Abstract

Abstract

A digital light modulation microscope (DLMM) that utilizes a digital micromirror device (DMD) on an epifluorescence microscope has been developed to modulate excitation light in spatial and temporal domains for phosphorescence lifetime detection. Local O2 concentration can be inferred through the detected lifetime around an O2-quenching phosphorescent porphyrin microsensor. Combined with microsensor arrays, the DLMM can sequentially address light to each microsensor element to construct a discrete lifetime image or O2 distribution. In contrast to conventional phosphorescence lifetime imaging, the new method eliminates the need for a pulsed light source and a time-gated camera. To demonstrate O2 sensing with lab-on-a-chip devices, an array of 150-µm-diameter micro-wells coated with phosphorescent porphyrin were observed. The locations of the sensor elements were automatically identified though image analysis. The goal of this platform is to measure the O2 consumption of individual cells trapped in the microwells.

© 2007 Optical Society of America

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  1. W. Trettnak, W. Gruber, F. Reininger, and I. Klimant, "Recent progress in optical oxygen sensor instrumentation," Sens. Actuators B,  1-3 (1995).
  2. F. Reininger, C. Kolle, W. Gruber, and W. Trettnak, "Current progress in O2 measurement by luminescence quenching," Med. Biol. Eng. Comput. 34, 113-114 (1996).
  3. L. J. Hornbeck, "The DMD (TM) projection display chip: A MEMS-based technology," Mrs. Bull. 26, 325-327 (2001).
    [CrossRef]
  4. M. Liang, R. L. Stehr, and A. W. Krause, "Confocal microscope system that uses a binary spatial light modulator," in Proceedings of the 1997 Conference on Lasers and Electro-Optics, CLEO, May 18-23 1997 (Optical Society of America, Washington DC, 1997), p. 154.
  5. S. Cha, P. C. Lin, L. Zhu, E. L. Botvinick, P.-C. Sun, and Y. Fainman, "3D profilometry using a dynamically configurable confocal microscope," Proceedings of SPIE of the Three-Dimensional Image Capture and Applications II, Jan 25-Jan 26 1999 3640, 246-253 (1999).
  6. C. Sungdo, P. C. Lin, Z. Lijun, E. L. Botvinick, and S. Pang Chen, "3D profilometry using a dynamically configurable confocal microscope," Proceedings of the SPIE The International Society for Optical, for-Optical (1999).
  7. P. J. Verveer, Q. S. Hanley, P. W. Verbeek, L. J. Van Vliet, and T. M. Jovin, "Theory of confocal fluorescence imaging in the programmable array microscope (PAM)," J. Microsc. 189, 192-198 (1998).
    [CrossRef]
  8. Q. S. Hanley, P. J. Verveer, M. J. Gemkow, D. Arndt Jovin, and T. M. Jovin, "An optical sectioning programmable array microscope implemented with a digital micromirror device," J. Microsc. 196, 317-331 (1999).
    [CrossRef] [PubMed]
  9. Q. S. Hanley, P. J. Verveer, and T. M. Jovin, "Optical sectioning fluorescence spectroscopy in a programmable array microscope," Appl. Spectrosc. 52, 783-789 (1998).
    [CrossRef]
  10. V. Bansal, S. Patel, and P. Saggau, "High-speed addressable confocal microscopy for functional imaging of cellular activity," J. Biomed. Opt. 11, 34003-34011 (2006).
