SPAD imagers for super resolution localization microscopy enable analysis of fast fluorophore blinking

Area of Science:

• Biophysics
• Optical Microscopy
• Materials Science

Background:

• sCMOS imagers are standard for high-speed super-resolution microscopy.
• Single-photon avalanche diode (SPAD) imagers offer comparable frame rates and unique timing resolution.
• SPADs generate microsecond 1-bit frames with no readout noise, enabling time-resolved analysis.

Purpose of the Study:

• To present a methodology for analyzing fluorescent dye blinking using SPAD data.
• To demonstrate the feasibility of SPAD imagers in super-resolution localization microscopy.
• To evaluate the impact of microlenses on SPAD imager sensitivity.

Main Methods:

• Developed a blinking analysis methodology for fluorescent dyes using experimental data.
• Integrated microlenses to enhance SPAD imager sensitivity by 12-fold.
• Acquired super-resolution localization microscopy data using the enhanced SPAD imager.

Main Results:

• Successfully recorded super-resolution localization microscopy data with a SPAD imager.
• Achieved a localization uncertainty of 20 nm and a resolution of 80 nm.
• Demonstrated the potential of SPAD imagers for time-resolved image analysis and photophysical studies.

Conclusions:

• SPAD imagers are a viable alternative to sCMOS for super-resolution microscopy.
• Enhanced SPAD sensitivity and timing resolution open avenues for advanced imaging and photophysical characterization.
• Microlens integration significantly boosts SPAD imager performance for demanding applications.