OSA's Digital Library

Biomedical Optics Express

Biomedical Optics Express

  • Editor: Joseph A. Izatt
  • Vol. 2, Iss. 7 — Jul. 1, 2011
  • pp: 2035–2046

Fast two-layer two-photon imaging of neuronal cell populations using an electrically tunable lens

Benjamin F. Grewe, Fabian F. Voigt, Marcel van ’t Hoff, and Fritjof Helmchen  »View Author Affiliations

Biomedical Optics Express, Vol. 2, Issue 7, pp. 2035-2046 (2011)

View Full Text Article

Enhanced HTML    Acrobat PDF (1500 KB)

Browse Journals / Lookup Meetings

Browse by Journal and Year


Lookup Conference Papers

Close Browse Journals / Lookup Meetings

Article Tools



Functional two-photon Ca2+-imaging is a versatile tool to study the dynamics of neuronal populations in brain slices and living animals. However, population imaging is typically restricted to a single two-dimensional image plane. By introducing an electrically tunable lens into the excitation path of a two-photon microscope we were able to realize fast axial focus shifts within 15 ms. The maximum axial scan range was 0.7 mm employing a 40x NA0.8 water immersion objective, plenty for typically required ranges of 0.2–0.3 mm. By combining the axial scanning method with 2D acousto-optic frame scanning and random-access scanning, we measured neuronal population activity of about 40 neurons across two imaging planes separated by 40 μm and achieved scan rates up to 20–30 Hz. The method presented is easily applicable and allows upgrading of existing two-photon microscopes for fast 3D scanning.

© 2011 OSA

OCIS Codes
(170.0180) Medical optics and biotechnology : Microscopy
(170.2520) Medical optics and biotechnology : Fluorescence microscopy
(180.6900) Microscopy : Three-dimensional microscopy
(180.4315) Microscopy : Nonlinear microscopy

ToC Category:

Original Manuscript: March 28, 2011
Revised Manuscript: June 10, 2011
Manuscript Accepted: June 10, 2011
Published: June 23, 2011

Benjamin F. Grewe, Fabian F. Voigt, Marcel van ’t Hoff, and Fritjof Helmchen, "Fast two-layer two-photon imaging of neuronal cell populations using an electrically tunable lens," Biomed. Opt. Express 2, 2035-2046 (2011)

