Abstract

A spatial-spectral multiplexing two-photon excited fluorescence (TPEF) hyperspectral imaging system was demonstrated at 17 frames per second (fps) with over 2,000 effective spectral channels. The system exploited the hardware used in beam-scanning microscopy to perform multiplexing of spatial and spectral information, leading to two orders of magnitude reduction in data throughput. An iterative demultiplexing algorithm enabled robust classification and full spectral recovery without requiring prior knowledge of the emission spectra of the fluorophores. Proof-of-concept demonstrations were performed using spatially heterogeneous multicomponent laser dyes and live genetically encoded fluorescent C. elegans. This instrument requires minimal hardware modifications for most existing multiphoton microscope and provides the ability to acquire hyperspectral TPEF images at video rate. It has the potential for dynamic studies in deep tissue with multiple spectrally overlapping fluorescence tags.

© 2017 Optical Society of America under the terms of the OSA Open Access Publishing Agreement

Full Article  |  PDF Article
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References

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2017 (6)

F. Cutrale, V. Trivedi, L. A. Trinh, C. L. Chiu, J. M. Choi, M. S. Artiga, and S. E. Fraser, “Hyperspectral phasor analysis enables multiplexed 5D in vivo imaging,” Nat. Methods 14(2), 149–152 (2017).
[PubMed]

S. Arrigoni, G. Turra, and A. Signoroni, “Hyperspectral image analysis for rapid and accurate discrimination of bacterial infections: A benchmark study,” Comput. Biol. Med. 88, 60–71 (2017).
[PubMed]

C. L. Rink, M. M. Wernke, H. M. Powell, M. Tornero, S. C. Gnyawali, R. M. Schroeder, J. Y. Kim, J. A. Denune, A. W. Albury, G. M. Gordillo, J. M. Colvin, and C. K. Sen, “Standardized Approach to Quantitatively Measure Residual Limb Skin Health in Individuals with Lower Limb Amputation,” Adv. Wound Care (New Rochelle) 6(7), 225–232 (2017).
[PubMed]

T. Nöbauer, O. Skocek, A. J. Pernía-Andrade, L. Weilguny, F. M. Traub, M. I. Molodtsov, and A. Vaziri, “Video rate volumetric Ca2+ imaging across cortex using seeded iterative demixing (SID) microscopy,” Nat. Methods 14(8), 811–818 (2017).
[PubMed]

A. S. Luthman, S. Dumitru, I. Quiros-Gonzalez, J. Joseph, and S. E. Bohndiek, “Fluorescence hyperspectral imaging (fHSI) using a spectrally resolved detector array,” J. Biophotonics 10(6-7), 840–853 (2017).
[PubMed]

J. G. Dwight and T. S. Tkaczyk, “Lenslet array tunable snapshot imaging spectrometer (LATIS) for hyperspectral fluorescence microscopy,” Biomed. Opt. Express 8(3), 1950–1964 (2017).
[PubMed]

2016 (2)

L. Gao and L. V. Wang, “A review of snapshot multidimensional optical imaging: Measuring photon tags in parallel,” Phys. Rep. 616, 1–37 (2016).
[PubMed]

F. R. Bertani, E. Botti, L. Ferrari, V. Mussi, A. Costanzo, M. D’Alessandro, F. Cilloco, and S. Selci, “Label-free and non-invasive discrimination of HaCaT and melanoma cells in a co-culture model by hyperspectral confocal reflectance microscopy,” J. Biophotonics 9(6), 619–625 (2016).
[PubMed]

2015 (1)

L. Gao and R. T. Smith, “Optical hyperspectral imaging in microscopy and spectroscopy - a review of data acquisition,” J. Biophotonics 8(6), 441–456 (2015).
[PubMed]

2014 (2)

R. Prevedel, Y. G. Yoon, M. Hoffmann, N. Pak, G. Wetzstein, S. Kato, T. Schrödel, R. Raskar, M. Zimmer, E. S. Boyden, and A. Vaziri, “Simultaneous whole-animal 3D imaging of neuronal activity using light-field microscopy,” Nat. Methods 11(7), 727–730 (2014).
[PubMed]

S. Z. Sullivan, R. D. Muir, J. A. Newman, M. S. Carlsen, S. Sreehari, C. Doerge, N. J. Begue, R. M. Everly, C. A. Bouman, and G. J. Simpson, “High frame-rate multichannel beam-scanning microscopy based on Lissajous trajectories,” Opt. Express 22(20), 24224–24234 (2014).
[PubMed]

2012 (1)

A. D. Elliott, L. Gao, A. Ustione, N. Bedard, R. Kester, D. W. Piston, and T. S. Tkaczyk, “Real-time hyperspectral fluorescence imaging of pancreatic β-cell dynamics with the image mapping spectrometer,” J. Cell Sci. 125(Pt 20), 4833–4840 (2012).
[PubMed]

2010 (2)

2008 (2)

N. Pujol, S. Cypowyj, K. Ziegler, A. Millet, A. Astrain, A. Goncharov, Y. Jin, A. D. Chisholm, and J. J. Ewbank, “Distinct innate immune responses to infection and wounding in the C. elegans epidermis,” Curr. Biol. 18(7), 481–489 (2008).
[PubMed]

N. Pujol, O. Zugasti, D. Wong, C. Couillault, C. L. Kurz, H. Schulenburg, and J. J. Ewbank, “Anti-fungal innate immunity in C. elegans is enhanced by evolutionary diversification of antimicrobial peptides,” PLoS Pathog. 4(7), e1000105 (2008).
[PubMed]

2006 (2)

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

P. M. Kasili and T. Vo-Dinh, “Hyperspectral imaging system using acousto-optic tunable filter for flow cytometry applications,” Cytometry A 69(8), 835–841 (2006).
[PubMed]

2001 (1)

R. Lansford, G. Bearman, and S. E. Fraser, “Resolution of multiple green fluorescent protein color variants and dyes using two-photon microscopy and imaging spectroscopy,” J. Biomed. Opt. 6(3), 311–318 (2001).
[PubMed]

2000 (1)

G. H. Patterson and D. W. Piston, “Photobleaching in two-photon excitation microscopy,” Biophys. J. 78(4), 2159–2162 (2000).
[PubMed]

1999 (1)

Y. Garini, A. Gil, I. Bar-Am, D. Cabib, and N. Katzir, “Signal to noise analysis of multiple color fluorescence imaging microscopy,” Cytometry 35(3), 214–226 (1999).
[PubMed]

1994 (1)

1975 (1)

G. A. Reynolds and K. H. Drexhage, “New coumarin dyes with rigidized structure for flashlamp-pumped dye lasers,” Opt. Commun. 13, 222–225 (1975).

Albury, A. W.

C. L. Rink, M. M. Wernke, H. M. Powell, M. Tornero, S. C. Gnyawali, R. M. Schroeder, J. Y. Kim, J. A. Denune, A. W. Albury, G. M. Gordillo, J. M. Colvin, and C. K. Sen, “Standardized Approach to Quantitatively Measure Residual Limb Skin Health in Individuals with Lower Limb Amputation,” Adv. Wound Care (New Rochelle) 6(7), 225–232 (2017).
[PubMed]

Arrigoni, S.

S. