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. 1998 Sep;75(3):1513–1521. doi: 10.1016/S0006-3495(98)74070-6

Continuous wave two-photon scanning near-field optical microscopy.

A K Kirsch 1, V Subramaniam 1, G Striker 1, C Schnetter 1, D J Arndt-Jovin 1, T M Jovin 1
PMCID: PMC1299826  PMID: 9726953

Abstract

We have implemented continuous-wave two-photon excitation of near-UV absorbing fluorophores in a scanning near-field optical microscope (SNOM). The 647-nm emission of an Ar-Kr mixed gas laser was used to excite the UV-absorbing DNA dyes DAPI, the bisbenzimidazole Hoechst 33342, and ethidium bromide in a shared aperture SNOM with uncoated fiber tips. Polytene chromosomes of Drosophila melanogaster and the nuclei of 3T3 Balb/c cells labeled with these dyes were readily imaged. The fluorescence intensity showed the expected nonlinear (second order) dependence on the excitation power in the range of 8-180 mW. We measured the fluorescence intensity as a function of the tip-sample displacement in the direction normal to the sample surface in the single- and two-photon excitation modes (SPE, TPE). The fluorescence intensity decayed faster in TPE than in SPE.

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Selected References

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  1. Akerman B., Tuite E. Single- and double-strand photocleavage of DNA by YO, YOYO and TOTO. Nucleic Acids Res. 1996 Mar 15;24(6):1080–1090. doi: 10.1093/nar/24.6.1080. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Arndt-Jovin D. J., Jovin T. M. Analysis and sorting of living cells according to deoxyribonucleic acid content. J Histochem Cytochem. 1977 Jul;25(7):585–589. doi: 10.1177/25.7.70450. [DOI] [PubMed] [Google Scholar]
  3. Barcellona M. L., Cardiel G., Gratton E. Time-resolved fluorescence of DAPI in solution and bound to polydeoxynucleotides. Biochem Biophys Res Commun. 1990 Jul 16;170(1):270–280. doi: 10.1016/0006-291x(90)91270-3. [DOI] [PubMed] [Google Scholar]
  4. Bennett B. D., Jetton T. L., Ying G., Magnuson M. A., Piston D. W. Quantitative subcellular imaging of glucose metabolism within intact pancreatic islets. J Biol Chem. 1996 Feb 16;271(7):3647–3651. doi: 10.1074/jbc.271.7.3647. [DOI] [PubMed] [Google Scholar]
  5. Booth M. J., Hell S. W. Continuous wave excitation two-photon fluorescence microscopy exemplified with the 647-nm ArKr laser line. J Microsc. 1998 Jun;190(Pt 3):298–304. doi: 10.1046/j.1365-2818.1998.00375.x. [DOI] [PubMed] [Google Scholar]
  6. Denk W., Strickler J. H., Webb W. W. Two-photon laser scanning fluorescence microscopy. Science. 1990 Apr 6;248(4951):73–76. doi: 10.1126/science.2321027. [DOI] [PubMed] [Google Scholar]
  7. Gerdes H. H., Kaether C. Green fluorescent protein: applications in cell biology. FEBS Lett. 1996 Jun 24;389(1):44–47. doi: 10.1016/0014-5793(96)00586-8. [DOI] [PubMed] [Google Scholar]
  8. Kapuscinski J. DAPI: a DNA-specific fluorescent probe. Biotech Histochem. 1995 Sep;70(5):220–233. doi: 10.3109/10520299509108199. [DOI] [PubMed] [Google Scholar]
  9. Köhler R. H., Cao J., Zipfel W. R., Webb W. W., Hanson M. R. Exchange of protein molecules through connections between higher plant plastids. Science. 1997 Jun 27;276(5321):2039–2042. doi: 10.1126/science.276.5321.2039. [DOI] [PubMed] [Google Scholar]
  10. König K., Simon U., Halbhuber K. J. 3D resolved two-photon fluorescence microscopy of living cells using a modified confocal laser scanning microscope. Cell Mol Biol (Noisy-le-grand) 1996 Dec;42(8):1181–1194. [PubMed] [Google Scholar]
  11. Masters B. R., So P. T., Gratton E. Multiphoton excitation fluorescence microscopy and spectroscopy of in vivo human skin. Biophys J. 1997 Jun;72(6):2405–2412. doi: 10.1016/S0006-3495(97)78886-6. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Pietrasanta L. I., Schaper A., Jovin T. M. Imaging subcellular structures of rat mammary carcinoma cells by scanning force microscopy. J Cell Sci. 1994 Sep;107(Pt 9):2427–2437. doi: 10.1242/jcs.107.9.2427. [DOI] [PubMed] [Google Scholar]
  13. Potter S. M., Wang C. M., Garrity P. A., Fraser S. E. Intravital imaging of green fluorescent protein using two-photon laser-scanning microscopy. Gene. 1996;173(1 Spec No):25–31. doi: 10.1016/0378-1119(95)00681-8. [DOI] [PubMed] [Google Scholar]
  14. Summers R. G., Piston D. W., Harris K. M., Morrill J. B. The orientation of first cleavage in the sea urchin embryo, Lytechinus variegatus, does not specify the axes of bilateral symmetry. Dev Biol. 1996 Apr 10;175(1):177–183. doi: 10.1006/dbio.1996.0105. [DOI] [PubMed] [Google Scholar]
  15. Svoboda K., Denk W., Kleinfeld D., Tank D. W. In vivo dendritic calcium dynamics in neocortical pyramidal neurons. Nature. 1997 Jan 9;385(6612):161–165. doi: 10.1038/385161a0. [DOI] [PubMed] [Google Scholar]

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