Abstract

The detrimental effects of a refractive-index mismatch on the image formation in a two-photon microscope were investigated. Point-spread functions (PSF’s) were recorded with an oil-immersion objective numerical aperture (NA) of 1.3 and a water-immersion objective NA of 1.2 in an aqueous sample at different depths. For the oil-immersion objective the enlargement of the PSF volume with increasing depth yields an axial and a lateral loss in resolution of approximately 380% and 160%, respectively, at a 90-µm depth in the sample. For the water-immersion objective no resolution decrease was found. Measurements on a thick aqueous biofilm sample shows the importance of matching the refractive index between immersion fluid and sample. With a good match, no loss in image resolution is observed.

© 1999 Optical Society of America

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References

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  1. W. Denk, J. H. Stickler, W. W. Webb, “Two-photon laser scanning fluorescence microscopy,” Science 248, 73–76 (1990).
    [Crossref] [PubMed]
  2. 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]
  3. C. Xu, W. W. Webb, “Measurement of two-photon excitation cross sections of molecular fluorophores with data from 690 to 1050 nm,” J. Opt. Soc. Am. B 13, 481–491 (1996).
    [Crossref]
  4. K. Carlsson, “The influence of specimen refractive index, detector signal integration, and non-uniform scan speed on the imaging properties in confocal microscopy,” J. Microsc. 163, 167–178 (1991).
    [Crossref]
  5. H. Jacobsen, P. E. Hänninen, E. Soini, S. W. Hell, “Refractive-index-induced aberrations in two-photon confocal microscopy,” J. Microsc. 176, 226–230 (1994).
    [Crossref]
  6. J. Sytsma, J. M. Vroom, C. J. de Grauw, H. C. Gerritsen, “Time-gated fluorescence lifetime imaging and microvolume spectroscopy using two-photon excitation,” J. Microsc. 191, 39–51 (1998).
    [Crossref]
  7. D. J. Bradshaw, P. D. Marsh, K. M. Schilling, D. Cummins, “A modified chemostat system to study the ecology of oral biofilms,” J. Appl. Bacteriology 80, 124–130 (1996).
    [Crossref]
  8. H. T. M. van der Voort, K. C. Strasters, “Restoration of confocal images for quantitative image analysis,” J. Microsc. 178, 165–181 (1995).
    [Crossref]
  9. H. Kano, H. T. M. van der Voort, M. Schrader, G. M. P. van Kempen, S. W. Hell, “Avalanche photodiode detection with object scanning and image restoration provides 2–4 fold resolution increase in two-photon fluorescence microscopy,” Bioimaging 4, 187–197 (1996).
    [Crossref]
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    [Crossref]
  11. A. Entwistle, “The effects of total internal reflection on the spread function along the axis in confocal microscopy,” J. Microsc. 180, 148–157 (1995).
    [Crossref]
  12. T. Wilson, R. Juškaitis, “The axial response of confocal microscopes with high numerical aperture objective lenses,” Bioimaging 3, 35–38 (1995).
    [Crossref]
  13. H. Jacobsen, S. W. Hell, “Effect of the specimen refractive index on the imaging of a confocal fluorescence microscope employing high aperture oil immersion lenses,” Bioimaging 3, 39–47 (1995).
    [Crossref]
  14. S. Hell, G. Reiner, C. Cremer, E. H. K. Stelzer, “Aberrations in confocal fluorescence microscopy induced by mismatches in refractive index,” J. Microsc. 169, 391–405 (1993).
    [Crossref]
  15. J. G. Holt, ed., Bergey’s Manual of Systematic Bacteriology (Williams & Wilkins, Baltimore, 1984), Vol. 1.

