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Virtual Journal for Biomedical Optics

Virtual Journal for Biomedical Optics

| EXPLORING THE INTERFACE OF LIGHT AND BIOMEDICINE

  • Editors: Andrew Dunn and Anthony Durkin
  • Vol. 7, Iss. 10 — Oct. 5, 2012

Optics InfoBase > Virtual Journal for Biomedical Optics > Volume 7 > Issue 10 > Page 18525

Extending the fundamental imaging-depth limit of multi-photon microscopy by imaging with photo-activatable fluorophores

Zhixing Chen, Lu Wei, Xinxin Zhu, and Wei Min  »View Author Affiliations


Optics Express, Vol. 20, Issue 17, pp. 18525-18536 (2012)
http://dx.doi.org/10.1364/OE.20.018525


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Abstract

It is highly desirable to be able to optically probe biological activities deep inside live organisms. By employing a spatially confined excitation via a nonlinear transition, multiphoton fluorescence microscopy has become indispensable for imaging scattering samples. However, as the incident laser power drops exponentially with imaging depth due to scattering loss, the out-of-focus fluorescence eventually overwhelms the in-focal signal. The resulting loss of imaging contrast defines a fundamental imaging-depth limit, which cannot be overcome by increasing excitation intensity. Herein we propose to significantly extend this depth limit by multiphoton activation and imaging (MPAI) of photo-activatable fluorophores. The imaging contrast is drastically improved due to the created disparity of bright-dark quantum states in space. We demonstrate this new principle by both analytical theory and experiments on tissue phantoms labeled with synthetic caged fluorescein dye or genetically encodable photoactivatable GFP.

© 2012 OSA

OCIS Codes
(160.4330) Materials : Nonlinear optical materials
(170.5810) Medical optics and biotechnology : Scanning microscopy
(170.6280) Medical optics and biotechnology : Spectroscopy, fluorescence and luminescence
(180.2520) Microscopy : Fluorescence microscopy
(190.4180) Nonlinear optics : Multiphoton processes
(180.4315) Microscopy : Nonlinear microscopy

ToC Category:
Microscopy

History
Original Manuscript: April 12, 2012
Revised Manuscript: June 18, 2012
Manuscript Accepted: June 19, 2012
Published: July 30, 2012

Virtual Issues
Vol. 7, Iss. 10 Virtual Journal for Biomedical Optics

Citation
Zhixing Chen, Lu Wei, Xinxin Zhu, and Wei Min, "Extending the fundamental imaging-depth limit of multi-photon microscopy by imaging with photo-activatable fluorophores," Opt. Express 20, 18525-18536 (2012)
http://www.opticsinfobase.org/vjbo/abstract.cfm?URI=oe-20-17-18525


