Fluorescence Lifetime Multiplex Imaging in Expansion Microscopy with Tunable Donor-Acceptor Polymer Dots

Area of Science:

• Nanotechnology
• Microscopy
• Biophysics

Background:

• Fluorescence lifetime imaging microscopy (FLIM) is crucial for detecting biomolecules but faces diffraction limits.
• Conventional dyes in FLIM have limited photon output and struggle to surpass the diffraction limit.
• Expansion microscopy (ExM) offers super-resolution but is photon-starved, challenging for traditional FLIM.

Purpose of the Study:

• To introduce semiconducting polymer dots (Pdots) as novel probes for fluorescence lifetime imaging in expansion microscopy.
• To leverage Pdots' tunable lifetimes and high photon budget to overcome FLIM limitations in ExM.
• To demonstrate multiplex lifetime imaging capability for enhanced subcellular structure resolution.

Main Methods:

• Development of three fluorescent Pdots with average lifetimes from 0.4 to 5 ns by tuning polymer composition.
• Utilizing Pdots in expansion microscopy for fluorescence lifetime imaging.
• Analyzing Pdot distributions in the lifetime domain despite spectral overlap.

Main Results:

• Distinct Pdot distributions were resolved in the lifetime domain, enabling multiplex imaging.
• Pdots' high brightness and photon output facilitated imaging in photon-starved ExM.
• Subcellular structures were resolved with a spatial resolution of approximately 49 nm.

Conclusions:

• Semiconducting polymer dots (Pdots) are effective probes for fluorescence lifetime imaging in expansion microscopy.
• Tunable Pdots overcome diffraction limits and photon-starved conditions in super-resolution microscopy.
• This approach shows significant potential for multiplex lifetime imaging applications in cell biology.