Abstract

We report on the experimental investigation into the characterization of two-photon fluorescence microscopy based on the separation distance of a single-mode optical fiber coupler and a gradient-index (GRIN) rod lens. The collected two-photon fluorescence signal exhibits a maximum intensity at a defined separation distance (gap length) where the increasing effective excitation numerical aperture is balanced by the decreasing confocal emission collection. A maximum signal is found at gap lengths of approximately 2, 1.25, and 1.75 mm for GRIN lenses with pitches of 0.23, 0.25, and 0.29 wavelength at 830 nm. The maximum two-photon fluorescence signal collected corresponds to a threefold reduction of axial resolution (38.5 µm at 1.25 mm), compared with the maximum resolution (11.6 µm at 5.5 mm), as shown by the three-dimensional imaging of 10 µm beads. These results demonstrate an intrinsic trade-off between signal collection and axial resolution.

© 2005 Optical Society of America

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  1. E. B. Brown, R. B. Campbell, Y. Tsuzuki, L. Xu, P. Carmeliet, D. Fukumura, R. K. Jain, “In vivo measurement of gene expression, angiogenesis and physiological function in tumors using multiphoton laser scanning microscopy,” Nat. Med. 7, 864–868 (2001).
    [CrossRef] [PubMed]
  2. C. Stosiek, O. Garaschuk, K. Holthoff, A. Konnerth, “In vivo two-photon calcium imaging of neuronal networks,” Proc. Natl. Acad. Sci. 100, 7319–7324 (2003).
  3. W. Denk, J. H. Strickler, W. W. Webb, “Two-photon laser scanning fluorescence microscopy,” Science 248, 73–75 (1990).
    [CrossRef] [PubMed]
  4. W. R. Zipfel, R. M. Williams, W. W. Webb, “Nonlinear magic: multiphoton microscopy in the biosciences,” Nat. Biotech. 21, 1369–1377 (2003).
    [CrossRef]
  5. J. C. Jung, M. J. Schnitzer, “Multiphoton endoscopy,” Opt. Lett. 28, 902–904 (2003).
    [CrossRef] [PubMed]
  6. D. Bird, M. Gu, “Two-photon fluorescence endoscopy with a micro-optic scanning head,” Opt. Lett. 28, 1552–1554 (2003).
    [CrossRef] [PubMed]
  7. J. Knittel, L. Schnieder, G. Buess, B. Messerschmidt, T. Possner, “Endoscope-compatible confocal microscope using a gradient index-lens system,” Opt. Commun. 188, 267–273 (2001).
    [CrossRef]
  8. D. Bird, M. Gu, “Fiber-optic two-photon scanning fluorescence microscopy,” J. Microsc. 208, 35–48 (2002).
    [CrossRef] [PubMed]
  9. D. Bird, M. Gu, “Compact two-photon fluorescence microscope based on a single-mode fiber coupler,” Opt. Lett. 27, 1031–1033 (2002).
    [CrossRef]
  10. B. E. A. Saleh, M. C. Teich, Fundamentals of Photonics (Wiley, New York, 1991).
    [CrossRef]
  11. M. Gu, Principles of Three-Dimensional Imaging in Confocal Microscopes (World Scientific, Singapore, 1996).
  12. C. Xu, W. W. Webb, “Measurement of two-photon excitation of cross sections of molecular fluorophores with data from 690 to 1050 nm,” J. Opt. Soc. Am. B 13, 481–491 (1996).
    [CrossRef]
  13. M. Gu, C. J. R. Sheppard, “Signal level of the fiber-optical confocal scanning microscope,” J. Mod. Opt. 38, 1621–1630 (1991).
    [CrossRef]
  14. M. Gu, D. Bird, “Three-dimensional optical-transfer-function analysis of fiber-optical two-photon fluorescence microscopy,” J. Opt. Soc. Am. A 20, 941–947 (2003).
    [CrossRef]

2003

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

J. C. Jung, M. J. Schnitzer, “Multiphoton endoscopy,” Opt. Lett. 28, 902–904 (2003).
[CrossRef] [PubMed]

D. Bird, M. Gu, “Two-photon fluorescence endoscopy with a micro-optic scanning head,” Opt. Lett. 28, 1552–1554 (2003).
[CrossRef] [PubMed]

C. Stosiek, O. Garaschuk, K. Holthoff, A. Konnerth, “In vivo two-photon calcium imaging of neuronal networks,” Proc. Natl. Acad. Sci. 100, 7319–7324 (2003).

