Fluorescence nanoscopy

The rhodamine derivative 55 serves as a chemical marker for fluorescence nanoscopy. Fluorescence at 580 nm can be switched on by UV (1 photon absorption) or red light (2 photon... 

STED microscopy has been applied to study the fate of synaptic vesicle proteins during exocytosis. The study revealed that, when the vesicles fuse with the presynaptic membrane, the protein synaptotagmin I forms integral nanosized clusters at the synapse [89]. Since the clusters are similar in size and molecular density as integral vesicles, the study indicated, that entire protein clusters are taken up from the neuronal membrane when forming new vesicles. By the same token, this initial application of far-held fluorescence nanoscopy... 

All diffraction-unlimited nanoscopy methods can provide improved axial resolution even when implemented with a single lens [77,83,120,131,132[. However, because it starts out from less favorable values, the -resolution usually remains worse than its focal plane counterpart. The coherent use of opposing lenses pioneered in 4Pi microscopy and FM, however, facilitates an independent resolution improvement factor by 3-7-fold along the optic axis as has already been demonstrated with STED [83,84,87]. In the stochastic single molecule switching modalities, a similar gain in resolution will take place by the coherent use of opposing lenses [133]. Thus, while 4Pi microscopy and FM did not break the diffraction barrier, they remain cornerstones of far-field fluorescence nanoscopy in the future. 

S.W. Hell, Toward fluorescence nanoscopy. Nature Biotechnol. 21(11), 1347-1355 (2003)... 

H. Bock et ah. Two-color far-fleld fluorescence nanoscopy based on photoswitching emitters. Appl. Phys. B 88, 161-165 (2007)... 

A. Egner et ah. Fluorescence nanoscopy in whole cells by asnychronous localization of photoswitching emitters. Biophys. J. 93, 3285-3290 (2007)... 

J. Foiling et ah. Fluorescence nanoscopy by ground-state depletion and singlemolecule return. Nat. Meth. 5, 943-945 (2008)... 

M. Bossi et ah. Multi-color far-field fluorescence nanoscopy through isolated detection of distinct molecular species. Nano Lett. 8(8), 2463-2468 (2008)... 

A. Egner, C. Geisler, C. von Middendorff, H. Bock, D. Wenzel, R. Medda, M. Andresen, A.C. Stiel, S. Jakobs, C. Eggehng, et al.. Fluorescence nanoscopy in whole cells by asynchronous localization of photoswitching emitters. Biophys. J. 93, 3285-3290 (2007)... 

H. Bock, C. Geisler, C.A. Wurm, C. Von Middendorff, S. Jakobs, A. Schonle, A. Egner, S.W. Hell, C. Eggehng, Two-color far-held fluorescence nanoscopy based on photoswitchable emitters. Appl. Phys. B 88, 161-165 (2007)... 

Photoswitching emission with rhodamine spiroamides for super-resolution fluorescence nanoscopies 13IJC267. 

FoUing J, Bossi M, Bock H et al (2008) Fluorescence nanoscopy by ground-state depletion and single-molecule retmn. Nat Methods 5 943-945... 

Testa 1, Wurm CA, Medda R et al (2010) Multicolor fluorescence nanoscopy in fixed and living cells by exciting conventional fluorophores... 

To switch fluorescence, it takes two states a bright (fluorescent) state A and a dark state B that are connected by a transition which is the actual switch. Several states in a fluorophore are suitable for such transitions (Fig. 19.3). The fluorescent singlet state and the ground state used in STED microscopy is the most basic and obvious pair of bright and dark states [39], but other examples will be given later, when discussing various nanoscopy implementations. 

B. Hein, K. WiUig, S.W. HeU, Stimulated emission depletion (STED) nanoscopy of a fluorescent protein - labeled organelle inside a living cell. Proc. Natl. Acad. Sci. USA 105(38), 14271-14276 (2008)... 

Normanno, D., van Zanten, X, and Garcia-Parajo, M. (2010) in Nanoscopy and Multidimensional Optical Fluorescence Microscopy (ed. A. Diaspro), CRC Press, Boca Raton, FL, pp. 18-1-18-28. 

Ear-field nanoscopic measuring technique Ear-field optical nanoscopic measuring technique Laser-induced fluorescence photobleaching anemometer Nanoscopy Stimulated emission depletion... 

An important trend in microscopy has been towards chemical distribution and state mapping, such as provided by infrared (IR), ultraviolet (UV), Raman (and fluorescence) microscopies. All of these allow detailed viewing of a specific chemical species either by natural contrast or by some labelling mechanism. Optical nanoscopy is approaching. 

---