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Fluorescence lifetime multiplexing enables simultaneous imaging of multiple cellular targets. This study identifies suitable synthetic probes for multiplexing, significantly advancing live-cell imaging capabilities.

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Area of Science:

  • Cell biology
  • Microscopy techniques
  • Biochemistry

Background:

  • Multicolor live-cell fluorescence microscopy is crucial for studying cellular processes.
  • Existing synthetic probes often lack characterization for fluorescence lifetime multiplexing.
  • Distinct fluorescence lifetimes are required for probes in multiplexing applications.

Purpose of the Study:

  • To identify suitable synthetic fluorescent probes for fluorescence lifetime multiplexing.
  • To expand the number of simultaneously observable biological targets in live-cell imaging.
  • To demonstrate the utility of combining synthetic probes with fluorescence lifetime multiplexing.

Main Methods:

  • Screening a panel of 18 synthetic fluorescent probes.
  • Characterizing probe suitability for fluorescence lifetime multiplexing in mammalian cell lines.
  • Utilizing self-labeling protein tags to enhance multiplexing capacity.

Main Results:

  • Eight pairwise combinations of synthetic probes were found suitable for fluorescence lifetime multiplexing.
  • Multiplexing enabled imaging of four biological targets using combined probe pairs.
  • Integration with self-labeling protein tags allowed imaging of up to eight targets.

Conclusions:

  • Synthetic probes are effective tools for fluorescence lifetime multiplexing.
  • This approach significantly increases the number of targets observable in live-cell imaging.
  • Fluorescence lifetime multiplexing offers a powerful strategy for advanced live-cell microscopy.