Abstract

We introduce a fast spectral imaging system using an electron-multiplying charge-coupled device (EM-CCD) as a detector. Our system is combined with a custom-built two-photon excitation laser scanning microscope and has 80 detection channels, which allow for high spectral resolution and fast frame acquisition without any loss of spectral information. To demonstrate the efficiency of our approach, we applied this technology to monitor fluorescent proteins and quantum dot-labeled G protein-coupled receptors in living cells as well as autofluorescence in tissue samples.

©2010 Optical Society of America

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  1. J. A. Conchello and J. W. Lichtman, “Optical sectioning microscopy,” Nat. Methods 2(12), 920–931 (2005).
    [Crossref] [PubMed]
  2. E. Wang, C. M. Babbey, and K. W. Dunn, “Performance comparison between the high-speed Yokogawa spinning disc confocal system and single-point scanning confocal systems,” J. Microsc. 218(2), 148–159 (2005).
    [Crossref] [PubMed]
  3. F. Helmchen and W. Denk, “Deep tissue two-photon microscopy,” Nat. Methods 2(12), 932–940 (2005).
    [Crossref] [PubMed]
  4. M. Wachsmuth, and K. Weisshart, “Fluorescence photobleaching and fluorescence correlation spectroscopy: two complementary technologies to study molecular dynamics in living cells,” in Imaging Cellular and Molecular Biological Functions (Springer Verlag, Heidelberg, 2007).
  5. T. Misteli and R. D. Phair, “High mobility of proteins in the mammalian cell nucleus,” Nature 404(6778), 604–609 (2000).
    [Crossref] [PubMed]
  6. P. Indovina, M. Collini, G. Chirico, and M. T. Santini, “Three-dimensional cell organization leads to almost immediate HRE activity as demonstrated by molecular imaging of MG-63 spheroids using two-photon excitation microscopy,” FEBS Lett. 581(4), 719–726 (2007).
    [Crossref] [PubMed]
  7. A. Hayek, A. Grichine, T. Huault, C. Ricard, F. Bolze, B. Van Der Sanden, J. C. Vial, Y. Mély, A. Duperray, P. L. Baldeck, and J. F. Nicoud, “Cell-permeant cytoplasmic blue fluorophores optimized for in vivo two-photon microscopy with low-power excitation,” Microsc. Res. Tech. 70(10), 880–885 (2007).
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    [Crossref] [PubMed]
  11. R. H. Berg, “Evaluation of spectral imaging for plant cell analysis,” J. Microsc. 214(2), 174–181 (2004).
    [Crossref] [PubMed]
  12. 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]
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    [Crossref] [PubMed]
  17. G. Heuvelman, F. Erdel, M. Wachsmuth, and K. Rippe, “Analysis of protein mobilities and interactions in living cells by multifocal fluorescence fluctuation microscopy,” Eur. Biophys. J. 38(6), 813–828 (2009).
    [Crossref] [PubMed]
  18. A. D. Keefe, D. S. Wilson, B. Seelig, and J. W. Szostak, “One-step purification of recombinant proteins using a nanomolar-affinity streptavidin-binding peptide, the SBP-Tag,” Protein Expr. Purif. 23(3), 440–446 (2001).
    [Crossref] [PubMed]
  19. 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. Express 17(25), 22747–22760 (2009).
    [Crossref]
  20. K. Svoboda and R. Yasuda, “Principles of two-photon excitation microscopy and its applications to neuroscience,” Neuron 50(6), 823–839 (2006).
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2010 (3)

2009 (2)

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. Express 17(25), 22747–22760 (2009).
[Crossref]

G. Heuvelman, F. Erdel, M. Wachsmuth, and K. Rippe, “Analysis of protein mobilities and interactions in living cells by multifocal fluorescence fluctuation microscopy,” Eur. Biophys. J. 38(6), 813–828 (2009).
[Crossref] [PubMed]

2007 (4)