1998 (2)

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]

J. Sytsma, J. M. Vroom, C. J. de Grauw, H. C. Gerritsen, “Time-gated fluorescence lifetime imaging and microvolume spectroscopy using two-photon excitation,” J. Microsc. 191, 39–51 (1998).
[Crossref]

1996 (3)

D. J. Bradshaw, P. D. Marsh, K. M. Schilling, D. Cummins, “A modified chemostat system to study the ecology of oral biofilms,” J. Appl. Bacteriology 80, 124–130 (1996).
[Crossref]

H. Kano, H. T. M. van der Voort, M. Schrader, G. M. P. van Kempen, S. W. Hell, “Avalanche photodiode detection with object scanning and image restoration provides 2–4 fold resolution increase in two-photon fluorescence microscopy,” Bioimaging 4, 187–197 (1996).
[Crossref]

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

1995 (4)

H. T. M. van der Voort, K. C. Strasters, “Restoration of confocal images for quantitative image analysis,” J. Microsc. 178, 165–181 (1995).
[Crossref]

A. Entwistle, “The effects of total internal reflection on the spread function along the axis in confocal microscopy,” J. Microsc. 180, 148–157 (1995).
[Crossref]

T. Wilson, R. Juškaitis, “The axial response of confocal microscopes with high numerical aperture objective lenses,” Bioimaging 3, 35–38 (1995).
[Crossref]

H. Jacobsen, S. W. Hell, “Effect of the specimen refractive index on the imaging of a confocal fluorescence microscope employing high aperture oil immersion lenses,” Bioimaging 3, 39–47 (1995).
[Crossref]

1994 (1)

H. Jacobsen, P. E. Hänninen, E. Soini, S. W. Hell, “Refractive-index-induced aberrations in two-photon confocal microscopy,” J. Microsc. 176, 226–230 (1994).
[Crossref]

1993 (1)

S. Hell, G. Reiner, C. Cremer, E. H. K. Stelzer, “Aberrations in confocal fluorescence microscopy induced by mismatches in refractive index,” J. Microsc. 169, 391–405 (1993).
[Crossref]

1991 (1)

K. Carlsson, “The influence of specimen refractive index, detector signal integration, and non-uniform scan speed on the imaging properties in confocal microscopy,” J. Microsc. 163, 167–178 (1991).
[Crossref]

1990 (2)

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

H. T. M. van der Voort, G. J. Brakenhoff, “3-D image formation in a high-aperture fluorescence confocal microscope: a numerical analysis,” J. Microsc. 158, 43–54 (1990).
[Crossref]

Bradshaw, D. J.

D. J. Bradshaw, P. D. Marsh, K. M. Schilling, D. Cummins, “A modified chemostat system to study the ecology of oral biofilms,” J. Appl. Bacteriology 80, 124–130 (1996).
[Crossref]

Brakenhoff, G. J.

H. T. M. van der Voort, G. J. Brakenhoff, “3-D image formation in a high-aperture fluorescence confocal microscope: a numerical analysis,” J. Microsc. 158, 43–54 (1990).
[Crossref]

Carlsson, K.

K. Carlsson, “The influence of specimen refractive index, detector signal integration, and non-uniform scan speed on the imaging properties in confocal microscopy,” J. Microsc. 163, 167–178 (1991).
[Crossref]

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]

Cremer, C.

S. Hell, G. Reiner, C. Cremer, E. H. K. Stelzer, “Aberrations in confocal fluorescence microscopy induced by mismatches in refractive index,” J. Microsc. 169, 391–405 (1993).
[Crossref]

Cummins, D.

D. J. Bradshaw, P. D. Marsh, K. M. Schilling, D. Cummins, “A modified chemostat system to study the ecology of oral biofilms,” J. Appl. Bacteriology 80, 124–130 (1996).
[Crossref]

de Grauw, C. J.

J. Sytsma, J. M. Vroom, C. J. de Grauw, H. C. Gerritsen, “Time-gated fluorescence lifetime imaging and microvolume spectroscopy using two-photon excitation,” J. Microsc. 191, 39–51 (1998).
[Crossref]

Denk, W.

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

Entwistle, A.