    [CrossRef] [PubMed]
  11. T. Fukano, and A. Miyawaki, "Whole-field fluorescence microscope with digital micromirror device: imaging of biological samples," Appl. Opt. 42,4119-4124 (2003).
    [CrossRef] [PubMed]
  12. C. MacAulay, and A. Dlugan, "Use of digital micro mirror devices in quantitative microscopy," in Optical Investigations of Cells In Vitro and In Vivo, Jan 25-28 1998 (The International Society for Optical Engineering, San Jose, CA), 201-206.
  13. P. M. Lane, A. L. P. Dlugan, R. Richards-Kortum, and C. E. MacAulay, "Fiber-optic confocal microscopy using a spatial light modulator," Opt. Lett. 25,1780-1782 (2000).
    [CrossRef]
  14. S.-H. Chao, M. R. Holl, S. C. McQuaide, and D. R. Meldrum, "Oxygen concentration measurement with a phosphorescence lifetime based micro-sensor array using a digital light modulation microscope," Proceedings of SPIE of Biomedical Optics and Imaging, 2006 (San Jose, CA), 6088, 60880S.
  15. S. C. McQuaide, M. R. Holl, L. Burgess, T. Molter, J. Dragavon, A. C. Young, T. Strovas, J. Anderson, A. Jen, B. Karlsgodt, M. Lidstrom, and R. D. Meldrum, "A Living Cell Array (LCA) for Multiparameter Cell Metabolism Studies," in IEEE / RAS-EMBS International Conference on Biomedical Robotics and Biomechatronics (Pisa, Italy, 2006), 971-976.
  16. M. E. Lidstrom, and D. R. Meldrum, "Life-on-a-chip," Nat. Rev. Microbiol. 1, 158-164 (2003).
    [CrossRef]
  17. D. B. Papkovsky, "Luminescent Porphyrins as Probes for Optical (Bio)Sensors," Sens. Actuators, B-Chemical 11, 293-300 (1993).Q1
    [CrossRef]
  18. T. W. Molter, M. R. Holl, J. M. Dragavon, S. C. McQuaide, J. B. Anderson, A. C. Young, L. W. Burgess, M. E. Lidstrom, and D. R. Meldrum, "A new approach for measuring single cell oxygen consumption rates," IEEE T. Automat. Sci. Eng. (to appear).
  19. "DMD discovery kit brochure," in Productivity systems Inc.(2002).
  20. R. D. Shonat, and A. C. Kight, "Oxygen tension imaging in the mouse retina," Ann. Biomed. Eng. 31, 1084-1096 (2003).
    [PubMed]
  21. G. Liebsch, I. Klimant, B. Frank, G. Holst, and O. S. Wolfbeis, "Luminescence lifetime imaging of oxygen, pH, and carbon dioxide," Appl. Spectrosc. 54, 548-559 (2000).
    [CrossRef]
  22. M. Shahidi, A. Shakoor, N. P. Blair, M. Mori, and R. D. Shonat, "A method for chorioretinal oxygen tension measurement," Curr. Eye. Res. 31, 357-366 (2006).
    [CrossRef] [PubMed]
  23. S.-H. Chao, T. T. H. Ren, S. A. Gales, M. R. Holl, S. C. McQuaide, and D. R. Meldrum, "Automated digital light modulation microscope (DLMM) for living cell array analysis: Pattern recognition and spatial alignment," in IEEE / RAS-EMBS International Conference on Biomedical Robotics and Biomechatronics (Pisa, Italy, 2006), 977-981.
  24. R. M. Haralick, L. G. Shapiro, "Computer and robot vision". Reading, Mass.: Addison-Wesley Pub. Co, (1992).
  25. G. Liebsch, I. Klimant, B. Frank, G. Holst, and O. S. Wolfbeis, "Luminescence lifetime imaging of oxygen, pH, and carbon dioxide," Appl. Spectrosc. 54, 548-559 (2000).
    [CrossRef]