P. Indovina, M. Collini, G. Chirico, and M. T. Santini, “Three-dimensional cell organization leads to almost immediate HRE activity as demonstrated by molecular imaging of MG-63 spheroids using two-photon excitation microscopy,” FEBS Lett. 581(4), 719–726 (2007).
[Crossref] [PubMed]

A. Hayek, A. Grichine, T. Huault, C. Ricard, F. Bolze, B. Van Der Sanden, J. C. Vial, Y. Mély, A. Duperray, P. L. Baldeck, and J. F. Nicoud, “Cell-permeant cytoplasmic blue fluorophores optimized for in vivo two-photon microscopy with low-power excitation,” Microsc. Res. Tech. 70(10), 880–885 (2007).
[Crossref] [PubMed]

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]

S. Kumazaki, M. Hasegawa, M. Ghoneim, Y. Shimizu, K. Okamoto, M. Nishiyama, H. Oh-Oka, and M. Terazima, “A line-scanning semi-confocal multi-photon fluorescence microscope with a simultaneous broadband spectral acquisition and its application to the study of the thylakoid membrane of a cyanobacterium Anabaena PCC7120,” J. Microsc. 228(2), 240–254 (2007).
[Crossref] [PubMed]

2006 (2)

K. Svoboda and R. Yasuda, “Principles of two-photon excitation microscopy and its applications to neuroscience,” Neuron 50(6), 823–839 (2006).
[Crossref] [PubMed]

M. B. Sinclair, D. M. Haaland, J. A. Timlin, and H. D. T. Jones, “Hyperspectral confocal microscope,” Appl. Opt. 45(24), 6283–6291 (2006).
[Crossref] [PubMed]

2005 (3)

J. A. Conchello and J. W. Lichtman, “Optical sectioning microscopy,” Nat. Methods 2(12), 920–931 (2005).
[Crossref] [PubMed]

E. Wang, C. M. Babbey, and K. W. Dunn, “Performance comparison between the high-speed Yokogawa spinning disc confocal system and single-point scanning confocal systems,” J. Microsc. 218(2), 148–159 (2005).
[Crossref] [PubMed]

F. Helmchen and W. Denk, “Deep tissue two-photon microscopy,” Nat. Methods 2(12), 932–940 (2005).
[Crossref] [PubMed]

2004 (1)

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

2002 (1)

Y. Hiraoka, T. Shimi, and T. Haraguchi, “Multispectral imaging fluorescence microscopy for living cells,” Cell Struct. Funct. 27(5), 367–374 (2002).
[Crossref] [PubMed]

2001 (1)

A. D. Keefe, D. S. Wilson, B. Seelig, and J. W. Szostak, “One-step purification of recombinant proteins using a nanomolar-affinity streptavidin-binding peptide, the SBP-Tag,” Protein Expr. Purif. 23(3), 440–446 (2001).
[Crossref] [PubMed]

2000 (1)

T. Misteli and R. D. Phair, “High mobility of proteins in the mammalian cell nucleus,” Nature 404(6778), 604–609 (2000).
[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]

Babbey, C. M.

E. Wang, C. M. Babbey, and K. W. Dunn, “Performance comparison between the high-speed Yokogawa spinning disc confocal system and single-point scanning confocal systems,” J. Microsc. 218(2), 148–159 (2005).
[Crossref] [PubMed]

Baldeck, P. L.

A. Hayek, A. Grichine, T. Huault, C. Ricard, F. Bolze, B. Van Der Sanden, J. C. Vial, Y. Mély, A. Duperray, P. L. Baldeck, and J. F. Nicoud, “Cell-permeant cytoplasmic blue fluorophores optimized for in vivo two-photon microscopy with low-power excitation,” Microsc. Res. Tech. 70(10), 880–885 (2007).
[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]

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]

Bolze, F.

A. Hayek, A. Grichine, T. Huault, C. Ricard, F. Bolze, B. Van Der Sanden, J. C. Vial, Y. Mély, A. Duperray, P. L. Baldeck, and J. F. Nicoud, “Cell-permeant cytoplasmic blue fluorophores optimized for in vivo two-photon microscopy with low-power excitation,” Microsc. Res. Tech. 70(10), 880–885 (2007).
[Crossref] [PubMed]

Carini, M.

