UPDATE : Superresolution imaging

20 06 2008

A couple of awesome new papers out on superresolution imaging.  

The first one is on PALM imaging in live tissues. Despite Eric’s promise that he’s “getting out of PALM imaging”, the Betzig lab continues to pump out papers on the topic. In a May Nature Methods paper, they do the logical extention of their previous work, demonstrating PALM imaging on live tissue. Frame rates of up to 1/25 Hz and spatial resolution of 60nm. Clearly the resolution is not optimal at these speeds, but they are now able to see dynamic processes. Improved photoconvertable fluorescent proteins could dramatically increase the speed. Don’t miss Mats Gustafsson’s informative commentary on the work. We ♥ this Mats, not this Mats.

Next up is a collaboration between Heinrich Leonhardt, John Sedat, and Gustafsson’s groups. Using 3D structured illumination (3D-SIM), they do multicolor 3D superresolution imaging in fixed tissue. SIM works by repeatedly illuminating a sample with gratings of interfering light, rotating the angle of the illumination pattern. The resulting dataset can be used to reconstruct the sample beyond the diffraction limit. Here the authors add an illumination pattern that varies in the z-dimension, allowing them to image with superresolution in all three axes. 3D-SIM seems to be a bit behind competing techniques on the typically achievable spatial resolution. However, it has a distinct advantage over PALM, STORM and STED. It uses standard fluorescent dyes, making it well suited for multicolor acquisition and compatible with the huge library of existing labels.

Finally, Stefan Hell’s group has managed to extend the STED technique into 3D. Using a 4Pi objective configuration (objective on the top and the bottom) with STED, they were able to sculpt their excitation spot into a 45nm sphere. Sweeping this across the sample allowed two-color 3D reconstruction of mitochondria morphology @ 40nm resolution in fixed mammalian cells. The excitation volume can theoretically be continuously tuned to arbitrary size.


3D and Multicolor Superresolution Imaging

19 02 2008

Progress in superresolution imaging is still moving very quickly. Here are two more great papers in the field.

First, Huang et al. from Xiaowei Zhuang’s group published a Science paper that moves superresolution imaging into three dimensions. Previously, STORM and PALM techniques were most useful for thin sections where the z-axis depth is well-constrained. Breaking the diffraction limit in the z-dimension was thought to possibly require recording from multiple angles, standing wave TIRF or optical lattice microscopy. Instead, the authors simply inserted a weak cylindrical mirror in between the imaging lens and the objective. This distorted the shape of the point spread function in the x- and y-dimensions, dependent on the z-axis distance from the focal plane. By examining the shape of each photoactivated molecule’s ‘photon cloud’, they were able to unambiguously assign a z-axis depth. This was a simple and clever way to map a third dimension of information on top of the two they were recording.


Due to increasing point spread widths with greater depth, the localization accuracy decreases with distance from the focal plane. Therefore, they only examined structures within a 500nm window around the focal depth. Z-scanning the focal plane could increase the depth range, though this might waste signal by photobleaching out of focus fluorophores. However, this is less of a concern in the STORM vs. PALM approach as the cyanine dyes used for STORM can be cycled on many times, while the Eos-FP used in PALM permanently bleaches. Of course, if a dye molecule moves position between on-cycles, this will degrade the effective resolution of the STORM approach.

PALM proponents also have a new paper out. Shroff et al. from Eric Betzig’s group show an alternative method of dual-color superresolution imaging. They co-express genes labeled with photoactivatable tandem dimer EosFP and with reversibly photoswitchable Dronpa or PS-CFP. The EosFP-tagged molecules are first photoactivated (405nm illumination), localized (561nm) and bleached. This process photoactivates a signficant population of the Dronpa or PS-CFP molecules. After all EosFP has been bleached, the activated second label is switched back to the dark state (Dronpa), or photobleached (PS-CFP) (488nm). The remaining second label can then be specifically photoactivated, localized and bleached.


A major advantage of this dual-color PALM technique over Zhuang or Hell’s two-color photoswitching approach is that all the fluorescent reagents are genetically encoded rather than antibody labeled. This permits more precise localization of the label to the target of interest. It also allows greater label packing density and more mild fixation. A disadvantage is that genetic overexpression could cause mislocalization of the target or artificial aggregation due to residual dimerization tendencies of the fluorescent tags. However, unnatural aggregation can also be induced with antibody labeling. Perhaps adaptation of Don Arnold’s FP tagged intrabodies could address this concern.