Abstract

We have developed a common-path multimodal optical microscopy system that is capable of using a single optical source and a single camera to image amplitude, phase, and fluorescence features of a biological specimen. This is achieved by varying either contrast enhancement filters at the Fourier plane and/or neutral density/fluorescence filters in front of the CCD camera. The feasibility of the technique is demonstrated by obtaining brightfield, fluorescence, phase-contrast, spatially filtered, brightfield+fluorescence, phase+fluorescence, and edge-enhanced+fluorescence images of the same Drosophila embryo without the need for image registration and fusion. This comprehensive microscope has the capability of providing both structural and functional information and may be used for applications such as studying live-cell dynamics and in high throughput microscopy and automated microscopy.

© 2009 Optical Society of America

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    [CrossRef] [PubMed]
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    [CrossRef] [PubMed]

2008 (2)

H. Kawano, T. Kogure, Y. Abe, H. Mizuno, and A. Miyawaki, Nat. Methods 5, 373 (2008).
[CrossRef] [PubMed]

T. B. Huff, Y. Shi, Y. Fu, H. Wang, and J.-X. Cheng, IEEE J. Sel. Top. Quantum Electron. 14, 4 (2008).
[CrossRef] [PubMed]

2007 (4)

W. C. Warger 2nd, G. S. Laevsky, D. J. Townsend, M. Rajadhyaksha, and C. A. DiMarzio, J. Biomed. Opt. 12, 044006 (2007).
[CrossRef] [PubMed]

J. A. Palero, H. S. de Bruijn, A. P. Heuvel, H. J. C. M. Sterenborg, and H. C. Gerritsen, Biophys. J. 93, 992 (2007).
[CrossRef] [PubMed]

W. J. Cottrell, J. D. Wilson, and T. H. Foster, Opt. Lett. 32, 2348 (2007).
[CrossRef] [PubMed]

P. S. P. Thong, K. W. Kho, W. Zheng, M. Harris, K. C. Soo, and M. Olivo, J. Mech. Med. Bio. 7, 11 (2007).
[CrossRef]

2006 (2)

C. S. Yelleswarapu, S.-R. Kothapalli, Y. R. Vaillancourt, F. J. Aranda, B. R. Kimball, and D. V. G. L. N. Rao, Appl. Phys. Lett. 89, 211116 (2006).
[CrossRef]

Y. Park, G. Popescu, K. Badizadegan, R. R. Dasari, and M. S. Feld, Opt. Express 14, 8263 (2006).
[CrossRef] [PubMed]

2005 (2)

2003 (1)

D. J. Stephens and V. J. Allan, Science 300, 82 (2003).
[CrossRef] [PubMed]

2001 (1)

P. T. Tran and F. Chang, Biol. Bull. 201, 253 (2001).
[CrossRef]

1994 (1)

E. M. O'Neill, I. Rebay, R. Tijan, and G. M. Rubin, Cell 78, 137 (1994).
[CrossRef] [PubMed]

1991 (1)

J. W. Goodman, Opt. Photonics News 2, 11 (1991).
[CrossRef]

1955 (2)

F. Zernike, Science 121, 345 (1955).
[CrossRef] [PubMed]

G. Nomarski, J. Phys. Radium 16, 9 (1955).

Abe, Y.

H. Kawano, T. Kogure, Y. Abe, H. Mizuno, and A. Miyawaki, Nat. Methods 5, 373 (2008).
[CrossRef] [PubMed]

Allan, V. J.

D. J. Stephens and V. J. Allan, Science 300, 82 (2003).
[CrossRef] [PubMed]

Aranda, F. J.

C. S. Yelleswarapu, S.-R. Kothapalli, Y. R. Vaillancourt, F. J. Aranda, B. R. Kimball, and D. V. G. L. N. Rao, Appl. Phys. Lett. 89, 211116 (2006).
[CrossRef]

Badizadegan, K.

Bernet, S.

Campos-Ortega, J. A.

J. A. Campos-Ortega and V. Hartenstein, The Embryonic Development of Drosophila Melanogaster (Springer-Verlag, 1985).

Chang, F.

P. T. Tran and F. Chang, Biol. Bull. 201, 253 (2001).
[CrossRef]

Cheng, J.-X.

T. B. Huff, Y. Shi, Y. Fu, H. Wang, and J.-X. Cheng, IEEE J. Sel. Top. Quantum Electron. 14, 4 (2008).
[CrossRef] [PubMed]

Conchello, J.