Chirico, G.

P. Indovina, M. Collini, G. Chirico, and M. T. Santini, “Three-dimensional cell organization leads to almost immediate HRE activity as demonstrated by molecular imaging of MG-63 spheroids using two-photon excitation microscopy,” FEBS Lett. 581(4), 719–726 (2007).
[Crossref] [PubMed]

Cicchi, R.

Collini, M.

P. Indovina, M. Collini, G. Chirico, and M. T. Santini, “Three-dimensional cell organization leads to almost immediate HRE activity as demonstrated by molecular imaging of MG-63 spheroids using two-photon excitation microscopy,” FEBS Lett. 581(4), 719–726 (2007).
[Crossref] [PubMed]

Conchello, J. A.

J. A. Conchello and J. W. Lichtman, “Optical sectioning microscopy,” Nat. Methods 2(12), 920–931 (2005).
[Crossref] [PubMed]

Cosci, A.

Côté, D.

Crisci, A.

Denk, W.

F. Helmchen and W. Denk, “Deep tissue two-photon microscopy,” Nat. Methods 2(12), 932–940 (2005).
[Crossref] [PubMed]

Dunn, K. W.

E. Wang, C. M. Babbey, and K. W. Dunn, “Performance comparison between the high-speed Yokogawa spinning disc confocal system and single-point scanning confocal systems,” J. Microsc. 218(2), 148–159 (2005).
[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]

Duperray, A.

A. Hayek, A. Grichine, T. Huault, C. Ricard, F. Bolze, B. Van Der Sanden, J. C. Vial, Y. Mély, A. Duperray, P. L. Baldeck, and J. F. Nicoud, “Cell-permeant cytoplasmic blue fluorophores optimized for in vivo two-photon microscopy with low-power excitation,” Microsc. Res. Tech. 70(10), 880–885 (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]

Erdel, F.

G. Heuvelman, F. Erdel, M. Wachsmuth, and K. Rippe, “Analysis of protein mobilities and interactions in living cells by multifocal fluorescence fluctuation microscopy,” Eur. Biophys. J. 38(6), 813–828 (2009).
[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]

Ghoneim, M.

S. Kumazaki, M. Hasegawa, M. Ghoneim, Y. Shimizu, K. Okamoto, M. Nishiyama, H. Oh-Oka, and M. Terazima, “A line-scanning semi-confocal multi-photon fluorescence microscope with a simultaneous broadband spectral acquisition and its application to the study of the thylakoid membrane of a cyanobacterium Anabaena PCC7120,” J. Microsc. 228(2), 240–254 (2007).
[Crossref] [PubMed]

Giancane, S.

Grichine, A.

A. Hayek, A. Grichine, T. Huault, C. Ricard, F. Bolze, B. Van Der Sanden, J. C. Vial, Y. Mély, A. Duperray, P. L. Baldeck, and J. F. Nicoud, “Cell-permeant cytoplasmic blue fluorophores optimized for in vivo two-photon microscopy with low-power excitation,” Microsc. Res. Tech. 70(10), 880–885 (2007).
[Crossref] [PubMed]

Gröner, N.

Haaland, D. M.

Haraguchi, T.

Y. Hiraoka, T. Shimi, and T. Haraguchi, “Multispectral imaging fluorescence microscopy for living cells,” Cell Struct. Funct. 27(5), 367–374 (2002).
[Crossref] [PubMed]

Hasegawa, M.

S. Kumazaki, M. Hasegawa, M. Ghoneim, Y. Shimizu, K. Okamoto, M. Nishiyama, H. Oh-Oka, and M. Terazima, “A line-scanning semi-confocal multi-photon fluorescence microscope with a simultaneous broadband spectral acquisition and its application to the study of the thylakoid membrane of a cyanobacterium Anabaena PCC7120,” J. Microsc. 228(2), 240–254 (2007).
[Crossref] [PubMed]

Hayek, A.

