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.

© 2010 OSA

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]

Adv. Opt. Electron Microsc.

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

Appl. Opt.

• E. Beaurepaire and J. Mertz, “Epifluorescence collection in two-photon microscopy,” Appl. Opt. 41(25), 5376–5382 (2002). [CrossRef] [PubMed]

Biophys. J.

• 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.

• 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.

• 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.

• 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

• 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]

Nat. Biotechnol.

• 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

• 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]
• 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]
• 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]

Opt. Lett.

• 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]
• 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]

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

• 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

• 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

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

Other

• 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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