Plenary Special Lectures doi:10.1017/S1431927611000705 NANOSCOPY WITH FOCUSED LIGHT


StefanW.Hell Max Planck Institute for Biophysical Chemistry, Department of NanoBiophotonics, 37077 Göttingen & German Cancer Research Center (DKFZ), Optical Nanoscopy Division, 69120 Heidelberg, Germany


For more than a century, it has been generally accepted that the resolution of a lens-based optical microscope is limited to about d  l/~2NA!. 200 nm in the focal plane and .500 nm along the optic axis, with NA denoting the numerical aperture of the lens and l the wavelength of light. The discovery in the 1990s that elementary transitions between the states of a fluorophore can be used to eliminate the limiting role of diffraction has led to light microscopy concepts with resolution on the nanometer scale @1, 2#. Currently, all existing and successfully applied nanoscopy methods share a common enabling element: they switch the fluorescence capability of fluorophores on and off, so that adjacent features are registered sequentially in time @3, 4#. For example, in a typical Stimulated Emission Depletion ~STED! microscope @1#, the fluorophores are switched off


registration. The resolution is now given by the smaller diameter d  l/~2NAM1I/Is! of this area in which the fluorophores are still fluorescent. I is the intensity of the STED beam, which, for I 


~ kept dark! by overlapping the excitation beam with a de-exciting ~STED! beam which effectively confines the fluorophores to the ground state everywhere in the focal region except at a tiny area where the STED beam is close to zero. Fluorophores that are located in this subdiffraction-sized smaller area are registered. Scanning the beams further in space registers those fluorophores that had been switched off initially. An image of the whole object is assembled by sequential


 Is, entails d r 0, meaning that the


resolution is conceptually no longer limited by l. An unprecedented, all-physics-based, far-field optical resolution of ,6nm was realized with nitrogen-vacancy centers in diamonds ~Fig. 1!@5#. The combination with 4Pi microscopy enabled an axial spatial resolution ,35 nm @2#. STED microscopy has been used to investigate the fate of synaptic vesicle proteins after exocytosis @6#, to reveal nanoscale patterns of synaptophysin on endosomes @7#, or to study the nanoscale distributions of TOM-complexes in the outer membrane of mitochondria @8#, thus demonstrating the potential of emerging “fluorescence nanoscopy” for the life sciences.A video-rate STED microscope was used to describe the mobility of vesicles inside the axons of cultured living neurons @9#. Live-cell STED microscopy has also been used to image activity-dependent morphological plasticity of dendritic spines @10#, while it also revealed that single sphingolipids, but not phospholipids, are transiently ~,10 ms! and locally ~,20 nm! trapped in a living cell membrane, mediated by cholesterol @11#.Multicolor operation @12, 13# facilitates colocalization of biomolecules. Importantly, the concept of STED microscopy has been expanded to lowintensity operation by switching the fluorophore


to a long-lived dark ~triplet! state or between a “fluorescence activated” and a “deactivated” ~conformational! state @2# as encountered in switchable fluorescent proteins @14#.More recent but seminal nanoscopy schemes such as PALM,STORM,and also GSDIM switch the molecules individually and stochastically. Switching is realized to a state that emits m .


. 1 detectable


photons in a rowbefore returning to a dark state, allowing the calculation of their position. These single fluorophore switching concepts @15–19# require only one switching cycle @3, 4# per fluorophore. This greatly extends the power of the switching concept for subdiffraction separation. These schemes have also been expanded tomulticolor operation ~Fig. 2! and combined with 4Pi-microscopy to also boost the axial resolution @20#. Altogether, lens-based optical nanoscopy is an unexpected and fascinating development in the physical sciences that is poised to impact many areas, in particular the life sciences.


References @1# S.W. Hell et al., Opt. Lett. 19 ~1994! 780. @2# S.W. Hell, Nature Biotech. 21 ~2003! 1347. @3# S.W. Hell, Science 316 ~2007! 1153. @4# S.W. Hell, Nature Meth. 6 ~2009! 24. @5# E. Rittweger et al., Nature Phot. 3 ~2009! 144. @6# K.I.Willig et al., Nature 440 ~2006! 935. @7# R. Schmidt et al., Nature Meth. 5 ~2008! 539. @8# G. Donnert et al., PNAS 103 ~2006! 11440. @9# V.Westphal et al., Science 320 ~2008! 246. @10# U.V. Nagerl et al., PNAS 105 ~2008! 18982.


