A sensitive and versatile laser scanning confocal optical microscope for single-molecule fluorescence at 77 K

Area of Science:

• Biophysics
• Materials Science
• Spectroscopy

Background:

• High numerical aperture objectives are crucial for efficient light collection in microscopy.
• Studying biomolecules at cryogenic temperatures can reveal unique photophysical properties.
• In situ preparation of amorphous water layers is challenging but important for cryo-studies.

Purpose of the Study:

• To develop a cryostat compatible with laser scanning confocal microscopy for high-efficiency measurements.
• To enable in situ preparation of amorphous water thin films at cryogenic temperatures.
• To investigate photophysics and photochemistry of model systems and biomolecules at 77 K.

Main Methods:

• A novel cryostat design allowing short working distances and high numerical aperture objectives.
• In situ preparation of amorphous water layers using a liquid-nitrogen immersed spin-coater within a Dewar vessel.
• Fluorescence resonance energy transfer (FRET) and alternating laser excitation (ALEX) measurements at ambient and 77 K.
• Line-scanning modification of ALEX (slow ALEX) for optimized sorting of labeled molecules.

Main Results:

• Successful demonstration of the cryostat's capability with a perylenemonoimide/polymethyl methacrylate model system.
• Applicability of the method for FRET measurements on freeze-quenched polyproline chains, indicating suitability for biomolecules.
• Investigation of photophysics and photochemistry at liquid nitrogen temperature (77 K).

Conclusions:

• The developed cryostat facilitates high-efficiency measurements at cryogenic temperatures.
• The setup is suitable for studying the photophysics of amorphous water and biomolecules.
• The modified slow ALEX technique enhances the analysis of labeled molecular systems at low temperatures.