2006 (2)

V. Bansal, S. Patel, and P. Saggau, "High-speed addressable confocal microscopy for functional imaging of cellular activity," J. Biomed. Opt. 11, 34003-34011 (2006).
[CrossRef] [PubMed]

M. Shahidi, A. Shakoor, N. P. Blair, M. Mori, and R. D. Shonat, "A method for chorioretinal oxygen tension measurement," Curr. Eye. Res. 31, 357-366 (2006).
[CrossRef] [PubMed]

2003 (3)

M. E. Lidstrom, and D. R. Meldrum, "Life-on-a-chip," Nat. Rev. Microbiol. 1, 158-164 (2003).
[CrossRef]

T. Fukano, and A. Miyawaki, "Whole-field fluorescence microscope with digital micromirror device: imaging of biological samples," Appl. Opt. 42,4119-4124 (2003).
[CrossRef] [PubMed]

R. D. Shonat, and A. C. Kight, "Oxygen tension imaging in the mouse retina," Ann. Biomed. Eng. 31, 1084-1096 (2003).
[PubMed]

2001 (1)

L. J. Hornbeck, "The DMD (TM) projection display chip: A MEMS-based technology," Mrs. Bull. 26, 325-327 (2001).
[CrossRef]

2000 (3)

1999 (1)

Q. S. Hanley, P. J. Verveer, M. J. Gemkow, D. Arndt Jovin, and T. M. Jovin, "An optical sectioning programmable array microscope implemented with a digital micromirror device," J. Microsc. 196, 317-331 (1999).
[CrossRef] [PubMed]

1998 (2)

P. J. Verveer, Q. S. Hanley, P. W. Verbeek, L. J. Van Vliet, and T. M. Jovin, "Theory of confocal fluorescence imaging in the programmable array microscope (PAM)," J. Microsc. 189, 192-198 (1998).
[CrossRef]

Q. S. Hanley, P. J. Verveer, and T. M. Jovin, "Optical sectioning fluorescence spectroscopy in a programmable array microscope," Appl. Spectrosc. 52, 783-789 (1998).
[CrossRef]

1996 (1)

F. Reininger, C. Kolle, W. Gruber, and W. Trettnak, "Current progress in O2 measurement by luminescence quenching," Med. Biol. Eng. Comput. 34, 113-114 (1996).

1993 (1)

D. B. Papkovsky, "Luminescent Porphyrins as Probes for Optical (Bio)Sensors," Sens. Actuators, B-Chemical 11, 293-300 (1993).Q1
[CrossRef]

Arndt Jovin, D.

Q. S. Hanley, P. J. Verveer, M. J. Gemkow, D. Arndt Jovin, and T. M. Jovin, "An optical sectioning programmable array microscope implemented with a digital micromirror device," J. Microsc. 196, 317-331 (1999).
[CrossRef] [PubMed]

Bansal, V.

V. Bansal, S. Patel, and P. Saggau, "High-speed addressable confocal microscopy for functional imaging of cellular activity," J. Biomed. Opt. 11, 34003-34011 (2006).
[CrossRef] [PubMed]

Blair, N. P.

M. Shahidi, A. Shakoor, N. P. Blair, M. Mori, and R. D. Shonat, "A method for chorioretinal oxygen tension measurement," Curr. Eye. Res. 31, 357-366 (2006).
[CrossRef] [PubMed]

Dlugan, A. L. P.

Frank, B.

Fukano, T.

Gemkow, M. J.

Q. S. Hanley, P. J. Verveer, M. J. Gemkow, D. Arndt Jovin, and T. M. Jovin, "An optical sectioning programmable array microscope implemented with a digital micromirror device," J. Microsc. 196, 317-331 (1999).
[CrossRef] [PubMed]

Gruber, W.

F. Reininger, C. Kolle, W. Gruber, and W. Trettnak, "Current progress in O2 measurement by luminescence quenching," Med. Biol. Eng. Comput. 34, 113-114 (1996).

Hanley, Q. S.

Q. S. Hanley, P. J. Verveer, M. J. Gemkow, D. Arndt Jovin, and T. M. Jovin, "An optical sectioning programmable array microscope implemented with a digital micromirror device," J. Microsc. 196, 317-331 (1999).
[CrossRef] [PubMed]

P. J. Verveer, Q. S. Hanley, P. W. Verbeek, L. J. Van Vliet, and T. M. Jovin, "Theory of confocal fluorescence imaging in the programmable array microscope (PAM)," J. Microsc. 189, 192-198 (1998).
[CrossRef]

Q. S. Hanley, P. J. Verveer, and T. M. Jovin, "Optical sectioning fluorescence spectroscopy in a programmable array microscope," Appl. Spectrosc. 52, 783-789 (1998).
[CrossRef]

Holst, G.

Hornbeck, L. J.

L. J. Hornbeck, "The DMD (TM) projection display chip: A MEMS-based technology," Mrs. Bull. 26, 325-327 (2001).
[CrossRef]

Jovin, T. M.

