Abstract

Most commercial laser-scanning imaging systems used for confocal fluorescence microscopy can be readily adapted for use with two-photon fluorescence excitation. We report here on the details of the conversion of the Nikon C1 (product released November 2001) with two channels of nondescanned detection of two-photon-excited fluorescence. One of the goals of the design was to utilize off-the-shelf components as much as possible to minimize the use of custom machining and electronics assembly. We also give some initial characterization of the imaging properties of the system.

© 2004 Optical Society of America

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  1. W. Denk, J. H. Strickler, W. W. Webb, “Two-photon laser scanning fluorescence microscopy,” Science 248, 73–76 (1990).
    [CrossRef] [PubMed]
  2. W. Denk, D. W. Piston, W. W. Webb, “Two-photon molecular excitation in laser-scanning microscopy,” in Handbook of Biological Confocal Microscopy, 2nd ed., J. B. Pawley, ed. (Plenum, New York, 1995), pp. 445–458.
    [CrossRef]
  3. G. H. Patterson, D. W. Piston, “Photobleaching in two-photon excitation microscopy,” Biophys. J. 78, 2159–2162 (2000).
    [CrossRef] [PubMed]
  4. H. J. Koester, D. Baur, R. Uhl, S. W. Hell, “Ca2+ fluorescence imaging with pico- and femtosecond two-photon excitation: signal and photodamage,” Biophys. J. 77, 2226–2236 (1999).
    [CrossRef] [PubMed]
  5. A. Hopt, E. Neher, “Highly nonlinear photodamage in two-photon fluorescence microscopy,” Biophys. J. 80, 2029–2036 (2001).
    [CrossRef] [PubMed]
  6. K. König, P. T. C. So, W. W. Mantulin, E. Gratton, “Cellular response to near-infrared femtosecond laser pulses in two-photon microscopy,” Opt. Lett. 22, 135–137 (1997).
    [CrossRef] [PubMed]
  7. V. E. Centonze, J. G. White, “Multiphoton excitation provides optical sections from deeper within scattering specimens than confocal imaging,” Biophys. J. 75, 2015–2024 (1998).
    [CrossRef] [PubMed]
  8. E. Beaurepaire, J. Mertz, “Epifluorescence collection in two-photon microscopy,” Appl. Opt. 41, 5376–5382 (2002).
    [CrossRef] [PubMed]
  9. M. Oheim, E. Beaurepaire, E. Chaigneau, J. Mertz, S. Charpak, “Two-photon microscopy in brain tissue: parameters influencing the imaging depth,” J. Neurosci. Methods 111, 29–37 (2001).
    [CrossRef] [PubMed]
  10. A. Diaspro, M. Corosu, P. Ramoino, M. Robello, “Adapting a compact confocal microscope system to a two-photon excitation fluorescence imaging architecture,” Microsc. Res. Tech. 47, 196–205 (1999).
    [CrossRef] [PubMed]
  11. A. Majewska, G. Yiu, R. Yuste, “A custom-made two-photon microscope and deconvolution system,” Pfluegers Arch. Eur. J. Physiol. 441, 398–408 (2000).
    [CrossRef]
  12. M. Gu, C. J. R. Sheppard, “Comparison of three-dimensional imaging properties between two-photon and single-photon fluorescence microscopy,” J. Microsc. 177, 128–137 (1994).
    [CrossRef]
  13. C. J. R. Sheppard, M. Gu, “Image formation in two-photon fluorescence microscopy,” Optik (Stuttgart) 86, 104–106 (1990).
  14. M. Kozubek, “Theoretical versus experimental resolution in optical microscopy,” Microsc. Res. Tech. 53, 157–166 (2001).
    [CrossRef] [PubMed]

2002 (1)

2001 (3)

A. Hopt, 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, S. Charpak, “Two-photon microscopy in brain tissue: parameters influencing the imaging depth,” J. Neurosci. Methods 111, 29–37 (2001).
[CrossRef] [PubMed]

M. Kozubek, “Theoretical versus experimental resolution in optical microscopy,” Microsc. Res. Tech. 53, 157–166 (2001).
[CrossRef] [PubMed]

2000 (2)

