Abstract

We present a handheld dual-axes confocal microscope that is based on a two-dimensional microelectromechanical systems (MEMS) scanner. It performs reflectance and fluorescence imaging at 488 nm wavelength, with three-dimensional imaging capability. The fully packaged microscope has a diameter of 10 mm and acquires images at 4 Hz frame rate with a maximum field of view of 400 µm×260 µm. The transverse and axial resolutions of the handheld probe are 1.7 µm and 5.8 µm, respectively. Capability to perform real time small animal imaging is demonstrated in vivo in transgenic mice.

© 2008 Optical Society of America

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  1. J. Pawley, ed., Handbook of Biological Confocal Microscopy, 3rd ed. (Plenum, New York, 1996).
  2. D. L. Dickensheets and G. S. Kino, "Silicon-micromachined scanning confocal microscope," J. Microelectromech. Syst. 7, 38-47 (1998).
    [CrossRef]
  3. W. Piyawattanametha, H. Toshiyoshi, J. LaCosse, and M. C. Wu, "Surface-micromachined confocal scanning optical microscope," in Conference on Lasers and Electro-Optics, Technical Digest (Optical Society of America, 2000), pp. 447-448.
  4. K. Murakami, A. Murata, T. Suga, H. Kitagawa, Y. Kamiya, M. Kubo, K. Matsumoto, H. Miyajima, and M. Katashiro, "A miniature confocal optical microscope with MEMS gimbal scanner," in Technical Digest of Transducers (Boston, 2003), pp. 587-590.
  5. S. Kwon and L. P. Lee, "Micromachined transmissive scanning confocal microscope," Opt. Lett. 29, 706-708 (2004).
    [CrossRef] [PubMed]
  6. H.-J. Shin, M. C. Pierce, D. Lee, H. Ra, O. Solgaard, and R. Richards-Kortum, "Fiber-optic confocal microscope using a MEMS scanner and miniature objective lens," Opt. Express 15, 9113-9122 (2007), http://www.opticsinfobase.org/abstract.cfm?URI=oe-15-15-9113
    [CrossRef] [PubMed]
  7. T. D. Wang, M. J. Mandella, C. H. Contag, and G. S. Kino, "Dual-axis confocal microscope for high-resolution in vivo imaging," Opt. Lett. 28, 414-416 (2003).
    [CrossRef] [PubMed]
  8. L. K. Wong, M. J. Mandella, G. S. Kino, and T. D. Wang, "Improved rejection of multiply scattered photons in confocal microscopy using dual-axes architecture," Opt. Lett. 32, 1674-1676 (2007).
    [CrossRef] [PubMed]
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    [CrossRef]
  10. W. Piyawattanametha, J. T. C. Liu, M. J. Mandella, H. Ra, L. K. Wong, P. Hsiung, T. D. Wang, G. S. Kino, and O. Solgaard, "MEMS Based Dual-axes Confocal Reflectance Handheld Microscope for in vivo Imaging," in Proceedings of IEEE International Conference on Optical MicroElectroMechanical Systems, (Institute of Electrical and Electronics Engineers, Montana, August, 2006), pp. 164-165.
  11. J. T. C. Liu, M. J. Mandella, H. Ra, L. K. Wong, O. Solgaard, G. S. Kino, W. Piyawattanametha, C. H. Contag, and T. D. Wang, "Miniature near-infrared dual-axes confocal microscope utilizing a two-dimensional microelectromechanical systems scanner," Opt. Lett. 32, 256-258 (2007).
    [CrossRef] [PubMed]
  12. W. Piyawattanametha, H. Ra, M. J. Mandella, J. T. C. Liu, L. K. Wong, C. B. Du, T. D. Wang, C. H. Contag, G. S. Kino, and O. Solgaard, "Three-dimensional in vivo Real Time Imaging by a Miniature Dual-axes Confocal Microscope based on a Two-dimensional MEMS Scanner," in Proceedings of International Conference on Solid-State Sensors, Actuators, and Microsystems, (Lyon, France, June, 2007), pp. 439-442.
  13. H. Ra, W. Piyawattanametha, M. J. Mandella, J. T. C. Liu, L. K. Wong, T. D. Wang, C. H. Contag, G. S. Kino, and O. Solgaard, "Three-Dimensional in vivo Reflectance and Fluorescence Imaging by a Handheld Dual-Axes Confocal Microscope," in Conference on Lasers and Electro-Optics /Quantum Electronics and Laser Science and Photonic Applications Systems Technologies, Technical Digest (CD) (Optical Society of America, May, 2007), paper CTuEE1, http://www.opticsinfobase.org/abstract.cfm?URI=CLEO-2007-CTuEE1
  14. A.-K. Hadjantonakis, M. Gertsenstein, M. Ikawa, M. Okabe, and A. Nagy, "Generating green fluorescent mice by germline transmission of green fluorescent ES cells," Mech. Dev. 76, 79-90 (1998).
    [CrossRef] [PubMed]
  15. A. E. Siegman, M. W. Sasnett, and T. F. Johnston, Jr., "Choice of clip levels for beam width measurements using knife-edge techniques," IEEE J. Quantum Electron. 27, 1098-1104 (1991).
    [CrossRef]

