Multicolor Caged dSTORM Resolves the Ultrastructure of Synaptic Vesicles in the Brain

Area of Science:

• Biophysics
• Microscopy
• Cell Biology

Background:

• Super-resolution microscopy techniques like direct Stochastic Optical Reconstruction Microscopy (dSTORM) rely on photon detection for precision.
• Increasing photon yield per localization enhances localization accuracy in single-color dSTORM.

Purpose of the Study:

• To develop and validate novel fluorescent dyes for multicolor super-resolution microscopy.
• To achieve high-precision multicolor imaging using caged dyes and spectral demixing.

Main Methods:

• Screening 39 fluorescent dyes for caging and recovery kinetics.
• Implementing caged dSTORM with spectral demixing (SD) for multicolor imaging.
• Applying the technique to image synaptic vesicles in brain sections.

Main Results:

• Identified novel dyes suitable for multicolor caged dSTORM.
• Achieved multicolor localization precision below 15 nm using a spectral demixing dye pair.
• Resolved ultrastructure of 40 nm synaptic vesicles with improved label density in two channels.

Conclusions:

• Caged spectral demixing dSTORM offers a powerful tool for high-precision multicolor super-resolution imaging.
• This method rivals immuno-electron microscopy in resolving ultrastructure while improving label density.
• Enables detailed visualization of nanoscale structures like synaptic vesicles in biological tissues.