Abstract

A two-photon hyperspectral microscope with non-de-scanned geometry and parallel recording scheme was constructed which had a low dwell time, high spectral resolution, and high spatial resolution. The dwell time was effectively decreased to reduce photodamage through parallel recording, while the non-de-scanned geometry led to a considerable reduction in the signal loss and spectrum distortion. Two experiments were performed to show that this system can solve crosstalk issues and spectrally resolve the intrinsic fluorophores in optically-thick tissues without staining and sectioning.

© 2014 Optical Society of America

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2012 (1)

H. J. Yang, C. L. Hsu, J. Y. Yang, and W. Y. Yang, “Monodansylpentane as a blue-fluorescent lipid-droplet marker for multi-color live-cell imaging,” PLoS ONE7(3), e32693 (2012).
[CrossRef] [PubMed]

2011 (2)

2010 (2)

2009 (2)

M. C. Pedroso, M. B. Sinclair, H. D. T. Jones, and D. M. Haaland, “Hyperspectral confocal fluorescence microscope: a new look into the cell,”Microsc. Microanal.15, 880–881 (2009).

S. Schlachter, S. Schwedler, A. Esposito, G. S. Kaminski Schierle, G. D. Moggridge, and C. F. Kaminski, “A method to unmix multiple fluorophores in microscopy images with minimal a priori information,” Opt. Express17(25), 22747–22760 (2009).
[CrossRef] [PubMed]

2008 (3)

M. H. Van Benthem, M. R. Keenan, R. David, P. Liu, H. D. T. Jones, D. M. Haaland, M. B. Sinclair, and A. R. Brasier, “Trilinear analysis of images obtained with a hyperspectral imaging confocal microscope,” J. Chemometr.22(9), 491–499 (2008).
[CrossRef]

A. J. Radosevich, M. B. Bouchard, S. A. Burgess, B. R. Chen, and E. M. C. Hillman, “Hyperspectral in vivo two-photon microscopy of intrinsic contrast,” Opt. Lett.33(18), 2164–2166 (2008).
[CrossRef] [PubMed]

I. Song and D.-G. Gweon, “A spectral detector using the dispersion of an acousto-optic tunable filter for confocal spectral imaging microscopy,” Meas. Sci. Technol.19(8), 085504 (2008).
[CrossRef]

2007 (1)

P. D. Beule, D. M. Owen, H. B. Manning, C. B. Talbot, J. Requejo-Isidro, C. Dunsby, J. McGinty, R. K. P. Benninger, D. S. Elson, I. Munro, M. John Lever, P. Anand, M. A. A. Neil, and P. M. W. French, “Rapid hyperspectral fluorescence lifetime imaging,” Microsc. Res. Tech.70(5), 481–484 (2007).
[CrossRef] [PubMed]

2006 (1)

Y. Garini, I. T. Young, and G. McNamara, “Spectral imaging: Principles and applications,” Cytometry A69(8), 735–747 (2006).
[CrossRef] [PubMed]

2005 (3)

X. H. Gao, L. L. Yang, J. A. Petros, F. F. Marshall, J. W. Simons, and S. M. Nie, “In vivo molecular and cellular imaging with quantum dots,” Curr. Opin. Biotechnol.16(1), 63–72 (2005).
[CrossRef] [PubMed]

F. Helmchen and W. Denk, “Deep tissue two-photon microscopy,” Nat. Methods2(12), 932–940 (2005).
[CrossRef] [PubMed]

J. A. Conchello and J. W. Lichtman, “Optical sectioning microscopy,” Nat. Methods2(12), 920–931 (2005).
[CrossRef] [PubMed]

2004 (2)

R. H. Berg, “Evaluation of spectral imaging for plant cell analysis,” J. Microsc.214(2), 174–181 (2004).
[CrossRef] [PubMed]

M. Rubart, “Two-photon microscopy of cells and tissue,” Circ. Res.95(12), 1154–1166 (2004).
[CrossRef] [PubMed]

2001 (3)

J. A. Squier and M. Müller, “High resolution nonlinear microscopy: A review of sources and methods for achieving optimal imaging,” Rev. Sci. Instrum.72(7), 2855–2867 (2001).
[CrossRef]

M. Oheim, E. Beaurepaire, E. Chaigneau, J. Mertz, and S. Charpak, “Two-photon microscopy in brain tissue: parameters influencing the imaging depth,” J. Neurosci. Methods111(1), 29–37 (2001).
[CrossRef] [PubMed]

R. A. Schultz, T. Nielsen, J. R. Zavaleta, R. Ruch, R. Wyatt, and H. R. Garner, “Hyperspectral imaging: a novel approach for microscopic analysis,” Cytometry43(4), 239–247 (2001).
[CrossRef] [PubMed]