A. Entwistle, “The effects of total internal reflection on the spread function along the axis in confocal microscopy,” J. Microsc. 180, 148–157 (1995).
[Crossref]

Gerritsen, H. C.

J. Sytsma, J. M. Vroom, C. J. de Grauw, H. C. Gerritsen, “Time-gated fluorescence lifetime imaging and microvolume spectroscopy using two-photon excitation,” J. Microsc. 191, 39–51 (1998).
[Crossref]

Hänninen, P. E.

H. Jacobsen, P. E. Hänninen, E. Soini, S. W. Hell, “Refractive-index-induced aberrations in two-photon confocal microscopy,” J. Microsc. 176, 226–230 (1994).
[Crossref]

Hell, S.

S. Hell, G. Reiner, C. Cremer, E. H. K. Stelzer, “Aberrations in confocal fluorescence microscopy induced by mismatches in refractive index,” J. Microsc. 169, 391–405 (1993).
[Crossref]

Hell, S. W.

H. Kano, H. T. M. van der Voort, M. Schrader, G. M. P. van Kempen, S. W. Hell, “Avalanche photodiode detection with object scanning and image restoration provides 2–4 fold resolution increase in two-photon fluorescence microscopy,” Bioimaging 4, 187–197 (1996).
[Crossref]

H. Jacobsen, S. W. Hell, “Effect of the specimen refractive index on the imaging of a confocal fluorescence microscope employing high aperture oil immersion lenses,” Bioimaging 3, 39–47 (1995).
[Crossref]

H. Jacobsen, P. E. Hänninen, E. Soini, S. W. Hell, “Refractive-index-induced aberrations in two-photon confocal microscopy,” J. Microsc. 176, 226–230 (1994).
[Crossref]

Jacobsen, H.

H. Jacobsen, S. W. Hell, “Effect of the specimen refractive index on the imaging of a confocal fluorescence microscope employing high aperture oil immersion lenses,” Bioimaging 3, 39–47 (1995).
[Crossref]

H. Jacobsen, P. E. Hänninen, E. Soini, S. W. Hell, “Refractive-index-induced aberrations in two-photon confocal microscopy,” J. Microsc. 176, 226–230 (1994).
[Crossref]

Juškaitis, R.

T. Wilson, R. Juškaitis, “The axial response of confocal microscopes with high numerical aperture objective lenses,” Bioimaging 3, 35–38 (1995).
[Crossref]

Kano, H.

H. Kano, H. T. M. van der Voort, M. Schrader, G. M. P. van Kempen, S. W. Hell, “Avalanche photodiode detection with object scanning and image restoration provides 2–4 fold resolution increase in two-photon fluorescence microscopy,” Bioimaging 4, 187–197 (1996).
[Crossref]

Marsh, P. D.

D. J. Bradshaw, P. D. Marsh, K. M. Schilling, D. Cummins, “A modified chemostat system to study the ecology of oral biofilms,” J. Appl. Bacteriology 80, 124–130 (1996).
[Crossref]

Reiner, G.

S. Hell, G. Reiner, C. Cremer, E. H. K. Stelzer, “Aberrations in confocal fluorescence microscopy induced by mismatches in refractive index,” J. Microsc. 169, 391–405 (1993).
[Crossref]

Schilling, K. M.

D. J. Bradshaw, P. D. Marsh, K. M. Schilling, D. Cummins, “A modified chemostat system to study the ecology of oral biofilms,” J. Appl. Bacteriology 80, 124–130 (1996).
[Crossref]

Schrader, M.

H. Kano, H. T. M. van der Voort, M. Schrader, G. M. P. van Kempen, S. W. Hell, “Avalanche photodiode detection with object scanning and image restoration provides 2–4 fold resolution increase in two-photon fluorescence microscopy,” Bioimaging 4, 187–197 (1996).
[Crossref]

Soini, E.

H. Jacobsen, P. E. Hänninen, E. Soini, S. W. Hell, “Refractive-index-induced aberrations in two-photon confocal microscopy,” J. Microsc. 176, 226–230 (1994).
[Crossref]

Stelzer, E. H. K.