A. Hayek, A. Grichine, T. Huault, C. Ricard, F. Bolze, B. Van Der Sanden, J. C. Vial, Y. Mély, A. Duperray, P. L. Baldeck, and J. F. Nicoud, “Cell-permeant cytoplasmic blue fluorophores optimized for in vivo two-photon microscopy with low-power excitation,” Microsc. Res. Tech. 70(10), 880–885 (2007).
[Crossref] [PubMed]

Helmchen, F.

F. Helmchen and W. Denk, “Deep tissue two-photon microscopy,” Nat. Methods 2(12), 932–940 (2005).
[Crossref] [PubMed]

Heuvelman, G.

G. Heuvelman, F. Erdel, M. Wachsmuth, and K. Rippe, “Analysis of protein mobilities and interactions in living cells by multifocal fluorescence fluctuation microscopy,” Eur. Biophys. J. 38(6), 813–828 (2009).
[Crossref] [PubMed]

Hiraoka, Y.

Y. Hiraoka, T. Shimi, and T. Haraguchi, “Multispectral imaging fluorescence microscopy for living cells,” Cell Struct. Funct. 27(5), 367–374 (2002).
[Crossref] [PubMed]

Huault, T.

A. Hayek, A. Grichine, T. Huault, C. Ricard, F. Bolze, B. Van Der Sanden, J. C. Vial, Y. Mély, A. Duperray, P. L. Baldeck, and J. F. Nicoud, “Cell-permeant cytoplasmic blue fluorophores optimized for in vivo two-photon microscopy with low-power excitation,” Microsc. Res. Tech. 70(10), 880–885 (2007).
[Crossref] [PubMed]

Im, K. B.

Indovina, P.

P. Indovina, M. Collini, G. Chirico, and M. T. Santini, “Three-dimensional cell organization leads to almost immediate HRE activity as demonstrated by molecular imaging of MG-63 spheroids using two-photon excitation microscopy,” FEBS Lett. 581(4), 719–726 (2007).
[Crossref] [PubMed]

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.

Kaminski, C. F.

Kaminski Schierle, G. S.

Kang, M. S.

Kapsokalyvas, D.

Keefe, A. D.

A. D. Keefe, D. S. Wilson, B. Seelig, and J. W. Szostak, “One-step purification of recombinant proteins using a nanomolar-affinity streptavidin-binding peptide, the SBP-Tag,” Protein Expr. Purif. 23(3), 440–446 (2001).
[Crossref] [PubMed]

Kumazaki, S.

S. Kumazaki, M. Hasegawa, M. Ghoneim, Y. Shimizu, K. Okamoto, M. Nishiyama, H. Oh-Oka, and M. Terazima, “A line-scanning semi-confocal multi-photon fluorescence microscope with a simultaneous broadband spectral acquisition and its application to the study of the thylakoid membrane of a cyanobacterium Anabaena PCC7120,” J. Microsc. 228(2), 240–254 (2007).
[Crossref] [PubMed]

Lee, J. Y.

Li, C.

Lichtman, J. W.

J. A. Conchello and J. W. Lichtman, “Optical sectioning microscopy,” Nat. Methods 2(12), 920–931 (2005).
[Crossref] [PubMed]

Lin, C. P.

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]

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]

Mély, Y.

A. Hayek, A. Grichine, T. Huault, C. Ricard, F. Bolze, B. Van Der Sanden, J. C. Vial, Y. Mély, A. Duperray, P. L. Baldeck, and J. F. Nicoud, “Cell-permeant cytoplasmic blue fluorophores optimized for in vivo two-photon microscopy with low-power excitation,” Microsc. Res. Tech. 70(10), 880–885 (2007).
[Crossref] [PubMed]

Misteli, T.

T. Misteli and R. D. Phair, “High mobility of proteins in the mammalian cell nucleus,” Nature 404(6778), 604–609 (2000).
[Crossref] [PubMed]

Moggridge, G. D.

