So far in this series I have given you an overview of how STED works and how to design your STED experiments. In this final article, I will tell you how to do two color STED and go over some tips and tricks to acquire the best images from your sample.

Dual Color STED Fluorophore Considerations

When trying to decide which fluorophores you should use when performing STED in two colours, the rules of confocal microscopy apply: The best dyes are those that are ‘spectrally-separated’. In confocal this would mean choosing fluorophores that are excited at completely different wavelengths – with well separated emission spectra – that do not display any cross-excitation. For STED, however, it is required that part of the emission spectra overlap with the depletion wavelength. Therefore, you often get slight cross-over between the two dyes that you choose.

The best combination tends to be a classic dye, and one with a large ‘Stokes shift’ (i.e. a large gap between the absorption and emission spectra). An example of a dye with a large Stokes shift is BD Horizon V500, which has an absorption spectra in the violet/blue range but an emission spectra which peaks at 500 nm.

Checklist for choosing your dyes:

  1. You need to be able to excite both dyes using the lasers available on your microscope.
  2. There should be some emission at the STED depletion laser wavelength.
  3. You need dyes that have high photo stability when subjected to the STED laser (i.e. Your dye shouldn’t bleach easily).
  4. You want minimal cross-excitation between your two dyes.
  5. You want to maximize the difference between your dyes’ emission spectrums.
  6. There should be no background excitation (referred to as ‘anti-Stokes’ excitation) at the STED wavelength.

Dye Combinations That Fulfil These Requirements

Finding combinations of dye that fulfil these requirements all depends on the wavelength of your STED depletion laser! Here are some examples…

592 nm depletion

  • BD Horizon V500 and Oregon Green 488. (You can also replace Oregon Green 488 with Chromeo 505 in this combination.)
  • Abberior STAR 440SX and Oregon Green 488. (You can also replace Oregon Green 488 with Chromeo 505 in this combination.)
  • Pacific Orange and Alexa 488. (You can NOT replace Oregon Green 488 with Chromeo 505 in this combination.)

660 nm depletion

  • Alexa Fluor 532 and TMR or Alexa Fluor 568
  • Oregon Green 488 and TMR or Alexa Fluor 568

Just remember: The most important thing is that both of the dyes DO NOT absorb light at the STED depletion wavelength or they will be bleached. So always check the absorption wavelength spectra of prospective dyes carefully!

STED Tips and Tricks

Hopefully you now have everything you need to create the perfect STED sample. But before I leave you, I want to go over some tips and tricks for getting the most out of your sample (whether it is one colour STED, or more!):

  • Sub Optimal Excitation of Dyes

    If background noise is your problem, consider not exciting your dye at peak wavelength for excitation. This can help you avoid cross-talk between dyes.

  • STED Depletion Power

    While it is tempting to use your STED laser at 100% power in an attempt to get the best resolution you can, you might not want to. In many cases 100% power will completely bleach your fluorophores. Instead try using your STED laser at 30% power and see what you get! It might not be the best improvement in point spread function (PSF) but it might work best for your sample. So do play with different levels of depletion level power to optimize your sample.

  • Gated STED

    – If your microscope system has the potential to use gated STED, then definitely use this! By gating out the first nanosecond of fluorescence collected by the detectors, you can dramatically reduce background.

  • Frame Accumulation

    – Some microscope systems will allow you to build an image by accumulating the fluorescence from multiple frames. This can be useful in STED, since the fluorescence that remains after STED depletion can be very low, especially if the protein you are interested in is not very abundant.

  • Deconvolution

    – Once you have your image acquired, I recommend that you do a bit of post-acquisition processing in the form of image deconvolution. This process will further improve the resolution of your image.


So that’s it for our STED article series! But keep a look out for future articles by me on the subject of super-resolution microscopy and beyond.

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