Abstract

We report the design and implementation of a laser scanning confocal fluorescence system with spectroscopy and anisotropy imaging capabilities. Confocal spectroscopy is achieved with a fiber pinhole that is inserted into and removed from the detection path as needed. Fluorescence anisotropy imaging is accomplished with a polarizing beam splitter placed after the conventional pinhole. Two orthogonal polarizations are detected simultaneously with balanced photomultiplier tubes. The quality of the axial sectioning that is achieved in the confocal fluorescence spectroscopy mode is demonstrated experimentally, and examples of polarization-sensitive fluorescence imaging are demonstrated in tumor cell monolayers.

© 2003 Optical Society of America

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    [CrossRef]
  2. A. W. Knight, N. J. Goddard, P. R. Fielden, M. G. Barker, N. Billinton, and R. M. Walmsley, Anal. Commun. 36, 113 (1999).
    [CrossRef]
  3. A. W. Knight, N. J. Goddard, N. Billinton, P. A. Cahill, and R. M. Walmsley, J. Biochem. Biophys. Methods 51, 165 (2002).
    [CrossRef] [PubMed]
  4. S. M. Blackman, C. E. Cobb, A. H. Beth, and D. W. Piston, Biophys. J. 71, 194 (1996).
    [CrossRef] [PubMed]
  5. C. E. Bigelow, C. J. Harkrider, D. L. Conover, T. H. Foster, I. Georgakoudi, S. Mitra, M. G. Nichols, and M. Rajadhyaksha, Rev. Sci. Instrum. 72, 3407 (2001).
    [CrossRef]
  6. R. Richards-Kortum, A. Durkin, and J. Zeng, Appl. Spectrosc. 48, 350 (1994).
    [CrossRef]
  7. R. Swaminathan, C. P. Hoang, and A. S. Verkman, Biophys. J. 72, 1900 (1997).
    [CrossRef] [PubMed]

2002 (1)

A. W. Knight, N. J. Goddard, N. Billinton, P. A. Cahill, and R. M. Walmsley, J. Biochem. Biophys. Methods 51, 165 (2002).
[CrossRef] [PubMed]

2001 (1)

C. E. Bigelow, C. J. Harkrider, D. L. Conover, T. H. Foster, I. Georgakoudi, S. Mitra, M. G. Nichols, and M. Rajadhyaksha, Rev. Sci. Instrum. 72, 3407 (2001).
[CrossRef]

1999 (1)

A. W. Knight, N. J. Goddard, P. R. Fielden, M. G. Barker, N. Billinton, and R. M. Walmsley, Anal. Commun. 36, 113 (1999).
[CrossRef]

1997 (1)

R. Swaminathan, C. P. Hoang, and A. S. Verkman, Biophys. J. 72, 1900 (1997).
[CrossRef] [PubMed]

1996 (1)

S. M. Blackman, C. E. Cobb, A. H. Beth, and D. W. Piston, Biophys. J. 71, 194 (1996).
[CrossRef] [PubMed]

1994 (1)

Barker, M. G.

A. W. Knight, N. J. Goddard, P. R. Fielden, M. G. Barker, N. Billinton, and R. M. Walmsley, Anal. Commun. 36, 113 (1999).
[CrossRef]

Beth, A. H.

S. M. Blackman, C. E. Cobb, A. H. Beth, and D. W. Piston, Biophys. J. 71, 194 (1996).
[CrossRef] [PubMed]

Bigelow, C. E.

C. E. Bigelow, C. J. Harkrider, D. L. Conover, T. H. Foster, I. Georgakoudi, S. Mitra, M. G. Nichols, and M. Rajadhyaksha, Rev. Sci. Instrum. 72, 3407 (2001).
[CrossRef]

Billinton, N.

A. W. Knight, N. J. Goddard, N. Billinton, P. A. Cahill, and R. M. Walmsley, J. Biochem. Biophys. Methods 51, 165 (2002).
[CrossRef] [PubMed]

A. W. Knight, N. J. Goddard, P. R. Fielden, M. G. Barker, N. Billinton, and R. M. Walmsley, Anal. Commun. 36, 113 (1999).
[CrossRef]

Blackman, S. M.

S. M. Blackman, C. E. Cobb, A. H. Beth, and D. W. Piston, Biophys. J. 71, 194 (1996).
[CrossRef] [PubMed]

Cahill, P. A.

