Successful FCS experiment in nonstandard conditions

Area of Science:

• Biophysics
• Spectroscopy
• Materials Science

Background:

• Fluorescence correlation spectroscopy (FCS) is a key technique for measuring the self-diffusion coefficient of fluorescent probes.
• Standard FCS experiments typically require specific immersion objectives and thin (0.13-0.19 mm) sample window thicknesses.

Purpose of the Study:

• To investigate the feasibility of FCS measurements with non-standard sample cell window thicknesses and refractive indices.
• To explore the adaptability of FCS under conditions beyond nominal requirements for improved experimental flexibility.

Main Methods:

• Utilized various immersion objectives (water, oil, silicon oil) and long working distance dry objectives with correction collars.
• Tested FCS measurements with sample cells featuring window thicknesses exceeding standard specifications.
• Compared experimental results across different objective types and immersion media.

Main Results:

• Demonstrated successful FCS measurements with sample cells having significantly thicker windows than conventionally allowed.
• Confirmed that FCS requirements are less stringent than high-resolution confocal imaging.
• Achieved reliable relative FCS measurements even when operating beyond the objectives' designed compensation range.

Conclusions:

• FCS is robust and adaptable to variations in sample cell window thickness and refractive index.
• The findings relax stringent experimental constraints, broadening the scope of FCS applications.
• Opens possibilities for developing custom high-temperature and high-pressure cells for advanced FCS studies.