Abstract
Lens-based or far-field fluorescence microscopy is a very popular technique for investigating the living cell. However, the spatial resolution of its standard versions is limited to about 200 nm due to diffraction, impeding the imaging of molecular assemblies at smaller scales. The turn of the twenty-first century has witnessed the advent of far-field fluorescence super-resolution microscopy or nanoscopy, a fluorescence microscopy featuring a spatial resolution down to molecular scales. STED microscopy was the first of such nanoscopy techniques, but was for a long time considered as a very complex technique, hard to apply in everyday biological research. Based on developments in label and laser technology, recent years have however seen major improvements of the STED nanoscopy approach, one of which is gated continuous-wave STED (gCW-STED) microscopy. gCW-STED microscopy reduces complexity by combining STED laser operating in CW with pulsed excitation and time-gated photon detection. Here, we describe the physical principles of gCW-STED, formulate the theoretical framework which characterizes its main benefits and limitations, as well as show experimental data.
This is a preview of subscription content, log in via an institution.
Buying options
Tax calculation will be finalised at checkout
Purchases are for personal use only
Learn about institutional subscriptionsReferences
Masters BR (2010) The development of fluorescence microscopy. Wiley, Chichester
Cella Zanacchi F, Bianchini P, Vicidomini G (2014) Fluorescence microscopy in the spotlight. Microsc Res Tech 77(7):479–482
Abbe E (1873) Beiträge zur theorie des mikroskops und der mikroskopischen wahrnehmung. Archiv für Mikroskopische Anatomie 9:413–468
Rost FWD (1995) Fluorescence microscopy, vol 2. Cambridge University Press, Cambridge
Pohl DW, Denk W, Lanz M (1984) Optical stethoscopy: image recording with resolution lambda/20. Appl Phys Lett 44:651–653
Mivelle M, Van Zanten TS, Manzo C, Garcia-Parajo MF (2014) Nanophotonic approaches for nanoscale imaging and single-molecule detection at ultrahigh concentrations. Microsc Res Tech 77(7):537–545
Hell SW, Wichmann J (1994) Breaking the diffraction resolution limit by stimulated emission: stimulated-emission-depletion fluorescence microscopy. Opt Lett 19(11):780–782
Hell SW, Kroug M (1995) Ground-state depletion fluorescence microscopy, a concept for breaking the diffraction resolution limit. Appl Phys B 60:495–497
Hell SW (2009) Microscopy and its focal switch. Nat Methods 6(1):24–32
Diaspro A (ed) (2009) Nanoscopy and multidimensional optical fluorescence microscopy. Chapman & Hall, New York
Huang B, Babcock H, Zhuang X (2010) Breaking the diffraction barrier: super-resolution imaging of cells. Cell 143(7):1047–1058
Hell SW, Jakobs S, Kastrup L (2003) Imaging and writing at the nanoscale with focused visible light through saturable optical transitions. Appl Phys A Mater Sci Process 77(7):859–860
Hell SW (2007) Far-field optical nanoscopy. Science 316(5828):1153–1158
Eggeling C, Heilemann M (2014) Editorial overview: molecular imaging. Curr Opin Chem Biol 20:v–vii
Eggeling C, Willig KI, Barrantes FJ (2013) STED microscopy of living cells: new frontiers in membrane and neurobiology. J Neurochem 126(2):203–212
Blom H, Widengren J (2014) STED microscopy: towards broadened use and scope of applications. Curr Opin Chem Biol 20:127–133
