Abstract

We present a de novo design of an objective for use in multi-photon (MPM) and second harmonic generation (SHG) microscopy. This objective was designed to have a large field of view (FOV), while maintaining a moderate numerical aperture (NA) and relative straight forward construction. A dichroic beam splitter was incorporated within the objective itself allowing for an increase in the front aperture of the objective and corresponding enhancement of the solid angle of collected emission by an order of magnitude over existing designs.

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  1. W. Denk, J. H. Strickler, and W. W. Webb, “Two-photon laser scanning fluorescence microscopy,” Science 248(4951), 73–76 (1990).
    [CrossRef] [PubMed]
  2. W. R. Zipfel, R. M. Williams, and W. W. Webb, “Nonlinear magic: multiphoton microscopy in the biosciences,” Nat. Biotechnol. 21(11), 1369–1377 (2003).
    [CrossRef] [PubMed]
  3. P. Theer and W. Denk, “On the fundamental imaging-depth limit in two-photon microscopy,” J. Opt. Soc. Am. A 23(12), 3139–3149 (2006).
    [CrossRef]
  4. 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(Pt 3), 330–337 (2007).
    [CrossRef] [PubMed]
  5. J. D. McMullen and W. Zipfel, “A Scheme for Increasing the Collection Efficiency of Multiphoton Microscopy,” Biophys. J. 96(3), 639a (2009).
    [CrossRef]
  6. C. J. Engelbrecht, W. Göbel, and F. Helmchen, “Enhanced fluorescence signal in nonlinear microscopy through supplementary fiber-optic light collection,” Opt. Express 17(8), 6421–6435 (2009).
    [CrossRef] [PubMed]
  7. D. Vucinić, T. M. Bartol, and T. J. Sejnowski, “Hybrid reflecting objectives for functional multiphoton microscopy in turbid media,” Opt. Lett. 31(16), 2447–2449 (2006).
    [CrossRef] [PubMed]
  8. M. Lelek, E. Suran, F. Louradour, A. Barthelemy, B. Viellerobe, and F. Lacombe, “Coherent femtosecond pulse shaping for the optimization of a non-linear micro-endoscope,” Opt. Express 15(16), 10154–10162 (2007).
    [CrossRef] [PubMed]
  9. W. Göbel, J. N. D. Kerr, A. Nimmerjahn, and F. Helmchen, “Miniaturized two-photon microscope based on a flexible coherent fiber bundle and a gradient-index lens objective,” Opt. Lett. 29(21), 2521–2523 (2004).
    [CrossRef] [PubMed]
  10. L. Fu, X. Gan, and M. Gu, “Nonlinear optical microscopy based on double-clad photonic crystal fibers,” Opt. Express 13(14), 5528–5534 (2005).
    [CrossRef] [PubMed]
  11. W. R. Zipfel, R. M. Williams, R. Christie, A. Y. Nikitin, B. T. Hyman, and W. W. Webb, “Live tissue intrinsic emission microscopy using multiphoton-excited native fluorescence and second harmonic generation,” Proc. Natl. Acad. Sci. U.S.A. 100(12), 7075–7080 (2003).
    [CrossRef] [PubMed]
  12. J. N. Rogart, J. Nagata, C. S. Loeser, R. D. Roorda, H. Aslanian, M. E. Robert, W. R. Zipfel, and M. H. Nathanson, “Multiphoton imaging can be used for microscopic examination of intact human gastrointestinal mucosa ex vivo,” Clin. Gastroenterol. Hepatol. 6(1), 95–101 (2008).
    [CrossRef]
  13. S. Mukherjee, J. S. Wysock, C. K. Ng, M. Akhtar, S. Perner, M. M. Lee, M. A. Rubin, F. R. Maxfield, W. W. Webb, and D. S. Scherr, “Human bladder cancer diagnosis using Multiphoton Microscopy,” Proc. SPIE 7161, 1–10 (2009).
  14. Y. Shimizu and H. Takenaka, “Microscope Objective Design,” Adv. Opt. Electron Microsc. 14, 249–334 (1994).
  15. W. J. Smith, “Modern Optical Engineering”, (McGraw Hill, 2000) Chapter 13.
  16. H. Kazuhiro, Olympus Optical Company, Ltd., “Microscope Objective” Japanese Patent 11–231224 (1999)
  17. W. F. Cheong, S. A. Prahl, and A. J. Welch, “A Review of the Optical Properties of Biological Tissues,” IEEE J. Quantum Electron. 26(12), 2166–2185 (1990).
    [CrossRef]
  18. E. Beaurepaire and J. Mertz, “Epifluorescence collection in two-photon microscopy,” Appl. Opt. 41(25), 5376–5382 (2002).
    [CrossRef] [PubMed]

