Doubling the resolution of a confocal spinning-disk microscope using image scanning microscopy

Area of Science:

• Cell Biology
• Microscopy
• Biophysics

Background:

• Fluorescence microscopy is crucial for cell biology.
• Super-resolution techniques offer high detail but are often costly and complex.
• Accessible methods are needed to overcome the diffraction limit.

Purpose of the Study:

• To present a low-cost, step-by-step protocol for converting commercial confocal spinning-disk (CSD) microscopes into CSD-image scanning microscopes (CSD-ISM).
• To enable high-resolution imaging of dynamic biological processes using accessible instrumentation.

Main Methods:

• Developed a protocol for hardware modification of existing CSD microscopes.
• Integrated image scanning microscopy (ISM) principles using structured illumination.
• Utilized a field programmable gate array and Micro-Manager software for operation and image reconstruction.

Main Results:

• Achieved a theoretical resolution increase of approximately 1.41-fold (√2), further improvable to twofold with deconvolution.
• Enabled imaging speeds of up to ~1 frame per second, suitable for dynamic processes.
• The CSD-ISM upgrade requires moderate hardware changes and can be completed in approximately 3 days.

Conclusions:

• The CSD-ISM conversion offers a cost-effective solution for enhancing microscope resolution.
• This accessible upgrade democratizes super-resolution imaging for studying live cellular dynamics.
• The provided protocol and software simplify the complex algorithmic and hardware requirements of ISM.