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Fluorescence Imaging with One-nanometer Accuracy FIONA
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MINFLUX fluorescence nanoscopy in biological tissue.

Thea Moosmayer1,2, Kamila A Kiszka1, Volker Westphal1

  • 1Department of NanoBiophotonics, Max Planck Institute for Multidisciplinary Sciences, Göttingen 37077, Germany.

Proceedings of the National Academy of Sciences of the United States of America
|December 20, 2024
PubMed
Summary

MINFLUX nanoscopy achieves nanoscale protein imaging in intact brain tissue, overcoming scattering challenges. This superresolution technique precisely localizes molecules deep within tissue, enabling detailed synaptic structure analysis.

Keywords:
3D nanoscopypostsynapsesuper-resolution microscopytissue imaging

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Area of Science:

  • Neuroscience
  • Biophysics
  • Optical Microscopy

Background:

  • Achieving nanometer-level protein distribution imaging in intact tissue is crucial for understanding cellular functions in physiological contexts.
  • Superresolution fluorescence microscopy faces challenges in scattering and absorbing biological tissues, limiting molecular localization precision.
  • MINFLUX nanoscopy, utilizing a confocal pinhole, is hypothesized to overcome these limitations by improving signal-to-background ratios.

Purpose of the Study:

  • To evaluate the performance of MINFLUX nanoscopy for imaging protein distributions in mouse brain tissue sections.
  • To assess the localization precision of single fluorophores within challenging tissue environments and at depth.
  • To investigate the potential of two-color MINFLUX for simultaneously visualizing different synaptic protein targets.

Main Methods:

  • MINFLUX (Minimal Emissionྜ Detection) nanoscopy was applied to fixed mouse brain tissue sections.
  • Different synaptic protein targets were labeled with specific fluorescent probes.
  • Two-color imaging was performed to analyze the relative localization of multiple proteins and cellular structures.

Main Results:

  • Single fluorophores were localized with precision below 5 nm, even at depths up to 80 µm within the tissue.
  • MINFLUX imaging enabled visualization of postsynaptic density protein 95 (PSD95) relative to spine head morphology.
  • The technique successfully resolved clustering of vesicular glutamate transporter 1 (VGlut1) and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPARs) at the postsynapse.
  • Achieved 3D fluorophore localization precision below 10 nm in two-color experiments.

Conclusions:

  • MINFLUX nanoscopy demonstrates high precision and depth penetration for nanoscale protein imaging in scattering biological tissues.
  • The study validates MINFLUX for detailed, multi-color visualization of synaptic protein organization at the single-synapse level.
  • This technique offers significant potential for advancing research on synaptic structure and function in both fixed and live brain slices.