Abstract

Nonlinear microscopy techniques crucially rely on efficient signal detection. Here, we present a ring of large-core optical fibers for epi-collection of fluorescence photons that are not transmitted through the objective and thus normally wasted. Theoretical treatments indicated that such a supplementary fiber-optic light collection system (SUFICS) can provide an up to 4-fold signal gain. In typical in vivo imaging experiments, the fiber-ring channel was brighter than the objective channel down to 800 μm depth, thus providing a gain >2. Moreover, SUFICS reduced noise levels in calcium imaging experiments by about 23%. We recommend SUFICS as a generally applicable, effective add-on to nonlinear microscopes for enhancing fluorescence signals.

© 2009 Optical Society of America

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2009

J. D. McMullen and W. Zipfel, "A Scheme for Increasing the Collection Efficiency of Multiphoton Microscopy," Biophys. J. 96, 639a (2009).
[CrossRef]

2008

A. Leray, C. Odin, and Y. Le Grand, "Out-of-focus fluorescence collection in two-photon microscopy of scattering media," Opt. Commun. 281, 6139-6144 (2008).
[CrossRef]

N. Ji, J. C. Magee, and E. Betzig, "High-speed, low-photodamage nonlinear imaging using passive pulse splitters," Nat. Methods 5, 197-202 (2008).
[CrossRef] [PubMed]

C. J. Engelbrecht, R. S. Johnston, E. J. Seibel, and F. Helmchen, "Ultra-compact fiber-optic two-photon microscope for functional fluorescence imaging in vivo," Opt. Express 16, 5556-5564 (2008).
[CrossRef] [PubMed]

2007

D. Debarre, N. Olivier, and E. Beaurepaire, "Signal epidetection in third-harmonic generation microscopy of turbid media," Opt. Express 15, 8913-8924 (2007).
[CrossRef] [PubMed]

G. Donnert, C. Eggeling, and S. W. Hell, "Major signal increase in fluorescence microscopy through dark-state relaxation," Nat. Methods 4, 81-86 (2007).
[CrossRef]

C. A. Combs, A. V. Smirnov, J. D. Riley, A. H. Gandjbakhche, J. R. Knutson, and R. S. Balaban, "Optimization of multiphoton excitation microscopy by total emission detection using a parabolic light reflector," J. Microsc. 228, 330-337 (2007).
[CrossRef] [PubMed]

W. Göbel, B. M. Kampa, and F. Helmchen, "Imaging cellular network dynamics in three dimensions using fast 3D laser scanning," Nat. Methods 4, 73-79 (2007).
[CrossRef]

W. Göbel and F. Helmchen, "In vivo calcium imaging of neural network function," Physiology (Bethesda) 22, 358-365 (2007).
[CrossRef]

2006

P. Theer and W. Denk, "On the fundamental imaging-depth limit in two-photon microscopy," J. Opt. Soc. Am. A Opt. Image Sci. Vis. 23, 3139-3149 (2006).
[CrossRef] [PubMed]

K. Svoboda and R. Yasuda, "Principles of two-photon excitation microscopy and its applications to neuroscience," Neuron 50, 823-839 (2006).
[CrossRef] [PubMed]

D. Vucinic, T. M. Bartol, and T. J. Sejnowski, "Hybrid reflecting objectives for functional multiphoton microscopy in turbid media," Opt. Lett. 31, 2447-2449 (2006).
[CrossRef] [PubMed]

E. J. Seibel, R. S. Johnston, and C. D. Melville, "A full-color scanning fiber endoscope," Optical Fibers and Sensors for Medical Diagnostics and Treatment Applications VI.Proc. SPIE. 6083, 9-16 (2006).

2005

F. Helmchen and W. Denk, "Deep tissue two-photon microscopy," Nat. Methods 2, 932-940 (2005).
[CrossRef] [PubMed]

J. N. D. Kerr, D. Greenberg, and F. Helmchen, "Imaging input and output of neocortical networks in vivo " Proc. Natl. Acad. Sci. U.S.A. 102, 14063-14068 (2005).
[CrossRef] [PubMed]

B. A. Flusberg, E. D. Cocker, W. Piyawattanametha, J. C. Jung, E. L. M. Cheung, and M. J. Schnitzer, "Fiber-optic fluorescence imaging," Nat. Methods 2, 941-950 (2005).
[CrossRef] [PubMed]

2004

J. Mertz, "Nonlinear microscopy: new techniques and applications," Curr. Opin. Neurobiol. 14, 610-616 (2004).
[CrossRef] [PubMed]

2003

W. R. Zipfel, R. M. Williams, and W. W. Webb, "Nonlinear magic: multiphoton microscopy in the biosciences," Nat. Biotechnol. 21, 1369-1377 (2003).
[CrossRef] [PubMed]

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, 7319-7324 (2003).
[CrossRef] [PubMed]

2002

J. X. Cheng, Y. K. Jia, G. Zheng, and X. S. Xie, "Laser-scanning coherent anti-Stokes Raman scattering microscopy and applications to cell biology," Biophys. J. 83, 502-509 (2002).
[CrossRef] [PubMed]