Klar TA, Hell SW (1999) Subdiffraction resolution in far-field fluorescence microscopy. Opt Lett 24(14):954–956
Klar TA, Jakobs S, Dyba M, Egner A, Hell SW (2000) Fluorescence microscopy with diffraction resolution barrier broken by stimulated emission. Proc Natl Acad Sci U S A 97(15):8206–8210
Clausen MP, Galiani S, Bernardino de la Serna J, Fritzsche M, Chojnacki J, Gehmlich K, Lagerholm BC, Eggeling C (2013) Pathways to optical STED microscopy. NanoBioImaging 1(1):1–12
Dyba M, Hell SW (2003) Photostability of a fluorescent marker under pulsed excited-state depletion through stimulated emission. Appl Optics 42(25):5123–5129
J-i H, Fron E, Dedecker P, Janssen KPF, Li C, Müllen K, Harke B, Bückers J, Hell SW, Hofkens J (2010) Spectroscopic rationale for efficient stimulated-emission depletion microscopy fluorophores. J Am Chem Soc 132(14):5021–5023
Leutenegger M, Eggeling C, Hell SW (2010) Analytical description of STED microscopy performance. Opt Express 18(25):26417–26429
Vicidomini G, Moneron G, Eggeling C, Rittweger E, Hell SW (2012) STED with wavelengths closer to the emission maximum. Opt Express 20(5):5225–5236
Willig KI, Rizzoli SO, Westphal V, Jahn R, Hell SW (2006) STED microscopy reveals that synaptotagmin remains clustered after synaptic vesicle exocytosis. Nature 440(7086):935–939
Wildanger D, Rittweger E, Kastrup L, Hell SW (2008) STED microscopy with a supercontinuum laser source. Opt Express 16(13):9614–9621
Bückers J, Wildanger D, Vicidomini G, Kastrup L, Hell SW (2011) Simultaneous multi-lifetime multi-color STED imaging for colocalization analyses. Opt Express 19(4):3130–3143
Galiani S, Harke B, Vicidomini G, Lignani G, Benfenati F, Diaspro A, Bianchini P (2012) Strategies to maximize the performance of a STED microscope. Opt Express 20(7):7362–7374
Rankin BR, Hell SW (2009) STED microscopy with a MHz pulsed stimulated-Raman-scattering source. Opt Express 17(18):15679–15684
Rittweger E, Han KY, Irvine SE, Eggeling C, Hell SW (2009) STED microscopy reveals crystal colour centres with nanometric resolution. Nat Photonics 3:144–147
Schrof S, Staudt T, Rittweger E, Wittenmayer N, Dresbach T, Engelhardt J, Hell SW (2011) STED nanoscopy with mass-produced laser diodes. Opt Express 19(9):8066–8072
Göttfert F, Wurm CA, Mueller V, Berning S, Cordes VC, Honigmann A, Hell SW (2013) Coaligned dual-channel STED nanoscopy and molecular diffusion analysis at 20 nm resolution. Biophys J 105:L01–L03
Willig KI, Harke B, Medda R, Hell SW (2007) STED microscopy with continuous wave beams. Nat Methods 4(11):915–918
Honigmann A, Eggeling C, Schulze M, Lepert A (2012) Super-resolution STED microscopy advances with yellow CW OPSL. Laser Focus World 48(1):75–79
Honigmann A, Mueller V, Fernando UP, Eggeling C, Sperling J (2013) Simplifying STED microscopy of photostable red-emitting labels. Laser + Potonik 5:40–42
Coto Hernàndez I, d’Amora M, Diaspro A, Vicidomini G (2014) Influence of laser intensity noise on gated CW-STED microscopy. Laser Phys Lett 11(9):095603
Moffitt JR, Osseforth C, Michaelis J (2011) Time-gating improves the spatial resolution of STED microscopy. Opt Express 19(5):4242
Vicidomini G, Moneron G, Han KY, Westphal V, Ta H, Reuss M, Engelhardt J, Eggeling C, Hell SW (2011) Sharper low-power STED nanoscopy by time gating. Nat Methods 8(7):571–573
Vicidomini G, Schönle A, Ta H, Han KY, Moneron G, Eggeling C, Hell SW (2013) STED nanoscopy with time-gated detection: theoretical and experimental aspects. PLoS One 8(1):e54421
