Using an Andor Ixon+ EMCCD camera, researchers have developed a 3D imaging technique that resolves single fluorescent molecules with 10 times more 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 nanometer-scale intracellular structures that were previously too small to see.
This 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), with a double-helix point spread function (DH-PSF) to provide accurate z-position information.
Prof Rafael Piestun at the University of Colorado and his students developed a PSF with two rotating lobes, where the angle of rotation depends on the axial position of the emitting molecule.
This means the PSF appears as a double helix along the z-axis of the microscope, lending it the name 'Double Helix PSF'.
Prof W E Moerner, of Stanford University, and his team realised that the DH-PSF could be used for super-resolution imaging with single molecules.
With the DH-PSF, a single emitting fluorescent molecule, emits a pattern corresponding to a standard PSF, but the image this creates is convolved with the DH-PSF using Fourier optics and a reflective mask outside the microscope.
At the detector the image from a single molecule appears as two spots, rather than one.
The orientation of the pair can be used to decode the z-location of a molecule, which, combined with the 2D localisation data, enables the 3D position to be accurately defined.
The DH-PSF approach has been shown to extend the depth of field to 2um in the specimen - approximately twice that which has been achieved in other 3D super-resolution techniques.
The DH-PSF was recently used in a 3D localisation experiment involving imaging of a single molecule of the new fluorogen, DCDHF-V-PF4 azide2.
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.
Prof Moerner and his team have called this new technique single-molecule Double Helix Photoactivated Localization Microscopy (DH-PALM) and said 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 biomolecules in cells or other environments,' Moerner said.