32


@11# C. Eggeling et al., Nature 457 ~2009! 1159. @12# I. Testa et al., Biophys. J. 99 ~2010! 2686. @13# J. Bückers et al., Opt. Express 19 ~2011! 3130. @14# M. Hofmann et al., PNAS 102 ~2005! 17565. @15# E. Betzig et al., Science 313 ~2006! 1642. @16# M.J. Rust et al., Nature Meth. 3 ~2006! 793. @17# S.T. Hess et al., Biophys. J. 91 ~2006! 4258. @18# A. Egner et al., Biophys. J. 93 ~2007! 3285. @19# J. Fölling et al., Nature Meth. 5 ~2008! 943. @20# D. Acquino et al., Nature Meth. 8 ~2011! 353.


Microscopy Microanalysis


AND © MICROSCOPY SOCIETY OF AMERICA 2011


Page 1  |  Page 2  |  Page 3  |  Page 4  |  Page 5  |  Page 6  |  Page 7  |  Page 8  |  Page 9  |  Page 10  |  Page 11  |  Page 12  |  Page 13  |  Page 14  |  Page 15  |  Page 16  |  Page 17  |  Page 18  |  Page 19  |  Page 20  |  Page 21  |  Page 22  |  Page 23  |  Page 24  |  Page 25  |  Page 26  |  Page 27  |  Page 28  |  Page 29  |  Page 30  |  Page 31  |  Page 32  |  Page 33  |  Page 34  |  Page 35  |  Page 36  |  Page 37  |  Page 38  |  Page 39  |  Page 40  |  Page 41  |  Page 42  |  Page 43  |  Page 44  |  Page 45  |  Page 46  |  Page 47  |  Page 48  |  Page 49  |  Page 50  |  Page 51  |  Page 52  |  Page 53  |  Page 54  |  Page 55  |  Page 56  |  Page 57  |  Page 58  |  Page 59  |  Page 60  |  Page 61  |  Page 62  |  Page 63  |  Page 64  |  Page 65  |  Page 66  |  Page 67  |  Page 68  |  Page 69  |  Page 70  |  Page 71  |  Page 72  |  Page 73  |  Page 74  |  Page 75  |  Page 76  |  Page 77  |  Page 78  |  Page 79  |  Page 80  |  Page 81  |  Page 82  |  Page 83  |  Page 84  |  Page 85  |  Page 86  |  Page 87  |  Page 88  |  Page 89  |  Page 90  |  Page 91  |  Page 92  |  Page 93  |  Page 94  |  Page 95  |  Page 96  |  Page 97  |  Page 98  |  Page 99  |  Page 100  |  Page 101  |  Page 102  |  Page 103  |  Page 104  |  Page 105  |  Page 106  |  Page 107  |  Page 108  |  Page 109  |  Page 110  |  Page 111  |  Page 112  |  Page 113  |  Page 114  |  Page 115  |  Page 116  |  Page 117  |  Page 118  |  Page 119  |  Page 120  |  Page 121  |  Page 122  |  Page 123  |  Page 124  |  Page 125  |  Page 126  |  Page 127  |  Page 128  |  Page 129  |  Page 130  |  Page 131  |  Page 132  |  Page 133  |  Page 134  |  Page 135  |  Page 136  |  Page 137  |  Page 138  |  Page 139  |  Page 140  |  Page 141  |  Page 142  |  Page 143  |  Page 144  |  Page 145  |  Page 146  |  Page 147  |  Page 148  |  Page 149  |  Page 150  |  Page 151  |  Page 152  |  Page 153  |  Page 154  |  Page 155  |  Page 156  |  Page 157  |  Page 158  |  Page 159  |  Page 160  |  Page 161  |  Page 162  |  Page 163  |  Page 164  |  Page 165  |  Page 166  |  Page 167  |  Page 168  |  Page 169  |  Page 170  |  Page 171  |  Page 172  |  Page 173  |  Page 174  |  Page 175  |  Page 176  |  Page 177  |  Page 178  |  Page 179  |  Page 180  |  Page 181  |  Page 182  |  Page 183  |  Page 184  |  Page 185  |  Page 186  |  Page 187  |  Page 188  |  Page 189  |  Page 190  |  Page 191  |  Page 192  |  Page 193  |  Page 194  |  Page 195  |  Page 196  |  Page 197  |  Page 198  |  Page 199  |  Page 200  |  Page 201  |  Page 202  |  Page 203  |  Page 204