2000 (2)

P. T. C. So, C. Y. Dong, B. R. Masters, and K. M. Berland, “Two-photon excitation fluorescence microscopy,” Annu. Rev. Biomed. Eng.2(1), 399–429 (2000).
[CrossRef] [PubMed]

K. König, “Multiphoton microscopy in life sciences,” J. Microsc.200(2), 83–104 (2000).
[CrossRef] [PubMed]

1997 (1)

N. Subhash and C. N. Mohanan, “Curve-fit analysis of chlorophyll fluorescence spectra: Application to nutrient stress detection in sunflower,” Remote Sens. Environ.60(3), 347–356 (1997).
[CrossRef]

1996 (1)

R. H. Webb, “Confocal optical microscopy,” Rep. Prog. Phys.59(3), 427–471 (1996).
[CrossRef]

1990 (1)

W. Denk, J. H. Strickler, and W. W. Webb, “Two-photon laser scanning fluorescence microscopy,” Science248(4951), 73–76 (1990).
[CrossRef] [PubMed]

Anand, P.

P. D. Beule, D. M. Owen, H. B. Manning, C. B. Talbot, J. Requejo-Isidro, C. Dunsby, J. McGinty, R. K. P. Benninger, D. S. Elson, I. Munro, M. John Lever, P. Anand, M. A. A. Neil, and P. M. W. French, “Rapid hyperspectral fluorescence lifetime imaging,” Microsc. Res. Tech.70(5), 481–484 (2007).
[CrossRef] [PubMed]

Arsov, Z.

Beaurepaire, E.

M. Oheim, E. Beaurepaire, E. Chaigneau, J. Mertz, and S. Charpak, “Two-photon microscopy in brain tissue: parameters influencing the imaging depth,” J. Neurosci. Methods111(1), 29–37 (2001).
[CrossRef] [PubMed]

Benninger, R. K. P.

P. D. Beule, D. M. Owen, H. B. Manning, C. B. Talbot, J. Requejo-Isidro, C. Dunsby, J. McGinty, R. K. P. Benninger, D. S. Elson, I. Munro, M. John Lever, P. Anand, M. A. A. Neil, and P. M. W. French, “Rapid hyperspectral fluorescence lifetime imaging,” Microsc. Res. Tech.70(5), 481–484 (2007).
[CrossRef] [PubMed]

Berg, R. H.

R. H. Berg, “Evaluation of spectral imaging for plant cell analysis,” J. Microsc.214(2), 174–181 (2004).
[CrossRef] [PubMed]

Berland, K. M.

P. T. C. So, C. Y. Dong, B. R. Masters, and K. M. Berland, “Two-photon excitation fluorescence microscopy,” Annu. Rev. Biomed. Eng.2(1), 399–429 (2000).
[CrossRef] [PubMed]

Bestvater, F.

Beule, P. D.

P. D. Beule, D. M. Owen, H. B. Manning, C. B. Talbot, J. Requejo-Isidro, C. Dunsby, J. McGinty, R. K. P. Benninger, D. S. Elson, I. Munro, M. John Lever, P. Anand, M. A. A. Neil, and P. M. W. French, “Rapid hyperspectral fluorescence lifetime imaging,” Microsc. Res. Tech.70(5), 481–484 (2007).
[CrossRef] [PubMed]

Biglino, D.

Bish, S.

Bouchard, M. B.

Brasier, A. R.

M. H. Van Benthem, M. R. Keenan, R. David, P. Liu, H. D. T. Jones, D. M. Haaland, M. B. Sinclair, and A. R. Brasier, “Trilinear analysis of images obtained with a hyperspectral imaging confocal microscope,” J. Chemometr.22(9), 491–499 (2008).
[CrossRef]

Burgess, S. A.

Chaigneau, E.

M. Oheim, E. Beaurepaire, E. Chaigneau, J. Mertz, and S. Charpak, “Two-photon microscopy in brain tissue: parameters influencing the imaging depth,” J. Neurosci. Methods111(1), 29–37 (2001).
[CrossRef] [PubMed]

Charpak, S.

M. Oheim, E. Beaurepaire, E. Chaigneau, J. Mertz, and S. Charpak, “Two-photon microscopy in brain tissue: parameters influencing the imaging depth,” J. Neurosci. Methods111(1), 29–37 (2001).
[CrossRef] [PubMed]

Chen, B. R.

Conchello, J. A.

J. A. Conchello and J. W. Lichtman, “Optical sectioning microscopy,” Nat. Methods2(12), 920–931 (2005).
[CrossRef] [PubMed]

David, R.