Q. S. Hanley, P. J. Verveer, M. J. Gemkow, D. Arndt Jovin, and T. M. Jovin, "An optical sectioning programmable array microscope implemented with a digital micromirror device," J. Microsc. 196, 317-331 (1999).
[CrossRef] [PubMed]

P. J. Verveer, Q. S. Hanley, P. W. Verbeek, L. J. Van Vliet, and T. M. Jovin, "Theory of confocal fluorescence imaging in the programmable array microscope (PAM)," J. Microsc. 189, 192-198 (1998).
[CrossRef]

Q. S. Hanley, P. J. Verveer, and T. M. Jovin, "Optical sectioning fluorescence spectroscopy in a programmable array microscope," Appl. Spectrosc. 52, 783-789 (1998).
[CrossRef]

Kight, A. C.

R. D. Shonat, and A. C. Kight, "Oxygen tension imaging in the mouse retina," Ann. Biomed. Eng. 31, 1084-1096 (2003).
[PubMed]

Klimant, I.

Kolle, C.

F. Reininger, C. Kolle, W. Gruber, and W. Trettnak, "Current progress in O2 measurement by luminescence quenching," Med. Biol. Eng. Comput. 34, 113-114 (1996).

Lane, P. M.

Lidstrom, M. E.

M. E. Lidstrom, and D. R. Meldrum, "Life-on-a-chip," Nat. Rev. Microbiol. 1, 158-164 (2003).
[CrossRef]

Liebsch, G.

MacAulay, C. E.

Meldrum, D. R.

M. E. Lidstrom, and D. R. Meldrum, "Life-on-a-chip," Nat. Rev. Microbiol. 1, 158-164 (2003).
[CrossRef]

Miyawaki, A.

Mori, M.

M. Shahidi, A. Shakoor, N. P. Blair, M. Mori, and R. D. Shonat, "A method for chorioretinal oxygen tension measurement," Curr. Eye. Res. 31, 357-366 (2006).
[CrossRef] [PubMed]

Papkovsky, D. B.

D. B. Papkovsky, "Luminescent Porphyrins as Probes for Optical (Bio)Sensors," Sens. Actuators, B-Chemical 11, 293-300 (1993).Q1
[CrossRef]

Patel, S.

V. Bansal, S. Patel, and P. Saggau, "High-speed addressable confocal microscopy for functional imaging of cellular activity," J. Biomed. Opt. 11, 34003-34011 (2006).
[CrossRef] [PubMed]

Reininger, F.

F. Reininger, C. Kolle, W. Gruber, and W. Trettnak, "Current progress in O2 measurement by luminescence quenching," Med. Biol. Eng. Comput. 34, 113-114 (1996).

Richards-Kortum, R.

Saggau, P.

V. Bansal, S. Patel, and P. Saggau, "High-speed addressable confocal microscopy for functional imaging of cellular activity," J. Biomed. Opt. 11, 34003-34011 (2006).
[CrossRef] [PubMed]

Shahidi, M.

M. Shahidi, A. Shakoor, N. P. Blair, M. Mori, and R. D. Shonat, "A method for chorioretinal oxygen tension measurement," Curr. Eye. Res. 31, 357-366 (2006).
[CrossRef] [PubMed]

Shakoor, A.

M. Shahidi, A. Shakoor, N. P. Blair, M. Mori, and R. D. Shonat, "A method for chorioretinal oxygen tension measurement," Curr. Eye. Res. 31, 357-366 (2006).
[CrossRef] [PubMed]

Shonat, R. D.

M. Shahidi, A. Shakoor, N. P. Blair, M. Mori, and R. D. Shonat, "A method for chorioretinal oxygen tension measurement," Curr. Eye. Res. 31, 357-366 (2006).
[CrossRef] [PubMed]

R. D. Shonat, and A. C. Kight, "Oxygen tension imaging in the mouse retina," Ann. Biomed. Eng. 31, 1084-1096 (2003).
[PubMed]

Trettnak, W.

F. Reininger, C. Kolle, W. Gruber, and W. Trettnak, "Current progress in O2 measurement by luminescence quenching," Med. Biol. Eng. Comput. 34, 113-114 (1996).

Van Vliet, L. J.

P. J. Verveer, Q. S. Hanley, P. W. Verbeek, L. J. Van Vliet, and T. M. Jovin, "Theory of confocal fluorescence imaging in the programmable array microscope (PAM)," J. Microsc. 189, 192-198 (1998).
[CrossRef]

Verbeek, P. W.

