US researchers have developed a super-resolution, 3D imaging technique that can resolve single fluorescent molecules with 10 times greater precision than conventional optical microscopy. By being able to locate molecules to within 12-20nm in all three axes, the researchers hope to be able to observe interactions between nanometre-scale intracellular structures previously too small to see.
This major advance in 3D super-resolution imaging has been achieved by combining two concepts: super-resolution imaging by sparse photoactivation of single-molecule labels (PALM, STORM, F-PALM), coupled with a double-helix point spread function (DH-PSF) to provide accurate z-position information. The technique uses the highly sensitive Andor iXon+ EMCCD camera.
Professor W. E. Moerner at Stanford University and his team have called the technique single molecule Double Helix Photoactivated Localisation Microscopy (DH-PALM), and are confident that it will provide far more useful information than is the case for other approaches to extracting 3D positional information. 'We expect that the DH-PSF optics will become a regular attachment on advanced microscopes, either for super-resolution 3D imaging of structures, or for 3D super-resolution tracking of individually labelled bio-molecules in cells or other environments.'
Commenting on the role played by the Andor iXon+ EMCCD camera in this breakthrough, Professor Moerner said: 'As the localisation precision of our super-resolution technique improves at a rate of one over the square root of the number of photons detected, it was essential to use a camera that allowed us to detect every possible photon from each single molecule. Put simply, the more photons we detected, the greater the x, y, and z precision. However, the speed of imaging is also important. Since we need to acquire multiple images for each reconstruction, it is always best to record the images as fast as possible.'
The DH-PSF's usefulness was recently validated in a 3D localisation experiment involving imaging of a single molecule of the new fluorogen, DCDHF-V-PF4 azide. This photoactivatable molecule was chosen as it emits a large number of photons before it bleaches, and is easily excited. By operating the EMCCD camera at a constant EM gain setting of x250, to eliminate the read noise detection limit, it was possible to acquire many images of the single photoactivated molecule. From these images, the xyz position of the fluorophore could be determined with 12-20nm precision, depending on the dimension of interest.
