OSA's Digital Library

Biomedical Optics Express

Biomedical Optics Express

  • Editor: Joseph A. Izatt
  • Vol. 4, Iss. 11 — Nov. 1, 2013
  • pp: 2396–2410

Enhancement of lateral resolution and optical sectioning capability of two-photon fluorescence microscopy by combining temporal-focusing with structured illumination

Keisuke Isobe, Takanori Takeda, Kyohei Mochizuki, Qiyuan Song, Akira Suda, Fumihiko Kannari, Hiroyuki Kawano, Akiko Kumagai, Atsushi Miyawaki, and Katsumi Midorikawa  »View Author Affiliations

Biomedical Optics Express, Vol. 4, Issue 11, pp. 2396-2410 (2013)

View Full Text Article

Enhanced HTML    Acrobat PDF (8281 KB)

Browse Journals / Lookup Meetings

Browse by Journal and Year


Lookup Conference Papers

Close Browse Journals / Lookup Meetings

Article Tools



We demonstrate super-resolution imaging with background fluorescence rejection by interferometric temporal focusing microscopy, in which temporal focusing is combined with structured illumination. The lateral resolution and the optical sectioning capability are simultaneously improved by factors of 1.6 and 1.4, respectively, compared to conventional temporal focusing microscopy. Fluorescent beads (200 nm diameter) that are difficult to distinguish from the background fluorescence in conventional temporal focusing microscopy, are clearly visualized by interferometric temporal focusing microscopy.

© 2013 Optical Society of America

OCIS Codes
(190.4180) Nonlinear optics : Multiphoton processes
(180.4315) Microscopy : Nonlinear microscopy

ToC Category:

Original Manuscript: August 26, 2013
Revised Manuscript: September 20, 2013
Manuscript Accepted: September 23, 2013
Published: October 10, 2013

Keisuke Isobe, Takanori Takeda, Kyohei Mochizuki, Qiyuan Song, Akira Suda, Fumihiko Kannari, Hiroyuki Kawano, Akiko Kumagai, Atsushi Miyawaki, and Katsumi Midorikawa, "Enhancement of lateral resolution and optical sectioning capability of two-photon fluorescence microscopy by combining temporal-focusing with structured illumination," Biomed. Opt. Express 4, 2396-2410 (2013)