P. J. Verveer, Q. S. Hanley, P. W. Verbeek, L. J. Van Vliet, and T. M. Jovin, "Theory of confocal fluorescence imaging in the programmable array microscope (PAM)," J. Microsc. 189, 192-198 (1998).
[CrossRef]

Verveer, P. J.

Q. S. Hanley, P. J. Verveer, M. J. Gemkow, D. Arndt Jovin, and T. M. Jovin, "An optical sectioning programmable array microscope implemented with a digital micromirror device," J. Microsc. 196, 317-331 (1999).
[CrossRef] [PubMed]

P. J. Verveer, Q. S. Hanley, P. W. Verbeek, L. J. Van Vliet, and T. M. Jovin, "Theory of confocal fluorescence imaging in the programmable array microscope (PAM)," J. Microsc. 189, 192-198 (1998).
[CrossRef]

Q. S. Hanley, P. J. Verveer, and T. M. Jovin, "Optical sectioning fluorescence spectroscopy in a programmable array microscope," Appl. Spectrosc. 52, 783-789 (1998).
[CrossRef]

Wolfbeis, O. S.

Ann. Biomed. Eng. (1)

R. D. Shonat, and A. C. Kight, "Oxygen tension imaging in the mouse retina," Ann. Biomed. Eng. 31, 1084-1096 (2003).
[PubMed]

Appl. Opt. (1)

Appl. Spectrosc. (3)

B-Chemical (1)

D. B. Papkovsky, "Luminescent Porphyrins as Probes for Optical (Bio)Sensors," Sens. Actuators, B-Chemical 11, 293-300 (1993).Q1
[CrossRef]

Curr. Eye. Res. (1)

M. Shahidi, A. Shakoor, N. P. Blair, M. Mori, and R. D. Shonat, "A method for chorioretinal oxygen tension measurement," Curr. Eye. Res. 31, 357-366 (2006).
[CrossRef] [PubMed]

J. Biomed. Opt. (1)

V. Bansal, S. Patel, and P. Saggau, "High-speed addressable confocal microscopy for functional imaging of cellular activity," J. Biomed. Opt. 11, 34003-34011 (2006).
[CrossRef] [PubMed]

J. Microsc. (2)

P. J. Verveer, Q. S. Hanley, P. W. Verbeek, L. J. Van Vliet, and T. M. Jovin, "Theory of confocal fluorescence imaging in the programmable array microscope (PAM)," J. Microsc. 189, 192-198 (1998).
[CrossRef]

Q. S. Hanley, P. J. Verveer, M. J. Gemkow, D. Arndt Jovin, and T. M. Jovin, "An optical sectioning programmable array microscope implemented with a digital micromirror device," J. Microsc. 196, 317-331 (1999).
[CrossRef] [PubMed]

Med. Biol. Eng. Comput. (1)

F. Reininger, C. Kolle, W. Gruber, and W. Trettnak, "Current progress in O2 measurement by luminescence quenching," Med. Biol. Eng. Comput. 34, 113-114 (1996).

Mrs. Bull. (1)

L. J. Hornbeck, "The DMD (TM) projection display chip: A MEMS-based technology," Mrs. Bull. 26, 325-327 (2001).
[CrossRef]

Nat. Rev. Microbiol. (1)

M. E. Lidstrom, and D. R. Meldrum, "Life-on-a-chip," Nat. Rev. Microbiol. 1, 158-164 (2003).
[CrossRef]

Opt. Lett. (1)

Other (11)

W. Trettnak, W. Gruber, F. Reininger, and I. Klimant, "Recent progress in optical oxygen sensor instrumentation," Sens. Actuators B,  1-3 (1995).

T. W. Molter, M. R. Holl, J. M. Dragavon, S. C. McQuaide, J. B. Anderson, A. C. Young, L. W. Burgess, M. E. Lidstrom, and D. R. Meldrum, "A new approach for measuring single cell oxygen consumption rates," IEEE T. Automat. Sci. Eng. (to appear).

"DMD discovery kit brochure," in Productivity systems Inc.(2002).

C. MacAulay, and A. Dlugan, "Use of digital micro mirror devices in quantitative microscopy," in Optical Investigations of Cells In Vitro and In Vivo, Jan 25-28 1998 (The International Society for Optical Engineering, San Jose, CA), 201-206.