2009 (3)

J. D. McMullen and W. Zipfel, “A Scheme for Increasing the Collection Efficiency of Multiphoton Microscopy,” Biophys. J. 96(3), 639a (2009).
[CrossRef]

S. Mukherjee, J. S. Wysock, C. K. Ng, M. Akhtar, S. Perner, M. M. Lee, M. A. Rubin, F. R. Maxfield, W. W. Webb, and D. S. Scherr, “Human bladder cancer diagnosis using Multiphoton Microscopy,” Proc. SPIE 7161, 1–10 (2009).

C. J. Engelbrecht, W. Göbel, and F. Helmchen, “Enhanced fluorescence signal in nonlinear microscopy through supplementary fiber-optic light collection,” Opt. Express 17(8), 6421–6435 (2009).
[CrossRef] [PubMed]

2008 (1)

J. N. Rogart, J. Nagata, C. S. Loeser, R. D. Roorda, H. Aslanian, M. E. Robert, W. R. Zipfel, and M. H. Nathanson, “Multiphoton imaging can be used for microscopic examination of intact human gastrointestinal mucosa ex vivo,” Clin. Gastroenterol. Hepatol. 6(1), 95–101 (2008).
[CrossRef]

2007 (2)

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(Pt 3), 330–337 (2007).
[CrossRef] [PubMed]

M. Lelek, E. Suran, F. Louradour, A. Barthelemy, B. Viellerobe, and F. Lacombe, “Coherent femtosecond pulse shaping for the optimization of a non-linear micro-endoscope,” Opt. Express 15(16), 10154–10162 (2007).
[CrossRef] [PubMed]

2006 (2)

2005 (1)

2004 (1)

2003 (2)

W. R. Zipfel, R. M. Williams, R. Christie, A. Y. Nikitin, B. T. Hyman, and W. W. Webb, “Live tissue intrinsic emission microscopy using multiphoton-excited native fluorescence and second harmonic generation,” Proc. Natl. Acad. Sci. U.S.A. 100(12), 7075–7080 (2003).
[CrossRef] [PubMed]

W. R. Zipfel, R. M. Williams, and W. W. Webb, “Nonlinear magic: multiphoton microscopy in the biosciences,” Nat. Biotechnol. 21(11), 1369–1377 (2003).
[CrossRef] [PubMed]

2002 (1)

1994 (1)

Y. Shimizu and H. Takenaka, “Microscope Objective Design,” Adv. Opt. Electron Microsc. 14, 249–334 (1994).

1990 (2)

W. F. Cheong, S. A. Prahl, and A. J. Welch, “A Review of the Optical Properties of Biological Tissues,” IEEE J. Quantum Electron. 26(12), 2166–2185 (1990).
[CrossRef]

W. Denk, J. H. Strickler, and W. W. Webb, “Two-photon laser scanning fluorescence microscopy,” Science 248(4951), 73–76 (1990).
[CrossRef] [PubMed]

Akhtar, M.

S. Mukherjee, J. S. Wysock, C. K. Ng, M. Akhtar, S. Perner, M. M. Lee, M. A. Rubin, F. R. Maxfield, W. W. Webb, and D. S. Scherr, “Human bladder cancer diagnosis using Multiphoton Microscopy,” Proc. SPIE 7161, 1–10 (2009).

Aslanian, H.

J. N. Rogart, J. Nagata, C. S. Loeser, R. D. Roorda, H. Aslanian, M. E. Robert, W. R. Zipfel, and M. H. Nathanson, “Multiphoton imaging can be used for microscopic examination of intact human gastrointestinal mucosa ex vivo,” Clin. Gastroenterol. Hepatol. 6(1), 95–101 (2008).
[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(Pt 3), 330–337 (2007).
[CrossRef] [PubMed]

Barthelemy, A.