M. H. Van Benthem, M. R. Keenan, R. David, P. Liu, H. D. T. Jones, D. M. Haaland, M. B. Sinclair, and A. R. Brasier, “Trilinear analysis of images obtained with a hyperspectral imaging confocal microscope,” J. Chemometr.22(9), 491–499 (2008).
[CrossRef]

Denk, W.

F. Helmchen and W. Denk, “Deep tissue two-photon microscopy,” Nat. Methods2(12), 932–940 (2005).
[CrossRef] [PubMed]

W. Denk, J. H. Strickler, and W. W. Webb, “Two-photon laser scanning fluorescence microscopy,” Science248(4951), 73–76 (1990).
[CrossRef] [PubMed]

Dong, C. Y.

P. T. C. So, C. Y. Dong, B. R. Masters, and K. M. Berland, “Two-photon excitation fluorescence microscopy,” Annu. Rev. Biomed. Eng.2(1), 399–429 (2000).
[CrossRef] [PubMed]

Dunsby, C.

P. D. Beule, D. M. Owen, H. B. Manning, C. B. Talbot, J. Requejo-Isidro, C. Dunsby, J. McGinty, R. K. P. Benninger, D. S. Elson, I. Munro, M. John Lever, P. Anand, M. A. A. Neil, and P. M. W. French, “Rapid hyperspectral fluorescence lifetime imaging,” Microsc. Res. Tech.70(5), 481–484 (2007).
[CrossRef] [PubMed]

Elson, D. S.

P. D. Beule, D. M. Owen, H. B. Manning, C. B. Talbot, J. Requejo-Isidro, C. Dunsby, J. McGinty, R. K. P. Benninger, D. S. Elson, I. Munro, M. John Lever, P. Anand, M. A. A. Neil, and P. M. W. French, “Rapid hyperspectral fluorescence lifetime imaging,” Microsc. Res. Tech.70(5), 481–484 (2007).
[CrossRef] [PubMed]

Esposito, A.

French, P. M. W.

P. D. Beule, D. M. Owen, H. B. Manning, C. B. Talbot, J. Requejo-Isidro, C. Dunsby, J. McGinty, R. K. P. Benninger, D. S. Elson, I. Munro, M. John Lever, P. Anand, M. A. A. Neil, and P. M. W. French, “Rapid hyperspectral fluorescence lifetime imaging,” Microsc. Res. Tech.70(5), 481–484 (2007).
[CrossRef] [PubMed]

Gao, X. H.

X. H. Gao, L. L. Yang, J. A. Petros, F. F. Marshall, J. W. Simons, and S. M. Nie, “In vivo molecular and cellular imaging with quantum dots,” Curr. Opin. Biotechnol.16(1), 63–72 (2005).
[CrossRef] [PubMed]

Garini, Y.

Y. Garini, I. T. Young, and G. McNamara, “Spectral imaging: Principles and applications,” Cytometry A69(8), 735–747 (2006).
[CrossRef] [PubMed]

Garner, H. R.

R. A. Schultz, T. Nielsen, J. R. Zavaleta, R. Ruch, R. Wyatt, and H. R. Garner, “Hyperspectral imaging: a novel approach for microscopic analysis,” Cytometry43(4), 239–247 (2001).
[CrossRef] [PubMed]

Garvas, M.

Grailhe, R.

Gweon, D.-G.

I. Song and D.-G. Gweon, “A spectral detector using the dispersion of an acousto-optic tunable filter for confocal spectral imaging microscopy,” Meas. Sci. Technol.19(8), 085504 (2008).
[CrossRef]

Haaland, D. M.

M. C. Pedroso, M. B. Sinclair, H. D. T. Jones, and D. M. Haaland, “Hyperspectral confocal fluorescence microscope: a new look into the cell,”Microsc. Microanal.15, 880–881 (2009).

M. H. Van Benthem, M. R. Keenan, R. David, P. Liu, H. D. T. Jones, D. M. Haaland, M. B. Sinclair, and A. R. Brasier, “Trilinear analysis of images obtained with a hyperspectral imaging confocal microscope,” J. Chemometr.22(9), 491–499 (2008).
[CrossRef]

Helmchen, F.

F. Helmchen and W. Denk, “Deep tissue two-photon microscopy,” Nat. Methods2(12), 932–940 (2005).
[CrossRef] [PubMed]

Hillman, E. M. C.

Hsu, C. L.

H. J. Yang, C. L. Hsu, J. Y. Yang, and W. Y. Yang, “Monodansylpentane as a blue-fluorescent lipid-droplet marker for multi-color live-cell imaging,” PLoS ONE7(3), e32693 (2012).
[CrossRef] [PubMed]

Im, K.-B.

John Lever, M.

