Total internal reflection fluorescence anisotropy imaging microscopy: setup, calibration, and data processing for...

Area of Science:

• Biophysics
• Cell Biology
• Microscopy

Background:

• Fluorescence anisotropy imaging microscopy (FAIM) is a technique used to study molecular environments by measuring light depolarization.
• Accurate FAIM requires careful system calibration, including the determination of the G factor, which corrects for system-induced polarization errors.

Purpose of the Study:

• To present a novel measurement strategy for G factor calibration in FAIM, specifically for high numerical aperture objectives in total internal reflection fluorescence (TIRF) illumination.
• To introduce AniCalc, an ImageJ/Fiji plugin for processing FAIM data.
• To demonstrate the application of the developed TIRF-FAIM system for studying cellular processes.

Main Methods:

• A new G factor calibration method utilizing evanescent fields with perpendicular polarization excitation was developed for TIRF-FAIM.
• The AniCalc plugin was created for automated FAIM data analysis.
• The system's performance was validated by measuring [Formula: see text]-actin polymerization in cellular models.

Main Results:

• The novel calibration strategy effectively determines the G factor for FAIM systems with high numerical aperture objectives in TIRF mode.
• The AniCalc plugin provides a user-friendly interface for processing FAIM data.
• The TIRF-FAIM system successfully visualized and quantified [Formula: see text]-actin polymerization dynamics in human embryonic kidney cells and retinal neurons.

Conclusions:

• The presented G factor calibration method and AniCalc plugin enhance the accuracy and applicability of FAIM, particularly in TIRF illumination.
• The developed system is capable of resolving dynamic molecular events like actin polymerization in living cells.
• This work provides a valuable tool for researchers investigating molecular dynamics in cell biology and biophysics.