E. Beaurepaire and J. Mertz, "Epifluorescence collection in two-photon microscopy," Appl. Opt. 41, 5376-5382 (2002).
[CrossRef] [PubMed]

2001

A. Hopt and E. Neher, "Highly nonlinear photodamage in two-photon fluorescence microscopy," Biophys. J. 80, 2029-2036 (2001).
[CrossRef] [PubMed]

M. Oheim, E. Beaurepaire, E. Chaigneau, J. Mertz, and S. Charpak, "Two-photon microscopy in brain tissue: parameters influencing the imaging depth (Vol. 111, pg 29, 2001)," J. Neurosci. Meth. 112, 205 (2001).
[CrossRef]

1999

H. J. Koester, D. Baur, R. Uhl, and S. W. Hell, "Ca2+ fluorescence imaging with pico- and femtosecond two-photon excitation: signal and photodamage," Biophys. J. 77, 2226-2236 (1999).
[CrossRef] [PubMed]

Z. F. Mainen, M. Maletic-Savatic, S. H. Shi, Y. Hayashi, R. Malinow, and K. Svoboda, "Two-photon imaging in living brain slices," Methods 18, 231-239 (1999).
[CrossRef] [PubMed]

1995

L. Wang, S. L. Jacques, and L. Zheng, "MCML--Monte Carlo modeling of light transport in multi-layered tissues," Comput. Methods Programs Biomed. 47, 131-146 (1995).
[CrossRef] [PubMed]

1990

W. Denk, J. H. Strickler, and W. W. Webb, "Two-photon laser scanning fluorescence microscopy," Science 248, 73-76 (1990).
[CrossRef] [PubMed]

1941

L. G. Henyey and J. L. Greenstein, "Diffuse radiation in the galaxy," Astrophys. J. 93, 70-83 (1941).
[CrossRef]

Balaban, R. S.

C. A. Combs, A. V. Smirnov, J. D. Riley, A. H. Gandjbakhche, J. R. Knutson, and R. S. Balaban, "Optimization of multiphoton excitation microscopy by total emission detection using a parabolic light reflector," J. Microsc. 228, 330-337 (2007).
[CrossRef] [PubMed]

Bartol, T. M.

Baur, D.

H. J. Koester, D. Baur, R. Uhl, and S. W. Hell, "Ca2+ fluorescence imaging with pico- and femtosecond two-photon excitation: signal and photodamage," Biophys. J. 77, 2226-2236 (1999).
[CrossRef] [PubMed]

Beaurepaire, E.

Betzig, E.

N. Ji, J. C. Magee, and E. Betzig, "High-speed, low-photodamage nonlinear imaging using passive pulse splitters," Nat. Methods 5, 197-202 (2008).
[CrossRef] [PubMed]

Chaigneau, E.

M. Oheim, E. Beaurepaire, E. Chaigneau, J. Mertz, and S. Charpak, "Two-photon microscopy in brain tissue: parameters influencing the imaging depth (Vol. 111, pg 29, 2001)," J. Neurosci. Meth. 112, 205 (2001).
[CrossRef]

Charpak, S.

M. Oheim, E. Beaurepaire, E. Chaigneau, J. Mertz, and S. Charpak, "Two-photon microscopy in brain tissue: parameters influencing the imaging depth (Vol. 111, pg 29, 2001)," J. Neurosci. Meth. 112, 205 (2001).
[CrossRef]

Cheng, J. X.

J. X. Cheng, Y. K. Jia, G. Zheng, and X. S. Xie, "Laser-scanning coherent anti-Stokes Raman scattering microscopy and applications to cell biology," Biophys. J. 83, 502-509 (2002).
[CrossRef] [PubMed]

Cheung, E. L. M.

B. A. Flusberg, E. D. Cocker, W. Piyawattanametha, J. C. Jung, E. L. M. Cheung, and M. J. Schnitzer, "Fiber-optic fluorescence imaging," Nat. Methods 2, 941-950 (2005).
[CrossRef] [PubMed]

Cocker, E. D.

B. A. Flusberg, E. D. Cocker, W. Piyawattanametha, J. C. Jung, E. L. M. Cheung, and M. J. Schnitzer, "Fiber-optic fluorescence imaging," Nat. Methods 2, 941-950 (2005).
[CrossRef] [PubMed]

Combs, C. A.

C. A. Combs, A. V. Smirnov, J. D. Riley, A. H. Gandjbakhche, J. R. Knutson, and R. S. Balaban, "Optimization of multiphoton excitation microscopy by total emission detection using a parabolic light reflector," J. Microsc. 228, 330-337 (2007).
[CrossRef] [PubMed]

Debarre, D.

Denk, W.

