Fluorescence Lifetime-Based Separation of FAST-Labeled Cellular Compartment

Area of Science:

• Cell biology
• Microscopy techniques
• Biotechnology

Background:

• Fluorescence lifetime imaging microscopy (FLIM) enables multiplexing by separating fluorescent species based on their fluorescence lifetimes.
• Previous methods utilized synthetic probes, fluorescent proteins, or self-labeling tags for FLIM multiplexing.
• Distinguishing co-localized fluorescent labels in live cells remains a challenge.

Purpose of the Study:

• To present a protocol for implementing fluorogen-activating protein (FAST) technology in FLIM for enhanced cellular compartment separation.
• To demonstrate the ability of FAST-fluorogen complexes to differentiate signals within the same spectral channel using fluorescence lifetime.
• To provide a user-friendly method for visualizing overlapping cellular structures in live mammalian cells.

Main Methods:

• Utilized engineered FAST point mutation variants that bind the fluorogen HBR-2,5-DM.
• Engineered FAST variants and fluorogen complexes with similar protein sizes (~14 kDa) and comparable optical properties.
• Expressed FAST variants with localization signals to target specific cellular compartments (nucleus and cytoskeleton).
• Employed fluorescence lifetime measurements to differentiate FAST-fluorogen complexes exhibiting distinct fluorescence decay kinetics.

Main Results:

• FAST-fluorogen complexes displayed monoexponential fluorescence decay kinetics and distinct fluorescence lifetimes.
• Robust signal separation was achieved for co-localized or spatially overlapping labels in the green emission channel (~500-550 nm).
• The protocol successfully differentiated between nuclear and cytoskeletal labels in live mammalian cells.
• The method is adaptable for separating other overlapping compartments like the nucleus/Golgi or mitochondria/cytoskeleton.

Conclusions:

• The FAST protein technology combined with FLIM offers a powerful approach for multiplexed imaging in live cells.
• This protocol provides a straightforward method for resolving overlapping fluorescent signals without requiring coding skills.
• The technique enhances the ability to study cellular structures and dynamics by enabling precise spatial localization of multiple labels.