Abstract

We present a low-cost, high-speed, retrofitted laser scanning module for microscopy. The cage-mounted system, with various available fiber-coupled sources, offers a real-time imaging alternative to costly commercial systems with capabilities for conventional or confocal reflectance and fluorescence applications as well as advanced laser scanning microscopy implementations. Reflectance images of a resolution target and confocal images of fluorescent polystyrene beads are presented for system characterization. Confocal fluorescence image stacks of T84 epithelial cancer cells are presented to demonstrate application to biological studies. This laser scanning module is a flexible, scalable, high-speed alternative to commercial laser scanning systems suitable for applications requiring a simple imaging tool and for teaching laboratories.

© 2005 Optical Society of America

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References

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    [CrossRef] [PubMed]
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    [CrossRef]
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    [CrossRef] [PubMed]
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    [CrossRef]
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    [CrossRef] [PubMed]
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    [CrossRef]
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2004 (2)

2003 (4)

2002 (1)

2001 (3)

C. Liang, M. R. Descour, K. B. Sung, R. Richards-Kortum, “Fiber confocal reflectance microscope (FCRM) for in-vivo imaging,” Opt. Express 9, 821–830 (2001), http://www.opticsexpress.org .
[CrossRef] [PubMed]

V. Andresen, A. Egner, S. W. Hell, “Time-multiplexed multifocal multiphoton microscope,” Opt. Lett. 26, 75–77 (2001).
[CrossRef]

C. E. Bigelow, C. J. Harkrider, D. L. Conover, T. H. Foster, I. Georgakoudi, S. Mitra, M. G. Nichols, M. Rajadhyaksha, “Retrofitted confocal laser scanner for a commercial inverted fluorescence microscope,” Rev. Sci. Instrum. 72, 3407–3410 (2001).
[CrossRef]

2000 (4)

S. W. Paddock, “Principles and practices of laser scanning confocal microscopy,” Mol. Biotechnol. 16, 127–149 (2000).
[CrossRef] [PubMed]

C. P. Lin, R. H. Webb, “Fiber-coupled multiplexed confocal microscope,” Opt. Lett. 25, 954–956 (2000).
[CrossRef]

P. M. Lane, A. L. P. Dlugan, R. Richards-Kortum, C. E. MacAulay, “Fiber-optic confocal microscopy using a spatial light modulator,” Opt. Lett. 25, 1780–1782 (2000).
[CrossRef]

K. Fujita, O. Nakamura, T. Kaneko, M. Oyamada, T. Taka-matsu, S. Kawata, “Confocal multipoint multiphoton excitation microscope with microlens and pinhole arrays,” Opt. Commun. 174, 7–12 (2000).
[CrossRef]

1999 (2)

1998 (2)

1994 (1)

1992 (1)

M. Laurent, G. Johannin, H. Leguyader, A. Fleury, “Con-focal scanning optical microscopy and 3-dimensional imaging,” Biol. Cell 76, 113–124 (1992).
[CrossRef]

1990 (1)

W. Denk, J. H. Strickler, W. W. Webb, “2-photon laser scanning fluorescence microscopy,” Science 248, 73–76 (1990).
[CrossRef] [PubMed]

1987 (1)

1985 (1)

Aguirre, A. D.

Andresen, V.

Arndt-Jovin, D. J.

Aslund, N.

Bigelow, C. E.

C. E. Bigelow, C. J. Harkrider, D. L. Conover, T. H. Foster, I. Georgakoudi, S. Mitra, M. G. Nichols, M. Rajadhyaksha, “Retrofitted confocal laser scanner for a commercial inverted fluorescence microscope,” Rev. Sci. Instrum. 72, 3407–3410 (2001).
[CrossRef]

Booth, M.

Bouma, B. E.

Carlsson, K.

Conover, D. L.