M. Gu, D. Bird, “Three-dimensional optical-transfer-function analysis of fiber-optical two-photon fluorescence microscopy,” J. Opt. Soc. Am. A 20, 941–947 (2003).
[CrossRef]

2002

D. Bird, M. Gu, “Fiber-optic two-photon scanning fluorescence microscopy,” J. Microsc. 208, 35–48 (2002).
[CrossRef] [PubMed]

D. Bird, M. Gu, “Compact two-photon fluorescence microscope based on a single-mode fiber coupler,” Opt. Lett. 27, 1031–1033 (2002).
[CrossRef]

2001

J. Knittel, L. Schnieder, G. Buess, B. Messerschmidt, T. Possner, “Endoscope-compatible confocal microscope using a gradient index-lens system,” Opt. Commun. 188, 267–273 (2001).
[CrossRef]

E. B. Brown, R. B. Campbell, Y. Tsuzuki, L. Xu, P. Carmeliet, D. Fukumura, R. K. Jain, “In vivo measurement of gene expression, angiogenesis and physiological function in tumors using multiphoton laser scanning microscopy,” Nat. Med. 7, 864–868 (2001).
[CrossRef] [PubMed]

1996

1991

M. Gu, C. J. R. Sheppard, “Signal level of the fiber-optical confocal scanning microscope,” J. Mod. Opt. 38, 1621–1630 (1991).
[CrossRef]

1990

W. Denk, J. H. Strickler, W. W. Webb, “Two-photon laser scanning fluorescence microscopy,” Science 248, 73–75 (1990).
[CrossRef] [PubMed]

Bird, D.

Brown, E. B.

E. B. Brown, R. B. Campbell, Y. Tsuzuki, L. Xu, P. Carmeliet, D. Fukumura, R. K. Jain, “In vivo measurement of gene expression, angiogenesis and physiological function in tumors using multiphoton laser scanning microscopy,” Nat. Med. 7, 864–868 (2001).
[CrossRef] [PubMed]

Buess, G.

J. Knittel, L. Schnieder, G. Buess, B. Messerschmidt, T. Possner, “Endoscope-compatible confocal microscope using a gradient index-lens system,” Opt. Commun. 188, 267–273 (2001).
[CrossRef]

Campbell, R. B.

E. B. Brown, R. B. Campbell, Y. Tsuzuki, L. Xu, P. Carmeliet, D. Fukumura, R. K. Jain, “In vivo measurement of gene expression, angiogenesis and physiological function in tumors using multiphoton laser scanning microscopy,” Nat. Med. 7, 864–868 (2001).
[CrossRef] [PubMed]

Carmeliet, P.

E. B. Brown, R. B. Campbell, Y. Tsuzuki, L. Xu, P. Carmeliet, D. Fukumura, R. K. Jain, “In vivo measurement of gene expression, angiogenesis and physiological function in tumors using multiphoton laser scanning microscopy,” Nat. Med. 7, 864–868 (2001).
[CrossRef] [PubMed]

Denk, W.

W. Denk, J. H. Strickler, W. W. Webb, “Two-photon laser scanning fluorescence microscopy,” Science 248, 73–75 (1990).
[CrossRef] [PubMed]

Fukumura, D.

E. B. Brown, R. B. Campbell, Y. Tsuzuki, L. Xu, P. Carmeliet, D. Fukumura, R. K. Jain, “In vivo measurement of gene expression, angiogenesis and physiological function in tumors using multiphoton laser scanning microscopy,” Nat. Med. 7, 864–868 (2001).
[CrossRef] [PubMed]

Garaschuk, O.

C. Stosiek, O. Garaschuk, K. Holthoff, A. Konnerth, “In vivo two-photon calcium imaging of neuronal networks,” Proc. Natl. Acad. Sci. 100, 7319–7324 (2003).

Gu, M.