P. D. Beule, D. M. Owen, H. B. Manning, C. B. Talbot, J. Requejo-Isidro, C. Dunsby, J. McGinty, R. K. P. Benninger, D. S. Elson, I. Munro, M. John Lever, P. Anand, M. A. A. Neil, and P. M. W. French, “Rapid hyperspectral fluorescence lifetime imaging,” Microsc. Res. Tech.70(5), 481–484 (2007).
[CrossRef] [PubMed]

Jones, H. D. T.

M. C. Pedroso, M. B. Sinclair, H. D. T. Jones, and D. M. Haaland, “Hyperspectral confocal fluorescence microscope: a new look into the cell,”Microsc. Microanal.15, 880–881 (2009).

M. H. Van Benthem, M. R. Keenan, R. David, P. Liu, H. D. T. Jones, D. M. Haaland, M. B. Sinclair, and A. R. Brasier, “Trilinear analysis of images obtained with a hyperspectral imaging confocal microscope,” J. Chemometr.22(9), 491–499 (2008).
[CrossRef]

Kaminski, C. F.

Kaminski Schierle, G. S.

Kang, M.-S.

Keenan, M. R.

M. H. Van Benthem, M. R. Keenan, R. David, P. Liu, H. D. T. Jones, D. M. Haaland, M. B. Sinclair, and A. R. Brasier, “Trilinear analysis of images obtained with a hyperspectral imaging confocal microscope,” J. Chemometr.22(9), 491–499 (2008).
[CrossRef]

Kim, J.

Koklic, T.

König, K.

K. König, “Multiphoton microscopy in life sciences,” J. Microsc.200(2), 83–104 (2000).
[CrossRef] [PubMed]

Li, D.

Lichtman, J. W.

J. A. Conchello and J. W. Lichtman, “Optical sectioning microscopy,” Nat. Methods2(12), 920–931 (2005).
[CrossRef] [PubMed]

Liu, P.

M. H. Van Benthem, M. R. Keenan, R. David, P. Liu, H. D. T. Jones, D. M. Haaland, M. B. Sinclair, and A. R. Brasier, “Trilinear analysis of images obtained with a hyperspectral imaging confocal microscope,” J. Chemometr.22(9), 491–499 (2008).
[CrossRef]

Ljubetic, A.

Luo, Y.

Manning, H. B.

P. D. Beule, D. M. Owen, H. B. Manning, C. B. Talbot, J. Requejo-Isidro, C. Dunsby, J. McGinty, R. K. P. Benninger, D. S. Elson, I. Munro, M. John Lever, P. Anand, M. A. A. Neil, and P. M. W. French, “Rapid hyperspectral fluorescence lifetime imaging,” Microsc. Res. Tech.70(5), 481–484 (2007).
[CrossRef] [PubMed]

Marshall, F. F.

X. H. Gao, L. L. Yang, J. A. Petros, F. F. Marshall, J. W. Simons, and S. M. Nie, “In vivo molecular and cellular imaging with quantum dots,” Curr. Opin. Biotechnol.16(1), 63–72 (2005).
[CrossRef] [PubMed]

Masters, B. R.

P. T. C. So, C. Y. Dong, B. R. Masters, and K. M. Berland, “Two-photon excitation fluorescence microscopy,” Annu. Rev. Biomed. Eng.2(1), 399–429 (2000).
[CrossRef] [PubMed]

McGinty, J.

P. D. Beule, D. M. Owen, H. B. Manning, C. B. Talbot, J. Requejo-Isidro, C. Dunsby, J. McGinty, R. K. P. Benninger, D. S. Elson, I. Munro, M. John Lever, P. Anand, M. A. A. Neil, and P. M. W. French, “Rapid hyperspectral fluorescence lifetime imaging,” Microsc. Res. Tech.70(5), 481–484 (2007).
[CrossRef] [PubMed]

McNamara, G.

Y. Garini, I. T. Young, and G. McNamara, “Spectral imaging: Principles and applications,” Cytometry A69(8), 735–747 (2006).
[CrossRef] [PubMed]

Mertz, J.

M. Oheim, E. Beaurepaire, E. Chaigneau, J. Mertz, and S. Charpak, “Two-photon microscopy in brain tissue: parameters influencing the imaging depth,” J. Neurosci. Methods111(1), 29–37 (2001).
[CrossRef] [PubMed]

Moggridge, G. D.

Mohanan, C. N.

N. Subhash and C. N. Mohanan, “Curve-fit analysis of chlorophyll fluorescence spectra: Application to nutrient stress detection in sunflower,” Remote Sens. Environ.60(3), 347–356 (1997).
[CrossRef]

Müller, M.

J. A. Squier and M. Müller, “High resolution nonlinear microscopy: A review of sources and methods for achieving optimal imaging,” Rev. Sci. Instrum.72(7), 2855–2867 (2001).
[CrossRef]

Munro, I.

