OSA's Digital Library

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

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


View Full Text Article

Enhanced HTML    Acrobat PDF (1950 KB) Open Access





Browse Journals / Lookup Meetings

Browse by Journal and Year


   


Lookup Conference Papers

Close Browse Journals / Lookup Meetings

Article Tools

Share
Citations

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


Sort:  Author  |  Year  |  Journal  |  Reset  

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]

Cited By

Alert me when this paper is cited

OSA is able to provide readers links to articles that cite this paper by participating in CrossRef's Cited-By Linking service. CrossRef includes content from more than 3000 publishers and societies. In addition to listing OSA journal articles that cite this paper, citing articles from other participating publishers will also be listed.


Next Article »

OSA is a member of CrossRef.

CrossCheck Deposited