P. Theer and W. Denk, "On the fundamental imaging-depth limit in two-photon microscopy," J. Opt. Soc. Am. A Opt. Image Sci. Vis. 23, 3139-3149 (2006).
[CrossRef] [PubMed]

F. Helmchen and W. Denk, "Deep tissue two-photon microscopy," Nat. Methods 2, 932-940 (2005).
[CrossRef] [PubMed]

W. Denk, J. H. Strickler, and W. W. Webb, "Two-photon laser scanning fluorescence microscopy," Science 248, 73-76 (1990).
[CrossRef] [PubMed]

Donnert, G.

G. Donnert, C. Eggeling, and S. W. Hell, "Major signal increase in fluorescence microscopy through dark-state relaxation," Nat. Methods 4, 81-86 (2007).
[CrossRef]

Eggeling, C.

G. Donnert, C. Eggeling, and S. W. Hell, "Major signal increase in fluorescence microscopy through dark-state relaxation," Nat. Methods 4, 81-86 (2007).
[CrossRef]

Engelbrecht, C. J.

Flusberg, B. A.

B. A. Flusberg, E. D. Cocker, W. Piyawattanametha, J. C. Jung, E. L. M. Cheung, and M. J. Schnitzer, "Fiber-optic fluorescence imaging," Nat. Methods 2, 941-950 (2005).
[CrossRef] [PubMed]

Gandjbakhche, A. H.

C. A. Combs, A. V. Smirnov, J. D. Riley, A. H. Gandjbakhche, J. R. Knutson, and R. S. Balaban, "Optimization of multiphoton excitation microscopy by total emission detection using a parabolic light reflector," J. Microsc. 228, 330-337 (2007).
[CrossRef] [PubMed]

Garaschuk, O.

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, 7319-7324 (2003).
[CrossRef] [PubMed]

Göbel, W.

W. Göbel and F. Helmchen, "In vivo calcium imaging of neural network function," Physiology (Bethesda) 22, 358-365 (2007).
[CrossRef]

W. Göbel, B. M. Kampa, and F. Helmchen, "Imaging cellular network dynamics in three dimensions using fast 3D laser scanning," Nat. Methods 4, 73-79 (2007).
[CrossRef]

Greenberg, D.

J. N. D. Kerr, D. Greenberg, and F. Helmchen, "Imaging input and output of neocortical networks in vivo " Proc. Natl. Acad. Sci. U.S.A. 102, 14063-14068 (2005).
[CrossRef] [PubMed]

Greenstein, J. L.

L. G. Henyey and J. L. Greenstein, "Diffuse radiation in the galaxy," Astrophys. J. 93, 70-83 (1941).
[CrossRef]

Hayashi, Y.

Z. F. Mainen, M. Maletic-Savatic, S. H. Shi, Y. Hayashi, R. Malinow, and K. Svoboda, "Two-photon imaging in living brain slices," Methods 18, 231-239 (1999).
[CrossRef] [PubMed]

Hell, S. W.

G. Donnert, C. Eggeling, and S. W. Hell, "Major signal increase in fluorescence microscopy through dark-state relaxation," Nat. Methods 4, 81-86 (2007).
[CrossRef]

H. J. Koester, D. Baur, R. Uhl, and S. W. Hell, "Ca2+ fluorescence imaging with pico- and femtosecond two-photon excitation: signal and photodamage," Biophys. J. 77, 2226-2236 (1999).
[CrossRef] [PubMed]

Helmchen, F.

C. J. Engelbrecht, R. S. Johnston, E. J. Seibel, and F. Helmchen, "Ultra-compact fiber-optic two-photon microscope for functional fluorescence imaging in vivo," Opt. Express 16, 5556-5564 (2008).
[CrossRef] [PubMed]

W. Göbel and F. Helmchen, "In vivo calcium imaging of neural network function," Physiology (Bethesda) 22, 358-365 (2007).
[CrossRef]

W. Göbel, B. M. Kampa, and F. Helmchen, "Imaging cellular network dynamics in three dimensions using fast 3D laser scanning," Nat. Methods 4, 73-79 (2007).
[CrossRef]

J. N. D. Kerr, D. Greenberg, and F. Helmchen, "Imaging input and output of neocortical networks in vivo " Proc. Natl. Acad. Sci. U.S.A. 102, 14063-14068 (2005).
[CrossRef] [PubMed]

F. Helmchen and W. Denk, "Deep tissue two-photon microscopy," Nat. Methods 2, 932-940 (2005).
[CrossRef] [PubMed]

Henyey, L. G.

L. G. Henyey and J. L. Greenstein, "Diffuse radiation in the galaxy," Astrophys. J. 93, 70-83 (1941).
[CrossRef]

Holthoff, K.

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, 7319-7324 (2003).
[CrossRef] [PubMed]

Hopt, A.

A. Hopt and E. Neher, "Highly nonlinear photodamage in two-photon fluorescence microscopy," Biophys. J. 80, 2029-2036 (2001).
[CrossRef] [PubMed]

Jacques, S. L.

L. Wang, S. L. Jacques, and L. Zheng, "MCML--Monte Carlo modeling of light transport in multi-layered tissues," Comput. Methods Programs Biomed. 47, 131-146 (1995).
[CrossRef] [PubMed]

Ji, N.