Leutenegger M, Rao R, Leitgeb RA, Lasser T (2006) Fast focus field calculations. Opt Express 14(23):11277–11291
Westphal V, Hell SW (2005) Nanoscale resolution in the focal plane of an optical microscope. Phys Rev Lett 94:143903
Vicidomini G, Coto Hernández I, d’Amora M, Cella Zanacchi F, Bianchini P, Diaspro A (2014) Gated CW-STED microscopy: a versatile tool for biological nanometer scale investigation. Methods 66(2):124–130
Moneron G, Medda R, Hein B, Giske A, Westphal V, Hell SW (2010) Fast STED microscopy with continuous wave fiber lasers. Opt Express 18(2):1302–1309
Eggeling C, Ringemann C, Medda R, Schwarzmann G, Sandhoff K, Polyakova S, Belov VN, Hein B, von Middendorff C, Schonle A, Hell SW (2009) Direct observation of the nanoscale dynamics of membrane lipids in a living cell. Nature 457(7233):1159–1162
Westin L, Reuss M, Lindskog M, Aperia A, Brismar H (2014) Nanoscopic spine localization of Norbin, an mGluR5 accessory protein. BMC Neurosci 15(1):45
Eggeling C, Widengren J, Rigler R, Seidel CAM (1998) Photobleaching of fluorescent dyes under conditions used for single-molecule detection: evidence of two-step photolysis. Anal Chem 70:2651–2659
Bülter A (2014) Single-photon counting detectors for the visible range between 300 and 1,000 nm. Springer Ser Fluoresc. doi:10.1007/4243_2014_63
Wahl M (2014) Modern TCSPC electronics: principles and acquisition modes. Springer Ser Fluoresc. doi:10.1007/4243_2014_62
Ronzitti E, Harke B, Diaspro A (2013) Frequency dependent detection in a STED microscope using modulated excitation light. Opt Express 21(1):210–219
Coto Hernàndez I, Peres C, Cella Zanacchi F, d’Amora M, Christodoulou S, Bianchini P, Diaspro A, Vicidomini G (2014) A new filtering technique for removing anti-stokes emission background in gated CW-STED microscopy. J Biophotonics 7(6):376–380
Wang Y, Kuang C, Gu Z, Xu Y, Li S, Hao X, Liu X (2013) Time-gated stimulated emission depletion nanoscopy. Opt Eng 52(9):093107
Bertero M, Boccacci P, Desiderá G, Vicidomini G (2009) Image deblurring with Poisson data: from cells to galaxies. Inverse Probl 25(12):123006
Zanella R, Zanghirati G, Cavicchioli R, Zanni L, Boccacci P, Bertero M, Vicidomini G (2013) Towards real-time image deconvolution: application to confocal and STED microscopy. Sci Rep 3:2523
Donnert G, Eggeling C, Hell SW (2007) Major signal increase in fluorescence microscopy through dark-state relaxation. Nat Methods 4(1):81–86
Donnert G, Eggeling C, Hell SW (2009) Triplet-relaxation microscopy with bunched pulsed excitation. Photochem Photobiol 8:481–485
Donnert G, Keller J, Medda R, Andrei MA, Rizzoli SO, Lührmann R, Jahn R, Eggeling C, Hell SW (2006) Macromolecular-scale resolution in biological fluorescence microscopy. Proc Natl Acad Sci 103(31):11440–11445
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2014 Springer International Publishing Switzerland
About this chapter
Cite this chapter
Vicidomini, G., Coto Hernàndez, I., Diaspro, A., Galiani, S., Eggeling, C. (2014). The Importance of Photon Arrival Times in STED Microscopy. In: Kapusta, P., Wahl, M., Erdmann, R. (eds) Advanced Photon Counting. Springer Series on Fluorescence, vol 15. Springer, Cham. https://doi.org/10.1007/4243_2014_73
Download citation
DOI: https://doi.org/10.1007/4243_2014_73
Published:
Publisher Name: Springer, Cham
Print ISBN: 978-3-319-15635-4
Online ISBN: 978-3-319-15636-1
eBook Packages: Chemistry and Materials ScienceChemistry and Material Science (R0)