D. Bird, M. Gu, “Two-photon fluorescence endoscopy with a micro-optic scanning head,” Opt. Lett. 28, 1552–1554 (2003).
[CrossRef] [PubMed]

M. Gu, D. Bird, “Three-dimensional optical-transfer-function analysis of fiber-optical two-photon fluorescence microscopy,” J. Opt. Soc. Am. A 20, 941–947 (2003).
[CrossRef]

D. Bird, M. Gu, “Fiber-optic two-photon scanning fluorescence microscopy,” J. Microsc. 208, 35–48 (2002).
[CrossRef] [PubMed]

D. Bird, M. Gu, “Compact two-photon fluorescence microscope based on a single-mode fiber coupler,” Opt. Lett. 27, 1031–1033 (2002).
[CrossRef]

M. Gu, C. J. R. Sheppard, “Signal level of the fiber-optical confocal scanning microscope,” J. Mod. Opt. 38, 1621–1630 (1991).
[CrossRef]

M. Gu, Principles of Three-Dimensional Imaging in Confocal Microscopes (World Scientific, Singapore, 1996).

Holthoff, K.

C. Stosiek, O. Garaschuk, K. Holthoff, A. Konnerth, “In vivo two-photon calcium imaging of neuronal networks,” Proc. Natl. Acad. Sci. 100, 7319–7324 (2003).

Jain, R. K.

E. B. Brown, R. B. Campbell, Y. Tsuzuki, L. Xu, P. Carmeliet, D. Fukumura, R. K. Jain, “In vivo measurement of gene expression, angiogenesis and physiological function in tumors using multiphoton laser scanning microscopy,” Nat. Med. 7, 864–868 (2001).
[CrossRef] [PubMed]

Jung, J. C.

Knittel, J.

J. Knittel, L. Schnieder, G. Buess, B. Messerschmidt, T. Possner, “Endoscope-compatible confocal microscope using a gradient index-lens system,” Opt. Commun. 188, 267–273 (2001).
[CrossRef]

Konnerth, A.

C. Stosiek, O. Garaschuk, K. Holthoff, A. Konnerth, “In vivo two-photon calcium imaging of neuronal networks,” Proc. Natl. Acad. Sci. 100, 7319–7324 (2003).

Messerschmidt, B.

J. Knittel, L. Schnieder, G. Buess, B. Messerschmidt, T. Possner, “Endoscope-compatible confocal microscope using a gradient index-lens system,” Opt. Commun. 188, 267–273 (2001).
[CrossRef]

Possner, T.

J. Knittel, L. Schnieder, G. Buess, B. Messerschmidt, T. Possner, “Endoscope-compatible confocal microscope using a gradient index-lens system,” Opt. Commun. 188, 267–273 (2001).
[CrossRef]

Saleh, B. E. A.

B. E. A. Saleh, M. C. Teich, Fundamentals of Photonics (Wiley, New York, 1991).
[CrossRef]

Schnieder, L.

J. Knittel, L. Schnieder, G. Buess, B. Messerschmidt, T. Possner, “Endoscope-compatible confocal microscope using a gradient index-lens system,” Opt. Commun. 188, 267–273 (2001).
[CrossRef]

Schnitzer, M. J.

Sheppard, C. J. R.

M. Gu, C. J. R. Sheppard, “Signal level of the fiber-optical confocal scanning microscope,” J. Mod. Opt. 38, 1621–1630 (1991).
[CrossRef]

Stosiek, C.

C. Stosiek, O. Garaschuk, K. Holthoff, A. Konnerth, “In vivo two-photon calcium imaging of neuronal networks,” Proc. Natl. Acad. Sci. 100, 7319–7324 (2003).

Strickler, J. H.

W. Denk, J. H. Strickler, W. W. Webb, “Two-photon laser scanning fluorescence microscopy,” Science 248, 73–75 (1990).
[CrossRef] [PubMed]

Teich, M. C.

B. E. A. Saleh, M. C. Teich, Fundamentals of Photonics (Wiley, New York, 1991).
[CrossRef]

Tsuzuki, Y.

E. B. Brown, R. B. Campbell, Y. Tsuzuki, L. Xu, P. Carmeliet, D. Fukumura, R. K. Jain, “In vivo measurement of gene expression, angiogenesis and physiological function in tumors using multiphoton laser scanning microscopy,” Nat. Med. 7, 864–868 (2001).
[CrossRef] [PubMed]

Webb, W. W.