N. Ji, J. C. Magee, and E. Betzig, "High-speed, low-photodamage nonlinear imaging using passive pulse splitters," Nat. Methods 5, 197-202 (2008).
[CrossRef] [PubMed]

Jia, Y. K.

J. X. Cheng, Y. K. Jia, G. Zheng, and X. S. Xie, "Laser-scanning coherent anti-Stokes Raman scattering microscopy and applications to cell biology," Biophys. J. 83, 502-509 (2002).
[CrossRef] [PubMed]

Johnston, R. S.

C. J. Engelbrecht, R. S. Johnston, E. J. Seibel, and F. Helmchen, "Ultra-compact fiber-optic two-photon microscope for functional fluorescence imaging in vivo," Opt. Express 16, 5556-5564 (2008).
[CrossRef] [PubMed]

E. J. Seibel, R. S. Johnston, and C. D. Melville, "A full-color scanning fiber endoscope," Optical Fibers and Sensors for Medical Diagnostics and Treatment Applications VI.Proc. SPIE. 6083, 9-16 (2006).

Jung, J. C.

B. A. Flusberg, E. D. Cocker, W. Piyawattanametha, J. C. Jung, E. L. M. Cheung, and M. J. Schnitzer, "Fiber-optic fluorescence imaging," Nat. Methods 2, 941-950 (2005).
[CrossRef] [PubMed]

Kampa, B. M.

W. Göbel, B. M. Kampa, and F. Helmchen, "Imaging cellular network dynamics in three dimensions using fast 3D laser scanning," Nat. Methods 4, 73-79 (2007).
[CrossRef]

Kerr, J. N. D.

J. N. D. Kerr, D. Greenberg, and F. Helmchen, "Imaging input and output of neocortical networks in vivo " Proc. Natl. Acad. Sci. U.S.A. 102, 14063-14068 (2005).
[CrossRef] [PubMed]

Knutson, J. R.

C. A. Combs, A. V. Smirnov, J. D. Riley, A. H. Gandjbakhche, J. R. Knutson, and R. S. Balaban, "Optimization of multiphoton excitation microscopy by total emission detection using a parabolic light reflector," J. Microsc. 228, 330-337 (2007).
[CrossRef] [PubMed]

Koester, H. J.

H. J. Koester, D. Baur, R. Uhl, and S. W. Hell, "Ca2+ fluorescence imaging with pico- and femtosecond two-photon excitation: signal and photodamage," Biophys. J. 77, 2226-2236 (1999).
[CrossRef] [PubMed]

Konnerth, A.

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, 7319-7324 (2003).
[CrossRef] [PubMed]

Le Grand, Y.

A. Leray, C. Odin, and Y. Le Grand, "Out-of-focus fluorescence collection in two-photon microscopy of scattering media," Opt. Commun. 281, 6139-6144 (2008).
[CrossRef]

Leray, A.

A. Leray, C. Odin, and Y. Le Grand, "Out-of-focus fluorescence collection in two-photon microscopy of scattering media," Opt. Commun. 281, 6139-6144 (2008).
[CrossRef]

Magee, J. C.

N. Ji, J. C. Magee, and E. Betzig, "High-speed, low-photodamage nonlinear imaging using passive pulse splitters," Nat. Methods 5, 197-202 (2008).
[CrossRef] [PubMed]

Mainen, Z. F.

Z. F. Mainen, M. Maletic-Savatic, S. H. Shi, Y. Hayashi, R. Malinow, and K. Svoboda, "Two-photon imaging in living brain slices," Methods 18, 231-239 (1999).
[CrossRef] [PubMed]

Maletic-Savatic, M.

Z. F. Mainen, M. Maletic-Savatic, S. H. Shi, Y. Hayashi, R. Malinow, and K. Svoboda, "Two-photon imaging in living brain slices," Methods 18, 231-239 (1999).
[CrossRef] [PubMed]

Malinow, R.

Z. F. Mainen, M. Maletic-Savatic, S. H. Shi, Y. Hayashi, R. Malinow, and K. Svoboda, "Two-photon imaging in living brain slices," Methods 18, 231-239 (1999).
[CrossRef] [PubMed]

McMullen, J. D.

J. D. McMullen and W. Zipfel, "A Scheme for Increasing the Collection Efficiency of Multiphoton Microscopy," Biophys. J. 96, 639a (2009).
[CrossRef]

Melville, C. D.

E. J. Seibel, R. S. Johnston, and C. D. Melville, "A full-color scanning fiber endoscope," Optical Fibers and Sensors for Medical Diagnostics and Treatment Applications VI.Proc. SPIE. 6083, 9-16 (2006).

Mertz, J.

J. Mertz, "Nonlinear microscopy: new techniques and applications," Curr. Opin. Neurobiol. 14, 610-616 (2004).
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E. Beaurepaire and J. Mertz, "Epifluorescence collection in two-photon microscopy," Appl. Opt. 41, 5376-5382 (2002).
[CrossRef] [PubMed]

M. Oheim, E. Beaurepaire, E. Chaigneau, J. Mertz, and S. Charpak, "Two-photon microscopy in brain tissue: parameters influencing the imaging depth (Vol. 111, pg 29, 2001)," J. Neurosci. Meth. 112, 205 (2001).
[CrossRef]

Neher, E.

