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Related Concept Videos

Protein Dynamics in Living Cells01:19

Protein Dynamics in Living Cells

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Different fluorescence-based techniques are used to study the protein dynamics in living cells. These techniques include FRAP, FRET, and PET.
Fluorescent recovery after photobleaching (FRAP) is a fluorescent-protein-based detection technique used to quantify protein movement rates within the cell. This method exposes a small portion of the cell to an intense laser beam. The laser beam causes permanent photobleaching of the fluorophore-tagged proteins in the exposed region. As the bleached...
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Visualizing Intracellular SNARE Trafficking by Fluorescence Lifetime Imaging Microscopy
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Fluorescence Lifetime-Based Separation of FAST-Labeled Cellular Compartment.

Aidar R Gilvanov1, Ilya D Solovyev2, Alexander P Savitsky2

  • 1Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry of the Russian Academy of Sciences, Moscow, Russia.

Bio-Protocol
|October 13, 2025
PubMed
Summary

This protocol uses fluorogen-activating protein (FAST) technology with fluorescence lifetime imaging microscopy (FLIM) to distinguish between fluorescent labels. This method enables clear separation of cellular compartments, even when they overlap, in live cells.

Keywords:
FAST variantsFLIMFluorescence lifetimeFluorogen-activating proteinLive cellMicroscopy

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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.