A. W. Knight, N. J. Goddard, N. Billinton, P. A. Cahill, and R. M. Walmsley, J. Biochem. Biophys. Methods 51, 165 (2002).
[CrossRef] [PubMed]

Cobb, C. E.

S. M. Blackman, C. E. Cobb, A. H. Beth, and D. W. Piston, Biophys. J. 71, 194 (1996).
[CrossRef] [PubMed]

Conover, D. L.

C. E. Bigelow, C. J. Harkrider, D. L. Conover, T. H. Foster, I. Georgakoudi, S. Mitra, M. G. Nichols, and M. Rajadhyaksha, Rev. Sci. Instrum. 72, 3407 (2001).
[CrossRef]

Durkin, A.

Fielden, P. R.

A. W. Knight, N. J. Goddard, P. R. Fielden, M. G. Barker, N. Billinton, and R. M. Walmsley, Anal. Commun. 36, 113 (1999).
[CrossRef]

Foster, T. H.

C. E. Bigelow, C. J. Harkrider, D. L. Conover, T. H. Foster, I. Georgakoudi, S. Mitra, M. G. Nichols, and M. Rajadhyaksha, Rev. Sci. Instrum. 72, 3407 (2001).
[CrossRef]

Georgakoudi, I.

C. E. Bigelow, C. J. Harkrider, D. L. Conover, T. H. Foster, I. Georgakoudi, S. Mitra, M. G. Nichols, and M. Rajadhyaksha, Rev. Sci. Instrum. 72, 3407 (2001).
[CrossRef]

Goddard, N. J.

A. W. Knight, N. J. Goddard, N. Billinton, P. A. Cahill, and R. M. Walmsley, J. Biochem. Biophys. Methods 51, 165 (2002).
[CrossRef] [PubMed]

A. W. Knight, N. J. Goddard, P. R. Fielden, M. G. Barker, N. Billinton, and R. M. Walmsley, Anal. Commun. 36, 113 (1999).
[CrossRef]

Harkrider, C. J.

C. E. Bigelow, C. J. Harkrider, D. L. Conover, T. H. Foster, I. Georgakoudi, S. Mitra, M. G. Nichols, and M. Rajadhyaksha, Rev. Sci. Instrum. 72, 3407 (2001).
[CrossRef]

Hoang, C. P.

R. Swaminathan, C. P. Hoang, and A. S. Verkman, Biophys. J. 72, 1900 (1997).
[CrossRef] [PubMed]

Knight, A. W.

A. W. Knight, N. J. Goddard, N. Billinton, P. A. Cahill, and R. M. Walmsley, J. Biochem. Biophys. Methods 51, 165 (2002).
[CrossRef] [PubMed]

A. W. Knight, N. J. Goddard, P. R. Fielden, M. G. Barker, N. Billinton, and R. M. Walmsley, Anal. Commun. 36, 113 (1999).
[CrossRef]

Lakowicz, J. R.

J. R. Lakowicz, in Principles of Fluorescence Spectroscopy, 2nd ed. (Kluwer, New York, 1999), pp. 291–319.
[CrossRef]

Mitra, S.

C. E. Bigelow, C. J. Harkrider, D. L. Conover, T. H. Foster, I. Georgakoudi, S. Mitra, M. G. Nichols, and M. Rajadhyaksha, Rev. Sci. Instrum. 72, 3407 (2001).
[CrossRef]

Nichols, M. G.

C. E. Bigelow, C. J. Harkrider, D. L. Conover, T. H. Foster, I. Georgakoudi, S. Mitra, M. G. Nichols, and M. Rajadhyaksha, Rev. Sci. Instrum. 72, 3407 (2001).
[CrossRef]

Piston, D. W.

S. M. Blackman, C. E. Cobb, A. H. Beth, and D. W. Piston, Biophys. J. 71, 194 (1996).
[CrossRef] [PubMed]

Rajadhyaksha, M.

C. E. Bigelow, C. J. Harkrider, D. L. Conover, T. H. Foster, I. Georgakoudi, S. Mitra, M. G. Nichols, and M. Rajadhyaksha, Rev. Sci. Instrum. 72, 3407 (2001).
[CrossRef]

Richards-Kortum, R.

Swaminathan, R.

R. Swaminathan, C. P. Hoang, and A. S. Verkman, Biophys. J. 72, 1900 (1997).
[CrossRef] [PubMed]

Verkman, A. S.