Sort:  Author  |  Year  |  Journal  |  Reset  


  1. F. Helmchen and W. Denk, “Deep tissue two-photon microscopy,” Nat. Methods 2(12), 932–940 (2005). [CrossRef] [PubMed]
  2. B. F. Grewe and F. Helmchen, “Optical probing of neuronal ensemble activity,” Curr. Opin. Neurobiol. 19(5), 520–529 (2009). [CrossRef] [PubMed]
  3. J. N. Kerr and W. Denk, “Imaging in vivo: watching the brain in action,” Nat. Rev. Neurosci. 9(3), 195–205 (2008). [CrossRef] [PubMed]
  4. W. Göbel, B. M. Kampa, and F. Helmchen, “Imaging cellular network dynamics in three dimensions using fast 3D laser scanning,” Nat. Methods 4(1), 73–79 (2007). [CrossRef] [PubMed]
  5. B. F. Grewe, D. Langer, H. Kasper, B. M. Kampa, and F. Helmchen, “High-speed in vivo calcium imaging reveals neuronal network activity with near-millisecond precision,” Nat. Methods 7(5), 399–405 (2010). [CrossRef] [PubMed]
  6. W. Göbel and F. Helmchen, “New angles on neuronal dendrites in vivo,” J. Neurophysiol. 98(6), 3770–3779 (2007). [CrossRef] [PubMed]
  7. A. M. Kerlin, M. L. Andermann, V. K. Berezovskii, and R. C. Reid, “Broadly tuned response properties of diverse inhibitory neuron subtypes in mouse visual cortex,” Neuron 67(5), 858–871 (2010). [CrossRef] [PubMed]
  8. E. Botcherby, C. Smith, M. Booth, R. Juskaitis, and T. Wilson, “Arbitrary-scan imaging for two-photon microscopy,” Proc. SPIE 7569(756917), 756917, 756917-8 (2010). [CrossRef]
  9. E. J. Botcherby, R. Juskaitis, M. J. Booth, and T. Wilson, “Aberration-free optical refocusing in high numerical aperture microscopy,” Opt. Lett. 32(14), 2007–2009 (2007). [CrossRef] [PubMed]
  10. E. J. Botcherby, R. Juskaitis, M. J. Booth, and T. Wilson, “An optical technique for remote focusing in microscopy,” Opt. Commun. 281(4), 880–887 (2008). [CrossRef]
  11. E. E. Hoover, M. D. Young, E. V. Chandler, A. Luo, J. J. Field, K. E. Sheetz, A. W. Sylvester, and J. A. Squier, “Remote focusing for programmable multi-layer differential multiphoton microscopy,” Biomed. Opt. Express 2(1), 113–122 (2011). [CrossRef] [PubMed]
  12. P. A. Kirkby, K. M. Srinivas Nadella, and R. A. Silver, “A compact Acousto-Optic Lens for 2D and 3D femtosecond based 2-photon microscopy,” Opt. Express 18(13), 13720–13745 (2010). [CrossRef] [PubMed]
  13. G. Duemani Reddy, K. Kelleher, R. Fink, and P. Saggau, “Three-dimensional random access multiphoton microscopy for functional imaging of neuronal activity,” Nat. Neurosci. 11(6), 713–720 (2008). [CrossRef] [PubMed]
  14. D. Vučinić and T. J. Sejnowski, “A compact multiphoton 3D imaging system for recording fast neuronal activity,” PLoS ONE 2(8), e699 (2007). [CrossRef] [PubMed]
  15. W. Amir, R. Carriles, E. E. Hoover, T. A. Planchon, C. G. Durfee, and J. A. Squier, “Simultaneous imaging of multiple focal planes using a two-photon scanning microscope,” Opt. Lett. 32(12), 1731–1733 (2007). [CrossRef] [PubMed]
  16. H. Oku, K. Hashimoto, and M. Ishikawa, “Variable-focus lens with 1-kHz bandwidth,” Opt. Express 12(10), 2138–2149 (2004). [CrossRef] [PubMed]
  17. B. H. W. Hendricks, S. Kuiper, M. A. J. Van As, C. A. Renders, and T. W. Tukker, “Electrowetting-based variable-focus lens for miniature systems,” Opt. Rev. 12(3), 255–259 (2005). [CrossRef]
  18. B. Berge and J. Peseux, “Variable focal lens controlled by an external voltage: An application of electrowetting,” Eur. Phys. J. E 3(2), 159–163 (2000). [CrossRef]
  19. D. Koyama, R. Isago, and K. Nakamura, “Compact, high-speed variable-focus liquid lens using acoustic radiation force,” Opt. Express 18(24), 25158–25169 (2010). [CrossRef] [PubMed]
  20. S. Liu and H. Hua, “Extended depth-of-field microscopic imaging with a variable focus microscope objective,” Opt. Express 19(1), 353–362 (2011). [CrossRef] [PubMed]
  21. K. S. Lee, P. Vanderwall, and J. P. Rolland, “Two-photon microscopy with dynamic focusing objective using a liquid lens,” Proc. SPIE 7569, 756923, 756923-7 (2010). [CrossRef]
  22. P. S. Tsai, B. Migliori, K. Campbell, T. N. Kim, K. Kam, A. Groisman, and D. Kleinfeld, “Spherical aberration correction in nonlinear microscopy and optical ablation using a transparent deformable membrane,” Appl. Phys. Lett. 91(19), 191102 (2007). [CrossRef]
  23. H. Gross, F. Blechinger, and B. Achtner, Handbook of Optical Systems, 1st ed. (Wiley-VCH, 2008), Vol. 4.
  24. A. Katsuyuki, “Embodiment 1,” Japanese Patent 8–292374 (Nov. 5, 1996).
  25. W. S. Rasband and J. Image, U. S. National Institutes of Health, Bethesda, Maryland, USA, 1997–2009, http://rsb.info.nih.gov/ij/ .
  26. C. Stosiek, O. Garaschuk, K. Holthoff, and A. Konnerth, “In vivo two-photon calcium imaging of neuronal networks,” Proc. Natl. Acad. Sci. U.S.A. 100(12), 7319–7324 (2003). [CrossRef] [PubMed]
  27. A. Nimmerjahn, F. Kirchhoff, J. N. Kerr, and F. Helmchen, “Sulforhodamine 101 as a specific marker of astroglia in the neocortex in vivo,” Nat. Methods 1(1), 31–37 (2004). [CrossRef] [PubMed]
  28. A. Cheng, J. T. Gonçalves, P. Golshani, K. Arisaka, and C. Portera-Cailliau, “Simultaneous two-photon calcium imaging at different depths with spatiotemporal multiplexing,” Nat. Methods 8(2), 139–142 (2011). [CrossRef] [PubMed]
  29. M. Blum and A. G. Optotune, Ueberlandstrasse 129, Dubendorf, Switzerland (personal communication, 2011).

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.


Fig. 1 Fig. 2 Fig. 3
Fig. 4

Supplementary Material

» Media 1: JPG (4474 KB)     
» Media 2: JPG (4433 KB)     
» Media 3: MOV (216 KB)     
» Media 4: MOV (3841 KB)     
» Media 5: MOV (563 KB)     

« Previous Article  |  Next Article »

OSA is a member of CrossRef.

CrossCheck Deposited