Direct characterization of the evanescent field in objective-type total internal reflection fluorescence microscopy

Area of Science:

• Biophysics
• Optical Microscopy
• Surface Science

Background:

• Total internal reflection fluorescence (TIRF) microscopy is vital for studying surface-associated molecules.
• The TIRF axial excitation profile is typically modeled as a single-exponential decay.
• Deviations from the ideal profile, including non-evanescent contributions, are observed in practice.

Purpose of the Study:

• To develop a method for direct characterization of the TIRF excitation field.
• To investigate the presence and nature of non-evanescent contributions in TIRF microscopy.
• To provide a tool for calibrating TIRF microscopy setups.

Main Methods:

• Development of a novel calibration slide with ten precisely fabricated height steps (25-550 nm).
• Utilizing a polymer with a refractive index matching water for the slide fabrication.
• Analyzing fluorescence intensity from fluorophores in aqueous solution at different heights above the slide surface.

Main Results:

• Directly measured TIRF excitation profiles confirmed the theoretically predicted exponential decay.
• The measurements also revealed a significant non-evanescent contribution to the excitation field.
• The calibration slide effectively characterized the TIRF excitation field across varying heights.

Conclusions:

• The developed calibration slide accurately characterizes the TIRF excitation field.
• Observed non-evanescent contributions in TIRF microscopy are experimentally confirmed.
• This method allows for precise calibration and better understanding of TIRF microscopy limitations.