R. Swaminathan, C. P. Hoang, and A. S. Verkman, Biophys. J. 72, 1900 (1997).
[CrossRef] [PubMed]

Walmsley, R. M.

A. W. Knight, N. J. Goddard, N. Billinton, P. A. Cahill, and R. M. Walmsley, J. Biochem. Biophys. Methods 51, 165 (2002).
[CrossRef] [PubMed]

A. W. Knight, N. J. Goddard, P. R. Fielden, M. G. Barker, N. Billinton, and R. M. Walmsley, Anal. Commun. 36, 113 (1999).
[CrossRef]

Zeng, J.

Anal. Commun. (1)

A. W. Knight, N. J. Goddard, P. R. Fielden, M. G. Barker, N. Billinton, and R. M. Walmsley, Anal. Commun. 36, 113 (1999).
[CrossRef]

Appl. Spectrosc. (1)

Biophys. J. (2)

R. Swaminathan, C. P. Hoang, and A. S. Verkman, Biophys. J. 72, 1900 (1997).
[CrossRef] [PubMed]

S. M. Blackman, C. E. Cobb, A. H. Beth, and D. W. Piston, Biophys. J. 71, 194 (1996).
[CrossRef] [PubMed]

J. Biochem. Biophys. Methods (1)

A. W. Knight, N. J. Goddard, N. Billinton, P. A. Cahill, and R. M. Walmsley, J. Biochem. Biophys. Methods 51, 165 (2002).
[CrossRef] [PubMed]

Rev. Sci. Instrum. (1)

C. E. Bigelow, C. J. Harkrider, D. L. Conover, T. H. Foster, I. Georgakoudi, S. Mitra, M. G. Nichols, and M. Rajadhyaksha, Rev. Sci. Instrum. 72, 3407 (2001).
[CrossRef]

Other (1)

J. R. Lakowicz, in Principles of Fluorescence Spectroscopy, 2nd ed. (Kluwer, New York, 1999), pp. 291–319.
[CrossRef]

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Figures (4)

Fig. 1
Fig. 1

Confocal fluorescence spectroscopy and anisotropy imaging system. For spectroscopy, mirror FM is flipped into the detection path. Sample fluorescence is de-scanned, reflected by dichroic mirror DM1, and focused by lens L6 into the 50µm core of a multimode fiber acting as the confocal limiting aperture. Fiber output is imaged through a grating spectrograph onto a cooled CCD. For polarization-sensitive imaging, a polarizing beam splitter (PBS) is placed in the detection path after the pinhole. Fluorescence parallel and perpendicular to the polarized excitation beam is directed simultaneously to two balanced photomultiplier tubes (PMT1, PMT2). For conventional simultaneous two-color fluorescence imaging, a dichroic mirror (DM) replaces the PBS. L1–L5, scanning lenses; M1, M2, scanning mirror; A/D, analog–digital.

Fig. 2
Fig. 2

Demonstration of confocal fluorescence spectroscopy. A red-fluorophore-embedded polymer rod was lowered into fluorescein solution in a coverslip dish. Fluorescence spectra were acquired as the focus was advanced axially from the coverslip through the fluid solution and into the bottom of the rod. (a) Sequence of individual fluorescence spectra versus depth into the sample. (b) Wavelength-resolved (516- and 599-nm) fluorescence edge responses are plotted for the fluorescein in solution (dashed curve) and for the fluorophore-embedded polymer rod (solid curve). Derivatives of these edge responses yield axial resolutions of 4.05.1 µm.

Fig. 3
Fig. 3

Polarization-sensitive imaging shows the orientation of a photosensitizing dye in mouse mammary carcinoma cells. A conventional confocal fluorescence image of the drug localization in viable cells is shown in (a). The anisotropy map computed from the polarization-sensitive images shown in (b) is consistent with the orientation of dye in the nuclear envelope. Images are 40 µm×40 µm.

Fig. 4
Fig. 4

Polarization-sensitive imaging discriminates between two fluorophores with similar emission spectra but different anisotropies. GFP-expressing cells were immersed in media containing 0.4µg/mL fluorescein. The conventional confocal fluorescence image excited at 488 nm is shown in (a). Subtraction of I-I yields the image shown in (b), where we exploit the large difference in anisotropy between GFP and fluorescein to discriminate against the fluorescein signal. The anisotropy map derived from the polarization-sensitive images is shown in (c). Images are 100 µm×100 µm.

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