Accessible STORM Imaging: An Optimized Workflow for Conventional Widefield Epifluorescence/TIRF Setups

Area of Science:

• Cell Biology
• Microscopy
• Biophysics

Background:

• Stochastic Optical Reconstruction Microscopy (STORM) is a super-resolution technique enabling visualization of cellular ultrastructure beyond the diffraction limit.
• STORM relies on sequential single-photon emission from photosensitive fluorophores for precise localization and high-resolution image reconstruction.

Purpose of the Study:

• To describe a simple and efficient STORM workflow for sample preparation, image acquisition, and quality control.
• To enable broader implementation of STORM by lowering technical barriers on widely available microscopy platforms.

Main Methods:

• Developed an optimized immunofluorescence protocol including washing steps and secondary antibody fixation for single and dual-color STORM.
• Adapted a conventional epifluorescence/TIRF microscope for STORM imaging with a 160×/1.43 NA objective and high-power laser mode.
• Implemented a 1.6× magnification lens and 4×4 camera binning for optimal 100 nm pixel size for molecule detection.

Main Results:

• Successfully visualized subcellular structures including mitochondria, microtubules, lysosomes, and the actin cytoskeleton.
• Achieved localization precision of up to ~20 nm, enabling detailed ultrastructural visualization.
• Demonstrated the protocol's effectiveness using Alexa Fluor 647 and Alexa Fluor 488-conjugated phalloidin.

Conclusions:

• The presented STORM workflow simplifies super-resolution microscopy, making it more accessible to the research community.
• This protocol facilitates the broader application of STORM for investigating cellular ultrastructure with nanoscale resolution.