Takahiro DEGUCHI^*, Sami KOHO, Tuomas Näreoja, and Pekka Hänninen

Laboratory of Biophysics, Department of Cell Biology and Anatomy, Institute of Biomedicine and Medicity Research Laboratories, University of Turku, Tykistökatu 6A, 20520 Turku, Finland

(Received October 9, 2013; Revised January 22, 2014; Accepted January 23, 2014)

In stimulated emission depletion (STED) microscopy, the lateral resolution is in the range of tens of nanometers depending on the sample and the instrument. The axial resolution, however, is in standard systems limited by diffraction to about 500 nm. We present an approach to three-dimensional diffraction-unlimited resolution by observing the sample at two optical angles. The system is realized by using an atomic force microscope (AFM) chip as a micro-reflector to deflect the STED beams near the region-of-interest (ROI), thus allowing observations at an angle ∠. Consequently, the superior lateral resolution can be utilized to resolve details in the axial direction of the main optical axis of the microscope. Here, fluorescent nanoparticles 90 nm apart and biological structures 80 nm apart along axial direction were distinguished by utilizing an off-the-shelf, commercial STED microscope, coupled with an AFM and an AFM chip micro-reflector.

Key words: fluorescence, confocal microscopy, STED microscopy, super-resolution, axial resolution

^*E-mail address: takdeg@utu.fi