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References

  1. S. W. Hell and J. Wichmann, “Breaking the diffraction resolution limit by stimulated emission: stimulated-emission-depletion fluorescence microscopy,” Opt. Lett.19(11), 780–782 (1994). [CrossRef] [PubMed]
  2. E. Betzig, G. H. Patterson, R. Sougrat, O. W. Lindwasser, S. Olenych, J. S. Bonifacino, M. W. Davidson, J. Lippincott-Schwartz, and H. F. Hess, “Imaging intracellular fluorescent proteins at nanometer resolution,” Science313(5793), 1642–1645 (2006). [CrossRef] [PubMed]
  3. S. T. Hess, T. P. Girirajan, and M. D. Mason, “Ultra-high resolution imaging by fluorescence photoactivation localization microscopy,” Biophys. J.91(11), 4258–4272 (2006). [CrossRef] [PubMed]
  4. M. J. Rust, M. Bates, and X. Zhuang, “Sub-diffraction-limit imaging by stochastic optical reconstruction microscopy (STORM),” Nat. Methods3(10), 793–796 (2006). [CrossRef] [PubMed]
  5. W. Denk, J. H. Strickler, and W. W. Webb, “Two-photon laser scanning fluorescence microscopy,” Science248(4951), 73–76 (1990). [CrossRef] [PubMed]
  6. W. R. Zipfel, R. M. Williams, and W. W. Webb, “Nonlinear magic: multiphoton microscopy in the biosciences,” Nat. Biotechnol.21(11), 1369–1377 (2003). [CrossRef] [PubMed]
  7. F. Helmchen and W. Denk, “Deep tissue two-photon microscopy,” Nat. Methods2(12), 932–940 (2005). [CrossRef] [PubMed]
  8. R. Yuste, ed., Imaging: A Laboratory Manual (Cold Spring Harbor Laboratory Press, 2010)
  9. P. Theer, M. T. Hasan, and W. Denk, “Two-photon imaging to a depth of 1000 microm in living brains by use of a Ti:Al2O3 regenerative amplifier,” Opt. Lett.28(12), 1022–1024 (2003). [CrossRef] [PubMed]
  10. P. Theer and W. Denk, “On the fundamental imaging-depth limit in two-photon microscopy,” J. Opt. Soc. Am. A23(12), 3139–3149 (2006). [CrossRef] [PubMed]
  11. D. Kobat, N. G. Horton, and C. Xu, “In vivo two-photon microscopy to 1.6-mm depth in mouse cortex,” J. Biomed. Opt.16(10), 106014 (2011). [CrossRef] [PubMed]
  12. N. J. Durr, C. T. Weisspfennig, B. A. Holfeld, and A. Ben-Yakar, “Maximum imaging depth of two-photon autofluorescence microscopy in epithelial tissues,” J. Biomed. Opt.16(2), 026008 (2011). [CrossRef] [PubMed]
  13. J. Ying, F. Liu, and R. R. Alfano, “Spatial distribution of two-photon-excited fluorescence in scattering media,” Appl. Opt.38(1), 224–229 (1999). [CrossRef] [PubMed]
  14. N. Ji, D. E. Milkie, and E. Betzig, “Adaptive optics via pupil segmentation for high-resolution imaging in biological tissues,” Nat. Methods7(2), 141–147 (2010). [CrossRef] [PubMed]
  15. M. Rueckel, J. A. Mack-Bucher, and W. Denk, “Adaptive wavefront correction in two-photon microscopy using coherence-gated wavefront sensing,” Proc. Natl. Acad. Sci. U.S.A.103(46), 17137–17142 (2006). [CrossRef] [PubMed]
  16. D. Kobat, M. E. Durst, N. Nishimura, A. W. Wong, C. B. Schaffer, and C. Xu, “Deep tissue multiphoton microscopy using longer wavelength excitation,” Opt. Express17(16), 13354–13364 (2009). [CrossRef] [PubMed]
  17. H. Hama, H. Kurokawa, H. Kawano, R. Ando, T. Shimogori, H. Noda, K. Fukami, A. Sakaue-Sawano, and A. Miyawaki, “Scale: a chemical approach for fluorescence imaging and reconstruction of transparent mouse brain,” Nat. Neurosci.14(11), 1481–1488 (2011). [CrossRef] [PubMed]
  18. A. Leray, K. Lillis, and J. Mertz, “Enhanced background rejection in thick tissue with differential-aberration two-photon microscopy,” Biophys. J.94(4), 1449–1458 (2008). [CrossRef] [PubMed]
  19. M. Fernández-Suárez and A. Y. Ting, “Fluorescent probes for super-resolution imaging in living cells,” Nat. Rev. Mol. Cell Biol.9(12), 929–943 (2008). [CrossRef] [PubMed]
  20. J. Lippincott-Schwartz and G. H. Patterson, “Photoactivatable fluorescent proteins for diffraction-limited and super-resolution imaging,” Trends Cell Biol.19(11), 555–565 (2009). [CrossRef] [PubMed]
  21. M. Heilemann, P. Dedecker, J. Hofkens, and M. Sauer, “Photoswitches: key molecules for subdiffraction-resolution fluorescence imaging and molecular quantification,” Laser Photon. Rev.3(1-2), 180–202 (2009). [CrossRef]
  22. H. L. Lee, S. J. Lord, S. Iwanaga, K. Zhan, H. Xie, J. C. Williams, H. Wang, G. R. Bowman, E. D. Goley, L. Shapiro, R. J. Twieg, J. Rao, and W. E. Moerner, “Superresolution imaging of targeted proteins in fixed and living cells using photoactivatable organic fluorophores,” J. Am. Chem. Soc.132(43), 15099–15101 (2010). [CrossRef] [PubMed]
  23. J. Fölling, V. Belov, R. Kunetsky, R. Medda, A. Schönle, A. Egner, C. Eggeling, M. Bossi, and S. W. Hell, “Photochromic rhodamines provide nanoscopy with optical sectioning,” Angew. Chem. Int. Ed. Engl.46(33), 6266–6270 (2007). [CrossRef] [PubMed]
  24. D. Kleinfeld, P. P. Mitra, F. Helmchen, and W. Denk, “Fluctuations and stimulus-induced changes in blood flow observed in individual capillaries in layers 2 through 4 of rat neocortex,” Proc. Natl. Acad. Sci. U.S.A.95(26), 15741–15746 (1998). [CrossRef] [PubMed]
  25. T. J. Mitchison, K. E. Sawin, J. A. Theriot, K. Gee, and A. Mallavarapu, “Caged fluorescent probes,” Methods Enzymol.291, 63–78 (1998). [CrossRef] [PubMed]
  26. G. H. Patterson and J. Lippincott-Schwartz, “A photoactivatable GFP for selective photolabeling of proteins and cells,” Science297(5588), 1873–1877 (2002). [CrossRef] [PubMed]
  27. M. Schneider, S. Barozzi, I. Testa, M. Faretta, and A. Diaspro, “Two-photon activation and excitation properties of PA-GFP in the 720-920-nm region,” Biophys. J.89(2), 1346–1352 (2005). [CrossRef] [PubMed]
  28. Y. Zhao, Q. Zheng, K. Dakin, K. Xu, M. L. Martinez, and W.-H. Li, “New caged coumarin fluorophores with extraordinary uncaging cross sections suitable for biological imaging applications,” J. Am. Chem. Soc.126(14), 4653–4663 (2004). [CrossRef] [PubMed]
  29. R. Ando, H. Mizuno, and A. Miyawaki, “Regulated fast nucleocytoplasmic shuttling observed by reversible protein highlighting,” Science306(5700), 1370–1373 (2004). [CrossRef] [PubMed]
  30. M.-Q. Zhu, G.-F. Zhang, C. Li, M. P. Aldred, E. Chang, R. A. Drezek, and A. D. Q. Li, “Reversible two-photon photoswitching and two-photon imaging of immunofunctionalized nanoparticles Targeted to Cancer Cells,” J. Am. Chem. Soc.133(2), 365–372 (2011). [CrossRef]
  31. L. Wei, Z. Chen, and W. Min, “Stimulated emission reduced fluorescence microscopy: a concept for extending the fundamental depth limit of two-photon fluorescence imaging,” Biomed. Opt. Express3(6), 1465–1475 (2012). [CrossRef]

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