A. Hopt and E. Neher, "Highly nonlinear photodamage in two-photon fluorescence microscopy," Biophys. J. 80, 2029-2036 (2001).
[CrossRef] [PubMed]

Odin, C.

A. Leray, C. Odin, and Y. Le Grand, "Out-of-focus fluorescence collection in two-photon microscopy of scattering media," Opt. Commun. 281, 6139-6144 (2008).
[CrossRef]

Oheim, M.

M. Oheim, E. Beaurepaire, E. Chaigneau, J. Mertz, and S. Charpak, "Two-photon microscopy in brain tissue: parameters influencing the imaging depth (Vol. 111, pg 29, 2001)," J. Neurosci. Meth. 112, 205 (2001).
[CrossRef]

Olivier, N.

Piyawattanametha, W.

B. A. Flusberg, E. D. Cocker, W. Piyawattanametha, J. C. Jung, E. L. M. Cheung, and M. J. Schnitzer, "Fiber-optic fluorescence imaging," Nat. Methods 2, 941-950 (2005).
[CrossRef] [PubMed]

Riley, J. D.

C. A. Combs, A. V. Smirnov, J. D. Riley, A. H. Gandjbakhche, J. R. Knutson, and R. S. Balaban, "Optimization of multiphoton excitation microscopy by total emission detection using a parabolic light reflector," J. Microsc. 228, 330-337 (2007).
[CrossRef] [PubMed]

Schnitzer, M. J.

B. A. Flusberg, E. D. Cocker, W. Piyawattanametha, J. C. Jung, E. L. M. Cheung, and M. J. Schnitzer, "Fiber-optic fluorescence imaging," Nat. Methods 2, 941-950 (2005).
[CrossRef] [PubMed]

Seibel, E. J.

C. J. Engelbrecht, R. S. Johnston, E. J. Seibel, and F. Helmchen, "Ultra-compact fiber-optic two-photon microscope for functional fluorescence imaging in vivo," Opt. Express 16, 5556-5564 (2008).
[CrossRef] [PubMed]

E. J. Seibel, R. S. Johnston, and C. D. Melville, "A full-color scanning fiber endoscope," Optical Fibers and Sensors for Medical Diagnostics and Treatment Applications VI.Proc. SPIE. 6083, 9-16 (2006).

Sejnowski, T. J.

Shi, S. H.

Z. F. Mainen, M. Maletic-Savatic, S. H. Shi, Y. Hayashi, R. Malinow, and K. Svoboda, "Two-photon imaging in living brain slices," Methods 18, 231-239 (1999).
[CrossRef] [PubMed]

Smirnov, A. V.

C. A. Combs, A. V. Smirnov, J. D. Riley, A. H. Gandjbakhche, J. R. Knutson, and R. S. Balaban, "Optimization of multiphoton excitation microscopy by total emission detection using a parabolic light reflector," J. Microsc. 228, 330-337 (2007).
[CrossRef] [PubMed]

Stosiek, C.

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, 7319-7324 (2003).
[CrossRef] [PubMed]

Strickler, J. H.

W. Denk, J. H. Strickler, and W. W. Webb, "Two-photon laser scanning fluorescence microscopy," Science 248, 73-76 (1990).
[CrossRef] [PubMed]

Svoboda, K.

K. Svoboda and R. Yasuda, "Principles of two-photon excitation microscopy and its applications to neuroscience," Neuron 50, 823-839 (2006).
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Z. F. Mainen, M. Maletic-Savatic, S. H. Shi, Y. Hayashi, R. Malinow, and K. Svoboda, "Two-photon imaging in living brain slices," Methods 18, 231-239 (1999).
[CrossRef] [PubMed]

Theer, P.

P. Theer and W. Denk, "On the fundamental imaging-depth limit in two-photon microscopy," J. Opt. Soc. Am. A Opt. Image Sci. Vis. 23, 3139-3149 (2006).
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Uhl, R.

H. J. Koester, D. Baur, R. Uhl, and S. W. Hell, "Ca2+ fluorescence imaging with pico- and femtosecond two-photon excitation: signal and photodamage," Biophys. J. 77, 2226-2236 (1999).
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Wang, L.

L. Wang, S. L. Jacques, and L. Zheng, "MCML--Monte Carlo modeling of light transport in multi-layered tissues," Comput. Methods Programs Biomed. 47, 131-146 (1995).
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W. R. Zipfel, R. M. Williams, and W. W. Webb, "Nonlinear magic: multiphoton microscopy in the biosciences," Nat. Biotechnol. 21, 1369-1377 (2003).
[CrossRef] [PubMed]

W. Denk, J. H. Strickler, and W. W. Webb, "Two-photon laser scanning fluorescence microscopy," Science 248, 73-76 (1990).
[CrossRef] [PubMed]

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W. R. Zipfel, R. M. Williams, and W. W. Webb, "Nonlinear magic: multiphoton microscopy in the biosciences," Nat. Biotechnol. 21, 1369-1377 (2003).
[CrossRef] [PubMed]

Xie, X. S.

