Register Log in

Scientific Method / Science & Exploration

The diffraction limit gets blown away

It seems that the diffraction limit is no longer quite as limiting as it once …

I seem to be writing about microscopy fairly often these days. First there was the article about a new technique that was able to resolve features smaller than that allowed by the diffraction limit. Then a grainy, low resolution form of microscopy turned up, which was able to produce three dimensional images from a single picture and promised to make the formation and manipulation of three dimensional images much simpler. However, that seems to be nothing compared with this week, where we have three articles on sub diffraction limited microscopy.

Stimulated emission depletion (STED) florescence microscopy was first demonstrated a few months ago in Germany. In this follow up paper, the same group demonstrate that the technique also works with GFP, an autofluorescing protein that is commonly used to label biological samples. For most florescent microscopy techniques, this would not be a question worthy of a major journal. However, STED relies on being able to manipulate the florescent properties of the label with a second light source and it is not immediately apparent that this will be possible for all labels. For the biologists amongst us, it is good news that GFP makes the list of labels useful for STED microscopy.

In the resolution department, STED has nothing compared to stochastic optical reconstruction microscopy (STORM), which can obtain a resolution of 20nm or smaller. This technique is very interesting in that it uses a combination of very clever chemistry and good statistical analysis. Imagine imaging a sample which has a single glowing molecule. The spot in the microscope will be about 200nm in diameter, as given by the diffraction limit. However, as time goes on, the precise position of the emitter from within the spot can be obtained as long as it emits enough photons. Unfortunately, applying such a technique to a useful situation where there is never a single emitter has proved to be problematic.

This is where the clever chemistry comes in, a florescent label that is switched off by red light and switched on by green light has been developed. Now, the single emitter can be obtained by switching the labels on and off very rapidly. The sequence is very simple; first blast the sample with red light, switching off all the labels. Then illuminate with a very weak green light source, switching on only one or two of the labels. A follow up pulse of red light causes the switched on labels to glow as they switch off. On each repetition, different labels will be cycled and over time each emitter produces enough photons for its position to be precisely located. Within ten minutes or so the complete image is constructed, with features as small as 20nm resolved.

A second paper independently reports the same technique, though they call it photoactivated localization microscopy (PALM) and use a different form of microscope. They work with less artificial samples, however, their labels cannot be cycled as quickly resulting in a 2-12 hour exposure time.

It is immediately apparent that these statistical techniques are not going to win any speed races and you can certainly rule out observing cell dynamics. However, they are yet another very cool tool which the physicists will eventually hand over to the biologists to do something useful with.

Expand full story

You must to comment.

Need to register for a new account?

If you don't have an account yet it's free and easy.