We introduce a microscopic method that determines quantitative optical properties beyond the optical diffraction limit and allows direct imaging of unstained living biological specimens. In established holographic microscopy, complex fields are measured using interferometric detection, allowing diffraction-limited phase measurements. Here, we show that non-invasive optical nanoscopy can achieve a lateral resolution of 90 nm by using a quasi-2π-holographic detection scheme and complex deconvolution. We record holograms from different illumination directions on the sample plane and observe subwavelength tomographic variations of the specimen. Nanoscale apertures serve to calibrate the tomographic reconstruction and to characterize the imaging system by means of the coherent transfer function. This gives rise to realistic inverse filtering and guarantees true complex field reconstruction. The observations are shown for nanoscopic porous cell frustule (diatoms), for the direct study of bacteria (Escherichia coli), and for a time-lapse approach to explore the dynamics of living dendritic spines (neurones).
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Hell, S. W. Far-field optical nanoscopy. Science 316, 1153–1158 (2007).
Gustafsson, M. G. L. Surpassing the lateral resolution limit by a factor of two using structured illumination microscopy. J. Microsc. 198, 82–87 (2000).
Rappaz, B. et al. Measurement of the integral refractive index and dynamic cell morphometry of living cells with digital holographic microscopy. Opt. Express 13, 9361–9373 (2005).
Charrière, F. et al. Living specimen tomography by digital holographic microscopy: morphometry of testate amoeba. Opt. Express 14, 7005–7013 (2006).
Choi, W. et al. Tomographic phase microscopy. Nature Methods 4, 717–719 (2007).
Kemper, B. et al. Investigation of living pancreas tumor cells by digital holographic microscopy. J. Biomed. Opt. 11, 034005 (2006).
Pavillon, N. et al. Early cell death detection with digital holographic microscopy. PLoS ONE 7, e30912 (2012).
Mir, M. et al. Visualizing Escherichia coli sub-cellular structure using sparse deconvolution spatial light interference tomography. PLoS ONE 7, e39816 (2012).
Rappaz, B., Depeursinge, C. & Marquet, P. in Biomedical Optical Phase Microscopy and Nanoscopy (eds Shaked, N. T., Zalevskey, Z. & Satterwhite, L.) Ch. 5.1, 71–95 (Elsevier, 2012).
Cuche, E., Marquet, P. & Depeursinge, C. Simultaneous amplitude-contrast and quantitative phase-contrast microscopy by numerical reconstruction of Fresnel off-axis holograms. Appl. Opt. 38, 6994–7001 (1999).
Gureyev, T. E., Roberts, A. & Nugent, K. A. Phase retrieval with the transport-of-intensity equation: matrix solution with use of Zernike polynomials. J. Opt. Soc. Am. A 12, 1932–1941 (1995).
Popescu, G. in Methods in Nano Cell Biology vol. 90 (eds Jena, B. P.) 87–115 (Academic, 2008).
Bon, P., Maucort, G., Wattellier, B. & Monneret, S. Quadriwave lateral shearing interferometry for quantitative phase microscopy of living cells. Opt. Express 17, 13080–13094 (2009).
Lauer, V. New approach to optical diffraction tomography yielding a vector equation of diffraction tomography and a novel tomographic microscope. J. Microsc. 205, 165–176 (2002).
Paturzo, M. et al. Super-resolution in digital holography by a two-dimensional dynamic phase grating. Opt. Express 16, 17107–17118 (2008).
Mico, V., Zalevsky, Z., Ferreira, C. & García, J. Superresolution digital holographic microscopy for three-dimensional samples. Opt. Express 16, 19260–19270 (2008).
Debailleul, M., Georges, V., Simon, B., Morin, R. & Haeberle, O. High-resolution three-dimensional tomographic diffractive microscopy of transparent inorganic and biological samples. Opt. Lett. 34, 79–81 (2009).
Kuznetsova, Y., Neumann, A. & Brueck, S. R. J. Solid-immersion imaging interferometric nanoscopy to the limits of available frequency space. J. Opt. Soc. Am. A 29, 772–781 (2012).
Devaney, A. A filtered backpropagation algorithm for diffraction tomography. Ultrason. Imaging 4, 336–350 (1982).
Cotte, Y., Toy, M. F., Pavillon, N. & Depeursinge, C. Microscopy image resolution improvement by deconvolution of complex fields. Opt. Express 18, 19462–19478 (2010).
Cotte, Y. et al. Realistic 3D coherent transfer function inverse filtering of complex fields. Biomed. Opt. Express 2, 2216–2230 (2011).
Sheppard, C. J. R. & Gu, M. Imaging by a high aperture optical-system. J. Mod. Opt. 40, 1631–1651 (1993).
Vertu, S., Flagge, J., Delaunay, J.-J. & Haeberle, O. Improved and isotropic resolution in tomographic diffractive microscopy combining sample and illumination rotation. Cent. Eur. J. Phys. 9, 969–974 (2011).
den Dekker, A. J. & van den Bos, A. Resolution: a survey. J. Opt. Soc. Am. A 14, 547–557 (1997).
Cotte, Y., Toy, M. F. & Depeursinge, C. Beyond the lateral resolution limit by phase imaging. J. Biomed. Opt. 16, 106007 (2011).
Montfort, F. et al. Purely numerical compensation for microscope objective phase curvature in digital holographic microscopy: influence of digital phase mask position. J. Opt. Soc. Am. A 23, 2944–2953 (2006).
Choi, Y., Yang, T. D., Lee, K. J. & Choi, W. Full-field and single-shot quantitative phase microscopy using dynamic speckle illumination. Opt. Lett. 36, 2465–2467 (2011).
Hildebrand, M. et al. Nanoscale control of silica morphology and three-dimensional structure during diatom cell wall formation. J. Mater. Res. 21, 2689–2698 (2006).
Sheppard, C. J. R., Kou, S. S. & Depeursinge, C. Reconstruction in interferometric synthetic aperture microscopy: comparison with optical coherence tomography and digital holographic microscopy. J. Opt. Soc. Am. A 29, 244–250 (2012).
Portera-Cailliau, C., Pan, D. T. & Yuste, R. Activity-regulated dynamic behavior of early dendritic protrusions: evidence for different types of dendritic filopodia. J. Neurosci. 23, 7129–7142 (2003).
Tao, H.-z. et al. Selective presynaptic propagation of long-term potentiation in defined neural networks. J. Neurosci. 20, 3233–3243 (2000).
This work was funded by the Swiss National Science Foundation (SNSF, grant no. 205 320–130 543) and EPFL (innovation grant INNO 12-14). The authors acknowledge the Center of MicroNano-Technology (CMI) for cooperation regarding its research facilities. The authors give special thanks to M. Hildebrand for providing diatom samples, and to L. Pollaro for preparing the E. coli samples. The authors also thank P. Lara Rodrigo, C-M. Cotte and Cemico GmbH for assistance with graphics. Finally, the authors thank E. Cuche, CTO of Lyncée Tec, for helpful discussions and suggestions.
Yann Cotte, Nicolas Pavillon and Christian Depeursinge are named inventors on international patent WO/2011/121523 (publication date 06.10.2011, international filing date 28.03.2011), which is related to the techniques described in this Letter.
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Cotte, Y., Toy, F., Jourdain, P. et al. Marker-free phase nanoscopy. Nature Photon 7, 113–117 (2013). https://doi.org/10.1038/nphoton.2012.329
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