J. X. Cheng, Y. K. Jia, G. Zheng, and X. S. Xie, "Laser-scanning coherent anti-Stokes Raman scattering microscopy and applications to cell biology," Biophys. J. 83, 502-509 (2002).
[CrossRef] [PubMed]

Yasuda, R.

K. Svoboda and R. Yasuda, "Principles of two-photon excitation microscopy and its applications to neuroscience," Neuron 50, 823-839 (2006).
[CrossRef] [PubMed]

Zheng, G.

J. X. Cheng, Y. K. Jia, G. Zheng, and X. S. Xie, "Laser-scanning coherent anti-Stokes Raman scattering microscopy and applications to cell biology," Biophys. J. 83, 502-509 (2002).
[CrossRef] [PubMed]

Zheng, L.

L. Wang, S. L. Jacques, and L. Zheng, "MCML--Monte Carlo modeling of light transport in multi-layered tissues," Comput. Methods Programs Biomed. 47, 131-146 (1995).
[CrossRef] [PubMed]

Zipfel, W.

J. D. McMullen and W. Zipfel, "A Scheme for Increasing the Collection Efficiency of Multiphoton Microscopy," Biophys. J. 96, 639a (2009).
[CrossRef]

Zipfel, W. R.

W. R. Zipfel, R. M. Williams, and W. W. Webb, "Nonlinear magic: multiphoton microscopy in the biosciences," Nat. Biotechnol. 21, 1369-1377 (2003).
[CrossRef] [PubMed]

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Biophys. J.

J. D. McMullen and W. Zipfel, "A Scheme for Increasing the Collection Efficiency of Multiphoton Microscopy," Biophys. J. 96, 639a (2009).
[CrossRef]

A. Hopt and E. Neher, "Highly nonlinear photodamage in two-photon fluorescence microscopy," Biophys. J. 80, 2029-2036 (2001).
[CrossRef] [PubMed]

H. J. Koester, D. Baur, R. Uhl, and S. W. Hell, "Ca2+ fluorescence imaging with pico- and femtosecond two-photon excitation: signal and photodamage," Biophys. J. 77, 2226-2236 (1999).
[CrossRef] [PubMed]

J. X. Cheng, Y. K. Jia, G. Zheng, and X. S. Xie, "Laser-scanning coherent anti-Stokes Raman scattering microscopy and applications to cell biology," Biophys. J. 83, 502-509 (2002).
[CrossRef] [PubMed]

Comput. Methods Programs Biomed.

L. Wang, S. L. Jacques, and L. Zheng, "MCML--Monte Carlo modeling of light transport in multi-layered tissues," Comput. Methods Programs Biomed. 47, 131-146 (1995).
[CrossRef] [PubMed]

Curr. Opin. Neurobiol.

J. Mertz, "Nonlinear microscopy: new techniques and applications," Curr. Opin. Neurobiol. 14, 610-616 (2004).
[CrossRef] [PubMed]

J. Microsc.

C. A. Combs, A. V. Smirnov, J. D. Riley, A. H. Gandjbakhche, J. R. Knutson, and R. S. Balaban, "Optimization of multiphoton excitation microscopy by total emission detection using a parabolic light reflector," J. Microsc. 228, 330-337 (2007).
[CrossRef] [PubMed]

J. Neurosci. Meth.

M. Oheim, E. Beaurepaire, E. Chaigneau, J. Mertz, and S. Charpak, "Two-photon microscopy in brain tissue: parameters influencing the imaging depth (Vol. 111, pg 29, 2001)," J. Neurosci. Meth. 112, 205 (2001).
[CrossRef]

J. Opt. Soc. Am. A Opt. Image Sci. Vis.

P. Theer and W. Denk, "On the fundamental imaging-depth limit in two-photon microscopy," J. Opt. Soc. Am. A Opt. Image Sci. Vis. 23, 3139-3149 (2006).
[CrossRef] [PubMed]

Methods

Z. F. Mainen, M. Maletic-Savatic, S. H. Shi, Y. Hayashi, R. Malinow, and K. Svoboda, "Two-photon imaging in living brain slices," Methods 18, 231-239 (1999).
[CrossRef] [PubMed]

Nat. Biotechnol.

