Abstract

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

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2010 (2)

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]

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]

2009 (2)

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]

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]

2008 (6)

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]

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]

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]

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]

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]

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]

2007 (1)

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]

2006 (3)

2005 (3)

2004 (1)

2002 (1)

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]

1998 (1)

Agard, D. A.

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]

Angulo, M. C.

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]

Araya, R.

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]

Bednarkiewicz, A.

Bernet, S.

Betzig, E.

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]

Bouhifd, M.

Brakenhoff, G. J.

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]

Brocks, L.

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]

Burnham, D. R.

Cande, W. Z.

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]

Carlton, P. M.

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]

Charpak, S.

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]

Clark, R. L.

Cojoc, D.

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]

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]

Cole, D. G.

Cooper, J.

Coppey-Moisan, M.

Courtial, J.

de Sars, V.

DeSars, V.

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]

Di Fabrizio, E.

Di Fabrizio, E. M.

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]

Difato, F.

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]

DiGregorio, D. A.

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]

Dileonardo, R.

Durieux, C.

Emiliani, V.

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]

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]

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]

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]

Fassl, S.

Ferrari, E.

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]

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]

Fortin, D. L.

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]

Fricke, M.

Fu, Z.

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]

Garbin, V.

Gibson, G.

Golubovskaya, I. N.

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]

Gustafsson, M. G.

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]

Iyer, V.

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]

Jalink, K.

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]

Ji, N.

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]

Jordan, P.

Kelly, T. L.

Khan, S.

Kramer, R. H.

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]

Laczik, Z.

Laishram, J.

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]

Leach, J.

Losavio, B. E.

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]

Losi, G.

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]

Luo, J. H.

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]

Lutz, C.

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]

Maurer, C.

McGloin, D.

Mei, L.

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]

Munch, J.

Nielsen, T.

Nikolenko, V.

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]

Oomen, L.

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]

Oron, D.

Otis, T. S.

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]

Padgett, M.

Padgett, M. J.

Papagiakoumou, E.

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]

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]

Peterka, D. S.

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]

Prybylowski, K.

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]

Righi, M.

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]

Ritsch-Marte, M.

Saggau, P.

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]

P. Saggau, “New methods and uses for fast optical scanning,” Curr. Opin. Neurobiol. 16(5), 543–550 (2006).
[CrossRef] [PubMed]

Sedat, J. W.

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]

Shahapure, R. B.

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]

Shao, L.

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]

Shroff, H.

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]

Sinclair, G.

Tell, F.

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]

Torre, V.

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]

Trauner, D.

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]

Vélez-Fort, M.

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]

Ventalon, C.

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]

Vicini, S.

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]

Wang, C. J.

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]

Watson, B. O.

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]

Whelan, M. P.

Woodruff, A.

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]

Wulff, K. D.

Wurpel, G. W.

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]

Yao, E.

Yuste, R.

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]

Zahid, M.

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]

Zhong, H.

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]

Zwier, J. M.

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]

Appl. Opt. (3)

Biophys. J. (1)

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]

Curr. Opin. Neurobiol. (3)

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]

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]

P. Saggau, “New methods and uses for fast optical scanning,” Curr. Opin. Neurobiol. 16(5), 543–550 (2006).
[CrossRef] [PubMed]

Front Neural Circuits (1)

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]

J. Biomed. Opt. (1)

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]

J. Microsc. (1)

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]

J. Neurophysiol. (1)

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]

Nat. Methods (1)

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]

Opt. Express (6)

PLoS ONE (2)

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]

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]

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Figures (6)

Fig. 1
Fig. 1

Schematic representation of the two experimental configurations of the optical set-up. A) The imaging beam (solid red line) coming from a Coherent Ultra II Chameleon laser is deflected by a flipping mirror (FM1), reshaped and modulated by the “holographic module” (pink area of the optical layout) and then directed to a commercial Prairie Ultima IV scanhead (blue area of the optical layout) coupled with a motorized Olympus BX61 microscope. A first telescope (L1 and L2) is used to reshape the laser beam to fit the Hamamatsu LCOS-SLM active window, while the lenses L3 and L4 form a second telescope that optimizes the beam size according to the dimensions of the mirrors inside the scanhead. A half wave plate (λ/2) is placed before the LCOS-SLM to modulate the beam polarization (see Methods section). A small piece of aluminum foil (ZB) mounted on a glass coverslip is placed at the focal plane of the second telescope (between L3 and L4) to reflect the zero order light (see description of the optical set up in the main text). Legend: PC1, pockel cell of the imaging beam; PC2, pockel cell of the uncaging beam; M1, turning mirror; λ/2, half wave plate; L1, plano-convex lens (f = 30 mm); L2, plano-convex lens (f = 100 mm); SLM, spatial light modulator; L3, plano-convex lens (f = 300 mm); M2, turning mirror; M3, turning mirror; ZB, zero order block; L4, plano-convex lens (f = 100 mm); PBS1, polarizing beam splitter; GM1, galvo mirror of imaging beam; M4, turning mirror; DM0, 760 nm long-pass dichroic mirror; L5, proprietary scan lens (f = 75 mm); DM1, 660 nm short-pass dichroic mirror; L6, proprietary tube lens (f = 180 mm); DM2, 660 nm long-pass dichroic mirror; DM3, 575 nm long-pass dichroic mirror; PMT1, photomultiplier #1; PMT2, photomultiplier #2; CCD1, CCD camera; GM2, galvo mirror of the uncaging beam; OBJ, microscope objective. (B) The uncaging beam (dashed black line) coming from a Coherent Chameleon Ultra laser is deflected by a flipping mirror (FM2) in the “holographic module”. Beam path is the same as described in A) except that the laser light is deflected by a mirror (M6) into the uncaging pathway of the Prairie scanhead. The plano-convex lens L4 (f = 100 mm) is, in this experimental configuration, moved to the uncaging path. The blue circles point to the optical elements that are different between the configuration displayed in A) and that shown in B). Legend: FM2, flipping mirror; M6 turning mirror.

