Abstract
Far-field fluorescence techniques based on the precise determination of object positions have the potential to circumvent the optical resolution limit of direct imaging given by diffraction theory. In order to use localization to obtain structural information far below the diffraction limit, the ‘point-like’ components of the structure have to be detected independently, even if their distance is lower than the conventional optical resolution limit. This goal can be achieved by exploiting various photo-physical properties of the fluorescence labeling (‘spectral signatures’). In first experiments, spectral precision distance microscopy/spectral position determination microscopy (SPDM) was limited to a relatively small number of components to be resolved within the observation volume. Recently, the introduction of photoconvertable molecules has dramatically increased the number of components which can be independently localized. Here, we present an extension of the SPDM concept, exploiting the novel spectral signature offered by reversible photobleaching of fluorescent proteins. In combination with spatially modulated illumination (SMI) microscopy, at the present stage, we have achieved an estimated effective optical resolution of approximately 20 nm in the lateral and 50 nm in the axial direction, or about 1/25th–1/10th of the exciting wavelength.
Article PDF
Similar content being viewed by others
Avoid common mistakes on your manuscript.
References
T. Cremer, C. Cremer, Nat. Rev. Genet. 2, 292–301 (2001)
E.A. Jares-Erijman, T.M. Jovin, Nat. Biotechnol. 21, 1387–1395 (2003)
J. Braga, J.M.P. Desterro, M. Carmo-Fonseca, Mol. Biol. Cell 15, 4749–4760 (2004)
C. Cremer, T. Cremer, Microsc. Acta 81, 31–44 (1978)
S.W. Hell, E.H.K. Stelzer, S. Lindek, C. Cremer, Opt. Lett. 19, 222–224 (1994)
P.E. Hänninen, S.W. Hell, J. Salo, E. Soini, C. Cremer, Appl. Phys. Lett. 66, 1698–1700 (1995)
A. Egner, S. Jakobs, S.W. Hell, Proc. Natl. Acad. Sci. USA 99, 3370–3375 (2002)
J. Bewersdorf, B.T. Bennett, K.L. Knight, Proc. Natl. Acad. USA 103, 18137–1814 (2006)
D. Baddeley, C. Carl, C. Cremer, Appl. Opt. 45, 7056–7064 (2006)
B. Albrecht, A. Schweitzer, A.V. Failla, P. Edelmann, C. Cremer, Appl. Opt. 41, 80–87 (2002)
A.V. Failla, A. Cavallo, C. Cremer, Appl. Opt. 4, 6651–6659 (2002)
A.V. Failla, B. Albrecht, U. Spoeri, A. Kroll, C. Cremer, Appl. Opt. 41, 7275–7283 (2002)
D. Baddeley, C. Batram, Y. Weiland, C. Cremer, U. Birk, Nat. Protoc. 2, 2640–2646 (2007)
R. Heintzmann, T. Jovin, C. Cremer, J. Opt. Soc. Am. A 19, 1599–1609 (2002)
S. Martin, A.V. Failla, U. Spöri, C. Cremer, A. Pombo, Mol. Biol. Cell 15, 2449–2455 (2004)
G. Hildenbrand, A. Rapp, U. Spoeri, Ch. Wagner, C. Cremer, M. Hausmann, Biophys. J. 88, 4312–4318 (2005)
H. Mathee, D. Baddeley, C. Wotzlaw, J. Fandrey, C. Cremer, U. Birk, Histochem. Cell Biol. 125, 75–82 (2006)
G. Donnert, J. Keller, R. Medda, M.A. Andrei, S.O. Rizzoli, R. Lührmann, R. Jahn, C. Eggeling, S.W. Hell, Proc. Natl. Acad. Sci. USA 103, 11440–11445 (2006)
S.W. Hell, I. Wichmann, Opt. Lett. 19, 780–782 (1994)
M.A. Schwentker, H. Bock, M. Hofmann, S. Jakobs, J. Bewersdorf, C. Eggeling, S.W. Hell, Microsc. Res. Tech. 29, 17262791 (2007)
H. Bornfleth, K. Sätzler, R. Eils, C. Cremer, J. Microsc. 189, 118–136 (1998)
A.M. van Oijen, J. Köhler, J. Schmidt, G.J. Brakenhoff, Chem. Phys. Lett. 292, 183–187 (1998)
T.D. Lacoste, X. Michalet, F. Pinaud, D.S. Chemla, A.P. Alivistatos, S. Weiss, Proc. Natl. Acad. Sci. USA 97, 9461–9466 (2000)
M. Schmidt, M. Nagorni, S.W. Hell, Rev. Sci. Instrum. 71, 2742–2745 (2000)
C. Cremer, A.V. Failla, B. Albrecht, US Patent 7,298,461 B2, filed 9 Oct. 2001
