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Functional Fluorescence Microscopy Imaging: Quantitative Scanning-Free Confocal Fluorescence Microscopy for the

Aleksandar J Krmpot1,2, Stanko N Nikolić1,2, Sho Oasa1

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Functional fluorescence microscopy imaging (fFMI) offers quantitative analysis of fast molecular processes in cells. This scanning-free technique reveals location-specific molecular concentration and diffusion differences in live cells.

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Area of Science:

  • Biophysics
  • Cell Biology
  • Microscopy

Background:

  • Characterizing fast reaction-transport processes in biological systems is challenging.
  • Existing microscopy techniques often lack the speed and sensitivity for real-time molecular analysis.

Purpose of the Study:

  • To develop a novel, time-resolved imaging method for quantitative analysis of molecular dynamics.
  • To demonstrate the capability of this method in live cells and ex vivo.
  • To differentiate molecular behavior based on biological function and location.

Main Methods:

  • Functional fluorescence microscopy imaging (fFMI) using massively parallel fluorescence correlation spectroscopy (FCS).
  • Simultaneous excitation of 1024 spots using a diffractive optical element (DOE).
  • Detection with a 32x32 single-photon avalanche photodiode (SPAD) array and GPU-accelerated correlation analysis.

Main Results:

  • Achieved time-resolved imaging with single-molecule sensitivity (21 μs/frame).
  • Demonstrated location-specific differences in concentration and diffusion for functional molecules (transcription factors).
  • Showed uniform diffusion for non-functional molecules (eGFP).

Conclusions:

  • fFMI is a powerful tool for quantitative, time-resolved imaging of molecular processes in live cells.
  • The method can distinguish between functional and non-functional molecular behavior based on subcellular localization.
  • Revealed location-specific dynamics of glucocorticoid receptor and Sex combs reduced transcription factors.