P. D. Beule, D. M. Owen, H. B. Manning, C. B. Talbot, J. Requejo-Isidro, C. Dunsby, J. McGinty, R. K. P. Benninger, D. S. Elson, I. Munro, M. John Lever, P. Anand, M. A. A. Neil, and P. M. W. French, “Rapid hyperspectral fluorescence lifetime imaging,” Microsc. Res. Tech.70(5), 481–484 (2007).
[CrossRef] [PubMed]

Neil, M. A. A.

P. D. Beule, D. M. Owen, H. B. Manning, C. B. Talbot, J. Requejo-Isidro, C. Dunsby, J. McGinty, R. K. P. Benninger, D. S. Elson, I. Munro, M. John Lever, P. Anand, M. A. A. Neil, and P. M. W. French, “Rapid hyperspectral fluorescence lifetime imaging,” Microsc. Res. Tech.70(5), 481–484 (2007).
[CrossRef] [PubMed]

Nie, S. M.

X. H. Gao, L. L. Yang, J. A. Petros, F. F. Marshall, J. W. Simons, and S. M. Nie, “In vivo molecular and cellular imaging with quantum dots,” Curr. Opin. Biotechnol.16(1), 63–72 (2005).
[CrossRef] [PubMed]

Nielsen, T.

R. A. Schultz, T. Nielsen, J. R. Zavaleta, R. Ruch, R. Wyatt, and H. R. Garner, “Hyperspectral imaging: a novel approach for microscopic analysis,” Cytometry43(4), 239–247 (2001).
[CrossRef] [PubMed]

Oheim, M.

M. Oheim, E. Beaurepaire, E. Chaigneau, J. Mertz, and S. Charpak, “Two-photon microscopy in brain tissue: parameters influencing the imaging depth,” J. Neurosci. Methods111(1), 29–37 (2001).
[CrossRef] [PubMed]

Owen, D. M.

P. D. Beule, D. M. Owen, H. B. Manning, C. B. Talbot, J. Requejo-Isidro, C. Dunsby, J. McGinty, R. K. P. Benninger, D. S. Elson, I. Munro, M. John Lever, P. Anand, M. A. A. Neil, and P. M. W. French, “Rapid hyperspectral fluorescence lifetime imaging,” Microsc. Res. Tech.70(5), 481–484 (2007).
[CrossRef] [PubMed]

Pedroso, M. C.

M. C. Pedroso, M. B. Sinclair, H. D. T. Jones, and D. M. Haaland, “Hyperspectral confocal fluorescence microscope: a new look into the cell,”Microsc. Microanal.15, 880–881 (2009).

Petros, J. A.

X. H. Gao, L. L. Yang, J. A. Petros, F. F. Marshall, J. W. Simons, and S. M. Nie, “In vivo molecular and cellular imaging with quantum dots,” Curr. Opin. Biotechnol.16(1), 63–72 (2005).
[CrossRef] [PubMed]

Qu, J. Y.

Radosevich, A. J.

Requejo-Isidro, J.

P. D. Beule, D. M. Owen, H. B. Manning, C. B. Talbot, J. Requejo-Isidro, C. Dunsby, J. McGinty, R. K. P. Benninger, D. S. Elson, I. Munro, M. John Lever, P. Anand, M. A. A. Neil, and P. M. W. French, “Rapid hyperspectral fluorescence lifetime imaging,” Microsc. Res. Tech.70(5), 481–484 (2007).
[CrossRef] [PubMed]

Rubart, M.

M. Rubart, “Two-photon microscopy of cells and tissue,” Circ. Res.95(12), 1154–1166 (2004).
[CrossRef] [PubMed]

Ruch, R.

R. A. Schultz, T. Nielsen, J. R. Zavaleta, R. Ruch, R. Wyatt, and H. R. Garner, “Hyperspectral imaging: a novel approach for microscopic analysis,” Cytometry43(4), 239–247 (2001).
[CrossRef] [PubMed]

Schlachter, S.

Schultz, R. A.

R. A. Schultz, T. Nielsen, J. R. Zavaleta, R. Ruch, R. Wyatt, and H. R. Garner, “Hyperspectral imaging: a novel approach for microscopic analysis,” Cytometry43(4), 239–247 (2001).
[CrossRef] [PubMed]

Schwedler, S.

Seghiri, Z.

Simons, J. W.

X. H. Gao, L. L. Yang, J. A. Petros, F. F. Marshall, J. W. Simons, and S. M. Nie, “In vivo molecular and cellular imaging with quantum dots,” Curr. Opin. Biotechnol.16(1), 63–72 (2005).
[CrossRef] [PubMed]

Sinclair, M. B.