C. E. Bigelow, C. J. Harkrider, D. L. Conover, T. H. Foster, I. Georgakoudi, S. Mitra, M. G. Nichols, M. Rajadhyaksha, “Retrofitted confocal laser scanner for a commercial inverted fluorescence microscope,” Rev. Sci. Instrum. 72, 3407–3410 (2001).
[CrossRef]

Danielsson, P. E.

Denk, W.

W. Denk, J. H. Strickler, W. W. Webb, “2-photon laser scanning fluorescence microscopy,” Science 248, 73–76 (1990).
[CrossRef] [PubMed]

Descour, M. R.

Dlugan, A. L. P.

Egner, A.

Fleury, A.

M. Laurent, G. Johannin, H. Leguyader, A. Fleury, “Con-focal scanning optical microscopy and 3-dimensional imaging,” Biol. Cell 76, 113–124 (1992).
[CrossRef]

Follen, M.

Foster, T. H.

C. E. Bigelow, C. J. Harkrider, D. L. Conover, T. H. Foster, I. Georgakoudi, S. Mitra, M. G. Nichols, M. Rajadhyaksha, “Retrofitted confocal laser scanner for a commercial inverted fluorescence microscope,” Rev. Sci. Instrum. 72, 3407–3410 (2001).
[CrossRef]

Fujimoto, J. G.

Fujita, K.

M. Kobayashi, K. Fujita, T. Kaneko, T. Takamatsu, O. Naka-mura, S. Kawata, “Second-harmonic-generation microscope with a microlens array scanner,” Opt. Lett. 27, 1324–1326 (2002).
[CrossRef]

K. Fujita, O. Nakamura, T. Kaneko, M. Oyamada, T. Taka-matsu, S. Kawata, “Confocal multipoint multiphoton excitation microscope with microlens and pinhole arrays,” Opt. Commun. 174, 7–12 (2000).
[CrossRef]

Georgakoudi, I.

C. E. Bigelow, C. J. Harkrider, D. L. Conover, T. H. Foster, I. Georgakoudi, S. Mitra, M. G. Nichols, M. Rajadhyaksha, “Retrofitted confocal laser scanner for a commercial inverted fluorescence microscope,” Rev. Sci. Instrum. 72, 3407–3410 (2001).
[CrossRef]

Gröbler, B.

S. Wilhelm, B. Gröbler, M. Gulch, H. Heinz, Confocal Laser Scanning Microscopy: Principles (Carl Zeiss, Inc., Jena, Germany, 2003), p. 11.

Gulch, M.

S. Wilhelm, B. Gröbler, M. Gulch, H. Heinz, Confocal Laser Scanning Microscopy: Principles (Carl Zeiss, Inc., Jena, Germany, 2003), p. 11.

Halbhuber, K. J.

K. J. Halbhuber, K. Konig, “Modern laser scanning microscopy in biology, biotechnology and medicine,” Annals of Anatomy-Anatomischer Anzeiger 185, 1–20 (2003).
[CrossRef]

Harkrider, C. J.

C. E. Bigelow, C. J. Harkrider, D. L. Conover, T. H. Foster, I. Georgakoudi, S. Mitra, M. G. Nichols, M. Rajadhyaksha, “Retrofitted confocal laser scanner for a commercial inverted fluorescence microscope,” Rev. Sci. Instrum. 72, 3407–3410 (2001).
[CrossRef]

Hartl, I.

Hee, M. R.

Heinz, H.

S. Wilhelm, B. Gröbler, M. Gulch, H. Heinz, Confocal Laser Scanning Microscopy: Principles (Carl Zeiss, Inc., Jena, Germany, 2003), p. 11.

Hell, S. W.

Hsiung, P.

Inoue, S.

S. Inoue, “Foundations of confocal scanned imaging in light microscopy,” in Handbook of Biological Confocal Microscopy, J. B. Pawley, ed. (Plenum, New York, 1995), pp. 1–14.
[CrossRef]

Izatt, J. A.

Johannin, G.

M. Laurent, G. Johannin, H. Leguyader, A. Fleury, “Con-focal scanning optical microscopy and 3-dimensional imaging,” Biol. Cell 76, 113–124 (1992).
[CrossRef]

Jovin, T. M.