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]

Nesi, G.

Nicoud, J. F.

A. Hayek, A. Grichine, T. Huault, C. Ricard, F. Bolze, B. Van Der Sanden, J. C. Vial, Y. Mély, A. Duperray, P. L. Baldeck, and J. F. Nicoud, “Cell-permeant cytoplasmic blue fluorophores optimized for in vivo two-photon microscopy with low-power excitation,” Microsc. Res. Tech. 70(10), 880–885 (2007).
[Crossref] [PubMed]

Nishiyama, M.

S. Kumazaki, M. Hasegawa, M. Ghoneim, Y. Shimizu, K. Okamoto, M. Nishiyama, H. Oh-Oka, and M. Terazima, “A line-scanning semi-confocal multi-photon fluorescence microscope with a simultaneous broadband spectral acquisition and its application to the study of the thylakoid membrane of a cyanobacterium Anabaena PCC7120,” J. Microsc. 228(2), 240–254 (2007).
[Crossref] [PubMed]

Oh-Oka, H.

S. Kumazaki, M. Hasegawa, M. Ghoneim, Y. Shimizu, K. Okamoto, M. Nishiyama, H. Oh-Oka, and M. Terazima, “A line-scanning semi-confocal multi-photon fluorescence microscope with a simultaneous broadband spectral acquisition and its application to the study of the thylakoid membrane of a cyanobacterium Anabaena PCC7120,” J. Microsc. 228(2), 240–254 (2007).
[Crossref] [PubMed]

Okamoto, K.

S. Kumazaki, M. Hasegawa, M. Ghoneim, Y. Shimizu, K. Okamoto, M. Nishiyama, H. Oh-Oka, and M. Terazima, “A line-scanning semi-confocal multi-photon fluorescence microscope with a simultaneous broadband spectral acquisition and its application to the study of the thylakoid membrane of a cyanobacterium Anabaena PCC7120,” J. Microsc. 228(2), 240–254 (2007).
[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]

Pastila, R. K.

Pavone, F. S.

Phair, R. D.

T. Misteli and R. D. Phair, “High mobility of proteins in the mammalian cell nucleus,” Nature 404(6778), 604–609 (2000).
[Crossref] [PubMed]

Pitsillides, C.

Puoris’haag, M.

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]

Ricard, C.

A. Hayek, A. Grichine, T. Huault, C. Ricard, F. Bolze, B. Van Der Sanden, J. C. Vial, Y. Mély, A. Duperray, P. L. Baldeck, and J. F. Nicoud, “Cell-permeant cytoplasmic blue fluorophores optimized for in vivo two-photon microscopy with low-power excitation,” Microsc. Res. Tech. 70(10), 880–885 (2007).
[Crossref] [PubMed]

Rippe, K.

G. Heuvelman, F. Erdel, M. Wachsmuth, and K. Rippe, “Analysis of protein mobilities and interactions in living cells by multifocal fluorescence fluctuation microscopy,” Eur. Biophys. J. 38(6), 813–828 (2009).
[Crossref] [PubMed]

Runnels, J. M.

Santini, M. T.

P. Indovina, M. Collini, G. Chirico, and M. T. Santini, “Three-dimensional cell organization leads to almost immediate HRE activity as demonstrated by molecular imaging of MG-63 spheroids using two-photon excitation microscopy,” FEBS Lett. 581(4), 719–726 (2007).
[Crossref] [PubMed]

Schlachter, S.

Schwedler, S.

Seelig, B.

A. D. Keefe, D. S. Wilson, B. Seelig, and J. W. Szostak, “One-step purification of recombinant proteins using a nanomolar-affinity streptavidin-binding peptide, the SBP-Tag,” Protein Expr. Purif. 23(3), 440–446 (2001).
[Crossref] [PubMed]

Seghiri, Z.

Shimi, T.

Y. Hiraoka, T. Shimi, and T. Haraguchi, “Multispectral imaging fluorescence microscopy for living cells,” Cell Struct. Funct. 27(5), 367–374 (2002).
[Crossref] [PubMed]

Shimizu, Y.

