Breakthrough in Far-field Optical Nanoscopy

8 10 2007

Its thesis crunch time for me, so I have had limited time to do ‘extracurricular’ reading and reporting for Brainwindows. However, there have been some very exciting developments in the field of superresolution fluorescence imaging that deserve a mention.

First, let’s take a look at this excellent review of far-field superresolution imaging techniques by Stefan Hell. I was almost able to understand the basics of the current techniques after reading it. Hopefully my summary doesn’t contain too many errors ☺.

Axial resolution is particularly bad in conventional superresolution techniques. Confocal imaging and 2 photon imaging provides ~450 nm resolution at best, while 4Pi microscopy with immersion lenses above and below the sample has delivered ~100nm axial resolution images of fixed and live cells. Lens aperture is the limiting factor in 4Pi and I5M imaging systems. These systems do not break the diffraction limit; they just push it.

In the absence of bleaching, fluorescent molecules can be localized to arbitrary precision (1nm) provided there are no other spectrally identical molecules within <lamda/2n. How do we constrain the coordinates of excited molecules? The new far-field superresolution techniques rely on sequential recording of fluorescent markers in a light state that is switchable with a dark state. Hell’s approach of stimulated emission depletion (STED) illuminates a sample with two beams, a short-wavelength excitation beam surrounded by a longer-wavelength, donut-shaped beam. The donut beam produces stimulated emission that drives the fluorophores in the excited (S1) state back to the ground (S0) state by photon emission of identical wavelength. These long wavelength photons are discarded. This process competes with excitation from the short-wavelength laser. Only in the very center of the donut does the S1 excited state, driven by absorption of the short-wavelength excitation beam, last long enough to decay with a photon of wavelength between the excitation beam and the stimulated emission beam. This donut is scanned across the sample and the intermediate wavelength emission photons are collected. In practice, STEM has yielded axial resolution of 100nm with a single lens and 33-60nm in conjunction with 4Pi imaging. STED is expected to reach 10nm resolution with more advanced 4Pi setups.

Other STED like systems include:

Ground state depletion (GSD) – The depletion donut is produced by pumping the dye to metastable triplet state, which decays much more slowly than the S1 state. This requires far less laser power (100kW/cm2) than STED and only single-wavelength illumination, but has been practically limited by photobleaching in the triplet state.

Saturated structured illumination microscopy (SSIM/SPEM) – Sample is illuminated with structured sharp lines between saturated S1 states and dark S0 states. The illumination pattern is shifted and rotated. Superresolution images can be computationally reconstructed from the results. Demonstrated lateral resolution of 50nm with beads.

RESOLFT – Photochemical rather than photophysical state transitions using photoswitchable fluorescent proteins such as Dronpa and asFP595. Can be done with ultralow laser power (10W/cm2).

The practical limitation with these methods is that the rate of fluorophore bleaching is dependent on the number of state transitions it makes. All of these methods induce a large number of cycles per usable photon coming out. Therefore, the power of these techniques should be improved with more bleach-resistant dyes and fluorescent proteins.

Two competing methods that are not as sensitive to cycling induced bleaching are photoactivatable localization microscopy (PALM) and stochastic optical reconstruction microscopy (STORM). Rather than precisely define the location of fluorescence emission as in STED, these techniques use an ultradim laser to stochastically activate a constellation of well-spaced fluorophores throughout the sample. In PALM, these are then repeatedly excited with a different wavelength laser till bleached and their centroids are determined. The process is repeated and computationally summed till a superresolution (2-25nm) Seurat-like image is composed. A major benefit of this approach is that the fluorophores are efficiently used. Each fluorophore is switched on only once, and a maximal amount of photons is collected from it until bleaching. A limitation of the approach is that the sequential integration of images requires long imaging periods (hours), so far making it useful only for immobile proteins or fixed tissues. If background noise can be reduced enough to permit wide-field camera-based recording rather than laser-scanning, the acquisition rate should be greatly enhanced.

Now, let’s look at the new paper.

A major thrust of the PALM/STORM groups has been to develop multi-color labeling methods, so the interaction between two or more proteins can be studied. Xiaowei Zhuang’s group has recently demonstrated a system of photoactivatable dye pairs that theoretically allow up to nine color imaging. Long-wavelength ‘reporter’ cyanine dyes (Cy5, Cy5.5 or Cy7) are paired with shorter-wavelength ‘activator’ dyes (A405, Cy2, Cy3) on a single antibody. The reporter section of dye pairs can be selectively activated by laser pulses at 405, 457, or 532 nm, dependent on the activator section. Illumination with a red laser then produces a short period of fluorescence, whose emission wavelength is dependent on the reporter dye. Following this fluorescence, the reporter transitions to a dark state, which can be reactivated with another short-wavelength pulse.

Using dim activation pulses similar to PALM, the authors demonstrate three-color imaging of immobilized DNA molecules and two-color imaging of antibody-stained proteins in fixed cells with ~25nm resolution. The typical image used TIRF illumination and consisted of 2000-100,000 passes of recorded at 19Hz on a CCD camera. Thus, high quality images required as little as 2 minutes to record. There was some spectral crosstalk and false activation of the dye pairs that they were able to statistically correct. The spread in the localization of the antibody on a protein is now a significant contributor to the optical resolution limit.

These super-resolution techniques are getting very close to being usable in living samples, with both PALM and STORM making very quick progress. Be sure to check out the beautiful pictures in the paper figs.


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4 responses

16 10 2007
transmitter

You probably also need to mention the work from Paul R. Selvin’s group. One of his new PNAS paper is quite neat. My feeling is that computation work and new tag (such as Qdots) will make superresolution into the live cells very soon.

2 11 2007
christophe

For me another big breakthrough is SW Hell’s paper in Biophysical Journal :

Egner A, Geisler C, von Middendorff C, Bock H, Wenzel D, Medda R, Andresen M, Stiel AC, Jakobs S, Eggeling C, Schonle A, Hell SW.
Fluorescence nanoscopy in whole cells by asynchronous localization of photoswitching emitters.
Biophys J. 2007 Nov 1;93(9):3285-90. Epub 2007 Jul 27.

They greatly simplified and speeded up the process, now needing just one laser at 488 (trough the use of a Dronpa mutant) and continuous illumination. Acquisition time go from hours to several minutes. Moreover, they are not obliged to do it by TIRF / cell sections and show nice labeling of microtubules in intact cells.

2 11 2007
andrewhires

Thanks for the heads up on that paper, I’ll be sure to check it out. Just got back from the Janelia Farm imaging conference, which rocked…

Unfortunately, both Paul Selvin and Stefan Hell cancelled, so I didn’t get to see their latest stuff. But Hell’s STED system is getting commercialized by Leica!

The super-resolution session got pretty fiesty actually. There was quite a debate over the current limiting factors for achiving high resolution with PALM and STORM. Betzig demonstrated that, in theory, the primary limitation of high resolution is the packing of your fluorescent label onto the structure of interest, and that collecting large numbers of photons from each dye molecule actually hurts you, because it slows the acquisition rate, and the total number of points you can collect from. In contrast, Xiaowei asserted that in their hands, large photo counts per molecule helped their image resolution. This may be a brightness vs. photostability distinction, or there may be something else going on with noise sources…

Clearly (very clearly😉 the field is progressing amazingly fast.

5 11 2007
Vance Johnson

Cool blog. Glad to find it.
Crunch on thesis.

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