MINFLUX fluorescence nanoscopy in biological tissue

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.