Kaneko, T.

M. Kobayashi, K. Fujita, T. Kaneko, T. Takamatsu, O. Naka-mura, S. Kawata, “Second-harmonic-generation microscope with a microlens array scanner,” Opt. Lett. 27, 1324–1326 (2002).
[CrossRef]

K. Fujita, O. Nakamura, T. Kaneko, M. Oyamada, T. Taka-matsu, S. Kawata, “Confocal multipoint multiphoton excitation microscope with microlens and pinhole arrays,” Opt. Commun. 174, 7–12 (2000).
[CrossRef]

Kawata, S.

M. Kobayashi, K. Fujita, T. Kaneko, T. Takamatsu, O. Naka-mura, S. Kawata, “Second-harmonic-generation microscope with a microlens array scanner,” Opt. Lett. 27, 1324–1326 (2002).
[CrossRef]

K. Fujita, O. Nakamura, T. Kaneko, M. Oyamada, T. Taka-matsu, S. Kawata, “Confocal multipoint multiphoton excitation microscope with microlens and pinhole arrays,” Opt. Commun. 174, 7–12 (2000).
[CrossRef]

Kirsch, A. K.

Ko, T. H.

Kobayashi, M.

Konig, K.

K. J. Halbhuber, K. Konig, “Modern laser scanning microscopy in biology, biotechnology and medicine,” Annals of Anatomy-Anatomischer Anzeiger 185, 1–20 (2003).
[CrossRef]

Laiho, L. H.

Lane, P. M.

Laurent, M.

M. Laurent, G. Johannin, H. Leguyader, A. Fleury, “Con-focal scanning optical microscopy and 3-dimensional imaging,” Biol. Cell 76, 113–124 (1992).
[CrossRef]

Leguyader, H.

M. Laurent, G. Johannin, H. Leguyader, A. Fleury, “Con-focal scanning optical microscopy and 3-dimensional imaging,” Biol. Cell 76, 113–124 (1992).
[CrossRef]

Lenz, R.

Li, P.

Liang, C.

Liljeborg, A.

Lin, C. P.

Liu, Z. W.

MacAulay, C. E.

Majlof, L.

Malpica, A.

Mitra, S.

C. E. Bigelow, C. J. Harkrider, D. L. Conover, T. H. Foster, I. Georgakoudi, S. Mitra, M. G. Nichols, M. Rajadhyaksha, “Retrofitted confocal laser scanner for a commercial inverted fluorescence microscope,” Rev. Sci. Instrum. 72, 3407–3410 (2001).
[CrossRef]

Moses, E.

Nakamura, O.

K. Fujita, O. Nakamura, T. Kaneko, M. Oyamada, T. Taka-matsu, S. Kawata, “Confocal multipoint multiphoton excitation microscope with microlens and pinhole arrays,” Opt. Commun. 174, 7–12 (2000).
[CrossRef]

Naka-mura, O.

Nichols, M. G.

C. E. Bigelow, C. J. Harkrider, D. L. Conover, T. H. Foster, I. Georgakoudi, S. Mitra, M. G. Nichols, M. Rajadhyaksha, “Retrofitted confocal laser scanner for a commercial inverted fluorescence microscope,” Rev. Sci. Instrum. 72, 3407–3410 (2001).
[CrossRef]

Oron, D.

Owen, G. M.

Oyamada, M.

K. Fujita, O. Nakamura, T. Kaneko, M. Oyamada, T. Taka-matsu, S. Kawata, “Confocal multipoint multiphoton excitation microscope with microlens and pinhole arrays,” Opt. Commun. 174, 7–12 (2000).
[CrossRef]

Paddock, S. W.

S. W. Paddock, “Principles and practices of laser scanning confocal microscopy,” Mol. Biotechnol. 16, 127–149 (2000).
[CrossRef] [PubMed]

S. W. Paddock, “Confocal laser scanning microscopy,” Biotechniques 27, 992–1004 (1999).
[PubMed]

Ploem, J. S.

