Autocalibrated scanning-angle prism-type total internal reflection fluorescence microscopy for nanometer-precision axial position determination.

An automatic calibration and scanning-angle prism-type total internal reflection fluorescence microscope (TIRFM) was constructed and tested for the highest vertical resolution. The angle of the incident laser beam can be changed automatically and reliably from subcritical angles to nearly 90 degrees with intervals smaller than 0.2 degrees, and the laser illumination spot in the sample can be calibrated to automatically overlap with the center of the microscope's field of view. By scanning through a wide range of incident angles with different evanescent-field layer thicknesses, the fluorescence intensity decay curves of randomly distributed fluorescent nanospheres in agarose gel were obtained and fitted with the theoretical decay functions to determine their vertical positions. The best axial resolution was demonstrated to be better than 10 nm under the rigorous statistical analysis of confidence levels and by the Monte Carlo simulation. The new setup was further utilized to determine the tilting angle of the microtubules buried in agarose gel and to find the precise surface plasmon resonance (SPR) angle for gold film enhanced TIRFM. We demonstrate the new microscope's unique capability to find the best illumination configuration for complex systems automatically and reproducibly.