High Resolution Fluorescence Lifetime Maps from Minimal Photon Counts.

Area of Science:

• Biophotonics and Imaging
• Computational Statistics
• Molecular Spectroscopy

Background:

• Fluorescence Lifetime Imaging Microscopy (FLIM) provides subcellular spatial lifetime maps of molecular species.
• Current FLIM analysis requires high photon budgets, leading to increased acquisition times and photodamage.
• Limitations exist in resolving arbitrary lifetimes and accounting for experimental variations like the instrument response function (IRF).

Purpose of the Study:

• To develop a data-efficient method for constructing accurate lifetime maps in FLIM.
• To overcome limitations of high photon budgets and reduce sample photodamage.
• To enable blind unmixing of molecular species with high spatiotemporal resolution.

Main Methods:

• A Bayesian statistical approach is employed to update lifetime map knowledge directly from photon arrival data.
• The method accounts for arbitrary instrument response functions (IRF) and utilizes information from undetected photons.
• Standard raster scan FLIM images are analyzed for direct blind unmixing of lifetimes.

Main Results:

• The developed method achieves high data efficiency, requiring less data for accurate lifetime determination.
• Subnanosecond resolution in lifetime unmixing and subpixel spatial resolution were demonstrated.
• The approach successfully maps arbitrary lifetimes, from short (IRF-comparable) to long (exceeding interpulse times).

Conclusions:

• This novel Bayesian FLIM analysis significantly enhances data efficiency and reduces photodamage.
• It provides a powerful tool for high-resolution molecular lifetime mapping and unmixing.
• The method's versatility is validated across diverse simulated and experimental datasets.