Fluorescence lifetime multiplexing with fluorogen activating protein FAST variants.

Area of Science:

• Biotechnology
• Microscopy
• Molecular Biology

Background:

• Multiplexing in fluorescence microscopy is crucial for simultaneous visualization of multiple cellular components.
• Existing methods often face limitations in signal overlap and specificity.
• Genetically encoded fluorescent labeling offers advantages in targeted protein detection.

Purpose of the Study:

• To develop a novel fluorescence-lifetime imaging microscopy (FLIM) multiplexing system.
• To utilize fluorogen-activating protein (FAST) mutants for distinct fluorescence lifetime generation.
• To demonstrate simultaneous labeling and imaging of intracellular structures in live cells.

Main Methods:

• Development of FAST mutants activating the same fluorogen but yielding different fluorescence lifetimes.
• Design of chemogenetic probes with minimal size and similar optical properties.
• Application of the system in live mammalian cells for dual-target imaging.

Main Results:

• Successful simultaneous targeting of two intracellular structures with distinct FAST-based probes.
• Clear signal separation achieved using FLIM, distinguishing probes by their fluorescence lifetimes.
• Monophasic fluorescence decay observed, potentially enhancing multiplexing efficiency and reducing crosstalk.

Conclusions:

• The proposed FAST-based FLIM multiplexing system enables efficient and specific simultaneous imaging.
• The system offers a powerful tool for studying complex cellular processes in live cells.
• The unique probe properties facilitate robust signal separation, even in co-localized or overlapped labeling scenarios.