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

C. Xu, W. W. Webb, “Measurement of two-photon excitation of cross sections of molecular fluorophores with data from 690 to 1050 nm,” J. Opt. Soc. Am. B 13, 481–491 (1996).
[CrossRef]

W. Denk, J. H. Strickler, W. W. Webb, “Two-photon laser scanning fluorescence microscopy,” Science 248, 73–75 (1990).
[CrossRef] [PubMed]

Williams, R. M.

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

Xu, C.

Xu, L.

E. B. Brown, R. B. Campbell, Y. Tsuzuki, L. Xu, P. Carmeliet, D. Fukumura, R. K. Jain, “In vivo measurement of gene expression, angiogenesis and physiological function in tumors using multiphoton laser scanning microscopy,” Nat. Med. 7, 864–868 (2001).
[CrossRef] [PubMed]

Zipfel, W. R.

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

J. Microsc.

D. Bird, M. Gu, “Fiber-optic two-photon scanning fluorescence microscopy,” J. Microsc. 208, 35–48 (2002).
[CrossRef] [PubMed]

J. Mod. Opt.

M. Gu, C. J. R. Sheppard, “Signal level of the fiber-optical confocal scanning microscope,” J. Mod. Opt. 38, 1621–1630 (1991).
[CrossRef]

J. Opt. Soc. Am. A

J. Opt. Soc. Am. B

Nat. Biotech.

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

Nat. Med.

E. B. Brown, R. B. Campbell, Y. Tsuzuki, L. Xu, P. Carmeliet, D. Fukumura, R. K. Jain, “In vivo measurement of gene expression, angiogenesis and physiological function in tumors using multiphoton laser scanning microscopy,” Nat. Med. 7, 864–868 (2001).
[CrossRef] [PubMed]

Opt. Commun.

J. Knittel, L. Schnieder, G. Buess, B. Messerschmidt, T. Possner, “Endoscope-compatible confocal microscope using a gradient index-lens system,” Opt. Commun. 188, 267–273 (2001).
[CrossRef]

Opt. Lett.

Proc. Natl. Acad. Sci.

C. Stosiek, O. Garaschuk, K. Holthoff, A. Konnerth, “In vivo two-photon calcium imaging of neuronal networks,” Proc. Natl. Acad. Sci. 100, 7319–7324 (2003).

Science

W. Denk, J. H. Strickler, W. W. Webb, “Two-photon laser scanning fluorescence microscopy,” Science 248, 73–75 (1990).
[CrossRef] [PubMed]

Other

B. E. A. Saleh, M. C. Teich, Fundamentals of Photonics (Wiley, New York, 1991).
[CrossRef]

M. Gu, Principles of Three-Dimensional Imaging in Confocal Microscopes (World Scientific, Singapore, 1996).

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Figures (4)

Fig. 1
Fig. 1

Schematic diagram of the fiber-optic two-photon fluorescence microscope based on a fiber coupler and a GRIN rod lens.

Fig. 2
Fig. 2

One-photon reflection confocal axial response. (a) FWHM of the axial response to a plane mirror and effective NA of the GRIN rod lens as a function of gap d. (b) Transmission efficiency of the GRIN lens and peak intensity of the axial response to a plane mirror as a function of gap d.

Fig. 3
Fig. 3

Two-photon fluorescence axial response. (a) FWHM of the axial response to a fluorescence sheet as a function of gap d. (b) Peak intensity of the axial response to a fluorescence sheet as a function of gap d.

Fig. 4
Fig. 4

Series of x–y images of 10 µm fluorescence polymer microspheres acquired with a 0.25 pitch GRIN lens by (a) setting d = 1.25 mm with a slice spacing of 15 µm and (b) setting d = 5.5 mm with a slice spacing of 5 µm. The axial resolution for these two gap lengths are shown in Table 1. Each slice size is 100 µm × 100 µm. The average power on the sample is 10 mW.

Tables (1)

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Table 1 Characterization of the GRIN Rod Lens in a Fiber-Optic Two-Photon Fluorescence Microscope

Equations (2)

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u = ( 8 π / λ ) Δ z sin 2 ( α / 2 ) ,
η 2 = β A 4 C 2 ( l = 0 ) 1 exp ( A ) ,

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