Special Reviews

Far-Field Optical Nanoscopy

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Science  25 May 2007:
Vol. 316, Issue 5828, pp. 1153-1158
DOI: 10.1126/science.1137395
  • Fig. 1.

    Fluorescence nanoscopy schemes: single-point scanning (upper row) and parallelized versions (lower row). (A) Confocal microscopy. The excitation light wave (blue) formed by the lens to a spherical cap produces a 3D diffraction spot, generating fluorescence in the focal region. A pointlike detector (not shown) registers fluorescence mostly from the main maximum (shown in green), thus providing a slightly improved resolution over regular epifluorescence microscopy. Nevertheless, the confocal microscopy resolution is limited by diffraction to >200 nm in the focal plane (x, y) and to >450 nm along the optical (z) axis. (B) By combining the wavefront caps of two opposing lenses, 4Pi microscopy produces a narrower spot along the z axis and hence an improved z resolution of 80 to 150 nm. (C) A typical single-point scanning STED microscope uses a regularly focused excitation beam (blue) that is superimposed by a doughnut-shaped STED beam (orange) that instantly quenches excited molecules at the periphery of the excitation spot, thus confining fluorescence emission to the doughnut zero. Saturated quenching results in a fluorescent spot far below diffraction (green), here 20 nm, whose scanning across the sample yields a subdiffraction-resolution image. The spots represent measured data. (D) RESOLFT principle: A focal intensity distribution I(r) featuring zeros that are > λ/2n apart confines either the bright state A (left) or the dark state B (right) through a saturable or switching transition, corresponding to a parallelized STED, GSD, or photoswitching approach (on the left) and to the SPEM concept (on the right). In both cases, imaging onto a camera causes the subdiffraction features created by the bright state A (left) or the dark state B (right) at the sample to be blurred on the camera by diffraction. Left: The blur can be dealt with by summing up each diffraction blob individually and allocating the signal to the pertinent coordinate of the zero in the sample space. The image is gained by translating the zeros across the sample and reading out the fluorescence for each coordinate step. (Right) The same holds for SPEM in which the superresolved data are encoded in the narrow regions around the zeros in which the dark state B is deliberately established (“negative data set”). The image is obtained by mathematically converting the negative data set into a positive one. Both strategies rely on a targeted signal readout based on preset positions of the zeros, and both operate with fluorophore ensembles; pA(r) ≤ 1 defines the normalized probability of occurrence of A. Small boxes symbolize molecules making up the object (gray-shaded mountains). (E) PALM and STORM readout the fluorophore molecules stochastically; the molecules must be switchable. Weak illumination sparsely switches individual fluorophores to the bright state A so that they are further apart than λ/2n. Detection of N ≫ 1 photons enables the centroid calculation of the diffraction blob of individual fluorophores on the camera, and hence assembling an image with resolution depending on N. Concepts (C) to (E) are not diffraction-limited, meaning that they can resolve similar molecules at nanometer distances. The STED, PALM, SPEM, and RESOLFT recording process is sketched in movies S1 to S4.

  • Fig. 2.

    Targeted versus stochastic time-sequential readout of fluorophore markers of a nanostructured object within the diffraction zone whose lower bound is given by λ/2n. A and B denote a bright and a dark state, respectively. In the targeted readout mode, one of the two states (here A) is established at a subdiffraction-sized spot at the position of a zero to read out an unknown number of fluorophore molecules. The image is assembled by deliberate translation of the zero. The zero can also be a groove. In the stochastic readout mode, a single switchable fluorophore from a random position within the diffraction zone is switched to a stable state A, while the other molecules remain in B. The coordinate is calculated from the centroid of the diffraction fluorescence spot measured by a pixelated detector. The coordinate pops up stochastically depending on where the interrogated marker molecule is located.

  • Fig. 3.

    Bright (A) and dark (B) molecular states used to break the diffraction barrier. Whereas STED, GSD, and SPEM utilize photophysical transitions, the photoswitching version of the RESOLFT scheme, as well as PALM and STORM, exploit photochemical transitions in which atoms are relocated or bonds formed and broken. PALM and STORM rely on measuring single (or at least identifiable) molecules at a time, whereas the other concepts, although compatible with single-molecule imaging, principally read out ensembles. Ensemble techniques rely on reversible transitions between A and B, as indicated by the rates k. The probability pA of being in state A depends nonlinearly on the light intensity applied, as indicated by the equations, ensuring that either A or B is confined to a subdiffraction area at a targeted coordinate in space. The e–γI and the (1 + γI)–1 dependence entail nonlinearities of infinite order (γI)m; m→∞. By increasing the lifetime of the chosen states, γ strengthens the nonlinear dependence of pA, thus enabling huge nonlinearities at low I. This is radically different from m-photon processes that, depending on the concomitant action of m photons and hence just on Im, are firmly limited to order m (15), which in practice is only m < 4. Because it operates with single molecules in a known state, the probability concept breaks down in PALM and STORM, but reminiscent of nonlinearity is the optical switching.

  • Fig. 4.

    Side-by-side comparisons. (A) Confocal versus 4Pi axial (xz) image of microtubules in a neuron: 4Pi image displays 140-nm z resolution; lens of α = 74° and with two-photon excitation at 800 nm. The plain 4Pi image is due to a narrow solitary peak without lobes; mathematical lobe-removal is not required. (B) Unlike the confocal reference, the STED image reveals the spatial order of self-assembled fused silica nanobeads containing a fluorescence core (45). (C) Neurofilaments in human neuroblastoma recorded in the confocal mode (left) and with STED after nonlinear deconvolution (right) displaying a focal plane resolution of 20 to 30 nm (39). (D) Epifluorescence versus PALM recording of a cryoprepared section from a mammalian cell expressing a lysosomal transmembrane protein tagged with a photoswitchable protein; both images were recorded with a TIRF setup. PALM resolution ranges between 20 and 60 nm, whereas individual protein localizations can be 2 nm (12).

Additional Files

  • Far-Field Optical Nanoscopy
    Stefan W. Hell

    Supporting Online Material

    This supplement contains:
    Movies S1 to S4

    Movies S1 to S4. Four movies in AVI format, plus PDF file with movie captions, packaged as a compressed archive, in *.zip format. Users should download the compressed file to their machine and decompress the file on their local hard drive, using the instructions below):

    Instructions for downloading and decompressing files:

    1. Create a temporary folder on your machine's hard drive.
    2. Save the compressed archive to the temporary folder you created, using the links above.
    3. Decompress the compressed file in the temporary folder using decompression software such as WinZip (Windows; www.winzip.com) or StuffIt Expander (Windows and Mac; www.stuffit.com).
    4. To play movies, use the freely downloadable QuickTime viewer. It may be necessary to upgrade your viewer to version 7 or above to play these movies.

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