TIRF microscopy evanescent field calibration using tilted fluorescent microtubules

Area of Science:

• Biophysics
• Microscopy techniques
• Cellular dynamics

Background:

• Total internal reflection fluorescence microscopy (TIRFm) is vital for observing sub-cellular structures and molecules near surfaces.
• The penetration depth of the evanescent field in TIRFm is often undetermined, limiting spatial information.
• Accurate characterization of the evanescent field is essential for quantitative TIRFm studies.

Purpose of the Study:

• To present a novel method for quantitatively characterizing the evanescent field illumination in TIRFm.
• To establish a reliable in situ calibration tool for TIRFm.

Main Methods:

• Utilized tilted, fluorescently labeled microtubules as nanoscale probes.
• Measured the decay of the evanescent field using these probes.
• Analyzed the illumination profile to determine penetration depth.

Main Results:

• The evanescent field was accurately described by a single exponential decay function.
• Penetration depth values were close to theoretical predictions.
• Microtubules minimally perturbed the evanescent field, eliminating scattering and maintaining refractive index.

Conclusions:

• The presented method provides a quantitative and accurate way to characterize TIRFm illumination.
• In vitro microtubules serve as effective, non-perturbing nanoscale probes for calibration.
• This technique offers a generic tool for in situ calibration of the evanescent field in TIRFm.