A. Majewska, G. Yiu, R. Yuste, “A custom-made two-photon microscope and deconvolution system,” Pfluegers Arch. Eur. J. Physiol. 441, 398–408 (2000).
[CrossRef]

G. H. Patterson, D. W. Piston, “Photobleaching in two-photon excitation microscopy,” Biophys. J. 78, 2159–2162 (2000).
[CrossRef] [PubMed]

1999 (2)

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

A. Diaspro, M. Corosu, P. Ramoino, M. Robello, “Adapting a compact confocal microscope system to a two-photon excitation fluorescence imaging architecture,” Microsc. Res. Tech. 47, 196–205 (1999).
[CrossRef] [PubMed]

1998 (1)

V. E. Centonze, J. G. White, “Multiphoton excitation provides optical sections from deeper within scattering specimens than confocal imaging,” Biophys. J. 75, 2015–2024 (1998).
[CrossRef] [PubMed]

1997 (1)

1994 (1)

M. Gu, C. J. R. Sheppard, “Comparison of three-dimensional imaging properties between two-photon and single-photon fluorescence microscopy,” J. Microsc. 177, 128–137 (1994).
[CrossRef]

1990 (2)

C. J. R. Sheppard, M. Gu, “Image formation in two-photon fluorescence microscopy,” Optik (Stuttgart) 86, 104–106 (1990).

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

Baur, D.

H. J. Koester, D. Baur, R. Uhl, 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.

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

M. Oheim, E. Beaurepaire, E. Chaigneau, J. Mertz, S. Charpak, “Two-photon microscopy in brain tissue: parameters influencing the imaging depth,” J. Neurosci. Methods 111, 29–37 (2001).
[CrossRef] [PubMed]

Centonze, V. E.

V. E. Centonze, J. G. White, “Multiphoton excitation provides optical sections from deeper within scattering specimens than confocal imaging,” Biophys. J. 75, 2015–2024 (1998).
[CrossRef] [PubMed]

Chaigneau, E.

M. Oheim, E. Beaurepaire, E. Chaigneau, J. Mertz, S. Charpak, “Two-photon microscopy in brain tissue: parameters influencing the imaging depth,” J. Neurosci. Methods 111, 29–37 (2001).
[CrossRef] [PubMed]

Charpak, S.

M. Oheim, E. Beaurepaire, E. Chaigneau, J. Mertz, S. Charpak, “Two-photon microscopy in brain tissue: parameters influencing the imaging depth,” J. Neurosci. Methods 111, 29–37 (2001).
[CrossRef] [PubMed]

Corosu, M.

A. Diaspro, M. Corosu, P. Ramoino, M. Robello, “Adapting a compact confocal microscope system to a two-photon excitation fluorescence imaging architecture,” Microsc. Res. Tech. 47, 196–205 (1999).
[CrossRef] [PubMed]

Denk, W.

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

W. Denk, D. W. Piston, W. W. Webb, “Two-photon molecular excitation in laser-scanning microscopy,” in Handbook of Biological Confocal Microscopy, 2nd ed., J. B. Pawley, ed. (Plenum, New York, 1995), pp. 445–458.
[CrossRef]

Diaspro, A.

A. Diaspro, M. Corosu, P. Ramoino, M. Robello, “Adapting a compact confocal microscope system to a two-photon excitation fluorescence imaging architecture,” Microsc. Res. Tech. 47, 196–205 (1999).
[CrossRef] [PubMed]

Gratton, E.

Gu, M.

M. Gu, C. J. R. Sheppard, “Comparison of three-dimensional imaging properties between two-photon and single-photon fluorescence microscopy,” J. Microsc. 177, 128–137 (1994).
[CrossRef]

C. J. R. Sheppard, M. Gu, “Image formation in two-photon fluorescence microscopy,” Optik (Stuttgart) 86, 104–106 (1990).

Hell, S. W.

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

Hopt, A.

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

Koester, H. J.

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

König, K.

Kozubek, M.

M. Kozubek, “Theoretical versus experimental resolution in optical microscopy,” Microsc. Res. Tech. 53, 157–166 (2001).
[CrossRef] [PubMed]

Majewska, A.

A. Majewska, G. Yiu, R. Yuste, “A custom-made two-photon microscope and deconvolution system,” Pfluegers Arch. Eur. J. Physiol. 441, 398–408 (2000).
[CrossRef]

Mantulin, W. W.