Rajadhyaksha, M.

C. E. Bigelow, C. J. Harkrider, D. L. Conover, T. H. Foster, I. Georgakoudi, S. Mitra, M. G. Nichols, M. Rajadhyaksha, “Retrofitted confocal laser scanner for a commercial inverted fluorescence microscope,” Rev. Sci. Instrum. 72, 3407–3410 (2001).
[CrossRef]

Richards-Kortum, R.

Schnetter, C. M.

Shi, K. B.

Silberberg, Y.

So, P. T. C.

Strickler, J. H.

W. Denk, J. H. Strickler, W. W. Webb, “2-photon laser scanning fluorescence microscopy,” Science 248, 73–76 (1990).
[CrossRef] [PubMed]

Sung, K. B.

Swanson, E. A.

Takamatsu, T.

Taka-matsu, T.

K. Fujita, O. Nakamura, T. Kaneko, M. Oyamada, T. Taka-matsu, S. Kawata, “Confocal multipoint multiphoton excitation microscope with microlens and pinhole arrays,” Opt. Commun. 174, 7–12 (2000).
[CrossRef]

Tearney, G. J.

Thiberge, S.

Webb, R. H.

Webb, W. W.

W. Denk, J. H. Strickler, W. W. Webb, “2-photon laser scanning fluorescence microscopy,” Science 248, 73–76 (1990).
[CrossRef] [PubMed]

Wilhelm, S.

S. Wilhelm, B. Gröbler, M. Gulch, H. Heinz, Confocal Laser Scanning Microscopy: Principles (Carl Zeiss, Inc., Jena, Germany, 2003), p. 11.

Wilms, S.

Yazdanfar, S.

Yelin, D.

Yin, S. Z.

Annals of Anatomy-Anatomischer Anzeiger (1)

K. J. Halbhuber, K. Konig, “Modern laser scanning microscopy in biology, biotechnology and medicine,” Annals of Anatomy-Anatomischer Anzeiger 185, 1–20 (2003).
[CrossRef]

Appl. Opt. (1)

Biol. Cell (1)

M. Laurent, G. Johannin, H. Leguyader, A. Fleury, “Con-focal scanning optical microscopy and 3-dimensional imaging,” Biol. Cell 76, 113–124 (1992).
[CrossRef]

Biotechniques (1)

S. W. Paddock, “Confocal laser scanning microscopy,” Biotechniques 27, 992–1004 (1999).
[PubMed]

Mol. Biotechnol. (1)

S. W. Paddock, “Principles and practices of laser scanning confocal microscopy,” Mol. Biotechnol. 16, 127–149 (2000).
[CrossRef] [PubMed]

Opt. Commun. (1)

K. Fujita, O. Nakamura, T. Kaneko, M. Oyamada, T. Taka-matsu, S. Kawata, “Confocal multipoint multiphoton excitation microscope with microlens and pinhole arrays,” Opt. Commun. 174, 7–12 (2000).
[CrossRef]

Opt. Express (6)

Opt. Lett. (9)

A. D. Aguirre, P. Hsiung, T. H. Ko, I. Hartl, J. G. Fujimoto, “High-resolution optical coherence microscopy for high-speed, in vivo cellular imaging,” Opt. Lett. 28, 2064–2066 (2003).
[CrossRef] [PubMed]

M. Kobayashi, K. Fujita, T. Kaneko, T. Takamatsu, O. Naka-mura, S. Kawata, “Second-harmonic-generation microscope with a microlens array scanner,” Opt. Lett. 27, 1324–1326 (2002).
[CrossRef]

G. J. Tearney, R. H. Webb, B. E. Bouma, “Spectrally encoded confocal microscopy,” Opt. Lett. 23, 1152–1154 (1998).
[CrossRef]

S. W. Hell, M. Booth, S. Wilms, C. M. Schnetter, A. K. Kirsch, D. J. Arndt-Jovin, T. M. Jovin, “Two-photon near- and far-field fluorescence microscopy with continuous-wave excitation,” Opt. Lett. 23, 1238–1240 (1998).
[CrossRef]

