Abstract
We present a new instrument for nanoscopic investigations by coupling an atomic force microscope (AFM) with a super resolution stimulated emission depletion (STED) microscope. This nanoscopic tool allows high resolution fluorescence imaging, topographical imaging and nano-mechanical imaging, such as, stiffness. Results obtained from technical and biological samples are shown illustrating different functions and the versatility of the presented tool. We assert that, this highly precise tractable tool paves the way to a new set of comprehensive studies in medicine, biology and materials science.
Highlights
The classical diffraction limit for the resolution of an optical microscope can be overcome (Hell 2009) and that was demonstrated in the last years
It clearly shows the advantages of the stimulated emission depletion (STED) routine coupled to an atomic force microscope (AFM) in comparison to the images acquired by the confocal counterpart
A finer scan was performed by the AFM and the overlay of AFM image with the corresponding confocal image and STED image is shown in d and e, respectively
Summary
The classical diffraction limit for the resolution of an optical microscope can be overcome (Hell 2009) and that was demonstrated in the last years. Super-resolution techniques based on stimulated emission depletion (STED) and others (Schermelleh et al 2010) showed their potential in material science applications and biological sciences (Willig et al 2006). The basic idea of STED is to selectively switch off the periphery of the fluorescent focus by superimposing the diffraction limited excitation focus with a second beam, red shifted in wavelength with respect to the excitation wavelength. This second beam stimulates the excited dye molecule to release its energy across an energy transition which corresponds to the same energy, i.e. same wavelength, like the stimulation beam itself. By adding an appropriate phase mask, the focal distribution of the stimulating beam can be tuned into a doughnut-like shape featuring
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