Super-resolution Imaging

Super-resolution imaging and tracking of cellular proteins

The field of super-resolution imaging is littered with a confusing array of acronyms (STORM, D-STORM, PALM, (f)PALM, sptPALM etc) but they are in fact very similar and side-step the diffraction barrier in essentially the same way.

All approaches are based on the same principle – localizing individual molecules one at a time, by ensuring that only sparsely distributed molecules are fluorescing at a given time.  The differences lie only in the nature of the fluorescent probes.

(See end of this page for a quick breakdown of these techniques – all of which are compatible with the STORM imaging platform)

Diffraction limits the resolution of conventional light microscopy to 250 nm—a value not improved upon for a century after the original observation of Abbe in 1873. However, novel far-field fluorescence imaging approaches now allow this limit to be circumvented, and recent techniques such as PALM (photo-activated light microscopy) and STORM (stochastic optical reconstruction microscopy) theoretically have unlimited spatial resolution. These superresolution techniques rely on the principle that a single fluorescent source (e.g., a fluorescent molecule) can be located with high precision by fitting a 2-dimensional Gaussian function to determine the centroid of the blurred spot (point spread function) formed by the microscope. The precision with which the centroid can be determined depends only on the number of photons collected; in practice, this would be a few tens of nanometers or better. The task is then to ensure that the density of fluorescent molecules is sufficiently low that two fluorophores are unlikely to lie indistinguishably close to one another so that their point spread functions do not overlap. With regards to the STORM technique, this has been accomplished by utilizing photoswitchable fluorophores such as Alexa fluor 647 (A647, known as the reporter dye) conjugated to secondary antibodies. In this process, red laser light that produces fluorescent emission from A647 can also switch the dye back to a stable dark state. Exposure to UV light converts A647 back to the fluorescent state, where it can be localized before it switches back to stable dark state again.

[youtube]http://youtu.be/SaDBvDp-80M[/youtube]
Alexa fluor 647 phalloidin – F-actin probe. At 8 seconds 647 laser power increased. Single molecules then reactivated spontaneously.

In each imaging cycle, only a fraction of the fluorophores in the field of view are switched on (B), such that each of the active flurophores is optically resolvable from the rest. Localizations from thousands of such on-off cycles are then combined to reconstruct a superesolution image (C and D). Multi-color STORM is achieved by conjugating an activator dye such as Alexa 488 in close proximity to the reporter on the same secondary antibody. Reactivation of the reporter dye is then achieved by irradiating the specimen with a wavelength (488nm) appropriate to the activator dye.

STORM: STORM (Stochastic  optical reconstruction microscopy). Immunofluorescence technique using antibodies custom conjugated  with photoswitchable dye pairs.

3D STORM: Optical astigmatism is used to determine both axial and lateral positions of individual fluorophores with nanometer precision. A cylindrical lens is placed in front of the camera to achieve this process.

Multi-Color STORM: Each secondary antibody is labelled with a photo-switchable “reporter” fluorophore (typically alexa fluor 647) that can be cycled between fluorescent and dark states, and an “activator” that facilitates photo-activation of the reporter Combinatorial pairing of the reporter with different activators allows the creation of probes with many distinct colors.

Live-STORM imaging: Using modifications of the imaging buffer, STORM is compatible with live cell imaging. Cells are live.

D-STORM: This technique differs from its parent technique STORM in that it does not rely on the proximity of two fluorophores attached to the same targeting antibody in a specific ratio. Secondary antibodies are labelled with a conventional fluorescent probe such as cy2 or Atto 488 or A647. These probes require substantial laser power to ensure effective transition of the photoswitchable fluorophores into a dark state.

REDUCTIVE CAGING: A reducing agent such as sodium borohydride is used to convert dyes to a long-lived reduced or ‘caged’ form. Photoactivation of the reduced dye can be achieved by exposure to UV light. Many conventional commercially available dyes in the visible spectrum can be used to obtain super-resolved images with this approach.

PALM/(f)PALM: Differs from STORM in that these technaiques use genetically encoded photoactivatble/photoswitchable proteins instead of photoswitchable dyes. These photoactivatble/photoswitchable proteins are tagged onto your own protein of interest and expressed in your cell of choice. Essentially non-fluorescent in native state – single molecules turned on by irradiation with 405nm laser. We have experience with:

Photoactivatable:
Green: PAGFP
RED: PAmCherry1 – this is far superior to PAGFP!

Photoswitchable:
Blue to Green: mEos2
Fluorescent in blue channel (488nm), dark in green channel (561nm) – ‘green’ single molecules are turned on with irradiation of 405nm laser.

sptPALM – tracks the movement of single molecules in live cells tagged with a gentically encoded photoactivatble/photoswitchable protein at a super-resolved resolution. Currently we have all the Matlab code and tracking algorithms in place for this type of tracking.

[youtube]http://youtu.be/g3xTG2fK3mo[/youtube]
mEos2 tagged onto an ER protein – single mEos2 molecules stochastically activate upon irradiation with weak 405nm laser whereupon they can be seen to diffuse  before photobleaching.

 Tracking mEos2 molecules