J. W. Lichtman and J. Conchello, Nat. Methods 2, 910 (2005).
[CrossRef] [PubMed]

Cottrell, W. J.

Dasari, R. R.

de Bruijn, H. S.

J. A. Palero, H. S. de Bruijn, A. P. Heuvel, H. J. C. M. Sterenborg, and H. C. Gerritsen, Biophys. J. 93, 992 (2007).
[CrossRef] [PubMed]

DiMarzio, C. A.

W. C. Warger 2nd, G. S. Laevsky, D. J. Townsend, M. Rajadhyaksha, and C. A. DiMarzio, J. Biomed. Opt. 12, 044006 (2007).
[CrossRef] [PubMed]

Feld, M. S.

Foster, T. H.

Fu, Y.

T. B. Huff, Y. Shi, Y. Fu, H. Wang, and J.-X. Cheng, IEEE J. Sel. Top. Quantum Electron. 14, 4 (2008).
[CrossRef] [PubMed]

Fürhapter, S.

Gerritsen, H. C.

J. A. Palero, H. S. de Bruijn, A. P. Heuvel, H. J. C. M. Sterenborg, and H. C. Gerritsen, Biophys. J. 93, 992 (2007).
[CrossRef] [PubMed]

Goodman, J. W.

J. W. Goodman, Opt. Photonics News 2, 11 (1991).
[CrossRef]

Harris, M.

P. S. P. Thong, K. W. Kho, W. Zheng, M. Harris, K. C. Soo, and M. Olivo, J. Mech. Med. Bio. 7, 11 (2007).
[CrossRef]

Hartenstein, V.

J. A. Campos-Ortega and V. Hartenstein, The Embryonic Development of Drosophila Melanogaster (Springer-Verlag, 1985).

Heuvel, A. P.

J. A. Palero, H. S. de Bruijn, A. P. Heuvel, H. J. C. M. Sterenborg, and H. C. Gerritsen, Biophys. J. 93, 992 (2007).
[CrossRef] [PubMed]

Huff, T. B.

T. B. Huff, Y. Shi, Y. Fu, H. Wang, and J.-X. Cheng, IEEE J. Sel. Top. Quantum Electron. 14, 4 (2008).
[CrossRef] [PubMed]

Jesacher, A.

Kawano, H.

H. Kawano, T. Kogure, Y. Abe, H. Mizuno, and A. Miyawaki, Nat. Methods 5, 373 (2008).
[CrossRef] [PubMed]

Kho, K. W.

P. S. P. Thong, K. W. Kho, W. Zheng, M. Harris, K. C. Soo, and M. Olivo, J. Mech. Med. Bio. 7, 11 (2007).
[CrossRef]

Kimball, B. R.

C. S. Yelleswarapu, S.-R. Kothapalli, Y. R. Vaillancourt, F. J. Aranda, B. R. Kimball, and D. V. G. L. N. Rao, Appl. Phys. Lett. 89, 211116 (2006).
[CrossRef]

Kogure, T.

H. Kawano, T. Kogure, Y. Abe, H. Mizuno, and A. Miyawaki, Nat. Methods 5, 373 (2008).
[CrossRef] [PubMed]

Kothapalli, S.-R.

C. S. Yelleswarapu, S.-R. Kothapalli, Y. R. Vaillancourt, F. J. Aranda, B. R. Kimball, and D. V. G. L. N. Rao, Appl. Phys. Lett. 89, 211116 (2006).
[CrossRef]

Laevsky, G. S.

W. C. Warger 2nd, G. S. Laevsky, D. J. Townsend, M. Rajadhyaksha, and C. A. DiMarzio, J. Biomed. Opt. 12, 044006 (2007).
[CrossRef] [PubMed]

Lichtman, J. W.

J. W. Lichtman and J. Conchello, Nat. Methods 2, 910 (2005).
[CrossRef] [PubMed]

Miyawaki, A.

H. Kawano, T. Kogure, Y. Abe, H. Mizuno, and A. Miyawaki, Nat. Methods 5, 373 (2008).
[CrossRef] [PubMed]

Mizuno, H.

H. Kawano, T. Kogure, Y. Abe, H. Mizuno, and A. Miyawaki, Nat. Methods 5, 373 (2008).
[CrossRef] [PubMed]

Nomarski, G.

G. Nomarski, J. Phys. Radium 16, 9 (1955).

Olivo, M.

P. S. P. Thong, K. W. Kho, W. Zheng, M. Harris, K. C. Soo, and M. Olivo, J. Mech. Med. Bio. 7, 11 (2007).
[CrossRef]

O'Neill, E. M.