Fig. 6
Fig. 6

Simultaneous scanning fluorescence imaging and holographic uncaging. A) Bright field image of cerebellar granule cells in culture. Scale bar: 10 μm. (B) Epifluorescence image displaying Fluo-4 loaded neurons in the same field of view shown in A). Based on this image, an image mask (C) is generated to shape the laser wave front of the uncaging beam. (D) While uncaging is performed simultaneously on two identified cells (areas delimited by red lines), imaging is performed with conventional scanning microscopy at 0.54 Hz. ROIs (numbered from 1 to 7) corresponding to different cells are shown in green. E-F) Time course of ΔF/F0 values of Fluo-4 fluorescence in the 7 ROIs displayed in D) before and after the application of the holographic uncaging stimulus. Note the clear response of the neurons (ROIs #2 and #3) upon which MNI-glutamate was holographically uncaged. The arrows indicate the time of delivery of the photolysis stimulus.

Fig. 2
Fig. 2

Two-photon structured light illumination. A) Example of light patterns that can be projected onto the sample plane. Four diffraction-limited spots positioned at the four corners of the field of view (top image), four rectangular shapes (middle panel) and a donut-like shape (bottom image) are shown. (B) Computer-generated DOEs corresponding to the different light patterns shown in (A) are sent to the LCOS-SLM. Given that the LCOS-SLM is a phase-only modulator, black/white shades on the panel correspond to 0-2 π phase modulation. (C) Two-photon excitation light reflected by a mirror positioned in the focal plane of the objective measured for the different illumination patterns shown in (A). Scale bar 20 μm. (D) Fluorescence signals from a 115 nm thick layer of fluorescein and perylenediimid generated by the two-photon excitation patterns shown in (A). Scale bar 20 μm. These data were obtained with the instrument in the configuration shown in Fig. 1A but similar results were gathered when the uncaging laser was deflected into the “holographic module” (configuration shown in Fig. 1B). The objective used was an Olympus 20X. (E) Line profiles showing the variation of light within different holographic shapes. Profiles shown in E correspond to the red lines shown in (D). Values on the x axis are expressed in μm while the y axis shows normalized intensity values in arbitrary units.

Fig. 5
Fig. 5

Simultaneous holographic imaging and galvo-steered uncaging on living neurons. A) Fluorescence image of Fluo-4-loaded hippocampal neurons in culture. Based on this image, ROIs corresponding to different cells or portion of a given cell are identified (green spots numbered 1-9). These ROIs are used to generate a phase hologram which results in fluorescence imaging over time only in those ROIs. The location of the four uncaging spots is indicated by the red crosses. Scale bar: 20 μm. (B) Transmitted light image of the same field of view shown in A. Scale bar: 20 μm. (C) ΔF/F0 values of Fluo-4 fluorescence as a function of time for the nine regions of interest before and after the sequential uncaging of MNI-glutamate in four different locations (red crosses in A). The arrows indicate the time of delivery of the photolysis stimulus. (D) The fluorescence time course in three different regions is shown. Note that in ROI #1-2, but not ROI #5, a clear response to MNI-glutamate uncaging is observed. (E) Left: Time course of Fluo-4 fluorescence for three holographic dots showing spontaneous Ca2+ signals recorded at 71 frames/s. The position of the different ROIs is shown in the fluorescence image displayed on the right. Scale bar 15 μm.

Fig. 3
Fig. 3

Axial dimension of structured light and point spread function of the holographic microscope. A-C) x-y (top) and x-z (bottom) profiles for illumination of a 115 nm thick film of fluorescein and perylenediimid with three circular shapes of different diameter. Scale bars: 10 μm. D-E) The intensity profile in the axial direction is generated for five different patterns of illumination (D). The full width (in the axial direction) at half maximum (FWHM) is then measured and plotted as a function of the x dimension of the light pattern (E). Note that the x dimension is calculated as the width of the intensity profile (in the x direction) at a threshold level set to 5 times the standard deviation of the background. F-G) Intensity profiles in the x-y (F) and z (G) directions of sub-resolved (20 nm) fluorescent beads excited at 830 nm with the imaging beam passing (holo, red trace) and not passing (no holo, black trace) through the “holographic module”. Each trace is the average of four measurements on different beads. These recordings were performed with a 60X, water immersion objective with a numerical aperture of 1.1.

Fig. 4
Fig. 4

Illumination of a neuronal cell with complex light patterns. A) A two-photon fluorescence image of a Fluo-4-loaded neuron is acquired with the CCD camera (CCD1 in Fig. 1). Scale bar: 10 μm. B-C) Binary images containing only the regions of interest corresponding to the cell body (B1) or the neuronal processes (B and B2) are created a posteriori from the image shown in (A) and used to generate the DOEs displayed in C-C2). Scale bar: 10 μm. D-D2) Fluorescence images obtained with holographic illumination with the patterns shown in (B-B2). Scale bar: 10 μm.

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