Bartol, T. M.

Beaurepaire, E.

Cheong, W. F.

W. F. Cheong, S. A. Prahl, and A. J. Welch, “A Review of the Optical Properties of Biological Tissues,” IEEE J. Quantum Electron. 26(12), 2166–2185 (1990).
[CrossRef]

Christie, R.

W. R. Zipfel, R. M. Williams, R. Christie, A. Y. Nikitin, B. T. Hyman, and W. W. Webb, “Live tissue intrinsic emission microscopy using multiphoton-excited native fluorescence and second harmonic generation,” Proc. Natl. Acad. Sci. U.S.A. 100(12), 7075–7080 (2003).
[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(Pt 3), 330–337 (2007).
[CrossRef] [PubMed]

Denk, W.

P. Theer and W. Denk, “On the fundamental imaging-depth limit in two-photon microscopy,” J. Opt. Soc. Am. A 23(12), 3139–3149 (2006).
[CrossRef]

W. Denk, J. H. Strickler, and W. W. Webb, “Two-photon laser scanning fluorescence microscopy,” Science 248(4951), 73–76 (1990).
[CrossRef] [PubMed]

Engelbrecht, C. J.

Fu, L.

Gan, X.

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(Pt 3), 330–337 (2007).
[CrossRef] [PubMed]

Göbel, W.

Gu, M.

Helmchen, F.

Hyman, B. T.

W. R. Zipfel, R. M. Williams, R. Christie, A. Y. Nikitin, B. T. Hyman, and W. W. Webb, “Live tissue intrinsic emission microscopy using multiphoton-excited native fluorescence and second harmonic generation,” Proc. Natl. Acad. Sci. U.S.A. 100(12), 7075–7080 (2003).
[CrossRef] [PubMed]

Kerr, J. N. D.

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(Pt 3), 330–337 (2007).
[CrossRef] [PubMed]

Lacombe, F.

Lee, M. M.

S. Mukherjee, J. S. Wysock, C. K. Ng, M. Akhtar, S. Perner, M. M. Lee, M. A. Rubin, F. R. Maxfield, W. W. Webb, and D. S. Scherr, “Human bladder cancer diagnosis using Multiphoton Microscopy,” Proc. SPIE 7161, 1–10 (2009).

Lelek, M.

Loeser, C. S.

J. N. Rogart, J. Nagata, C. S. Loeser, R. D. Roorda, H. Aslanian, M. E. Robert, W. R. Zipfel, and M. H. Nathanson, “Multiphoton imaging can be used for microscopic examination of intact human gastrointestinal mucosa ex vivo,” Clin. Gastroenterol. Hepatol. 6(1), 95–101 (2008).
[CrossRef]

Louradour, F.

Maxfield, F. R.

S. Mukherjee, J. S. Wysock, C. K. Ng, M. Akhtar, S. Perner, M. M. Lee, M. A. Rubin, F. R. Maxfield, W. W. Webb, and D. S. Scherr, “Human bladder cancer diagnosis using Multiphoton Microscopy,” Proc. SPIE 7161, 1–10 (2009).

McMullen, J. D.

J. D. McMullen and W. Zipfel, “A Scheme for Increasing the Collection Efficiency of Multiphoton Microscopy,” Biophys. J. 96(3), 639a (2009).
[CrossRef]

Mertz, J.

Mukherjee, S.

S. Mukherjee, J. S. Wysock, C. K. Ng, M. Akhtar, S. Perner, M. M. Lee, M. A. Rubin, F. R. Maxfield, W. W. Webb, and D. S. Scherr, “Human bladder cancer diagnosis using Multiphoton Microscopy,” Proc. SPIE 7161, 1–10 (2009).

Nagata, J.

J. N. Rogart, J. Nagata, C. S. Loeser, R. D. Roorda, H. Aslanian, M. E. Robert, W. R. Zipfel, and M. H. Nathanson, “Multiphoton imaging can be used for microscopic examination of intact human gastrointestinal mucosa ex vivo,” Clin. Gastroenterol. Hepatol. 6(1), 95–101 (2008).
[CrossRef]

Nathanson, M. H.

