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Quantitative optical lock-in detection for quantitative imaging of switchable and non-switchable components.

Gerardo Abbandonato1, Barbara Storti1, Giovanni Signore2

  • 1NEST, Scuola Normale Superiore and Istituto Nanoscienze, piazza San Silvestro 12, I-56127, Italy.

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Summary

Quantitative optical-lock-in-detection (qOLID) enables precise measurement of switching and non-switching molecular components. This advanced method improves fluorescence imaging by accurately quantifying signals from specific molecules within complex backgrounds.

Keywords:
TRPV1optical lock-in detectionphotochromic FRETphotochromismreversibly switchable fluorescent proteins

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Area of Science:

  • Biophysics
  • Optical Imaging
  • Molecular Biology

Background:

  • Optical-lock-in-detection (OLID) identifies low-abundance molecules using photoswitching.
  • Standard OLID provides high contrast but lacks quantitative intensity information.
  • Distinguishing specific signals from background autofluorescence is challenging.

Purpose of the Study:

  • To develop a quantitative OLID (qOLID) method for measuring switching and non-switching molecular fractions.
  • To validate qOLID's performance using sample datasets and live-cell imaging.
  • To demonstrate qOLID's utility in Förster (Fluorescence) Resonance Energy Transfer (FRET) imaging.

Main Methods:

  • Modification of the optical-lock-in-detection (OLID) technique to yield quantitative data.
  • Application of qOLID to cells expressing the photochromic protein EYQ1.
  • Utilizing a photochromic donor and non-photochromic acceptor for qOLID-based FRET imaging.

Main Results:

  • qOLID successfully quantifies switching (fSW) and non-switching (fNS) molecular components.
  • The method effectively separates modulated signals from background and autofluorescence.
  • qOLID accurately highlights FRET intensity distribution using photochromic and non-photochromic fluorophores.

Conclusions:

  • qOLID provides crucial quantitative insights into molecular populations.
  • This technique enhances specificity in fluorescence imaging, particularly in complex biological samples.
  • qOLID offers a powerful tool for advanced FRET analysis and molecular imaging.