Mertz, J.

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

M. Oheim, E. Beaurepaire, E. Chaigneau, J. Mertz, S. Charpak, “Two-photon microscopy in brain tissue: parameters influencing the imaging depth,” J. Neurosci. Methods 111, 29–37 (2001).
[CrossRef] [PubMed]

Neher, E.

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

Oheim, M.

M. Oheim, E. Beaurepaire, E. Chaigneau, J. Mertz, S. Charpak, “Two-photon microscopy in brain tissue: parameters influencing the imaging depth,” J. Neurosci. Methods 111, 29–37 (2001).
[CrossRef] [PubMed]

Patterson, G. H.

G. H. Patterson, D. W. Piston, “Photobleaching in two-photon excitation microscopy,” Biophys. J. 78, 2159–2162 (2000).
[CrossRef] [PubMed]

Piston, D. W.

G. H. Patterson, D. W. Piston, “Photobleaching in two-photon excitation microscopy,” Biophys. J. 78, 2159–2162 (2000).
[CrossRef] [PubMed]

W. Denk, D. W. Piston, W. W. Webb, “Two-photon molecular excitation in laser-scanning microscopy,” in Handbook of Biological Confocal Microscopy, 2nd ed., J. B. Pawley, ed. (Plenum, New York, 1995), pp. 445–458.
[CrossRef]

Ramoino, P.

A. Diaspro, M. Corosu, P. Ramoino, M. Robello, “Adapting a compact confocal microscope system to a two-photon excitation fluorescence imaging architecture,” Microsc. Res. Tech. 47, 196–205 (1999).
[CrossRef] [PubMed]

Robello, M.

A. Diaspro, M. Corosu, P. Ramoino, M. Robello, “Adapting a compact confocal microscope system to a two-photon excitation fluorescence imaging architecture,” Microsc. Res. Tech. 47, 196–205 (1999).
[CrossRef] [PubMed]

Sheppard, C. J. R.

M. Gu, C. J. R. Sheppard, “Comparison of three-dimensional imaging properties between two-photon and single-photon fluorescence microscopy,” J. Microsc. 177, 128–137 (1994).
[CrossRef]

C. J. R. Sheppard, M. Gu, “Image formation in two-photon fluorescence microscopy,” Optik (Stuttgart) 86, 104–106 (1990).

So, P. T. C.

Strickler, J. H.

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

Uhl, R.

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

Webb, W. W.

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

W. Denk, D. W. Piston, W. W. Webb, “Two-photon molecular excitation in laser-scanning microscopy,” in Handbook of Biological Confocal Microscopy, 2nd ed., J. B. Pawley, ed. (Plenum, New York, 1995), pp. 445–458.
[CrossRef]

White, J. G.

V. E. Centonze, J. G. White, “Multiphoton excitation provides optical sections from deeper within scattering specimens than confocal imaging,” Biophys. J. 75, 2015–2024 (1998).
[CrossRef] [PubMed]

Yiu, G.

A. Majewska, G. Yiu, R. Yuste, “A custom-made two-photon microscope and deconvolution system,” Pfluegers Arch. Eur. J. Physiol. 441, 398–408 (2000).
[CrossRef]

Yuste, R.

A. Majewska, G. Yiu, R. Yuste, “A custom-made two-photon microscope and deconvolution system,” Pfluegers Arch. Eur. J. Physiol. 441, 398–408 (2000).
[CrossRef]

Appl. Opt. (1)

Biophys. J. (4)

V. E. Centonze, J. G. White, “Multiphoton excitation provides optical sections from deeper within scattering specimens than confocal imaging,” Biophys. J. 75, 2015–2024 (1998).
[CrossRef] [PubMed]

G. H. Patterson, D. W. Piston, “Photobleaching in two-photon excitation microscopy,” Biophys. J. 78, 2159–2162 (2000).
[CrossRef] [PubMed]

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

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

J. Microsc. (1)

M. Gu, C. J. R. Sheppard, “Comparison of three-dimensional imaging properties between two-photon and single-photon fluorescence microscopy,” J. Microsc. 177, 128–137 (1994).
[CrossRef]

