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Fluorescence Bar-Coding and Flowmetry Based on Dark State Transitions in Fluorescence Emitters.

Elin Sandberg1, Baris Demirbay1, Abhilash Kulkarni1

  • 1Royal Institute of Technology (KTH), Experimental Biomolecular Physics, Dept. Applied Physics, Albanova University Center, 106 91 Stockholm, Sweden.

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This study uses fluorophore blinking kinetics to distinguish Cyanine5 (Cy5) from rhodamine dyes. Transient state spectroscopy (TRAST) enables multiplexed imaging in live cells and microfluidic samples, offering a new bar-coding strategy.

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Area of Science:

  • Biophysics
  • Spectroscopy
  • Fluorescence Imaging

Background:

  • Reversible dark state transitions in fluorophores limit ultrasensitive spectroscopy and super-resolution imaging.
  • These transitions, however, can be leveraged for orthogonal fluorescence-based readout parameters.
  • Distinguishing fluorophores with overlapping emission spectra is crucial for multiplexed detection.

Purpose of the Study:

  • To analyze the blinking kinetics of Cyanine5 (Cy5) as a bar-coding feature.
  • To differentiate Cy5 from rhodamine fluorophores based on their distinct blinking behaviors.
  • To develop and apply transient state spectroscopy (TRAST) for multiplexed imaging and analysis.

Main Methods:

  • Fluorescence correlation spectroscopy (FCS) to analyze fluorophore mixtures in solution and on vesicles.
  • Transient state (TRAST) spectroscopy to determine fluorophore dark state kinetics via excitation modulation.
  • Wide-field imaging of live cells and microfluidic systems using TRAST for spatially resolved multiplexing.

Main Results:

  • FCS demonstrated that Cy5's reversible trans-cis isomerization allows distinction from rhodamines.
  • TRAST robustly determined dark state kinetics and enabled cell-level and spatially resolved discrimination of Cy5 and rhodamines.
  • A microfluidic TRAST concept successfully distinguished fluorophore mixtures on-the-fly.

Conclusions:

  • TRAST offers a robust method for distinguishing fluorophores based on blinking kinetics, independent of single-molecule detection or high time resolution.
  • This bar-coding concept provides a valuable strategy for fluorescence-based multiplexing in diverse biological samples.
  • TRAST is broadly applicable to both stationary and moving samples, enhancing multiplexed fluorescence analysis.