Abstract

The resolution of a far-field optical microscope is usually limited to d = λ/(2n sin α) > 200 nm, with n sin α denoting the numerical aperture of the lens and λ the wavelength of light. Here, I will discuss lens-based fluorescence microscopy concepts that feature a resolving power on the nanoscale. All these concepts share a common basis: exploiting selected (pairs of) states and transitions of the fluorescent marker to neutralize the limiting role of diffraction [1, 2]. Specifically, the marker is switched between a bright and a dark state (on or off) to detect the emission of adjacent features sequentially in time. The first viable concept of this kind was Stimulated Emission Depletion (STED) microscopy [3] where the diameter of the region (focal spot) in which the molecule can be in the fluorescent state follows $d=\lambda /\left(2n\sin \alpha \sqrt{1+I/I_s}\right)$. I is the intensity that drives a fluorophore from the bright fluorescent state to the dark ground state by stimulated emission. I_S depends (inversely) on the lifetime of the states. For I/I_s → ∞, it follows that d → 0, meaning that the resolution can be molecular [4-6]. Altogether, far-field optical ‘nanoscopy’ is a fascinating development in optics with high relevance to the many areas of sciences, in particular the life sciences. Since it has already been a key to answering important questions in biology, and owing to its simplicity and commercial availability, I expect far-field fluorescence ‘nanoscopes’ to enter most cell biology and many nanoscience laboratories the near future [7].

© 2009 IEEE