S. Kumazaki, M. Hasegawa, M. Ghoneim, Y. Shimizu, K. Okamoto, M. Nishiyama, H. Oh-Oka, and M. Terazima, “A line-scanning semi-confocal multi-photon fluorescence microscope with a simultaneous broadband spectral acquisition and its application to the study of the thylakoid membrane of a cyanobacterium Anabaena PCC7120,” J. Microsc. 228(2), 240–254 (2007).
[Crossref] [PubMed]

Sinclair, M. B.

Svoboda, K.

K. Svoboda and R. Yasuda, “Principles of two-photon excitation microscopy and its applications to neuroscience,” Neuron 50(6), 823–839 (2006).
[Crossref] [PubMed]

Szostak, J. W.

A. D. Keefe, D. S. Wilson, B. Seelig, and J. W. Szostak, “One-step purification of recombinant proteins using a nanomolar-affinity streptavidin-binding peptide, the SBP-Tag,” Protein Expr. Purif. 23(3), 440–446 (2001).
[Crossref] [PubMed]

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]

Terazima, M.

S. Kumazaki, M. Hasegawa, M. Ghoneim, Y. Shimizu, K. Okamoto, M. Nishiyama, H. Oh-Oka, and M. Terazima, “A line-scanning semi-confocal multi-photon fluorescence microscope with a simultaneous broadband spectral acquisition and its application to the study of the thylakoid membrane of a cyanobacterium Anabaena PCC7120,” J. Microsc. 228(2), 240–254 (2007).
[Crossref] [PubMed]

Timlin, J. A.

Van Der Sanden, B.

A. Hayek, A. Grichine, T. Huault, C. Ricard, F. Bolze, B. Van Der Sanden, J. C. Vial, Y. Mély, A. Duperray, P. L. Baldeck, and J. F. Nicoud, “Cell-permeant cytoplasmic blue fluorophores optimized for in vivo two-photon microscopy with low-power excitation,” Microsc. Res. Tech. 70(10), 880–885 (2007).
[Crossref] [PubMed]

Vial, J. C.

A. Hayek, A. Grichine, T. Huault, C. Ricard, F. Bolze, B. Van Der Sanden, J. C. Vial, Y. Mély, A. Duperray, P. L. Baldeck, and J. F. Nicoud, “Cell-permeant cytoplasmic blue fluorophores optimized for in vivo two-photon microscopy with low-power excitation,” Microsc. Res. Tech. 70(10), 880–885 (2007).
[Crossref] [PubMed]

Wachsmuth, M.

F. Bestvater, Z. Seghiri, M. S. Kang, N. Gröner, J. Y. Lee, K. B. Im, and M. Wachsmuth, “EMCCD-based spectrally resolved fluorescence correlation spectroscopy,” Opt. Express 18(23), 23818–23828 (2010).
[Crossref] [PubMed]

G. Heuvelman, F. Erdel, M. Wachsmuth, and K. Rippe, “Analysis of protein mobilities and interactions in living cells by multifocal fluorescence fluctuation microscopy,” Eur. Biophys. J. 38(6), 813–828 (2009).
[Crossref] [PubMed]

Wang, E.

E. Wang, C. M. Babbey, and K. W. Dunn, “Performance comparison between the high-speed Yokogawa spinning disc confocal system and single-point scanning confocal systems,” J. Microsc. 218(2), 148–159 (2005).
[Crossref] [PubMed]

Wilson, D. S.

A. D. Keefe, D. S. Wilson, B. Seelig, and J. W. Szostak, “One-step purification of recombinant proteins using a nanomolar-affinity streptavidin-binding peptide, the SBP-Tag,” Protein Expr. Purif. 23(3), 440–446 (2001).
[Crossref] [PubMed]

Yasuda, R.

