The power of STED microscopy, Part 1: How does it work?

Do you suspect that your favourite protein is doing something really cool? But you cannot see it because your confocal microscope’s resolution is limited. Then Stimulated Emission Depletion (STED) microscopy is what you need! With the power to smash through the diffraction limit of confocal microscopy, STED opens up a whole new world of improved sub-cellular resolution without the need for extensive post-image processing. Last month I gave you an overview of STED microscopy in part 1 of the ‘Who’s who of super-resolution microscopy’. In this series of articles on STED microscopy, I will cover more about how it works, optimising staining, and share some tips and tricks on how to get the most out of it. Today I will cover (in detail) how STED microscopy works.

How STED works… in detail!

A STED microscope is built on the basis of a confocal laser-scanning microscope. For those that do not know everything about a confocal laser-scanning microscope off the top of their head here is a brief reminder: Confocal laser-scanning microscopy works when an objective focuses a laser light onto a small spot on your sample causing all fluorophores within said spot to emit fluorescence. The light intensity of which distributes according to the point spread function (PSF) and limits the resolution of the image. This emitted fluorescence is then collected by the objective and sent to the detector, which is outputted as a single pixel. To collect more pixels the position of this focused spot needs to be moved. By either moving the ‘scanning mirror’ within the microscope, or the sample itself. This ‘scanning’ is what allows an image to be built up. In contrast to confocal laser-scanning microscope, in STED microscopy a second laser has been added. Now during image acquisition, the normal excitation laser pulse is closely followed by a doughnut-shaped pulse of a longer wavelength, termed the STED beam. The excited fluorophores that are exposed to the STED beam are instantaneously ‘bleached’ back to the ground state. Therefore, only molecules that are sitting in the centre of the STED beam (the hole in the doughnut) are able to emit fluorescence. This physically narrows the PSF, therefore increasing resolution beyond the diffraction limit.

Different ‘types’ of STED laser beam

Pulsed STED:

Originally, STED microscopy was demonstrated using ‘pulsed’ lasers for both the excitation and the STED beams. This is a very standard type of laser that works by repeatedly emitting pulses of the same duration. This means that the pulses of the two lasers have to be optimised in both the time both lasers hit the sample and the duration of the pulses in order to get the best resolution improvement.

Continuous wave (CW) STED:

This type of STED microscopy uses a continuous wave STED beam (where the output beam remains constant over time). This has been implemented by Leica for their commercially available STED microscope and results in a simpler system, as no optimisation and preparation of the laser pulse is required. However, the downside to this continuous wave is that the fluorophores might not have time to ‘see’ enough STED photons before starting to emit fluorescence. This can result in residual fluorescence within the doughnut shape around the central point (where all the fluorophores should be back in the ground state). This causes a slight blurring of the final images and therefore a reduced resolution.

Gated CW STED:

One way around this problem with CW STED is to include time-gated detection for recording fluorescence. This means that you tell the detector to not collect photons from, for example, the first nanosecond after the fluorophores have been excited. This allows you to exclude the residual photons that have appeared before the STED beam has had time to take full effect.   I know all of the information so far has been very technical but it is useful to have a small understanding of how the microscope works before you start designing your experiments. This can help you make the most of the system that you have access to! In part 2 of this article series I will go into detail about STED sample preparation, including which fluorophores are good to use and how to choose the right STED settings for you.