M. C. Pedroso, M. B. Sinclair, H. D. T. Jones, and D. M. Haaland, “Hyperspectral confocal fluorescence microscope: a new look into the cell,”Microsc. Microanal.15, 880–881 (2009).

M. H. Van Benthem, M. R. Keenan, R. David, P. Liu, H. D. T. Jones, D. M. Haaland, M. B. Sinclair, and A. R. Brasier, “Trilinear analysis of images obtained with a hyperspectral imaging confocal microscope,” J. Chemometr.22(9), 491–499 (2008).
[CrossRef]

So, P. T. C.

P. T. C. So, C. Y. Dong, B. R. Masters, and K. M. Berland, “Two-photon excitation fluorescence microscopy,” Annu. Rev. Biomed. Eng.2(1), 399–429 (2000).
[CrossRef] [PubMed]

Song, I.

I. Song and D.-G. Gweon, “A spectral detector using the dispersion of an acousto-optic tunable filter for confocal spectral imaging microscopy,” Meas. Sci. Technol.19(8), 085504 (2008).
[CrossRef]

Squier, J. A.

J. A. Squier and M. Müller, “High resolution nonlinear microscopy: A review of sources and methods for achieving optimal imaging,” Rev. Sci. Instrum.72(7), 2855–2867 (2001).
[CrossRef]

Strancar, J.

Strickler, J. H.

W. Denk, J. H. Strickler, and W. W. Webb, “Two-photon laser scanning fluorescence microscopy,” Science248(4951), 73–76 (1990).
[CrossRef] [PubMed]

Subhash, N.

N. Subhash and C. N. Mohanan, “Curve-fit analysis of chlorophyll fluorescence spectra: Application to nutrient stress detection in sunflower,” Remote Sens. Environ.60(3), 347–356 (1997).
[CrossRef]

Talbot, C. B.

P. D. Beule, D. M. Owen, H. B. Manning, C. B. Talbot, J. Requejo-Isidro, C. Dunsby, J. McGinty, R. K. P. Benninger, D. S. Elson, I. Munro, M. John Lever, P. Anand, M. A. A. Neil, and P. M. W. French, “Rapid hyperspectral fluorescence lifetime imaging,” Microsc. Res. Tech.70(5), 481–484 (2007).
[CrossRef] [PubMed]

Tunnell, J. W.

Urbancic, I.

Van Benthem, M. H.

M. H. Van Benthem, M. R. Keenan, R. David, P. Liu, H. D. T. Jones, D. M. Haaland, M. B. Sinclair, and A. R. Brasier, “Trilinear analysis of images obtained with a hyperspectral imaging confocal microscope,” J. Chemometr.22(9), 491–499 (2008).
[CrossRef]

Wachsmuth, M.

Wang, Y.

Webb, R. H.

R. H. Webb, “Confocal optical microscopy,” Rep. Prog. Phys.59(3), 427–471 (1996).
[CrossRef]

Webb, W. W.

W. Denk, J. H. Strickler, and W. W. Webb, “Two-photon laser scanning fluorescence microscopy,” Science248(4951), 73–76 (1990).
[CrossRef] [PubMed]

Wyatt, R.

R. A. Schultz, T. Nielsen, J. R. Zavaleta, R. Ruch, R. Wyatt, and H. R. Garner, “Hyperspectral imaging: a novel approach for microscopic analysis,” Cytometry43(4), 239–247 (2001).
[CrossRef] [PubMed]

Yang, H. J.

H. J. Yang, C. L. Hsu, J. Y. Yang, and W. Y. Yang, “Monodansylpentane as a blue-fluorescent lipid-droplet marker for multi-color live-cell imaging,” PLoS ONE7(3), e32693 (2012).
[CrossRef] [PubMed]

Yang, J. Y.

H. J. Yang, C. L. Hsu, J. Y. Yang, and W. Y. Yang, “Monodansylpentane as a blue-fluorescent lipid-droplet marker for multi-color live-cell imaging,” PLoS ONE7(3), e32693 (2012).
[CrossRef] [PubMed]

Yang, L. L.

X. H. Gao, L. L. Yang, J. A. Petros, F. F. Marshall, J. W. Simons, and S. M. Nie, “In vivo molecular and cellular imaging with quantum dots,” Curr. Opin. Biotechnol.16(1), 63–72 (2005).
[CrossRef] [PubMed]

Yang, W. Y.

H. J. Yang, C. L. Hsu, J. Y. Yang, and W. Y. Yang, “Monodansylpentane as a blue-fluorescent lipid-droplet marker for multi-color live-cell imaging,” PLoS ONE7(3), e32693 (2012).
[CrossRef] [PubMed]

Young, I. T.