E. M. O'Neill, I. Rebay, R. Tijan, and G. M. Rubin, Cell 78, 137 (1994).
[CrossRef] [PubMed]

Palero, J. A.

J. A. Palero, H. S. de Bruijn, A. P. Heuvel, H. J. C. M. Sterenborg, and H. C. Gerritsen, Biophys. J. 93, 992 (2007).
[CrossRef] [PubMed]

Park, Y.

Popescu, G.

Rajadhyaksha, M.

W. C. Warger 2nd, G. S. Laevsky, D. J. Townsend, M. Rajadhyaksha, and C. A. DiMarzio, J. Biomed. Opt. 12, 044006 (2007).
[CrossRef] [PubMed]

Rao, D. V. G. L. N.

C. S. Yelleswarapu, S.-R. Kothapalli, Y. R. Vaillancourt, F. J. Aranda, B. R. Kimball, and D. V. G. L. N. Rao, Appl. Phys. Lett. 89, 211116 (2006).
[CrossRef]

Rebay, I.

E. M. O'Neill, I. Rebay, R. Tijan, and G. M. Rubin, Cell 78, 137 (1994).
[CrossRef] [PubMed]

Ritsch-Marte, M.

Rubin, G. M.

E. M. O'Neill, I. Rebay, R. Tijan, and G. M. Rubin, Cell 78, 137 (1994).
[CrossRef] [PubMed]

Shi, Y.

T. B. Huff, Y. Shi, Y. Fu, H. Wang, and J.-X. Cheng, IEEE J. Sel. Top. Quantum Electron. 14, 4 (2008).
[CrossRef] [PubMed]

Soo, K. C.

P. S. P. Thong, K. W. Kho, W. Zheng, M. Harris, K. C. Soo, and M. Olivo, J. Mech. Med. Bio. 7, 11 (2007).
[CrossRef]

Stephens, D. J.

D. J. Stephens and V. J. Allan, Science 300, 82 (2003).
[CrossRef] [PubMed]

Sterenborg, H. J. C. M.

J. A. Palero, H. S. de Bruijn, A. P. Heuvel, H. J. C. M. Sterenborg, and H. C. Gerritsen, Biophys. J. 93, 992 (2007).
[CrossRef] [PubMed]

Thong, P. S. P.

P. S. P. Thong, K. W. Kho, W. Zheng, M. Harris, K. C. Soo, and M. Olivo, J. Mech. Med. Bio. 7, 11 (2007).
[CrossRef]

Tijan, R.

E. M. O'Neill, I. Rebay, R. Tijan, and G. M. Rubin, Cell 78, 137 (1994).
[CrossRef] [PubMed]

Townsend, D. J.

W. C. Warger 2nd, G. S. Laevsky, D. J. Townsend, M. Rajadhyaksha, and C. A. DiMarzio, J. Biomed. Opt. 12, 044006 (2007).
[CrossRef] [PubMed]

Tran, P. T.

P. T. Tran and F. Chang, Biol. Bull. 201, 253 (2001).
[CrossRef]

Vaillancourt, Y. R.

C. S. Yelleswarapu, S.-R. Kothapalli, Y. R. Vaillancourt, F. J. Aranda, B. R. Kimball, and D. V. G. L. N. Rao, Appl. Phys. Lett. 89, 211116 (2006).
[CrossRef]

Wang, H.

T. B. Huff, Y. Shi, Y. Fu, H. Wang, and J.-X. Cheng, IEEE J. Sel. Top. Quantum Electron. 14, 4 (2008).
[CrossRef] [PubMed]

Warger, W. C.

W. C. Warger 2nd, G. S. Laevsky, D. J. Townsend, M. Rajadhyaksha, and C. A. DiMarzio, J. Biomed. Opt. 12, 044006 (2007).
[CrossRef] [PubMed]

Wilson, J. D.

Yelleswarapu, C. S.

C. S. Yelleswarapu, S.-R. Kothapalli, Y. R. Vaillancourt, F. J. Aranda, B. R. Kimball, and D. V. G. L. N. Rao, Appl. Phys. Lett. 89, 211116 (2006).
[CrossRef]

Zernike, F.

F. Zernike, Science 121, 345 (1955).
[CrossRef] [PubMed]

Zheng, W.