J. N. Rogart, J. Nagata, C. S. Loeser, R. D. Roorda, H. Aslanian, M. E. Robert, W. R. Zipfel, and M. H. Nathanson, “Multiphoton imaging can be used for microscopic examination of intact human gastrointestinal mucosa ex vivo,” Clin. Gastroenterol. Hepatol. 6(1), 95–101 (2008).
[CrossRef]

Ng, C. K.

S. Mukherjee, J. S. Wysock, C. K. Ng, M. Akhtar, S. Perner, M. M. Lee, M. A. Rubin, F. R. Maxfield, W. W. Webb, and D. S. Scherr, “Human bladder cancer diagnosis using Multiphoton Microscopy,” Proc. SPIE 7161, 1–10 (2009).

Nikitin, A. Y.

W. R. Zipfel, R. M. Williams, R. Christie, A. Y. Nikitin, B. T. Hyman, and W. W. Webb, “Live tissue intrinsic emission microscopy using multiphoton-excited native fluorescence and second harmonic generation,” Proc. Natl. Acad. Sci. U.S.A. 100(12), 7075–7080 (2003).
[CrossRef] [PubMed]

Nimmerjahn, A.

Perner, S.

S. Mukherjee, J. S. Wysock, C. K. Ng, M. Akhtar, S. Perner, M. M. Lee, M. A. Rubin, F. R. Maxfield, W. W. Webb, and D. S. Scherr, “Human bladder cancer diagnosis using Multiphoton Microscopy,” Proc. SPIE 7161, 1–10 (2009).

Prahl, S. A.

W. F. Cheong, S. A. Prahl, and A. J. Welch, “A Review of the Optical Properties of Biological Tissues,” IEEE J. Quantum Electron. 26(12), 2166–2185 (1990).
[CrossRef]

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(Pt 3), 330–337 (2007).
[CrossRef] [PubMed]

Robert, M. E.

J. N. Rogart, J. Nagata, C. S. Loeser, R. D. Roorda, H. Aslanian, M. E. Robert, W. R. Zipfel, and M. H. Nathanson, “Multiphoton imaging can be used for microscopic examination of intact human gastrointestinal mucosa ex vivo,” Clin. Gastroenterol. Hepatol. 6(1), 95–101 (2008).
[CrossRef]

Rogart, J. N.

J. N. Rogart, J. Nagata, C. S. Loeser, R. D. Roorda, H. Aslanian, M. E. Robert, W. R. Zipfel, and M. H. Nathanson, “Multiphoton imaging can be used for microscopic examination of intact human gastrointestinal mucosa ex vivo,” Clin. Gastroenterol. Hepatol. 6(1), 95–101 (2008).
[CrossRef]

Roorda, R. D.

J. N. Rogart, J. Nagata, C. S. Loeser, R. D. Roorda, H. Aslanian, M. E. Robert, W. R. Zipfel, and M. H. Nathanson, “Multiphoton imaging can be used for microscopic examination of intact human gastrointestinal mucosa ex vivo,” Clin. Gastroenterol. Hepatol. 6(1), 95–101 (2008).
[CrossRef]

Rubin, M. A.

S. Mukherjee, J. S. Wysock, C. K. Ng, M. Akhtar, S. Perner, M. M. Lee, M. A. Rubin, F. R. Maxfield, W. W. Webb, and D. S. Scherr, “Human bladder cancer diagnosis using Multiphoton Microscopy,” Proc. SPIE 7161, 1–10 (2009).

Scherr, D. S.

S. Mukherjee, J. S. Wysock, C. K. Ng, M. Akhtar, S. Perner, M. M. Lee, M. A. Rubin, F. R. Maxfield, W. W. Webb, and D. S. Scherr, “Human bladder cancer diagnosis using Multiphoton Microscopy,” Proc. SPIE 7161, 1–10 (2009).

Sejnowski, T. J.

Shimizu, Y.

Y. Shimizu and H. Takenaka, “Microscope Objective Design,” Adv. Opt. Electron Microsc. 14, 249–334 (1994).

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(Pt 3), 330–337 (2007).
[CrossRef] [PubMed]

Strickler, J. H.

W. Denk, J. H. Strickler, and W. W. Webb, “Two-photon laser scanning fluorescence microscopy,” Science 248(4951), 73–76 (1990).
[CrossRef] [PubMed]

Suran, E.