Y. Garini, I. T. Young, and G. McNamara, “Spectral imaging: Principles and applications,” Cytometry A69(8), 735–747 (2006).
[CrossRef] [PubMed]

Zavaleta, J. R.

R. A. Schultz, T. Nielsen, J. R. Zavaleta, R. Ruch, R. Wyatt, and H. R. Garner, “Hyperspectral imaging: a novel approach for microscopic analysis,” Cytometry43(4), 239–247 (2001).
[CrossRef] [PubMed]

Zeng, Y.

Zhang, X.

Zheng, W.

Annu. Rev. Biomed. Eng. (1)

P. T. C. So, C. Y. Dong, B. R. Masters, and K. M. Berland, “Two-photon excitation fluorescence microscopy,” Annu. Rev. Biomed. Eng.2(1), 399–429 (2000).
[CrossRef] [PubMed]

Biomed. Opt. Express (2)

Circ. Res. (1)

M. Rubart, “Two-photon microscopy of cells and tissue,” Circ. Res.95(12), 1154–1166 (2004).
[CrossRef] [PubMed]

Curr. Opin. Biotechnol. (1)

X. H. Gao, L. L. Yang, J. A. Petros, F. F. Marshall, J. W. Simons, and S. M. Nie, “In vivo molecular and cellular imaging with quantum dots,” Curr. Opin. Biotechnol.16(1), 63–72 (2005).
[CrossRef] [PubMed]

Cytometry (1)

R. A. Schultz, T. Nielsen, J. R. Zavaleta, R. Ruch, R. Wyatt, and H. R. Garner, “Hyperspectral imaging: a novel approach for microscopic analysis,” Cytometry43(4), 239–247 (2001).
[CrossRef] [PubMed]

Cytometry A (1)

Y. Garini, I. T. Young, and G. McNamara, “Spectral imaging: Principles and applications,” Cytometry A69(8), 735–747 (2006).
[CrossRef] [PubMed]

J. Chemometr. (1)

M. H. Van Benthem, M. R. Keenan, R. David, P. Liu, H. D. T. Jones, D. M. Haaland, M. B. Sinclair, and A. R. Brasier, “Trilinear analysis of images obtained with a hyperspectral imaging confocal microscope,” J. Chemometr.22(9), 491–499 (2008).
[CrossRef]

J. Microsc. (2)

R. H. Berg, “Evaluation of spectral imaging for plant cell analysis,” J. Microsc.214(2), 174–181 (2004).
[CrossRef] [PubMed]

K. König, “Multiphoton microscopy in life sciences,” J. Microsc.200(2), 83–104 (2000).
[CrossRef] [PubMed]

J. Neurosci. Methods (1)

M. Oheim, E. Beaurepaire, E. Chaigneau, J. Mertz, and S. Charpak, “Two-photon microscopy in brain tissue: parameters influencing the imaging depth,” J. Neurosci. Methods111(1), 29–37 (2001).
[CrossRef] [PubMed]

Meas. Sci. Technol. (1)

I. Song and D.-G. Gweon, “A spectral detector using the dispersion of an acousto-optic tunable filter for confocal spectral imaging microscopy,” Meas. Sci. Technol.19(8), 085504 (2008).
[CrossRef]

Microsc. Microanal. (1)

M. C. Pedroso, M. B. Sinclair, H. D. T. Jones, and D. M. Haaland, “Hyperspectral confocal fluorescence microscope: a new look into the cell,”Microsc. Microanal.15, 880–881 (2009).

Microsc. Res. Tech. (1)

P. D. Beule, D. M. Owen, H. B. Manning, C. B. Talbot, J. Requejo-Isidro, C. Dunsby, J. McGinty, R. K. P. Benninger, D. S. Elson, I. Munro, M. John Lever, P. Anand, M. A. A. Neil, and P. M. W. French, “Rapid hyperspectral fluorescence lifetime imaging,” Microsc. Res. Tech.70(5), 481–484 (2007).
[CrossRef] [PubMed]

Nat. Methods (2)

F. Helmchen and W. Denk, “Deep tissue two-photon microscopy,” Nat. Methods2(12), 932–940 (2005).
[CrossRef] [PubMed]

J. A. Conchello and J. W. Lichtman, “Optical sectioning microscopy,” Nat. Methods2(12), 920–931 (2005).
[CrossRef] [PubMed]

Opt. Express (3)

Opt. Lett. (1)

PLoS ONE (1)

H. J. Yang, C. L. Hsu, J. Y. Yang, and W. Y. Yang, “Monodansylpentane as a blue-fluorescent lipid-droplet marker for multi-color live-cell imaging,” PLoS ONE7(3), e32693 (2012).
[CrossRef] [PubMed]