P. S. P. Thong, K. W. Kho, W. Zheng, M. Harris, K. C. Soo, and M. Olivo, J. Mech. Med. Bio. 7, 11 (2007).
[CrossRef]

Appl. Phys. Lett. (1)

C. S. Yelleswarapu, S.-R. Kothapalli, Y. R. Vaillancourt, F. J. Aranda, B. R. Kimball, and D. V. G. L. N. Rao, Appl. Phys. Lett. 89, 211116 (2006).
[CrossRef]

Biol. Bull. (1)

P. T. Tran and F. Chang, Biol. Bull. 201, 253 (2001).
[CrossRef]

Biophys. J. (1)

J. A. Palero, H. S. de Bruijn, A. P. Heuvel, H. J. C. M. Sterenborg, and H. C. Gerritsen, Biophys. J. 93, 992 (2007).
[CrossRef] [PubMed]

Cell (1)

E. M. O'Neill, I. Rebay, R. Tijan, and G. M. Rubin, Cell 78, 137 (1994).
[CrossRef] [PubMed]

IEEE J. Sel. Top. Quantum Electron. (1)

T. B. Huff, Y. Shi, Y. Fu, H. Wang, and J.-X. Cheng, IEEE J. Sel. Top. Quantum Electron. 14, 4 (2008).
[CrossRef] [PubMed]

J. Biomed. Opt. (1)

W. C. Warger 2nd, G. S. Laevsky, D. J. Townsend, M. Rajadhyaksha, and C. A. DiMarzio, J. Biomed. Opt. 12, 044006 (2007).
[CrossRef] [PubMed]

J. Mech. Med. Bio. (1)

P. S. P. Thong, K. W. Kho, W. Zheng, M. Harris, K. C. Soo, and M. Olivo, J. Mech. Med. Bio. 7, 11 (2007).
[CrossRef]

J. Phys. Radium (1)

G. Nomarski, J. Phys. Radium 16, 9 (1955).

Nat. Methods (2)

J. W. Lichtman and J. Conchello, Nat. Methods 2, 910 (2005).
[CrossRef] [PubMed]

H. Kawano, T. Kogure, Y. Abe, H. Mizuno, and A. Miyawaki, Nat. Methods 5, 373 (2008).
[CrossRef] [PubMed]

Opt. Express (2)

Opt. Lett. (1)

Opt. Photonics News (1)

J. W. Goodman, Opt. Photonics News 2, 11 (1991).
[CrossRef]

Science (2)

F. Zernike, Science 121, 345 (1955).
[CrossRef] [PubMed]

D. J. Stephens and V. J. Allan, Science 300, 82 (2003).
[CrossRef] [PubMed]

Other (1)

J. A. Campos-Ortega and V. Hartenstein, The Embryonic Development of Drosophila Melanogaster (Springer-Verlag, 1985).

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Figures (1)

Fig. 1
Fig. 1

CMOM system and its application in imaging amplitude, phase, and fluorescence features of a Drosophila embryo. All panels show the same embryo, stained with antielav primary antibody to visualize the nervous system, followed by FITC-conjugated secondary antibody. Scale bar, 50 μ m . a, Schematic of the experimental setup. S, sample; MO, microscope objective; L, lens; F 1 , Fourier plane; F 2 , filter plane. b, Conventional bright-field image. Internal structures of the embryo are not well defined. c, FPCM image obtained using a phase filter at F 1 and neutral density filter (NDF) at F 2 . Segmental boundaries are clearly visible in this sagittal image (arrows) as well as the invaginating foregut primordia at the stomodeum (arrowhead), the midgut area (asterisk), and other internal features. d, Edge-enhanced image obtained with a high-pass spatial filter at F 1 and NDF at F 2 . Inner yolk-containing cells are optically dense and appear as a lighter area in the middle of the embryo (asterisk). e, Fluorescence image with laser line notch filter (NF) at F 2 and no filter at F 1 . The segmented ventral nerve cord (vnc) and the brain region (br) are detected in the sagittal optical section of the embryo. Although the NF blocks most of the excitation source, a small amount of light leaks through, making it possible to observe the overall embryo morphology. The midgut is autofluorescent (asterisk). f, Phase + fluorescence image obtained with a phase filter at F 1 and NF at F 2 . Both the FPCM and trans-fluorescence information are recorded at the same time. Most of the information visible in part c is retained, with the nervous system and midgut fluorescence also detectable. g, Edge- enhanced + fluorescence image obtained with a spatial filter at F 1 and NF at F 2 . The boundary of the embryo is better resolved (compare to e), and the fluorescence signal is clearly visible.

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