Sort:  Author  |  Year  |  Journal  |  Reset  


  1. W. Denk, J. H. Strickler, W. W. Webb, “Two-photon laser scanning fluorescence microscopy,” Science 248(4951), 73–76 (1990). [CrossRef] [PubMed]
  2. K. König, “Multiphoton microscopy in life sciences,” J. Microsc. 200(2), 83–104 (2000). [CrossRef] [PubMed]
  3. W. R. Zipfel, R. M. Williams, W. W. Webb, “Nonlinear magic: multiphoton microscopy in the biosciences,” Nat. Biotechnol. 21(11), 1369–1377 (2003). [CrossRef] [PubMed]
  4. P. Theer, M. T. Hasan, 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]
  5. P. Theer, W. Denk, “On the fundamental imaging-depth limit in two-photon microscopy,” J. Opt. Soc. Am. A 23(12), 3139–3149 (2006). [CrossRef] [PubMed]
  6. D. Kobat, M. E. Durst, N. Nishimura, A. W. Wong, C. B. Schaffer, C. Xu, “Deep tissue multiphoton microscopy using longer wavelength excitation,” Opt. Express 17(16), 13354–13364 (2009). [CrossRef] [PubMed]
  7. D. Kobat, N. G. Horton, C. Xu, “In vivo two-photon microscopy to 1.6-mm depth in mouse cortex,” J. Biomed. Opt. 16(10), 106014 (2011). [CrossRef] [PubMed]
  8. N. G. Horton, K. Wang, D. Kobat, C. G. Clark, F. W. Wise, C. B. Schaffer, C. Xu, “In vivo three-photon microscopy of subcortical structures within an intact mouse brain,” Nat. Photonics 7(3), 205–209 (2013). [CrossRef]
  9. G. Zhu, J. van Howe, M. Durst, W. Zipfel, C. Xu, “Simultaneous spatial and temporal focusing of femtosecond pulses,” Opt. Express 13(6), 2153–2159 (2005). [CrossRef] [PubMed]
  10. D. Oron, E. Tal, Y. Silberberg, “Scanningless depth-resolved microscopy,” Opt. Express 13(5), 1468–1476 (2005). [CrossRef] [PubMed]
  11. M. E. Durst, A. A. Straub, C. Xu, “Enhanced axial confinement of sum-frequency generation in a temporal focusing setup,” Opt. Lett. 34(12), 1786–1788 (2009). [CrossRef] [PubMed]
  12. A. Leray, J. Mertz, “Rejection of two-photon fluorescence background in thick tissue by differential aberration imaging,” Opt. Express 14(22), 10565–10573 (2006). [CrossRef] [PubMed]
  13. N. Ji, D. E. Milkie, E. Betzig, “Adaptive optics via pupil segmentation for high-resolution imaging in biological tissues,” Nat. Methods 7(2), 141–147 (2010). [CrossRef] [PubMed]
  14. K. Isobe, H. Kawano, T. Takeda, A. Suda, A. Kumagai, H. Mizuno, A. Miyawaki, K. Midorikawa, “Background-free deep imaging by spatial overlap modulation nonlinear optical microscopy,” Biomed. Opt. Express 3(7), 1594–1608 (2012). [CrossRef] [PubMed]
  15. K. Isobe, H. Kawano, A. Kumagai, A. Miyawaki, K. Midorikawa, “Implementation of spatial overlap modulation nonlinear optical microscopy using an electro-optic deflector,” Biomed. Opt. Express 4(10), 1937–1945 (2013).
  16. Z. Chen, L. Wei, X. Zhu, W. Min, “Extending the fundamental imaging-depth limit of multi-photon microscopy by imaging with photo-activatable fluorophores,” Opt. Express 20(17), 18525–18536 (2012). [CrossRef] [PubMed]
  17. K. Isobe, A. Suda, H. Hashimoto, F. Kannari, H. Kawano, H. Mizuno, A. Miyawaki, K. Midorikawa, “High-resolution fluorescence microscopy based on a cyclic sequential multiphoton process,” Biomed. Opt. Express 1(3), 791–797 (2010). [CrossRef] [PubMed]
  18. M. A. A. Neil, R. Juskaitis, T. Wilson, “Method of obtaining optical sectioning by using structured light in a conventional microscope,” Opt. Lett. 22(24), 1905–1907 (1997). [CrossRef] [PubMed]
  19. N. Chen, C.-H. Wong, C. J. R. Sheppard, “Focal modulation microscopy,” Opt. Express 16(23), 18764–18769 (2008). [CrossRef] [PubMed]
  20. A. H. Buist, M. Muller, J. Squier, G. J. Brakenhoff, “Real time two-photon absorption microscopy using multi point excitation,” J. Microsc. 192(2), 217–226 (1998). [CrossRef]
  21. J. Bewersdorf, R. Pick, S. W. Hell, “Multifocal multiphoton microscopy,” Opt. Lett. 23(9), 655–657 (1998). [CrossRef] [PubMed]
  22. A. Egner, S. W. Hell, “Time multiplexing and parallelization in multifocal multiphoton microscopy,” J. Opt. Soc. Am. A 17(7), 1192–1201 (2000). [CrossRef] [PubMed]