S. Hell, G. Reiner, C. Cremer, E. H. K. Stelzer, “Aberrations in confocal fluorescence microscopy induced by mismatches in refractive index,” J. Microsc. 169, 391–405 (1993).
[Crossref]

Stickler, J. H.

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

Strasters, K. C.

H. T. M. van der Voort, K. C. Strasters, “Restoration of confocal images for quantitative image analysis,” J. Microsc. 178, 165–181 (1995).
[Crossref]

Sytsma, J.

J. Sytsma, J. M. Vroom, C. J. de Grauw, H. C. Gerritsen, “Time-gated fluorescence lifetime imaging and microvolume spectroscopy using two-photon excitation,” J. Microsc. 191, 39–51 (1998).
[Crossref]

van der Voort, H. T. M.

H. Kano, H. T. M. van der Voort, M. Schrader, G. M. P. van Kempen, S. W. Hell, “Avalanche photodiode detection with object scanning and image restoration provides 2–4 fold resolution increase in two-photon fluorescence microscopy,” Bioimaging 4, 187–197 (1996).
[Crossref]

H. T. M. van der Voort, K. C. Strasters, “Restoration of confocal images for quantitative image analysis,” J. Microsc. 178, 165–181 (1995).
[Crossref]

H. T. M. van der Voort, G. J. Brakenhoff, “3-D image formation in a high-aperture fluorescence confocal microscope: a numerical analysis,” J. Microsc. 158, 43–54 (1990).
[Crossref]

van Kempen, G. M. P.

H. Kano, H. T. M. van der Voort, M. Schrader, G. M. P. van Kempen, S. W. Hell, “Avalanche photodiode detection with object scanning and image restoration provides 2–4 fold resolution increase in two-photon fluorescence microscopy,” Bioimaging 4, 187–197 (1996).
[Crossref]

Vroom, J. M.

J. Sytsma, J. M. Vroom, C. J. de Grauw, H. C. Gerritsen, “Time-gated fluorescence lifetime imaging and microvolume spectroscopy using two-photon excitation,” J. Microsc. 191, 39–51 (1998).
[Crossref]

Webb, W. W.

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]

Wilson, T.

T. Wilson, R. Juškaitis, “The axial response of confocal microscopes with high numerical aperture objective lenses,” Bioimaging 3, 35–38 (1995).
[Crossref]

Xu, C.

Bioimaging (3)

H. Kano, H. T. M. van der Voort, M. Schrader, G. M. P. van Kempen, S. W. Hell, “Avalanche photodiode detection with object scanning and image restoration provides 2–4 fold resolution increase in two-photon fluorescence microscopy,” Bioimaging 4, 187–197 (1996).
[Crossref]

T. Wilson, R. Juškaitis, “The axial response of confocal microscopes with high numerical aperture objective lenses,” Bioimaging 3, 35–38 (1995).
[Crossref]

H. Jacobsen, S. W. Hell, “Effect of the specimen refractive index on the imaging of a confocal fluorescence microscope employing high aperture oil immersion lenses,” Bioimaging 3, 39–47 (1995).
[Crossref]

Biophys. J. (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]

J. Appl. Bacteriology (1)

D. J. Bradshaw, P. D. Marsh, K. M. Schilling, D. Cummins, “A modified chemostat system to study the ecology of oral biofilms,” J. Appl. Bacteriology 80, 124–130 (1996).
[Crossref]

J. Microsc. (7)

H. T. M. van der Voort, K. C. Strasters, “Restoration of confocal images for quantitative image analysis,” J. Microsc. 178, 165–181 (1995).
[Crossref]

H. T. M. van der Voort, G. J. Brakenhoff, “3-D image formation in a high-aperture fluorescence confocal microscope: a numerical analysis,” J. Microsc. 158, 43–54 (1990).
[Crossref]