K. Svoboda and R. Yasuda, “Principles of two-photon excitation microscopy and its applications to neuroscience,” Neuron 50(6), 823–839 (2006).
[Crossref] [PubMed]

Appl. Opt. (1)

Cell Struct. Funct. (1)

Y. Hiraoka, T. Shimi, and T. Haraguchi, “Multispectral imaging fluorescence microscopy for living cells,” Cell Struct. Funct. 27(5), 367–374 (2002).
[Crossref] [PubMed]

Eur. Biophys. J. (1)

G. Heuvelman, F. Erdel, M. Wachsmuth, and K. Rippe, “Analysis of protein mobilities and interactions in living cells by multifocal fluorescence fluctuation microscopy,” Eur. Biophys. J. 38(6), 813–828 (2009).
[Crossref] [PubMed]

FEBS Lett. (1)

P. Indovina, M. Collini, G. Chirico, and M. T. Santini, “Three-dimensional cell organization leads to almost immediate HRE activity as demonstrated by molecular imaging of MG-63 spheroids using two-photon excitation microscopy,” FEBS Lett. 581(4), 719–726 (2007).
[Crossref] [PubMed]

J. Microsc. (3)

E. Wang, C. M. Babbey, and K. W. Dunn, “Performance comparison between the high-speed Yokogawa spinning disc confocal system and single-point scanning confocal systems,” J. Microsc. 218(2), 148–159 (2005).
[Crossref] [PubMed]

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

S. Kumazaki, M. Hasegawa, M. Ghoneim, Y. Shimizu, K. Okamoto, M. Nishiyama, H. Oh-Oka, and M. Terazima, “A line-scanning semi-confocal multi-photon fluorescence microscope with a simultaneous broadband spectral acquisition and its application to the study of the thylakoid membrane of a cyanobacterium Anabaena PCC7120,” J. Microsc. 228(2), 240–254 (2007).
[Crossref] [PubMed]

Microsc. Res. Tech. (2)

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]

A. Hayek, A. Grichine, T. Huault, C. Ricard, F. Bolze, B. Van Der Sanden, J. C. Vial, Y. Mély, A. Duperray, P. L. Baldeck, and J. F. Nicoud, “Cell-permeant cytoplasmic blue fluorophores optimized for in vivo two-photon microscopy with low-power excitation,” Microsc. Res. Tech. 70(10), 880–885 (2007).
[Crossref] [PubMed]

Nat. Methods (2)

F. Helmchen and W. Denk, “Deep tissue two-photon microscopy,” Nat. Methods 2(12), 932–940 (2005).
[Crossref] [PubMed]

J. A. Conchello and J. W. Lichtman, “Optical sectioning microscopy,” Nat. Methods 2(12), 920–931 (2005).
[Crossref] [PubMed]

Nature (1)

T. Misteli and R. D. Phair, “High mobility of proteins in the mammalian cell nucleus,” Nature 404(6778), 604–609 (2000).
[Crossref] [PubMed]

Neuron (1)

K. Svoboda and R. Yasuda, “Principles of two-photon excitation microscopy and its applications to neuroscience,” Neuron 50(6), 823–839 (2006).
[Crossref] [PubMed]

Opt. Express (4)

Protein Expr. Purif. (1)

A. D. Keefe, D. S. Wilson, B. Seelig, and J. W. Szostak, “One-step purification of recombinant proteins using a nanomolar-affinity streptavidin-binding peptide, the SBP-Tag,” Protein Expr. Purif. 23(3), 440–446 (2001).
[Crossref] [PubMed]

Other (2)

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.

M. Wachsmuth, and K. Weisshart, “Fluorescence photobleaching and fluorescence correlation spectroscopy: two complementary technologies to study molecular dynamics in living cells,” in Imaging Cellular and Molecular Biological Functions (Springer Verlag, Heidelberg, 2007).

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Figures (7)

Fig. 1
Fig. 1 Overview and comparison of spectral acquisition using an EM-CCD with common schemes using linear PMT arrays and moving slits. The effective detection areas are indicated in gray.

