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Three-Dimensional Superresolution Imaging

The Abbe diffraction limit in optical microscopy restricts the dimensions of the point-spread function of a single emitter to approximately 200 nanometers in the lateral plane and 500 to 700 nanometers axially. A number of remarkable superresolution techniques have recently been defined that significantly reduce localization precision to much smaller values (often only tens of nanometers) in both dimensions. This emerging methodology should ultimately enable far-field fluorescence microscopy to have non-invasive access to the interior of living cells with high spatial resolution.

Hell, S. W., Schmidt, R. and Egner, A.

Diffraction-unlimited three-dimensional optical nanoscopy with opposing lenses.  Nature Photonics 3: 381-387 (2009).  Pioneer Stefan Hell and colleagues review the theoretical and practical aspects of nanoscale superresolution imaging in three dimensions by instruments that are equipped with opposing objectives. These techniques include STED, iPALM, and 3D-STORM.

Schrader, M., Hell, S. W. and van der Voort, H. T. M.

Three-dimensional super-resolution with a 4pi-confocal microscope using image restoration.  Journal of Applied Physics 84: 4033-4042 (1998).  Demonstration of three-dimensional superresolution using two-photon 4Pi confocal microscopy to image transparent, fluorescent specimens. By applying an illumination wavelength of 810 nanometers, the authors achieve lateral and axial resolutions of better than 140 and 100 nanometers, respectively.

Shtengel, G., Galbraith, J. A., Galbraith, C. G., Lippincott-Schwartz, J., Gillette, J. M., Manley, S., Sougrat, R., Waterman, C. M., Kanchanawong, P., Davidson, M. W., Fetter, R. D. and Hess, H. F.

Interferometric fluorescent super-resolution microscopy resolves 3D cellular ultrastructure.  Proceedings of the National Academy of Sciences (USA) 106: 3125-3130 (2009).  The original report on three-dimensional imaging using photoactivation localization microscopy (PALM) with opposing objectives (iPALM) to achieve axial resolution in the tens of nanometers.

Huang, B., Wang, W., Bates, M. and Zhuang, X.

Three-dimensional super-resolution imaging by scholastic optical reconstruction microscopy.  Science 319: 810-822 (2008).  Xiaowei Zhuang and her colleagues utilize three-dimensional stochastic optical reconstruction microscopy (STORM) coupled to an optical astigmatism lens system to determine both axial and lateral positions of individual fluorophores with nanometer accuracy.

Juette, M. F., Gould, T. J., Lessard, M. D., Mlodzianoski, M. J., Nagpure, B. S., Bennett, B. T., Hess, S. T. and Bewersdorf, J.

Three-dimensional sub-100nm resolution fluorescence microscopy of thick samples.  Nature Methods 5: 527-540 (2008).  The authors introduce a three-dimensional superresolution technique named biplane (BP) FPALM that combines a double-plane detection scheme with fluorescence photoactivation localization microscopy (FPALM) to produce sub-diffraction resolution at high speed and sensitivity.

Pavani, S. R. P., Thompson, M. A., Biteen, J. S., Lord, S. J., Liu, N., Twieg, R. J., Piestun, R. and Moerner, W. E.

Three-dimensional, single-molecule fluorescence imaging beyond the diffraction limit by using a double-helix point spread function.  Proceedings of the National Academy of Sciences (USA) 106: 2995-2999 (2009).  A clever demonstration of three-dimensional single-molecule imaging using a design that features high and uniform Fisher information and has two dominant lobes in the image plane whose angular orientation rotates with the axial position of the emitter.

Punge, A., Rizzoli, S. O., Jahn, R., Wildanger, J. D., Meyer, L., Schonle, A., Kastrup, L. and Hell, S. W.

3D reconstruction of high-resolution STED microscope images.  Microscopy Research and Technique 71: 644-650 (2008).  Stefan Hell and colleagues describe three-dimensional STED of fixed samples that are fluorescently labeled, embedded in a polymer resin, and cut into thin sections to generate images with greater than 80-nanometer resolution.

Vaziri, A., Tang, J., Shroff, H. and Shank, C. V.

Multilayer three-dimensional super resolution imaging of thick biological samples.  Proceedings of the National Academy of Sciences (USA) 105: 20221-20226 (2008).  The authors combine photoactivation localization microscopy with two-photon temporal focusing to provide optical sectioning over an axial range of approximately 10-micrometers with 50-nanometer lateral resolution.

Middendorff, C. V., Egner, A., Geisler, C., Hell, S. W. and Schonle, A.

Isotropic 3D nanoscopy based on single emitter switching.  Optics Express 16: 20774-20788 (2008).  A sophisticated theoretical analysis of isotropic resolution enhancement in superresolution imaging based on switching and mathematically localizing individual emitters. The authors provide Monte-Carlo simulations using the Fisher information matrix.

van Oijen, A. M., Kohler, J., Schmidt, J., Muller, M. and Brakenhoff, G. J.

3-dimensional super-resolution by spectrally selective imaging.  Chemical Physics Letters 292: 183-187 (1998).  A unique approach to superresolution that employs the unique spectral properties of individual fluorophores to segregate emitters confined to the same diffraction-limited area. Lateral and axial resolutions of 40 and 100 nanometers, respectively, are achieved.