2007

2004

2003

1998

D. L. Dickensheets and G. S. Kino, "Silicon-micromachined scanning confocal microscope," J. Microelectromech. Syst. 7, 38-47 (1998).
[CrossRef]

A.-K. Hadjantonakis, M. Gertsenstein, M. Ikawa, M. Okabe, and A. Nagy, "Generating green fluorescent mice by germline transmission of green fluorescent ES cells," Mech. Dev. 76, 79-90 (1998).
[CrossRef] [PubMed]

1991

A. E. Siegman, M. W. Sasnett, and T. F. Johnston, Jr., "Choice of clip levels for beam width measurements using knife-edge techniques," IEEE J. Quantum Electron. 27, 1098-1104 (1991).
[CrossRef]

Contag, C. H.

Dickensheets, D. L.

D. L. Dickensheets and G. S. Kino, "Silicon-micromachined scanning confocal microscope," J. Microelectromech. Syst. 7, 38-47 (1998).
[CrossRef]

Gertsenstein, M.

A.-K. Hadjantonakis, M. Gertsenstein, M. Ikawa, M. Okabe, and A. Nagy, "Generating green fluorescent mice by germline transmission of green fluorescent ES cells," Mech. Dev. 76, 79-90 (1998).
[CrossRef] [PubMed]

Hadjantonakis, A.-K.

A.-K. Hadjantonakis, M. Gertsenstein, M. Ikawa, M. Okabe, and A. Nagy, "Generating green fluorescent mice by germline transmission of green fluorescent ES cells," Mech. Dev. 76, 79-90 (1998).
[CrossRef] [PubMed]

Ikawa, M.

A.-K. Hadjantonakis, M. Gertsenstein, M. Ikawa, M. Okabe, and A. Nagy, "Generating green fluorescent mice by germline transmission of green fluorescent ES cells," Mech. Dev. 76, 79-90 (1998).
[CrossRef] [PubMed]

Johnston, T. F.

A. E. Siegman, M. W. Sasnett, and T. F. Johnston, Jr., "Choice of clip levels for beam width measurements using knife-edge techniques," IEEE J. Quantum Electron. 27, 1098-1104 (1991).
[CrossRef]

Kino, G. S.

Kwon, S.

Lee, D.

H.-J. Shin, M. C. Pierce, D. Lee, H. Ra, O. Solgaard, and R. Richards-Kortum, "Fiber-optic confocal microscope using a MEMS scanner and miniature objective lens," Opt. Express 15, 9113-9122 (2007), http://www.opticsinfobase.org/abstract.cfm?URI=oe-15-15-9113
[CrossRef] [PubMed]

H. Ra, W. Piyawattanametha, Y. Taguchi, D. Lee, M. J. Mandella, and O. Solgaard, "Two-Dimensional MEMS Scanner for Dual-Axes Confocal Microscopy," J. Microelectromech. Syst. 16, 969-976 (2007).
[CrossRef]

Lee, L. P.

Liu, J. T. C.

Mandella, M. J.

Nagy, A.

A.-K. Hadjantonakis, M. Gertsenstein, M. Ikawa, M. Okabe, and A. Nagy, "Generating green fluorescent mice by germline transmission of green fluorescent ES cells," Mech. Dev. 76, 79-90 (1998).
[CrossRef] [PubMed]

Okabe, M.

A.-K. Hadjantonakis, M. Gertsenstein, M. Ikawa, M. Okabe, and A. Nagy, "Generating green fluorescent mice by germline transmission of green fluorescent ES cells," Mech. Dev. 76, 79-90 (1998).
[CrossRef] [PubMed]

Pierce, M. C.

Piyawattanametha, W.

Ra, H.

Richards-Kortum, R.

Sasnett, M. W.

A. E. Siegman, M. W. Sasnett, and T. F. Johnston, Jr., "Choice of clip levels for beam width measurements using knife-edge techniques," IEEE J. Quantum Electron. 27, 1098-1104 (1991).
[CrossRef]

Shin, H.-J.

Siegman, A. E.

A. E. Siegman, M. W. Sasnett, and T. F. Johnston, Jr., "Choice of clip levels for beam width measurements using knife-edge techniques," IEEE J. Quantum Electron. 27, 1098-1104 (1991).
[CrossRef]

Solgaard, O.

Taguchi, Y.

H. Ra, W. Piyawattanametha, Y. Taguchi, D. Lee, M. J. Mandella, and O. Solgaard, "Two-Dimensional MEMS Scanner for Dual-Axes Confocal Microscopy," J. Microelectromech. Syst. 16, 969-976 (2007).
[CrossRef]

Wang, T. D.