S.-H. Chao, T. T. H. Ren, S. A. Gales, M. R. Holl, S. C. McQuaide, and D. R. Meldrum, "Automated digital light modulation microscope (DLMM) for living cell array analysis: Pattern recognition and spatial alignment," in IEEE / RAS-EMBS International Conference on Biomedical Robotics and Biomechatronics (Pisa, Italy, 2006), 977-981.

R. M. Haralick, L. G. Shapiro, "Computer and robot vision". Reading, Mass.: Addison-Wesley Pub. Co, (1992).

M. Liang, R. L. Stehr, and A. W. Krause, "Confocal microscope system that uses a binary spatial light modulator," in Proceedings of the 1997 Conference on Lasers and Electro-Optics, CLEO, May 18-23 1997 (Optical Society of America, Washington DC, 1997), p. 154.

S. Cha, P. C. Lin, L. Zhu, E. L. Botvinick, P.-C. Sun, and Y. Fainman, "3D profilometry using a dynamically configurable confocal microscope," Proceedings of SPIE of the Three-Dimensional Image Capture and Applications II, Jan 25-Jan 26 1999 3640, 246-253 (1999).

C. Sungdo, P. C. Lin, Z. Lijun, E. L. Botvinick, and S. Pang Chen, "3D profilometry using a dynamically configurable confocal microscope," Proceedings of the SPIE The International Society for Optical, for-Optical (1999).

S.-H. Chao, M. R. Holl, S. C. McQuaide, and D. R. Meldrum, "Oxygen concentration measurement with a phosphorescence lifetime based micro-sensor array using a digital light modulation microscope," Proceedings of SPIE of Biomedical Optics and Imaging, 2006 (San Jose, CA), 6088, 60880S.

S. C. McQuaide, M. R. Holl, L. Burgess, T. Molter, J. Dragavon, A. C. Young, T. Strovas, J. Anderson, A. Jen, B. Karlsgodt, M. Lidstrom, and R. D. Meldrum, "A Living Cell Array (LCA) for Multiparameter Cell Metabolism Studies," in IEEE / RAS-EMBS International Conference on Biomedical Robotics and Biomechatronics (Pisa, Italy, 2006), 971-976.

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Figures (7)

Fig. 1.
Fig. 1.

The microfabricated living cell array (LCA) with microwells etched on a glass substrate, where each well works as a cell trap with ring-shaped O2-sensitive phosphorescent material.

Fig. 2.
Fig. 2.

The schematic of the digital light modulation microscope (DLMM).

Fig. 3.
Fig. 3.

Images of two 150-µm diameter, ring-shaped phosphorescent patches imaged by the DLMM. The inserts on the lower-left of all images indicate the light pattern in the DMD (white areas represent switched-on micro-mirrors).

Fig. 4.
Fig. 4.

(a) The relationship of the coordinate system of the CCD camera to that of the DMD. (b-c) Calibration of the coordinate systems using a user interface that processes images from the CCD camera: (b) a DMD generated a horizontal line for calculating the rotation angle between two coordinate systems. (c) DMD generated squares for calculating the magnification between the two coordinate systems.

Fig. 5.
Fig. 5.

The procedure and demonstrated results of micro-well identification: a) raw fluorescence image of five micro-wells, b) thresholding the raw image into a binary image, c) morphological processing to remove undesired artifacts, and d) estimating the geometrical parameters (unit: pixel).

Fig. 6.
Fig. 6.

The phosphorescence decay curves of the detected O2 sensor patch (solid lines) and fit curves (dashed lines). (a) ambient conditions (~21% O2); (b) depleted O2 conditions (~0% O2).

Fig. 7.
Fig. 7.

(a) Images of five wells with O2 sensing phosphorescent rings. (b) The measured phosphorescence decay curves of the O2 sensors and the inferred decay time τ ^ .

Equations (3)

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I em ( t ) I ¯ em = e t τ ,
τ 0 τ = 1 + K sv [ O 2 ] ,
[ x y ] = M [ cos θ sin θ sin θ cos θ ] [ x ¯ x ¯ 0 y ¯ y ¯ 0 ]

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