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Fig. 2
Fig. 2 (A) Schematics of the 2PE spectral fluorescence imaging setup using an EM-CCD camera. (B) Upon 2PE point-scanning excitation of the fluorescent sample, a spectral data stack comprised of 80 images is generated. B: blue, G: green, R: red. (C) Only 80 pixels in the bottom-most line of the detection area of the EM-CCD chip are illuminated with the spectrally dispersed fluorescence light at least for the time required to amplify and read out 80 pixels. Then a rapid single line shift is applied to move the photoelectrons to the first line of the storage area while the galvanometer scanner moves the focus to the next pixel. Eventually the photoelectrons from the 80 illuminated pixels arrive at the bottom-most line of the storage area. Only the 80 pixels of interest instead of the full line of 656 pixels are transferred to the charge amplifiers, the analog-to-digital converter and to the frame grabber. This is repeated for all pixels of an image frame and the recorded spectra are assigned appropriately to the respective pixels. (D) Line readout rate as a function of pixels per line that are read out. The pixel number/readout rate combination used in this study is highlighted.

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Fig. 7
Fig. 7 Multiple QD pulse-chase staining of 5-HT2C receptors undergoing endocytosis and spectral images. (A) SBP-tagged 5-HT2C receptors were incubated sequentially, washed and kept in the chamber at 37°C until the next pulse-chase staining. This took place at 24 hours, 6 hours and 1 hour, and used three different streptavidin-conjugated QDs for 30 minutes. Image stacks were acquired 30 minutes after the last staining (B). The images representing (C) QD 525, (D) QD 585 and (E) QD 655 were obtained from the channels specific to each peak, ranging from 526 to 535 nm for QD 525, 576-588 nm for QD 585 and 635-647 nm for QD 655. The scale bar represents 20 μm. In the overlay image (B), spectrally different QD-labeled 5-HT2C can be found in intracellular compartments (F and G) and in the plasmalemma (H) based on the receptor internalization rate and depending on the pulse chase time point.

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Fig. 3
Fig. 3 Point spread function measurement. (A) Lateral section of a 0.17 μm diameter fluorescent bead excited at 900 nm. (B) Emission spectrum of the bead. The FWHM values of the beads obtained from axial (C) and lateral (D) line scans are 0.51 and 0.36 μm, respectively. The red curves indicate Gaussian fits.

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Fig. 4
Fig. 4 Calibration and characterization of the EM-CCD spectral imaging system. (A) Spectral sampling of our setup as calculated with Zemax (Zemax Development Corp.). The three wavelengths used for calibration are highlighted, anticipating a FWHM of 3.9, 4.8 and 9.6 nm when taking a diffraction-limited spot size of 1.2 pixels into consideration. The three laser lines (458, 488 and 633 nm) were measured using (B) a commercial spectrometer under the same conditions and (C) our setup. Based on (B), (C) was calibrated, and a wavelength range was assigned to each channel. A Gaussian function was fitted to the spectrum of each laser line to measure its bandwidth. The emission bandwidths of the three laser lines were sufficiently narrow (~1 nm) for a proper estimation of the spectral resolution of our system. The full widths at half maximum were 6, 5.7 and 13 nm at 458, 488 and 633 nm, respectively. In (D)-(F), the circles indicate the measured spectra from (A), and the red curves show Gaussian fits.

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Fig. 5
Fig. 5 Spectral images of HEK-293 cells expressing (A) CFP, (B) YFP and (C) DsRed were acquired at excitation wavelengths of (A) 800, (B) 900, and (C) 940 nm, and detected at (A) 486-560, (B) 508-588 and (C) 560-654 nm, respectively. The spectra in the regions of interest are shown in (D). The scale bar represents 20 μm.

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Fig. 6
Fig. 6 Spectral autofluorescence images of Convallaria Majalis tissue. Multiple spectral signatures reveal subcellular compartments (B-F) as seen in the overlay image (A) taken from single channels at (B) 639-647, (C) 661-669, (D) 693-701 and (E) 719-728 nm. The spectra of cell wall (a) and plasma membrane (b) are shown in (F). The scale bar represents 20 μm.

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