Wong, L. K.

IEEE J. Quantum Electron.

A. E. Siegman, M. W. Sasnett, and T. F. Johnston, Jr., "Choice of clip levels for beam width measurements using knife-edge techniques," IEEE J. Quantum Electron. 27, 1098-1104 (1991).
[CrossRef]

J. Microelectromech. Syst.

D. L. Dickensheets and G. S. Kino, "Silicon-micromachined scanning confocal microscope," J. Microelectromech. Syst. 7, 38-47 (1998).
[CrossRef]

H. Ra, W. Piyawattanametha, Y. Taguchi, D. Lee, M. J. Mandella, and O. Solgaard, "Two-Dimensional MEMS Scanner for Dual-Axes Confocal Microscopy," J. Microelectromech. Syst. 16, 969-976 (2007).
[CrossRef]

Mech. Dev.

A.-K. Hadjantonakis, M. Gertsenstein, M. Ikawa, M. Okabe, and A. Nagy, "Generating green fluorescent mice by germline transmission of green fluorescent ES cells," Mech. Dev. 76, 79-90 (1998).
[CrossRef] [PubMed]

Opt. Express

Opt. Lett.

Other

W. Piyawattanametha, H. Ra, M. J. Mandella, J. T. C. Liu, L. K. Wong, C. B. Du, T. D. Wang, C. H. Contag, G. S. Kino, and O. Solgaard, "Three-dimensional in vivo Real Time Imaging by a Miniature Dual-axes Confocal Microscope based on a Two-dimensional MEMS Scanner," in Proceedings of International Conference on Solid-State Sensors, Actuators, and Microsystems, (Lyon, France, June, 2007), pp. 439-442.

H. Ra, W. Piyawattanametha, M. J. Mandella, J. T. C. Liu, L. K. Wong, T. D. Wang, C. H. Contag, G. S. Kino, and O. Solgaard, "Three-Dimensional in vivo Reflectance and Fluorescence Imaging by a Handheld Dual-Axes Confocal Microscope," in Conference on Lasers and Electro-Optics /Quantum Electronics and Laser Science and Photonic Applications Systems Technologies, Technical Digest (CD) (Optical Society of America, May, 2007), paper CTuEE1, http://www.opticsinfobase.org/abstract.cfm?URI=CLEO-2007-CTuEE1

J. Pawley, ed., Handbook of Biological Confocal Microscopy, 3rd ed. (Plenum, New York, 1996).

W. Piyawattanametha, H. Toshiyoshi, J. LaCosse, and M. C. Wu, "Surface-micromachined confocal scanning optical microscope," in Conference on Lasers and Electro-Optics, Technical Digest (Optical Society of America, 2000), pp. 447-448.

K. Murakami, A. Murata, T. Suga, H. Kitagawa, Y. Kamiya, M. Kubo, K. Matsumoto, H. Miyajima, and M. Katashiro, "A miniature confocal optical microscope with MEMS gimbal scanner," in Technical Digest of Transducers (Boston, 2003), pp. 587-590.

W. Piyawattanametha, J. T. C. Liu, M. J. Mandella, H. Ra, L. K. Wong, P. Hsiung, T. D. Wang, G. S. Kino, and O. Solgaard, "MEMS Based Dual-axes Confocal Reflectance Handheld Microscope for in vivo Imaging," in Proceedings of IEEE International Conference on Optical MicroElectroMechanical Systems, (Institute of Electrical and Electronics Engineers, Montana, August, 2006), pp. 164-165.

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Figures (7)

Fig. 1.
Fig. 1.

Diagram of the imaging system.

Fig. 2.
Fig. 2.

(a) Schematic of the 10 mm diameter handheld probe with the packaged MEMS scanner in the inset. (b) Photograph of the fully assembled dual-axes confocal handheld probe.

Fig. 3.
Fig. 3.

SEM of the MEMS scanner.

Fig. 4.
Fig. 4.

Reflectance image of group 7 of USAF target. Scale bar is 20 µm.

Fig. 5.
Fig. 5.

Ex vivo images of freshly excised GFP mouse tissue and corresponding histology. (a) Adipocytes in the hind leg region. (b) Histology of adipocytes. (c) Villi in the small intestine. (d) Histology of small intestine.

Fig. 6.
Fig. 6.

Ex vivo images of freshly excised GFP mouse tissue. Three en face planes extracted from a 3-D volume rendering and movies of the entire image stack are shown in (a) and (c). (a) Muscle. (Movie size 2.9 MB) (b) One en face image within the stack. (c) Vessel-like structures in the brain. (Movie size 2.7 MB) (d) Maximum intensity projected image of the stack. [Media 1][Media 2]

Fig. 7.
Fig. 7.

In vivo image of blood vessels in an intact mouse ear. (a) 3-D volume rendering. (b) Maximum intensity projected image of the stack.

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