Takenaka, H.

Y. Shimizu and H. Takenaka, “Microscope Objective Design,” Adv. Opt. Electron Microsc. 14, 249–334 (1994).

Theer, P.

Viellerobe, B.

Vucinic, D.

Webb, W. W.

S. Mukherjee, J. S. Wysock, C. K. Ng, M. Akhtar, S. Perner, M. M. Lee, M. A. Rubin, F. R. Maxfield, W. W. Webb, and D. S. Scherr, “Human bladder cancer diagnosis using Multiphoton Microscopy,” Proc. SPIE 7161, 1–10 (2009).

W. R. Zipfel, R. M. Williams, and W. W. Webb, “Nonlinear magic: multiphoton microscopy in the biosciences,” Nat. Biotechnol. 21(11), 1369–1377 (2003).
[CrossRef] [PubMed]

W. R. Zipfel, R. M. Williams, R. Christie, A. Y. Nikitin, B. T. Hyman, and W. W. Webb, “Live tissue intrinsic emission microscopy using multiphoton-excited native fluorescence and second harmonic generation,” Proc. Natl. Acad. Sci. U.S.A. 100(12), 7075–7080 (2003).
[CrossRef] [PubMed]

W. Denk, J. H. Strickler, and W. W. Webb, “Two-photon laser scanning fluorescence microscopy,” Science 248(4951), 73–76 (1990).
[CrossRef] [PubMed]

Welch, A. J.

W. F. Cheong, S. A. Prahl, and A. J. Welch, “A Review of the Optical Properties of Biological Tissues,” IEEE J. Quantum Electron. 26(12), 2166–2185 (1990).
[CrossRef]

Williams, R. M.

W. R. Zipfel, R. M. Williams, R. Christie, A. Y. Nikitin, B. T. Hyman, and W. W. Webb, “Live tissue intrinsic emission microscopy using multiphoton-excited native fluorescence and second harmonic generation,” Proc. Natl. Acad. Sci. U.S.A. 100(12), 7075–7080 (2003).
[CrossRef] [PubMed]

W. R. Zipfel, R. M. Williams, and W. W. Webb, “Nonlinear magic: multiphoton microscopy in the biosciences,” Nat. Biotechnol. 21(11), 1369–1377 (2003).
[CrossRef] [PubMed]

Wysock, J. S.

S. Mukherjee, J. S. Wysock, C. K. Ng, M. Akhtar, S. Perner, M. M. Lee, M. A. Rubin, F. R. Maxfield, W. W. Webb, and D. S. Scherr, “Human bladder cancer diagnosis using Multiphoton Microscopy,” Proc. SPIE 7161, 1–10 (2009).

Zipfel, W.

J. D. McMullen and W. Zipfel, “A Scheme for Increasing the Collection Efficiency of Multiphoton Microscopy,” Biophys. J. 96(3), 639a (2009).
[CrossRef]

Zipfel, W. R.

J. N. Rogart, J. Nagata, C. S. Loeser, R. D. Roorda, H. Aslanian, M. E. Robert, W. R. Zipfel, and M. H. Nathanson, “Multiphoton imaging can be used for microscopic examination of intact human gastrointestinal mucosa ex vivo,” Clin. Gastroenterol. Hepatol. 6(1), 95–101 (2008).
[CrossRef]

W. R. Zipfel, R. M. Williams, and W. W. Webb, “Nonlinear magic: multiphoton microscopy in the biosciences,” Nat. Biotechnol. 21(11), 1369–1377 (2003).
[CrossRef] [PubMed]

W. R. Zipfel, R. M. Williams, R. Christie, A. Y. Nikitin, B. T. Hyman, and W. W. Webb, “Live tissue intrinsic emission microscopy using multiphoton-excited native fluorescence and second harmonic generation,” Proc. Natl. Acad. Sci. U.S.A. 100(12), 7075–7080 (2003).
[CrossRef] [PubMed]

Adv. Opt. Electron Microsc. (1)

Y. Shimizu and H. Takenaka, “Microscope Objective Design,” Adv. Opt. Electron Microsc. 14, 249–334 (1994).

Appl. Opt. (1)

Biophys. J. (1)