A. Entwistle, “The effects of total internal reflection on the spread function along the axis in confocal microscopy,” J. Microsc. 180, 148–157 (1995).
[Crossref]

K. Carlsson, “The influence of specimen refractive index, detector signal integration, and non-uniform scan speed on the imaging properties in confocal microscopy,” J. Microsc. 163, 167–178 (1991).
[Crossref]

H. Jacobsen, P. E. Hänninen, E. Soini, S. W. Hell, “Refractive-index-induced aberrations in two-photon confocal microscopy,” J. Microsc. 176, 226–230 (1994).
[Crossref]

J. Sytsma, J. M. Vroom, C. J. de Grauw, H. C. Gerritsen, “Time-gated fluorescence lifetime imaging and microvolume spectroscopy using two-photon excitation,” J. Microsc. 191, 39–51 (1998).
[Crossref]

S. Hell, G. Reiner, C. Cremer, E. H. K. Stelzer, “Aberrations in confocal fluorescence microscopy induced by mismatches in refractive index,” J. Microsc. 169, 391–405 (1993).
[Crossref]

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

Science (1)

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

Other (1)

J. G. Holt, ed., Bergey’s Manual of Systematic Bacteriology (Williams & Wilkins, Baltimore, 1984), Vol. 1.

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Figures (6)

Fig. 1
Fig. 1

XZ planes of the two-photon PSF recorded with the water-immersion objective (NA of 1.2) (a) at the surface (z = 0) and (b) at a 90-µm depth. XZ planes of the two-photon PSF recorded with the oil-immersion objective (NA of 1.3) (c) at surface (z = 0) and (d) at 90 µm depth. Image size: 1.5 × 5 µm.

Fig. 2
Fig. 2

Lateral- (×) and axial- (●) intensity profiles through the PSF’s of Fig. 1.

Fig. 3
Fig. 3

Lateral and axial width (FWHM) of the measured PSF’s as function of depth for the different objectives. Water-immersion objective (NA of 1.2) lateral (squares) and axial (circles), and the oil-immersion objective (NA of 1.3) lateral (up triangles) and axial (down triangles).

Fig. 4
Fig. 4

Axial fluorescence intensity profiles of the homogeneous fluorescent sample for (a) the water-immersion objective (NA of 1.2) and (b) the oil-immersion objective (NA of 1.3). The edge between cover glass and fluorescent solution is near 0 µm. Dots indicate the integrated intensity of the PSF’s measured with the oil-immersion objective at various depths, normalized to the value at a 0-µm depth.

Fig. 5
Fig. 5

XZ planes from the 3-D data sets of biofilm. (a) Measured data, recorded with the water-immersion objective and (b) the corresponding image after image restoration. (c) Measured data, recorded with the oil objective; (d) restored image, restored with one central PSF; (e) restored image with three, depth-dependent PSF’s. Image size 11 µm × 90 µm. The image intensities are scaled in the z direction to correct for the depth-dependent intensity decrease. Surface (z = 0) is located at the bottom of the images.

Fig. 6
Fig. 6

XY images (parallel to the biofilm surface) from the 3-D data set of biofilm. Left panels show the measured data, and the right panels show the images after restoration. (a) and (c) Water-immersion objective recorded at a 4- and a 80-µm depth, respectively, in the biofilm. (b) and (d) Oil-immersion objective recorded at a 4- and a 80-µm depth, respectively, and restored with their corresponding depth-dependent PSF. All images are 11 µm × 11 µm. Arrows show some of the oral bacteria Fusobacterium nucleatum.

Tables (2)

Tables Icon

Table 1 Theoretical and Experimental FWHM’s of PSF’s at the Sample Surface (z = 0) for Both Objectives

Tables Icon

Table 2 Diameter (FWHM) of Fusobacterium nucleatum Bacteria and Their Characteristic Signal Noisea in the Biofilm Images at Two Different Depths

Equations (2)

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

AFP = NFPn1/n2.
gr=hrfr, mr=Ngr,

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