyrin. We measure the phasor values of germ cells in C. Elegans germ line. Their metabolic fingerprint cluster according to their differentiation state, reflecting changes in FAD concentration and NADH binding during the differentiation pathway. The phasor approach to lifetime imaging provides a label-free, fit-free and sensitive method to identify different metabolic state of cells during differentiation, to sense small changes in the redox state of cells and may identify symmetric and asymmetric divisions and predict cell fate.