W. R. Zipfel, R. M. Williams, and W. W. Webb, "Nonlinear magic: multiphoton microscopy in the biosciences," Nat. Biotechnol. 21, 1369-1377 (2003).
[CrossRef] [PubMed]

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G. Donnert, C. Eggeling, and S. W. Hell, "Major signal increase in fluorescence microscopy through dark-state relaxation," Nat. Methods 4, 81-86 (2007).
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W. Göbel, B. M. Kampa, and F. Helmchen, "Imaging cellular network dynamics in three dimensions using fast 3D laser scanning," Nat. Methods 4, 73-79 (2007).
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F. Helmchen and W. Denk, "Deep tissue two-photon microscopy," Nat. Methods 2, 932-940 (2005).
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B. A. Flusberg, E. D. Cocker, W. Piyawattanametha, J. C. Jung, E. L. M. Cheung, and M. J. Schnitzer, "Fiber-optic fluorescence imaging," Nat. Methods 2, 941-950 (2005).
[CrossRef] [PubMed]

Neuron

K. Svoboda and R. Yasuda, "Principles of two-photon excitation microscopy and its applications to neuroscience," Neuron 50, 823-839 (2006).
[CrossRef] [PubMed]

Opt. Commun.

A. Leray, C. Odin, and Y. Le Grand, "Out-of-focus fluorescence collection in two-photon microscopy of scattering media," Opt. Commun. 281, 6139-6144 (2008).
[CrossRef]

Opt. Express

Opt. Lett.

Physiology (Bethesda)

W. Göbel and F. Helmchen, "In vivo calcium imaging of neural network function," Physiology (Bethesda) 22, 358-365 (2007).
[CrossRef]

Proc. Natl. Acad. Sci. U.S.A.

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, 7319-7324 (2003).
[CrossRef] [PubMed]

J. N. D. Kerr, D. Greenberg, and F. Helmchen, "Imaging input and output of neocortical networks in vivo " Proc. Natl. Acad. Sci. U.S.A. 102, 14063-14068 (2005).
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Proc. SPIE.

E. J. Seibel, R. S. Johnston, and C. D. Melville, "A full-color scanning fiber endoscope," Optical Fibers and Sensors for Medical Diagnostics and Treatment Applications VI.Proc. SPIE. 6083, 9-16 (2006).

Science

W. Denk, J. H. Strickler, and W. W. Webb, "Two-photon laser scanning fluorescence microscopy," Science 248, 73-76 (1990).
[CrossRef] [PubMed]

Other

W. Denk, D. W. Piston, and W. W. Webb, "Multi-photon molecular excitation in laser-scanning microscopy," in Handbook of Biological Confocal Microscopy, J. B. Pawley, ed., (Plenum Press, New York, 2005).

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E. W. Weisstein, "Circle-Circle Intersection," http://mathworld.wolfram.com/Circle-CircleIntersection.html.

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Figures (7)

Fig. 1.
Fig. 1.

Supplementary epifluorescence collection through a ring of optical fibers. (a) Top: CAD-drawing of a custom fiber-ring holder placed under an objective. Bottom: Closeup view showing the ring-like arrangement of the fiber tips. Only five of eight fibers are shown. Fluorophores are 2-photon excited in the focus of an infrared laser beam (red), causing isotropic fluorescence emission (green). (b) Left: Top view of the ring-like arrangement of eight 1-mm diameter fibers. Right: Dual-channel detection in a custom 2PLSM setup. Optical fibers were bundled and placed in front of a second PMT.

Fig. 2.
Fig. 2.

Theory of fiber-optic fluorescence collection. (a) Large-core optical fibers with a tilt angle ϕ positioned outside of the acceptance cone of a microscope objective. Maximum acceptance angles of objective and fibers are αobj and αf, respectively. (b) Fiber collection from an arbitrarily positioned source. The fiber solid angle Ωf depends on the core radius r0, the source distance R and the off-axis angle γ. See main text for further details. (c) The maximum acceptance angle αf determines the transmitted fraction of photons that hit the fiber core, indicated by the circle-circle intersection area (green). Top: fiber-limited case with a distal source; the fiber core size (red area) limits the maximum number of transmitted photons. Bottom: NA-limited case with a proximal source; the maximum solid angle determined by the NA (green area) limits light collection. (d) Theoretical dependence of single-fiber collection efficiency ηf on axial source position (top) and on lateral position (bottom) for three axial distances (0.5·z0 blue, 1.0·z0 black, 1.5·z0 red; vertical lines in d). Efficiencies are normalized to ηmax, z′ to z0, and r′ to r0. (e) Experimental data points for the axial (top) and lateral dependence (bottom) as obtained from fluorescent bead measurements (n = 5 beads; S.D. error bars). Data points are normalized to the plateau level for z′ < z0.

Fig. 3.
Fig. 3.

Large signal gain by epifluorescence collection through a ring of optical fibers. (a) Signal gain obtained with the fiber-ring. Pollen grain images in objective and fiber-ring channels at various axial offsets Δz. (b) Comparison of theoretical and experimental gains as a function of Δz (solid red curve: analytical prediction; solid red symbols: Monte Carlo simulation; black curve: experiment). (c) Objective channel intensity as a function of axial offset Δz of the fiber-ring. The signal only decreases when the elevated fiber-ring partially obstructs the objective’s light cone as schematically indicated.

Fig. 4.
Fig. 4.