J. Neurosci. Methods (1)

M. Oheim, E. Beaurepaire, E. Chaigneau, J. Mertz, S. Charpak, “Two-photon microscopy in brain tissue: parameters influencing the imaging depth,” J. Neurosci. Methods 111, 29–37 (2001).
[CrossRef] [PubMed]

Microsc. Res. Tech. (2)

A. Diaspro, M. Corosu, P. Ramoino, M. Robello, “Adapting a compact confocal microscope system to a two-photon excitation fluorescence imaging architecture,” Microsc. Res. Tech. 47, 196–205 (1999).
[CrossRef] [PubMed]

M. Kozubek, “Theoretical versus experimental resolution in optical microscopy,” Microsc. Res. Tech. 53, 157–166 (2001).
[CrossRef] [PubMed]

Opt. Lett. (1)

Optik (Stuttgart) (1)

C. J. R. Sheppard, M. Gu, “Image formation in two-photon fluorescence microscopy,” Optik (Stuttgart) 86, 104–106 (1990).

Pfluegers Arch. Eur. J. Physiol. (1)

A. Majewska, G. Yiu, R. Yuste, “A custom-made two-photon microscope and deconvolution system,” Pfluegers Arch. Eur. J. Physiol. 441, 398–408 (2000).
[CrossRef]

Science (1)

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

Other (1)

W. Denk, D. W. Piston, W. W. Webb, “Two-photon molecular excitation in laser-scanning microscopy,” in Handbook of Biological Confocal Microscopy, 2nd ed., J. B. Pawley, ed. (Plenum, New York, 1995), pp. 445–458.
[CrossRef]

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Figures (6)

Fig. 1
Fig. 1

Schematic side view of the top of the Nikon E800 (or E1000) upright microscope, showing the light path in the modified C1 laser-scanning system. A, beam-splitter module; B, trinocular tube; C, C1 scan head; D, objective lens; E, dichroic mirror and short-pass filter; F, filter cube slider with cover; G, dichroic mirror with filters to provide two channels; H, steering mirror inserted into scan head. The darker-shaded arrows indicate the laser path to the objective lens, and lighter-shaded arrows indicate fluorescence. Refer to the text for details.

Fig. 2
Fig. 2

Expanded schematic view of the nondescanned detector assembly onto the Nikon beam-splitter module. Note: The view is not to scale and only indicates the relative positions of the pieces. See text for details. A, PMT in housing (Hamamatsu TE7718); B, screw-on mounting flange for PMT housing (Hamamatsu A7719); C, custom-made cover; D, Nikon filter cube slider (Nikon MEV41000); E, custom-made dovetail fits the bottom of D; F, Lens mounting tube (Thorlabs SM1L10) of 1-in. (25.4 mm); G, custom-made 38-mm outside diameter (inside diameter friction fit for 1.2 in.) for back port of beam-splitter module; H, filter cube to direct fluorescence to back port (laser enters from top); I, beam-splitter module for Nikon E800 on E1000 (Nikon MAD56110).

Fig. 3
Fig. 3

Perspective view of the scan head showing the laser path inside the scan head. Galvos = galvanometers. Note: The view is not to scale and only indicates the relative positions of the beam-steering mirrors.

Fig. 4
Fig. 4

Sample image collected with the two-photon excitation of fluorescence. Pollen grain (mixed pollen grain sample slide, Carolina Biological Supply) excitation with 70 mW at 800 nm. The scale bar is 10 μm.

Fig. 5
Fig. 5

Sample images collected with the system by use of two-photon excitation. These images are collected by use of typical live-cell observation conditions. These images were recorded after a 50-min time-course experiment during which more than 50 images were taken from within the same volume. Fluorescein dextran was used to label the axoplasm of rat spinal cord axons. The laser power was 100 mW, and the data-collection rate was 3.36 μs/pixel. The scale bar is 10 μm.

Fig. 6
Fig. 6

Surface and volume rendering of the two channels. The image data set is the same as in Fig. 5 with the longer wavelength channel, X-Rhod-1, added as an isosurface to achieve some contrast in the gray-scale image. X-Rhod is cationic calcium indicator that accumulates in the myelin, which sheaths the axoplasm. The stars indicate a region in which this sheathing can be seen in the image. The scale bar is 10 μm.

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