J. D. McMullen and W. Zipfel, “A Scheme for Increasing the Collection Efficiency of Multiphoton Microscopy,” Biophys. J. 96(3), 639a (2009).
[CrossRef]

Clin. Gastroenterol. Hepatol. (1)

J. N. Rogart, J. Nagata, C. S. Loeser, R. D. Roorda, H. Aslanian, M. E. Robert, W. R. Zipfel, and M. H. Nathanson, “Multiphoton imaging can be used for microscopic examination of intact human gastrointestinal mucosa ex vivo,” Clin. Gastroenterol. Hepatol. 6(1), 95–101 (2008).
[CrossRef]

IEEE J. Quantum Electron. (1)

W. F. Cheong, S. A. Prahl, and A. J. Welch, “A Review of the Optical Properties of Biological Tissues,” IEEE J. Quantum Electron. 26(12), 2166–2185 (1990).
[CrossRef]

J. Microsc. (1)

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(Pt 3), 330–337 (2007).
[CrossRef] [PubMed]

J. Opt. Soc. Am. A (1)

Nat. Biotechnol. (1)

W. R. Zipfel, R. M. Williams, and W. W. Webb, “Nonlinear magic: multiphoton microscopy in the biosciences,” Nat. Biotechnol. 21(11), 1369–1377 (2003).
[CrossRef] [PubMed]

Opt. Express (3)

Opt. Lett. (2)

Proc. Natl. Acad. Sci. U.S.A. (1)

W. R. Zipfel, R. M. Williams, R. Christie, A. Y. Nikitin, B. T. Hyman, and W. W. Webb, “Live tissue intrinsic emission microscopy using multiphoton-excited native fluorescence and second harmonic generation,” Proc. Natl. Acad. Sci. U.S.A. 100(12), 7075–7080 (2003).
[CrossRef] [PubMed]

Proc. SPIE (1)

S. Mukherjee, J. S. Wysock, C. K. Ng, M. Akhtar, S. Perner, M. M. Lee, M. A. Rubin, F. R. Maxfield, W. W. Webb, and D. S. Scherr, “Human bladder cancer diagnosis using Multiphoton Microscopy,” Proc. SPIE 7161, 1–10 (2009).

Science (1)

W. Denk, J. H. Strickler, and W. W. Webb, “Two-photon laser scanning fluorescence microscopy,” Science 248(4951), 73–76 (1990).
[CrossRef] [PubMed]

Other (2)

W. J. Smith, “Modern Optical Engineering”, (McGraw Hill, 2000) Chapter 13.

H. Kazuhiro, Olympus Optical Company, Ltd., “Microscope Objective” Japanese Patent 11–231224 (1999)

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Figures (4)

Fig. 1
Fig. 1

The design of an objective incorporating a beam splitter to efficiently decouple the excitation and collection path (a) air immersion, (b) water immersion

Fig. 2
Fig. 2

Characterization of the excitation transmission for the air and water immersion objectives (a,d) modulation transfer function (b,e) two photon PSF at the greatest field angle - scale bar = 2μm (c,f) field curvature at the edge of the field for the tangential (dashed) and sagittal (solid) surfaces.

Fig. 3
Fig. 3

A cross-section of the water immersion objective modeled using LightTools a) receiver b) water c) tissue slab d) beam splitter e) point source (scale bar ≈1 cm)

Fig. 4
Fig. 4

Simulated light collection in a scattering material. a) Fractional collection as a function of depth of the (on-axis) photon origin in the scattering material. b-d) Spatial variance in the collection path. Collection of scattered light calculated for differing photon origins positions over the FOV relative to the optical axis. b) Olympus 4x objective with focal plane near surface, c) water immersion objective for focal plane near surface, and d) the water immersion objective with the focal plane 500 μm into the simulated tissue.

Tables (3)

Tables Icon

Table 1 Surface Properties of the Air and Water Immersion Objectives

Tables Icon

Table 2 A Summary of Objective Lens Parameters

Tables Icon

Table 3 A Summary of The Lens' Numerical Apertures

Equations (2)

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

ω x , y = 0.32 λ 2 N A           ω z = 0.532 λ 2 [ 1 n n 2 N A 2 ]
N A c = n sin ( cos 1 ( 1 2 Φ ) )

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