Spatial dependence of individual channel intensities and SUFICS signal gain. (a) Horizontal map of the fluorescence intensity for objective (left) and fiber-ring channel (right) as a function of lateral x- and y-offset of the fiber-ring (fiber-axes intersection aligned with objective’s focal plane). Fluorescence intensity decreases when the objective’s light cone is partially obstructed by fibers as indicated. (b) Left: Horizontal map of the gain factor determined relative to the central fluorescence intensity of the non-obstructed objective channel. Right: Surface map of the gain factor within the fiber-ring arrangement. A non-obstructed field with a diameter of ~0.8 mm was obtained.

Fig. 5.
Fig. 5.

Monte Carlo simulation of SUFICS in scattering tissue. (a) General setup used for Monte-Carlo simulations. Photons were launched from a fluorescence source at different imaging depths zS. After being scattered within the tissue (grainy area) photons either reached the objective (red trajectory) or one of the fiber cores (blue trajectory) or they missed any of them. (b) Collection efficiencies η from Monte-Carlo simulations for fiber-ring (blue) and objective (red) as a function of imaging depth zS for smooth tissue-surfaces. The fiber-ring position was fixed to the tissue surface in all simulations. Analytical curves for transparent samples are displayed for comparison. Collection efficiencies are normalized to 4π. (c) Gain factors as a function of imaging depth zS for different values of the anisotropy factor g (g = 0.95 black squares; g = 0.90 black triangles up; g = 0.85 black circles; g = 0.70 black triangles down). Values above the tissue surface (zS < 0) are academic (no fluorescence staining) and provided for consistency check only. The analytical curve for non-scattering tissue is provided for comparison (line).

Fig. 6.
Fig. 6.

Signal gain and deep imaging with SUFICS in vivo. (a) Photographs of the fiber-ring holder (top) and the fiber tips (bottom) centered above an exposed surface area of mouse neocortex. (b) Two-photon image stack of YFP-expressing neocortical pyramidal cells. Sample images for both, objective and fiber-ring channel, are shown at identical grey scales for 3 focal depths as indicated. (c) Maximum-intensity side projections of the image stacks shown in b for both objective and fiber-ring channel. (d) Signal gain as a function of imaging depth (black circles: experimental data; red: theoretical prediction by Monte-Carlo simulation with smooth surface and g = 0.85).

Fig. 7.
Fig. 7.

Noise reduction of neuronal calcium transients in vivo. (a) Images of OGB1-AM-stained layer 2/3 cells in mouse neocortex for the objective channel (top), the fiber-ring channel (middle), and the combined channel (bottom; all identical grey scales). (b) Examples of spontaneous calcium signals in the 4 cells marked in a. (c) Left: Close-up views of the calcium transient indicated by the box in b. Right: Histograms with Gaussian fits of high-frequency baseline noise (f > 0.5 Hz). (d) Mean widths of Gaussian fits to noise histograms (3 mice, n = 20 cells each) were reduced in the combined channel compared to the objective channel.

Equations (15)

Equations on this page are rendered with MathJax. Learn more.

η obj = Ω NA , obj 4 π = 1 2 ( 1 cos α obj )
Ω f ( r = 0 , z' ) = 2 π ( 1 cos β )
Ω f = ( r , z ) = 2 π ( 1 cos ( arctan ( r γ R ) ) = 2 π ( 1 cos ( arctan ( r 0 z ( cos γ ) 3 / 2 ) ) )
Ω f ( r , z ) = 2 π ( 1 cos β ) ( cos γ ) 2
η max = Ω NA , f 4 π = 1 2 ( 1 cos α f )
η f ( u , v ) = min ( Ω f ( u , v ) , Ω NA , f ) A f ( u , v ) 4 π
G = η obj + N η f ( u , v ) η obj = 1 + N min ( Ω f ( u , v ) Ω NA , f ) A f ( u , v ) Ω NA , obj
G max = η obj + N · η max η obj = 1 + N 1 cos α f 1 cos α obj = 1 + N 1 1 ( N A f / n ) 2 1 1 ( N A obj / n ) 2
A ( R 1 , R 2 , d ) = Re { R 1 2 arccos ( d 2 + R 1 2 R 2 2 2 d R 1 ) + R 2 2 arccos ( d 2 + R 2 2 R 1 2 2 d R 2 ) 1 2 ( R 1 2 ( R 2 d ) 2 ) ( ( R 2 + d ) 2 R 1 2 ) }
A f ( u , v ) = 1 π min ( 1 , u ) 2 Re { arccos ( ν 2 u 2 + 1 2 ν ) + u 2 arccos ( ν 2 + u 2 1 2 uv ) 1 2 ( 1 ( u ν ) 2 ) ( ( u + ν ) 2 1 ) }
for ν > max ( 1 , u ) min ( 1 , u ) ( partial overlap of circles )
A f ( u , ν ) = 1 ν max ( 1 , u ) min ( 1 , u ) ( full overlap of circles )
A f ( u , v ) = 0 for ν > u + 1 ( no overlap of circles )
A f ( ν , 1 ) = 1 π { 2 arccos ( ν 2 ) ν 2 4 ν 2 } ν < = 2
A f ( ν , 1 ) = 0 ν > 2

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