Charles T. Troy, email@example.com
BELFAST, UK – Researchers in the US have developed a superresolution
3-D imaging technique that resolves single fluorescent molecules with >10 times
the precision of conventional optical microscopy. Using an iXon+ electron multiplying
CCD (EMCCD) camera from Andor Technology plc, they can locate molecules to within
12 to 20 nm in all three axes and hope to be able to observe interactions between
nanometer-scale intracellular structures previously too small to see.
Combining the concepts of superresolution imaging by sparse photoactivation
of single-molecule labels, three-dimensional stochastic optical reconstruction microscopy
(STORM) and fluorescence photo-activation localization microscopy (F-PALM) together
with a double-helix point spread function (DH-PSF) to provide accurate Z-position
information resulted in the 3-D superresolution imaging.
Associate Professor Rafael Piestun of the University of Colorado
at Boulder and his students developed a point spread function 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.”
Professor William E. Moerner at 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 accurately
Furthermore, the DH-PSF approach has been shown to extend the
depth of field to ~2 μm in the specimen, approximately twice that achieved
with other 3-D superresolution techniques.
Commenting on the role played by the iXon+ camera in this breakthrough,
Moerner said, “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. 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 validated recently 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 because it is easily
excited and emits a large number of photons before photobleaching takes effect. By
operating the EMCCD camera at a constant electron multiplying 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 X-Y-Z position of
the fluorophore could be determined with 12- to 20-nm precision, depending upon
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.