M. Heilemann, D.P. Herten, R. Heintzmann, C. Cremer, C. Müller, Ph. Tinnefeld, K.D. Weston, J. Wolfrum, Anal. Chem. 74, 3511–3517 (2002)
C. Cremer, M. Hausmann, J. Bradl, B. Rinke, German Patent Application No. 196.54. 824.1/DE, filed 23 Dec. 23 1996; European Patent EP 1997953660, 8 Apr. 1999; Japanese Patent JP 1998528237, 23 June 1999; United States Patent US 09331644, 25 Aug. 1999
C. Cremer, P. Edelmann, H. Bornfleth, G. Kreth, H. Muench, H. Luz, M. Hausmann, in Handbook of Computer Vision and Applications, vol. 3 ed. by B. Jähne, H. Haußecker, P. Geißler (Academic Press, San Diego, 1999), pp. 839—885
A. Esa, P. Edelmann, L. Trakthenbrot, N. Amariglio, G. Rechavi, M. Hausmann, C. Cremer, J. Microsc. 199, 96–105 (2000)
E. Betzig, G.H. Patterson, R. Sougrat, O.W. Lindwassser, S. Olenych, J.S. Bonifacino, M.W. Davidson, J. Lippincott-Schwart, H.F. Hess, Sciencexpress 313, 1642–1645 (2006)
S.T. Hess, T.P.K. Girirajan, M.D. Mason, Biophys. J. 91, 4258–4272 (2006)
M. Bates, B. Huang, G.T. Dempsey, X. Zhuang, Science 317, 1749–1753 (2007)
C. Geisler, A. Schönle, C. von Middendorf, H. Bock, C. Eggeling, A. Egner, S.W. Hell, Appl. Phys. A 88, 223–226 (2007)
B. Huang, W. Wang, M. Bates, X. Zhuang, Science 319, 810–813 (2008)
A.K. Lidke, B. Rieger, T.M. Jovin, R. Heintzmann, Opt. Express 13, 7052–7062 (2005)
A. Esa, A.E. Coleman, P. Edelmann, S. Silva, C. Cremer, S. Janz, Cancer Genet. Cytogenet. 127, 168–173 (2001)
J. Rauch, T.A. Knoch, I. Solovei, K. Teller, S. Stein, K. Buiting, B. Horsthemke, J. Langowski, T. Cremer, M. Hausmann, C. Cremer, Differentiation 76, 66–82 (2008)
T.B. McAnaney, W. Zeng, C.F.E. Doe, N. Bhanji, S. Wakelin, D.S. Pearson, P. Abbyad, X. Shi, S.G. Boxer, C.R. Bagshaw, Biochemistry 44, 5510–5524 (2005)
C. Eggeling, J. Widengren, R. Rigler, C.A.M. Seidel, Anal. Chem. 70, 2651–2659 (1998)
J. Lippincott-Schwartz, N. Altan-Bonnet, G.H. Patterson, Nat. Biotechnol. 20, 87–90 (2002)
R.Y. Tsien, Ann. Rev. Biochem. 67, 509–544 (1998)
T. Nagai, K. Ibata, E.S. Park, M. Kubota, K. Mikoshiba, A. Miyawaki, Genetics 122, 19–27 (1989)
M. Schmidt, M. Nagorni, S.W. Hell, Rev. Sci. Instrum. 71, 2742–2745 (2000)
J. Enderlein, E. Toprak, P.R. Selvin, Opt. Express 14, 8111–8120 (2006)
F. Aguet, D. Van De Ville, M. Unser, Opt. Express 13, 10503–10522 (2005)
M.F. Juette, T.J. Gould, M.D. Lessard, M.J. Mlodzianoski, B.S. Nagpure, B.T. Bennett, S.T. Hess, J. Bewersdorf, Nat. Methods (2008)
J. Reymann, D. Baddeley, P. Lemmer, W. Stadter, T. Jegou, K. Rippe, C. Cremer, U. Birk, Chromosome Res. 16, 367–382 (2008)
P. Edelmann, C. Cremer, Proc. SPIE 3921, 313–320 (2000)
S. Fenz, S.H. Mathee, G. Kreth, D. Baddeley, Y. Weiland, J. Schwarz-Finsterle, C.G. Cremer, U.J. Birk, Proc. SPIE 6630, 663002-1 (2007)
B. Albrecht, A.V. Failla, R. Heintzmann, C. Cremer, J. Biomed. Opt. 6, 292–299 (2001)
D.H. Burns, J.B. Callis, G.D. Christian, E.R. Davidson, Appl. Opt. 24, 154–161 (1985)
E. Betzig, Opt. Lett. 20, 237–239 (1995)
R. Heintzmann, C. Cremer, Proc. SPIE 3658, 185–195 (1999)
J.T. Frohn, H.F. Knapp, A. Stemmer, Proc. Natl. Acad. Sci. USA 97, 7232–7236 (2000)
M. Gustafsson, L. Shao, P.M. Carlton, C.J.R. Wang, I.N. Golubovskaya, W.Z. Cande, D.A. Agard, J.W. Sedat, Biophys. J., 13 March (2008)
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
About this article
Cite this article
Lemmer, P., Gunkel, M., Baddeley, D. et al. SPDM: light microscopy with single-molecule resolution at the nanoscale. Appl. Phys. B 93, 1–12 (2008). https://doi.org/10.1007/s00340-008-3152-x
Received:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s00340-008-3152-x