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uFLIM - Unsupervised analysis of FLIM-FRET microscopy data
Francesco Masia1, Walter Dewitte2, Paola Borri2
1School of Physics and Astronomy, Cardiff University, The Parade, Cardiff CF24 3AA, UK; School of Biosciences, Cardiff University, Museum Avenue, Cardiff CF10 3AX, UK.
Medical Image Analysis
|September 1, 2022
Summary
We developed uFLIM, a fast fluorescence lifetime imaging microscopy (FLIM) analysis method. It accurately identifies multiple fluorescent components and their dynamics without prior knowledge, improving cell biology research.
Area of Science:
- Cell Biology
- Microscopy
- Biophysics
Background:
- Fluorescence Lifetime Imaging Microscopy (FLIM) data analysis is complex due to superimposed fluorescent components at each spatial position.
- Current methods often require prior knowledge of component dynamics or assume specific decay models (e.g., exponential decay).
Purpose of the Study:
- To present uFLIM, a novel, fast data analysis method for FLIM datasets.
- To determine spatial distributions and temporal dynamics of multiple fluorescent components without prior constraints.
- To enable real-time processing of FLIM data.
Main Methods:
- Development of uFLIM, a method utilizing the full photon budget.
- Implementation of an efficient non-negative matrix factorization algorithm for real-time processing.
- In silico validation using complex datasets with multiple fluorescing probes and Förster resonant energy transfer (FRET) scenarios.
Main Results:
- uFLIM successfully disentangles spatial distribution and spectral properties of five fluorescing probes with limited data (100 photons/pixel, 2 channels).
- The method accurately retrieves spatial and transfer rate distributions in FRET-exhibiting data without constraining donor/acceptor dynamics.
- Demonstrated capability for real-time data processing.
Conclusions:
- uFLIM offers a significant advancement in FLIM data analysis, overcoming limitations of existing approaches.
- The method provides accurate, model-independent characterization of complex biological systems.
- uFLIM enables efficient and comprehensive analysis of FLIM data, advancing cell biology and biophysics research.
