Stimulated emission depletion (STED) microscopy is a confocal based imaging technique that delivers a smaller effective PSF than traditional confocal to give a resolution of approximately 40 nm in the lateral plane and 100 nm in the axial plane. Example images using a STED microscope compared to diffraction-limited confocal microscopy are shown below:
The advantages of using STED over other super-resolution techniques, such as STORM or PALM, is that as STED is not a localisation based technique but a direct manipulation of the PSF it can operate just as conventional confocal microscopy for example in three dimensions, with two colours or on live samples. STED can also be used to minimise the focal volume in FCS. However, in addition to increased technical challenges, samples must be stained with specific dyes susceptible to effective stimulated emission, and these can be found at nanobiophotonics.
The STED principle:
STED microscopy works by minimising the volume in which fluorescent molecules emit fluorescence by depleting (turning off) the molecules in the outer part of the excitation point spread function through a process called stimulated-emission. Put simply, stimulated emission is the process by which an excited fluorophore is forced to rapidly relax to its lower energy configuration through the emission of a photon. This depletion is facilitated via an additional intense laser illumination, a depletion beam, typically of lower energy than the excitation and fluorescent emission wavelengths. By depleting a donut shaped ring around the outside of the excitation spot the effective emitted PSF is reduced and resolution enhanced. In the diagram below the upper panel describes the PSF of a single fluorescent molecule after each laser pulse, the lower panel illustrates how these correlate to the energy levels at which the electrons will reside.
Our system uses a pulsed excitation laser and a CW STED laser, which is advantageous becuase there is no complicated optimisation or preparation of the laser pulse. It does however mean that there are some photons that will not be depleted simply because they were emitted too quickly for the STED depletion laser to force them into stimulated emission, this is not advantageous and can create a ‘blur’ around your signal. One way around this is to use time gating, which not only improves your resolution but also can help reduce background in highly reflective sample mounts or resins and allow compromise on the STED depletion power.
All fluorescent molecules have a fluorescent lifetime and generally a fluorescent signal will have a longer fluorescent lifetime in the centre decreasing with distance from the centre toward the periphery. This means that the photons emitted further away from the zero-intensity point will have shorter lifetimes – they may be emitted too quickly for the STED depletion laser to have time to deplete them.
By gating the emission you can select which photons to collect and by gating off the photons with shorter lifetimes you are able to remove the ‘blur’ that you see in CW STED and therefore increase your resolution.
So gating significantly further improves the resolution from STED and gives you the option to use a lower STED depletion power, which can help in taking stacks or time series. The compromise with gating is that you will also lose signal across the image as fluorescence is spontaneous and so some short lifetimes will still be found in the central spot. This can be counterbalanced using accumulation settings or higher excitation power.
For more information on gated STED using CW lasers please visit the Leica Science Lab.