Quantitative comparison of spinning disk geometries for PAINT based super-resolution microscopy

Area of Science:

• Biophysics
• Microscopy
• Cell Biology

Background:

• DNA- or protein-based exchangeable probe PAINT methods enable super-resolution imaging.
• Combining PAINT with spinning disk confocal microscopy allows imaging of thicker biological samples.
• Standard spinning disk geometries may limit resolution and imaging speed in PAINT applications.

Purpose of the Study:

• To evaluate the performance of different spinning disk geometries for PAINT-based super-resolution imaging.
• To identify spinning disk characteristics that optimize PAINT applications.
• To improve imaging speed and resolution in thick biological samples using PAINT.

Main Methods:

• Utilized PAINT methods with DNA- or protein-based exchangeable probes.
• Employed spinning disk confocal microscopy for imaging.
• Studied four distinct spinning disk geometries using *Drosophila* egg chambers and the Lifeact peptide.

Main Results:

• Spinning disk geometries with higher light collection efficiency demonstrated superior performance for PAINT imaging.
• Increased photon numbers were detected with optimized disk geometries.
• A higher frequency of blinking events was observed, correlating with improved imaging.

Conclusions:

• Spinning disk geometry significantly impacts the efficiency of PAINT-based super-resolution imaging.
• Disk designs maximizing light collection enhance photon yield and blinking event detection.
• Optimized spinning disk microscopy is essential for advancing PAINT applications in biological research.