With the help of a highly sensitive Andor iXon+ electron multiplying CCD camera, US researchers have developed a superresolution 3-D imaging technique that can resolve single fluorescent molecules with greater than 10 times more precision than conventional optical microscopy. By locating molecules to within 12 to 20 nm on all three axes, the researchers hope to observe interactions between nanometer-scale intracellular structures previously too small to see.

This technological advance has been achieved by combining two concepts: superresolution 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.

Prof. Rafael Piestun of the University of Colorado at Boulder 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 that the PSF appears as a double helix along the Z-axis of the microscope, lending it the distinctive name of “double helix PSF.”

Prof. W.E. Moerner of Stanford University in California and his team realized that the DH-PSF could be used for superresolution 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 2-D localization data, enables the 3-D position to be defined accurately. Furthermore, the DH-PSF approach has been shown to extend the depth of field to about 2 µm in the specimen, approximately twice that achieved in other 3-D superresolution techniques.

“As the localization precision of our superresolution 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,” said Moerner, commenting on the role played by the iXon+ camera in this breakthrough. “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 3-D localization 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 electron multiplying (EM) CCD camera at a constant EM gain setting of ×250, to eliminate the read noise detection limit, it was possible to acquire many images of the single photoactivated molecule. From these images, the X-Y-Z position of the fluorophore could be determined with 12- to 20-nm precision, depending on the dimension of interest.

Moerner and his team have called this new technique single-molecule double-helix photoactivated localization microscopy (DH-PALM) and are confident that it will provide far more useful information than is the case for other approaches to extracting 3-D positional information.

“We expect that the DH-PSF optics will become a regular attachment on advanced microscopes, either for superresolution 3-D imaging of structures, or for 3-D superresolution tracking of individually labeled biomolecules in cells or other environments,” he said.

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