Ultra-stable and versatile widefield cryo-fluorescence microscope for single-molecule localization with sub-nanometer...

Area of Science:

• Physical Chemistry
• Biophysics
• Optical Microscopy

Background:

• Single-molecule spectroscopy requires stable, low-temperature environments.
• Existing setups can be complex and lack adaptability.
• Photostability of fluorescent dyes limits observation times and localization precision.

Purpose of the Study:

• To develop a user-friendly, adaptable cryostat for single-molecule fluorescence studies.
• To improve the photostability of single dye molecules.
• To achieve high-precision single-molecule localization.

Main Methods:

• Designed a stand-alone cryostat with optical access for epi-fluorescence microscopes.
• Cooled samples to cryogenic temperatures (89 K).
• Utilized an air objective (NA 0.7) for imaging and performed photo-bleaching studies on Atto647N dye molecules.

Main Results:

• Achieved excellent thermal and mechanical stability for observations over several hours.
• Demonstrated over a two-orders-of-magnitude improvement in Atto647N photostability.
• Enabled single-molecule localization accuracy of sub-nanometer due to increased photon counts.

Conclusions:

• The developed cryostat system is highly stable and adaptable for advanced single-molecule fluorescence imaging.
• Enhanced molecular photostability significantly improves data acquisition and localization precision.
• This system facilitates long-term, high-resolution studies of single molecules.