OSA's Digital Library

Optics Express

Optics Express

  • Editor: C. Martijn de Sterke
  • Vol. 18, Iss. 18 — Aug. 30, 2010
  • pp: 18720–18731

Simultaneous two-photon imaging and photo-stimulation with structured light illumination

Marco Dal Maschio, Francesco Difato, Riccardo Beltramo, Axel Blau, Fabio Benfenati, and Tommaso Fellin  »View Author Affiliations

Optics Express, Vol. 18, Issue 18, pp. 18720-18731 (2010)

View Full Text Article

Enhanced HTML    Acrobat PDF (2655 KB)

Browse Journals / Lookup Meetings

Browse by Journal and Year


Lookup Conference Papers

Close Browse Journals / Lookup Meetings

Article Tools



Holographic microscopy is increasingly recognized as a promising tool for the study of the central nervous system. Here we present a “holographic module”, a simple optical path that can be combined with commercial scanheads for simultaneous imaging and uncaging with structured two-photon light. The present microscope is coupled to two independently tunable lasers and has two principal configurations: holographic imaging combined with galvo-steered uncaging and holographic uncaging combined with conventional scanning imaging. We applied this flexible system for simultaneous two-photon imaging and photostimulation of neuronal cells with complex light patterns, opening new perspectives for the study of brain function in situ and in vivo.

© 2010 OSA

OCIS Codes
(180.2520) Microscopy : Fluorescence microscopy
(090.1995) Holography : Digital holography
(180.4315) Microscopy : Nonlinear microscopy
(070.6120) Fourier optics and signal processing : Spatial light modulators

ToC Category:

Original Manuscript: May 26, 2010
Revised Manuscript: July 29, 2010
Manuscript Accepted: August 7, 2010
Published: August 18, 2010

Virtual Issues
Vol. 5, Iss. 13 Virtual Journal for Biomedical Optics

Marco Dal Maschio, Francesco Difato, Riccardo Beltramo, Axel Blau, Fabio Benfenati, and Tommaso Fellin, "Simultaneous two-photon imaging and photo-stimulation with structured light illumination," Opt. Express 18, 18720-18731 (2010)

Sort:  Author  |  Year  |  Journal  |  Reset  


  1. R. H. Kramer, D. L. Fortin, and D. Trauner, “New photochemical tools for controlling neuronal activity,” Curr. Opin. Neurobiol. 19(5), 544–552 (2009). [CrossRef]
  2. P. Saggau, “New methods and uses for fast optical scanning,” Curr. Opin. Neurobiol. 16(5), 543–550 (2006). [CrossRef] [PubMed]
  3. A. Bednarkiewicz, M. Bouhifd, and M. P. Whelan, “Digital micromirror device as a spatial illuminator for fluorescence lifetime and hyperspectral imaging,” Appl. Opt. 47(9), 1193–1199 (2008). [CrossRef] [PubMed]
  4. M. G. Gustafsson, L. Shao, P. M. Carlton, C. J. Wang, I. N. Golubovskaya, W. Z. Cande, D. A. Agard, and J. W. Sedat, “Three-dimensional resolution doubling in wide-field fluorescence microscopy by structured illumination,” Biophys. J. 94(12), 4957–4970 (2008). [CrossRef] [PubMed]
  5. B. E. Losavio, V. Iyer, and P. Saggau, “Two-photon microscope for multisite microphotolysis of caged neurotransmitters in acute brain slices,” J. Biomed. Opt. 14(6), 064033 (2009). [CrossRef]
  6. C. Lutz, T. S. Otis, V. DeSars, S. Charpak, D. A. DiGregorio, and V. Emiliani, “Holographic photolysis of caged neurotransmitters,” Nat. Methods 5(9), 821–827 (2008). [CrossRef]
  7. V. Nikolenko, B. O. Watson, R. Araya, A. Woodruff, D. S. Peterka, and R. Yuste, “SLM microscopy: scanless two-photon imaging and photostimulation with spatial light modulators,” Front Neural Circuits 2, 5 (2008). [CrossRef]
  8. T. L. Kelly and J. Munch, “Phase-aberration correction with dual liquid-crystal spatial light modulators,” Appl. Opt. 37(22), 5184–5189 (1998). [CrossRef]
  9. K. D. Wulff, D. G. Cole, R. L. Clark, R. Dileonardo, J. Leach, J. Cooper, G. Gibson, and M. J. Padgett, “Aberration correction in holographic optical tweezers,” Opt. Express 14(9), 4170–4175 (2006). [CrossRef] [PubMed]
  10. D. R. Burnham and D. McGloin, “Holographic optical trapping of aerosol droplets,” Opt. Express 14(9), 4176–4182 (2006). [CrossRef] [PubMed]
  11. D. Cojoc, F. Difato, E. Ferrari, R. B. Shahapure, J. Laishram, M. Righi, E. M. Di Fabrizio, V. Torre, and L. Mei, “Properties of the force exerted by filopodia and lamellipodia and the involvement of cytoskeletal components,” PLoS ONE 2(10), e1072 (2007). [CrossRef] [PubMed]
  12. M. Zahid, M. Vélez-Fort, E. Papagiakoumou, C. Ventalon, M. C. Angulo, V. Emiliani, and F. Tell, “Holographic photolysis for multiple cell stimulation in mouse hippocampal slices,” PLoS ONE 5(2), e9431 (2010). [CrossRef] [PubMed]
  13. N. Ji, H. Shroff, H. Zhong, and E. Betzig, “Advances in the speed and resolution of light microscopy,” Curr. Opin. Neurobiol. 18(6), 605–616 (2008). [CrossRef]
  14. G. Losi, K. Prybylowski, Z. Fu, J. H. Luo, and S. Vicini, “Silent synapses in developing cerebellar granule neurons,” J. Neurophysiol. 87(3), 1263–1270 (2002). [PubMed]
  15. G. J. Brakenhoff, G. W. Wurpel, K. Jalink, L. Oomen, L. Brocks, and J. M. Zwier, “Characterization of sectioning fluorescence microscopy with thin uniform fluorescent layers: Sectioned Imaging Property or SIPcharts,” J. Microsc. 219(Pt 3), 122–132 (2005). [CrossRef] [PubMed]
  16. E. Papagiakoumou, V. de Sars, D. Oron, and V. Emiliani, “Patterned two-photon illumination by spatiotemporal shaping of ultrashort pulses,” Opt. Express 16(26), 22039–22047 (2008). [CrossRef] [PubMed]
  17. C. Maurer, S. Khan, S. Fassl, S. Bernet, and M. Ritsch-Marte, “Depth of field multiplexing in microscopy,” Opt. Express 18(3), 3023–3034 (2010). [CrossRef] [PubMed]
  18. G. Sinclair, J. Leach, P. Jordan, G. Gibson, E. Yao, Z. Laczik, M. Padgett, and J. Courtial, “Interactive application in holographic optical tweezers of a multi-plane Gerchberg-Saxton algorithm for three-dimensional light shaping,” Opt. Express 12(8), 1665–1670 (2004). [CrossRef] [PubMed]
  19. V. Emiliani, D. Cojoc, E. Ferrari, V. Garbin, C. Durieux, M. Coppey-Moisan, and E. Di Fabrizio, “Wave front engineering for microscopy of living cells,” Opt. Express 13(5), 1395–1405 (2005). [CrossRef] [PubMed]
  20. M. Fricke and T. Nielsen, “Two-dimensional imaging without scanning by multifocal multiphoton microscopy,” Appl. Opt. 44(15), 2984–2988 (2005). [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