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Related Concept Videos

Super-resolution Fluorescence Microscopy01:37

Super-resolution Fluorescence Microscopy

Super-resolution fluorescence microscopy (SRFM) provides a better resolution than conventional fluorescence microscopy by reducing the point spread function (PSF). PSF is the light intensity distribution from a point that causes it to appear blurred. Due to PSF, each fluorescing point appears bigger than its actual size, and it is the PSF interference of nearby fluorophores that causes the blurred image. Various approaches to achieving higher resolution through SRFM have recently been developed.

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Rational Control of Off-State Heterogeneity in a Photoswitchable Fluorescent Protein Provides Switching Contrast

Virgile Adam1, Kyprianos Hadjidemetriou1, Nickels Jensen2,3

  • 1Univ. Grenoble Alpes, CEA, CNRS, Institut de Biologie Structurale, F-38044, Grenoble, France.

Chemphyschem : a European Journal of Chemical Physics and Physical Chemistry
|August 12, 2022
PubMed
Summary

Researchers improved photoswitching contrast in fluorescent proteins by altering chromophore conformation. This enhances resolution in super-resolution microscopy (RESOLFT), enabling better biological imaging.

Keywords:
nanoscopyphotoswitchable fluorescent proteinsquantum chemistryserial femtosecond crystallographyswitching contrast

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Area of Science:

  • Biophysics
  • Structural Biology
  • Super-resolution Microscopy

Background:

  • Reversibly photoswitchable fluorescent proteins are crucial for advanced biological imaging techniques like RESOLFT microscopy.
  • Optimizing their photophysical properties is key to improving performance and enabling new applications.

Purpose of the Study:

  • To investigate the relationship between photoswitching contrast and chromophore conformation in the non-fluorescent state of rsEGFP2.
  • To engineer variants with enhanced switching contrast for improved nanoscopy resolution.

Main Methods:

  • Serial femtosecond crystallography for structural analysis.
  • High-level quantum chemical calculations.
  • In vitro spectroscopic and photophysical measurements.
  • Engineering of rsEGFP2 variants (V151A, V151L).

Main Results:

  • rsEGFP2's cis chromophore isomerizes into two off-state conformations (trans1 and trans2).
  • Modifying the V151 side chain (V151A, V151L) resulted in single off-state conformations with altered switching contrast.
  • The V151A variant showed over two-fold higher switching contrast due to the elimination of the trans2 conformation.
  • Demonstrated application of the rsFolder2-V151A variant in RESOLFT nanoscopy.

Conclusions:

  • Established a direct link between chromophore conformation and photoswitching contrast in rsEGFP2.
  • Demonstrated that rational structural modification can significantly enhance switching contrast.
  • The engineered variants offer improved performance for RESOLFT nanoscopy and other super-resolution imaging applications.