  23. D. N. Fittinghoff, P. W. Wiseman, J. Squier, “Widefield multiphoton and temporally decorrelated multifocal multiphoton microscopy,” Opt. Express 7(8), 273–279 (2000). [CrossRef] [PubMed]
  24. T. Nielsen, M. Fricke, D. Hellweg, P. Andresen, “High efficiency beam splitter for multifocal multiphoton microscopy,” J. Microsc. 201(3), 368–376 (2001). [CrossRef] [PubMed]
  25. L. Sacconi, E. Froner, R. Antolini, M. R. Taghizadeh, A. Choudhury, F. S. Pavone, “Multiphoton multifocal microscopy exploiting a diffractive optical element,” Opt. Lett. 28(20), 1918–1920 (2003). [CrossRef] [PubMed]
  26. K. H. Kim, C. Buehler, K. Bahlmann, T. Ragan, W.-C. A. Lee, E. Nedivi, E. L. Heffer, S. Fantini, P. T. C. So, “Multifocal multiphoton microscopy based on multianode photomultiplier tubes,” Opt. Express 15(18), 11658–11678 (2007). [CrossRef] [PubMed]
  27. O. D. Therrien, B. Aubé, S. Pagès, P. D. Koninck, D. Côté, “Wide-field multiphoton imaging of cellular dynamics in thick tissue by temporal focusing and patterned illumination,” Biomed. Opt. Express 2(3), 696–704 (2011). [CrossRef] [PubMed]
  28. L.-C. Cheng, C.-Y. Chang, C.-Y. Lin, K.-C. Cho, W.-C. Yen, N.-S. Chang, C. Xu, C. Y. Dong, S.-J. Chen, “Spatiotemporal focusing-based widefield multiphoton microscopy for fast optical sectioning,” Opt. Express 20(8), 8939–8948 (2012). [CrossRef] [PubMed]
  29. D. Lim, K. K. Chu, J. Mertz, “Wide-field fluorescence sectioning with hybrid speckle and uniform-illumination microscopy,” Opt. Lett. 33(16), 1819–1821 (2008). [CrossRef] [PubMed]
  30. H. Choi, E. Y. S. Yew, B. Hallacoglu, S. Fantini, C. J. R. Sheppard, P. T. C. So, “Improvement of axial resolution and contrast in temporally focused widefield two-photon microscopy with structured light illumination,” Biomed. Opt. Express 4(7), 995–1005 (2013). [CrossRef] [PubMed]
  31. M. G. L. Gustafsson, “Surpassing the lateral resolution limit by a factor of two using structured illumination microscopy,” J. Microsc. 198(2), 82–87 (2000). [CrossRef] [PubMed]
  32. J. T. Frohn, H. F. Knapp, A. Stemmer, “True optical resolution beyond the Rayleigh limit achieved by standing wave illumination,” Proc. Natl. Acad. Sci. U.S.A. 97(13), 7232–7236 (2000). [CrossRef] [PubMed]
  33. H. Mashiko, A. Suda, K. Midorikawa, “All-reflective interferometric autocorrelator for the measurement of ultra-short optical pulses,” Appl. Phys. B 76(5), 525–530 (2003). [CrossRef]
  34. Y. Nabekawa, T. Shimizu, Y. Furukawa, E. J. Takahashi, K. Midorikawa, “Interferometry of attosecond pulse trains in the extreme ultraviolet wavelength region,” Phys. Rev. Lett. 102(21), 213904 (2009). [CrossRef] [PubMed]
  35. R. Heintzmann, T. M. Jovin, C. Cremer, “Saturated patterned excitation microscopy--a concept for optical resolution improvement,” J. Opt. Soc. Am. A 19(8), 1599–1609 (2002). [CrossRef] [PubMed]
  36. R. Heintzmann, “Saturated patterned excitation microscopy with two-dimensional excitation patterns,” Micron 34(6-7), 283–291 (2003). [CrossRef] [PubMed]
  37. M. G. L. Gustafsson, “Nonlinear structured-illumination microscopy: Wide-field fluorescence imaging with theoretically unlimited resolution,” Proc. Natl. Acad. Sci. U.S.A. 102(37), 13081–13086 (2005). [CrossRef] [PubMed]
  38. E. H. Rego, L. Shao, J. J. Macklin, L. Winoto, G. A. Johansson, N. Kamps-Hughes, M. W. Davidson, M. G. L. Gustafsson, “Nonlinear structured-illumination microscopy with a photoswitchable protein reveals cellular structures at 50-nm resolution,” Proc. Natl. Acad. Sci. U.S.A. 109(3), E135–E143 (2012). [CrossRef] [PubMed]
  39. R. Fiolka, M. Beck, A. Stemmer, “Structured illumination in total internal reflection fluorescence microscopy using a spatial light modulator,” Opt. Lett. 33(14), 1629–1631 (2008). [CrossRef] [PubMed]
  40. B.-J. Chang, L.-J. Chou, Y.-C. Chang, S.-Y. Chiang, “Isotropic image in structured illumination microscopy patterned with a spatial light modulator,” Opt. Express 17(17), 14710–14721 (2009). [CrossRef] [PubMed]

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.

« Previous Article  |  Next Article »

OSA is a member of CrossRef.

CrossCheck Deposited