Global analysis of fluorescence lifetime imaging microscopy data.

Area of Science:

• Biophysics
• Microscopy
• Spectroscopy

Background:

• Frequency domain fluorescence lifetime imaging microscopy (FLIM) provides insights into molecular environments.
• Analyzing complex biological samples with FLIM often requires advanced data processing techniques.
• Prior knowledge of limited, spatially invariant fluorescent species can simplify analysis.

Purpose of the Study:

• To develop and present global analysis techniques for frequency domain FLIM data.
• To implement algorithms that exploit the constraint of spatially invariant lifetimes.
• To enhance the capability of FLIM for resolving multiple fluorescent species in biological samples.

Main Methods:

• Described two approaches: lifetime invariant fit and a superior global analysis technique.
• The global analysis method simultaneously fits fractional contributions and spatially invariant lifetimes across all pixels.
• Tested algorithms on simulated data before application to real biological samples.

Main Results:

• The global analysis method allows fitting twice the number of lifetimes compared to conventional approaches.
• It is possible to discern two lifetimes using a single-frequency FLIM setup.
• Successfully separated cellular distributions of coexpressed green fluorescent proteins in living cells.

Conclusions:

• Global analysis techniques offer a powerful approach for analyzing frequency domain FLIM data.
• These methods improve the resolution and accuracy of lifetime and fractional contribution estimations.
• The developed algorithms are effective for resolving complex biological structures and processes.