Single Molecule Projects:

Superresolution In Three Dimensions Using Photoswitching/Photoactivation and Single-Molecule Imaging

 

Three-Dimensional Super-resolution Imaging of Single Molecules using a Double-Helix Point Spread Function (DH-PSF)

We have developed a unique method for 3D super-resolution with single fluorescent molecules where the PSF of the microscope has been engineered to have 2 rotating lobes where the angle of rotation depends on the axial position of the emitting molecule. In other words, the PSF appears as a double-helix along the z axis of the microscope, so it is called the double-helix PSF (DH-PSF) for convenience. This method is based on earlier work of our collaborator Rafael Piestun at University of Colorado who showed that a rotating DH-PSF could be formed by a superposition of Gauss-Laguerre (GL) modes that form a line in the GL modal plane (1). His student, Prasanna Pavani, modified the PSFdesign to increase efficiency, and used it for both photon-unlimited scatterers and very bright moving fluorescent microspheres(2). The figure at the right shows the image of a single fluorescent sphere at different z-positions relative to the usual focal position of the microscope. You are not seeing double, but, rather, the actual behavior of the DH-PSF sampled by the fluorescent bead! Various z-slices of the PSF appear as pairs of two spots. The angle of the line between the two spots can be used to read out the z-position of the object; the lower part of the figure shows a calibration curve extracted from the bead images. The DH-PSF can be generated by inserting a phase mask in the Fourier transform plane of the microscope.

We have recently shown(3) that a particularly useful photon-limited source, a single fluorescent molecule, can be imaged far beyond the diffraction limit by using a DH-PSF. In thick samples, we have demonstrated super-localization of single fluorescent molecules with precisions as low as 10 nm laterally and 20 nm axially over axial ranges >2 µm. The DH-PSF imaging system can be used to identify the 3D position of many molecules in a single image as long as the PSFs from the different emitters do not appreciably overlap. We have demonstrated this capability by using a sample containing a low concentration of the fluorophore DCDHF-P embedded in a ≈2 µm-thick PMMA film. The figure at right, left side, compares the standard (upper) and the DH-PSF(lower) images of 2 single molecules at different 3D positions selected to be fairly close to the focal plane for purposes of illustration only. In general, molecules away from the focal plane appear quite blurry in the standard PSF image. In contrast, the DH-PSF image encodes the axial position of the molecules in the angular orientation of the molecules’ DH-PSF lobes, which are distinctly above the background with approximately the same intensity through the entire z range of interest. This increased depth-of-field is illustrated directly in the right side of the figure, which shows a representative DH-PSF image of multiple molecules in a volume. Each molecule is seen to exhibit 2 lobes oriented at an angle that is uniquely related to its axial position, and the x,y,z, positions of these molecules are shown in Ref. 3..

Finally, to demonstrate true superresolution, we used single-molecule photoactivated localization microscopy (PALM) to determine the 3D location of many single molecules in a polymer sample, where many pairs of molecules were much closer than the standard diffraction limit. Our method may thus be called DH-PALM, for Double-Helix PALM. The photoactivatable molecule is from the new class of aryl azide fluorogens we have recently developed(4). The resulting image is shown at the right below, and the inset illustrates localizations of two molecules only 36 nm apart. For full details, see Ref. (3). Our work illustrates a new and powerful method for 3D superresolution imaging, because the DH-PSF has far more Fisher information (changes more rapidly with z) than is the case in other approaches for extracting 3D position information.

(1) R. Piestun, Y. Y. Schechner, and J. Shamir, Journal of the Optical Society of America A 17, 294-303 (2000).

(2) S. R. P. Pavani and R. Piestun, Optics Express 16, 3484-3489 and 22048-22057 (2008).

(3) S. R. P. Pavani*, M. A. Thompson*, J. S. Biteen, S. J. Lord, N. Liu, R. J. Twieg, R. Piestun and W. E. Moerner, “Three-dimensional single-molecule fluorescence imaging beyond the diffraction limit using a double-helix point spread function,” PNAS 106, 2995-2999 (2009) [Journal Link]

(4) S. J. Lord, N. R. Conley, H.-l. D. Lee, R. Samuel, N. Liu, R. J. Twieg, W. E. Moerner, JACS 130, 9204 (2008) [Slide] [journal link: JACS]