Stimulated emission depletion (STED) microscopy is a confocal based super-resolution technique that delivers a smaller effective PSF than traditional confocal. The resulting resolution can be up to 40 nm laterally and 100 nm axially in biological samples. The improvement in resolution is demonstrated in the below movie of a Paramecium imaged using the new SP8 3X STED at ESRIC. The animation begins with the confocal 3D reconstructed image and progresses to reveal the corresponding STED image; a higher resolution, more detailed image. (credit: Dr. Rebecca Saleeb) 

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. This means:

  1. Acquisition times are no longer than a normal confocal image

  2. Standard dyes can be used and standard fixation/mounting techniques

  3. Super-resolution can be achieved in 3 Dimensions

  4. Super-resolution can be acheived in living samples

The STED principle:

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 – it is depleted. This relaxation occurs faster than spontaneous fluorescence and unlike this, stimulated emission results in the emission of a photon with the same wavelength as its excitation beam (see below). STED selectively depletes molecules in the outer part of the excitation point spread function using a donut shaped beam of intense laser light (see below). By aligning the excitation and depletion beams on top of each other the outer fluorescent molecules are (mostly all) depleted and the effective emitted PSF is reduced and resolution enhanced. Using the appropriate filters the photons arising from stimulated emission are supressed and photons arising from spontaneous emission are sent to the detector.

Lightpath

Image Credit: Leica Microsystems. Image above shows the light path of the excitation and depletion lasers and the process of spontaneous fluorescence (central spot) and stimulated emission (outer ring).

STED in 3 Dimensions:

STED beam shaping in the Leica SP8 3X STED system is achieved using phase plates, a vortex phase plate for the xy dimension and an annular phase plate for the z dimension. Using the vortex plate will give 100% increase in xy resolution with no axial improvement and vice versa with the annular plate. However cleverly this system allows you use both phase plates in combination to achieve increased resolution in both dimensions, with obvious trade-offs. Below is a set of images showing the z dimension of fluorescent beads, the first is the confocal image and the images following show the increase in resolution in the z axis as you increase the % toward the annular phase plate (z dimension), the final image at 100% toward z.

Oil lens 3DSTED

 Image credit: Dr Rebecca Saleeb. Gattaquant Atto647 70R rulers in Mowiol. Depletion line 775 nm.

Gated-STED:

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 (image). 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; 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. 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 with no gating, therefore increasing 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.

Gating with the 592 nm laser:

Gating when using the 592 nm depletion laser has a substantial effect on the effective PSF, this is due to the effect described above where some molecules will spontaneously fluoresce and be detected in the outer region before being exposed to enough STED photons to be depleted. Gating can remedy this however, when set to exclude photons emitted within the first ~1 or 2 ns the image appears much sharper and the resolution increased. The 592 nm enables you to perform 3 colour imaging on the Leica SP8 3X STED with sufficient dye separation. See sample preparation for more information.

Gating with the 775 nm laser:

 The 775 nm laser is often a preferred choice over the 592 nm laser for multiple reasons.

  1. The effects of irradiation on your sample are significantly less as the laser is pulsed and also of a lower energy wavelength.

  2. The choice of STED dyes is broader with options for 2 colour STED imaging using the 775.

  3. Live cell imaging is possible using this laser due to its reduced phototoxic effects.

Gating with the pulsed 775 laser has less of an impact on your resultant image but can still give some improvement if used correctly. As we only gate off signal when the depletion laser is on and in a pulsed set up this is only ~500 ps, excluding signal after this time will just result in a loss of detected fluorescence signal. So gating is often set to exclude everything below 0.5 ns. Using the same logic as gating with the 592 nm laser you would expect that molecules which weren’t depleted in time (after 500 ps) would be collected and the resolution would not be as good. This is however not the case due to the peak power of the 775 nm laser being higher than the 592 nm laser; nearly all of the molecules are depleted when the 775 nm laser is on. 

The image below shows the power of using the 775 nm depletion line with gating to resolve DNA nanorulers, acheiving a FWHM of <40 nm.

ConfocalvSTED graph

Image credit Dr. Rebecca Saleeb, Gattaquant Atto 647 70R rulers mounted in Mowiol. Depletion line 775 nm with gating. Scale bar 50 nm.

Sample Preparation:

Being a confocal based technique as described, the preparation of samples for STED is relatively similar to confocal imaging.

Mountant:

Mowiol and Prolong Gold. Do not use Vectashield or any moutants that contain DAPI.

Dyes:

The criteria for dyes that can be used for STED microscopy is that they have a stoke’s shifts long enough to be depleted by the laser of choice and that they are relatively bright and stable to withstand high intensity laser light (i.e. Atto/Alexa dyes). Below is a graph showing dyes which are suitable for the ESRIC 592/775 system. The Leica Sample Guide is extremely comprehensive however please note we do not have the 660 nm depletion line at present.

The ESRIC SP8 3X STED is available to use at the Heriot-Watt University site. For access, to book training and/or to discuss a project please contact Ali Dun at This email address is being protected from spambots. You need JavaScript enabled to view it.