Fig. 1.

Principle of FLIC microscopy demonstrated with fluorescently labeled MTs above a reflecting Si/SiO₂ surface. (A) Interference between direct and reflected light (for both, excitation and emission, thin lines with arrows) leads to a modulation of the observed fluorescence intensity depending on the distance to the surface. MTs were present in buffer solution between a glass coverslip and a reflecting Si/SiO₂ wafer. Illumination and imaging were performed through the glass coverslip. (B) Fluorescent images of immobilized MTs at varying tilt angles as captured on the CCD camera chip. i₀, i₁, and i₂ denote the measured fluorescence intensities of the first maximum, first minimum, and the second maximum, respectively. (C) Z-scan through a tilted MT (tilt angle α) fixed on a SiO₂ surface by a dilute agarose gel. Plotted is the fluorescence signal along the MT (horizontal axis) vs. z (vertical axis). (D) Height-calibrated FLIC curve of the imaging system used.