Remote Sens. Environ. (1)

N. Subhash and C. N. Mohanan, “Curve-fit analysis of chlorophyll fluorescence spectra: Application to nutrient stress detection in sunflower,” Remote Sens. Environ.60(3), 347–356 (1997).
[CrossRef]

Rep. Prog. Phys. (1)

R. H. Webb, “Confocal optical microscopy,” Rep. Prog. Phys.59(3), 427–471 (1996).
[CrossRef]

Rev. Sci. Instrum. (1)

J. A. Squier and M. Müller, “High resolution nonlinear microscopy: A review of sources and methods for achieving optimal imaging,” Rev. Sci. Instrum.72(7), 2855–2867 (2001).
[CrossRef]

Science (1)

W. Denk, J. H. Strickler, and W. W. Webb, “Two-photon laser scanning fluorescence microscopy,” Science248(4951), 73–76 (1990).
[CrossRef] [PubMed]

Other (1)

J. Rietdorf and E. H. K. Stelzer, “Special optical elements,” in Handbook of biological confocal microscopy, J. B. Pawley, 3ed. (Springer, 2006), pp. 43–58.

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Figures (6)

Fig. 1
Fig. 1

Conceptual diagram of the two-photon hyperspectral microscope with non-de-scanned geometry and parallel recording of spatial-spectral information.

Fig. 2
Fig. 2

(a) The configuration of the non-de-scanned 2PF hyperspectral imaging system using a 2D-CCD to perform parallel recording of one spatial dimension (x) and spectral dimension (λ). T1: telescope; T2: tube lenses; M: mirror; DBS: dichroic beamsplitter; Obj: objective; L1 and L2: telescope; L3: imaging lens; AS: adjustable slit. (b) The time sequential actions of the galvo mirror, 2D-CCD and y-stage. E: exposure/record; R: readout; M: movement.

Fig. 3
Fig. 3

(a) The relations between the wavelengths and the pixel numbers of the system; (b) calibrated spectra of the Hg-Ne lamp (546, 577, and 579 nm; black line), He-Ne laser (633 nm; red line), and 671-nm laser (671 nm; blue line); (c) and (d) show the spectra of three lines from the Hg-Ne lamp, enlarged from (b); (e) and (f) show the spectra of He-Ne laser at 633 nm and 671-nm laser at 671 nm, respectively. The red lines in (c)-(f) indicate the Gaussian fitting curves for the measured spectral data.

Fig. 4
Fig. 4

(a) The 3D image stack of F8801 fluorescent beads with a 100-s acquisition time. The dimensions along the x, y, and λ axis are 100 μm, 100 μm, and 300 nm, respectively. (b) The emission spectrum of the beads obtained at the position indicated by the arrow in (a). (c) The spatial distribution of the beads obtained at the peak emission wavelength, 610 nm, indicated by the arrow in (b). (d) and (e) show the averaged intensity profiles of the beads along the x- and y-directions, respectively. The corresponding spatial resolutions are 482 nm and 557 nm.

Fig. 5
Fig. 5

(a) The normalized two-photon fluorescence spectra of the F8800 (blue line) and F8801 (red line) fluorescent beads. (b) 3D image stack of the mixed fluorescent beads with a 100-s acquisition time. The dimensions are 100 μm (x) × 100 μm (y) × 300 nm (λ). (c) The sample spectrum (black line) taken from the image stack in (b). By applying the linear unmixing algorithm, the spectrum was decomposed into two spectra (blue line for F8800 and red line for F8801); (d) and (e) show the images of the F8800 and F8801 fluorescent beads obtained by simulated traditional fluorescence microscopy (separated at the dashed line indicating 590 nm, in (c)); (f) and (g) show images of the F8800 and F8801 fluorescent beads obtained by the linear unmixing algorithm. Scale bar: 1 μm.

Fig. 6
Fig. 6

(a) Two-photon fluorescence spectrum of the GL (black line). The spectrum was decomposed into PS II (blue line) and PS I (red line) spectra by applying Gaussian curve fitting (green line); (b), (c), and (d) show the PS II, PS I, and the superpositioned images of the GL with a 300-s acquisition time. (e) The two-photon fluorescence spectrum of the withered yellow leaf (black line). Based on the spectra shown in (a), the spectrum was decomposed into PS II (blue line) and PS I (red line) spectra by applying linear unmixing; (f), (g), and (h) show the PS II, PS I, and superpositioned image of the YL with a 300-s acquisition time. Both the hyperspectral images of GL and YL were obtained at a depth of 70 μm beneath the leaf surface. Scale bar: 25 μm.

Equations (1)

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I(λ)= i=1 n C i R i (λ) ,

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