Stimulated Emission Depletion (STED) Microscopy

Super-resolution imaging is the ability to resolve structures well below the diffraction limit. The pioneering technique, called STED microscopy was awarded the Nobel Prize in Physics in 2014.  In our lab we have a custom-built 3D dual color STED microscope with a super-continuum laser source.

The working principle of the STED technique is based on using a confocal laser scanning microscope equipped with an additional depletion beam, with a “donut shaped” intensity profile, which is overlaid onto the diffraction limited excitation spot. This allows the excited fluorophores to be depleted via stimulated emission preventing a spontaneous emission, while the fluorophores in the hole of the “donut-shaped” beam are stimulated only by the activation light and hence allowed to fluoresce spontaneously. By using this method, emission can be selectively silenced in the periphery of an excited spot, thus effectively reducing the area of detected fluorescence and improving the lateral resolution. Further implementation of a similar principle along the optical axis in the 3D STED increases the axial resolution as well.

We use the 3D STED microscope setup to investigate compositions of cellular architectures especially in neural synapses and for direct observation of other processes in bacterial, yeast or mammalian cellular systems. Also, we developed an implemented time-gating in the STED setup.

Recommended literature:

- Hell, S. W. and Wichmann, J. Optics Letters (1994), 19, 780-782
- Osseforth, C., Moffitt, J. R., Schermelleh, L. and Michaelis, J. Optics Express (2014), 22, 7028-7039

Published projects:

→ Simultaneous dual-color 3D STED microscopy

We describe the design and implementation of a stimulated emission depletion (STED) microscope which allows simultaneous three-dimensional super-resolution imaging in two colors. A super-continuum laser source is used to provide all spectral bands necessary for excitation and efficient depletion to achieve a lateral and axial resolution of ~35 nm and ~90 nm respectively. We characterize the systems' performance by imaging colloidal particles and single fluorescent molecules. Its biological applicability is demonstrated by dual-color imaging of nuclear pore complexes and of DNA replication sites in mammalian cells.
DOI: 10.1364/OE.22.007028

→ Time-gating improves the spatial resolution of STED microscopy

Stimulated-emission depletion (STED) microscopy improves image resolution by encoding additional spatial information in a second stimulated-decay channel with a spatially-varying strength. Here we demonstrate that spatial information is also encoded in the fluorophore lifetime and that this information can be used to improve the spatial resolution of STED microscopy. By solving a kinetic model for emission in the presence of a time-varying STED pulse, we derive the effective resolution as a function of fluorophore lifetime and pulse duration. We find that the best resolution for a given pulse power is achieved with a pulse of infinitesimally short duration; however, the maximum resolution can be restored for pulses of finite duration by time-gating the fluorescence signal. In parallel, we consider time-gating in the presence of a continuous-wave (CW) STED beam and find that time-gating produces theoretically unbounded resolution with finite laser power. In both cases, the cost of this improved resolution is a reduction in the brightness of the final image. We conclude by discussing situations in which time-gated STED microscopy (T-STED) may provide improved microscope performance beyond an increase in resolution.
Optics Express 19 (2011) 4242-4254