K. Carlsson, P. E. Danielsson, R. Lenz, A. Liljeborg, L. Majlof, N. Aslund, “Three-dimensional microscopy using a confo-cal laser scanning microscope,” Opt. Lett. 10, 53–55 (1985).
[CrossRef] [PubMed]

C. P. Lin, R. H. Webb, “Fiber-coupled multiplexed confocal microscope,” Opt. Lett. 25, 954–956 (2000).
[CrossRef]

P. M. Lane, A. L. P. Dlugan, R. Richards-Kortum, C. E. MacAulay, “Fiber-optic confocal microscopy using a spatial light modulator,” Opt. Lett. 25, 1780–1782 (2000).
[CrossRef]

V. Andresen, A. Egner, S. W. Hell, “Time-multiplexed multifocal multiphoton microscope,” Opt. Lett. 26, 75–77 (2001).
[CrossRef]

J. A. Izatt, M. R. Hee, G. M. Owen, E. A. Swanson, J. G. Fujimoto, “Optical coherence microscopy in scattering media,” Opt. Lett. 19, 590–592 (1994).
[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, M. Rajadhyaksha, “Retrofitted confocal laser scanner for a commercial inverted fluorescence microscope,” Rev. Sci. Instrum. 72, 3407–3410 (2001).
[CrossRef]

Science (1)

W. Denk, J. H. Strickler, W. W. Webb, “2-photon laser scanning fluorescence microscopy,” Science 248, 73–76 (1990).
[CrossRef] [PubMed]

Other (2)

S. Wilhelm, B. Gröbler, M. Gulch, H. Heinz, Confocal Laser Scanning Microscopy: Principles (Carl Zeiss, Inc., Jena, Germany, 2003), p. 11.

S. Inoue, “Foundations of confocal scanned imaging in light microscopy,” in Handbook of Biological Confocal Microscopy, J. B. Pawley, ed. (Plenum, New York, 1995), pp. 1–14.
[CrossRef]

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Figures (6)

Fig. 1
Fig. 1

Laser scanning module, optical schematic. OF, optical fiber; OFM, optical fiber mount; CL, collimating lens; BS, beam splitter; St, beam stop; Gx, x-direction resonant scanner; L1, L2, 4f translation lenses; Gy, y-direction optical scanner; FL, focusing lens; IP, image plane of microscope; DL, detector lens; F/PH, removable filter set or pinhole; PD/PMT, photodetector or photomultiplier tube; x,y directions shown indicate line and field scanning directions, respectively.

Fig. 2
Fig. 2

Confocal reflectance images, USAF resolution target. a, Groups 6 and 7, 10× (0.25 NA); b, Group 6 element 1, Group 7 elements 4–6, 40× (0.75 NA); c, Group 7 elements 4–6, 63× (1.4 NA/oil); d, Group 7 elements 1–4, Axiovert 100M commercial laser scanning confocal, 63× (1.2 NA/water).

Fig. 3
Fig. 3

Field-distortion analysis. Apparent size of imaged objects represented as distortion of 10 × 10 grid across the field of view. The distortion was less than 5% in the horizontal direction and remained under 10% in the vertical direction over 75% of the field of view.

Fig. 4
Fig. 4

Gaussian fit to axial position. Image intensity evaluated as mean intensity taken over multiple regions of interest of a reflective sample. Axial resolution determined as FWHM of Gaussian fit.

Fig. 5
Fig. 5

Fluorescent beads at 40× (0.75 NA). a, Confocal fluorescence with laser scanning system. b, Rhodamine-filtered image taken with CCD camera. c, DAPI-filtered image taken with CCD camera.

Fig. 6
Fig. 6

Confocal fluorescence images of T84 epithelial cells acquired with (a, f) a CCD camera and (b–e, g–j) a laser scanning system. Image sections taken at ascending 5-μm intervals with 40×(0.75 NA).

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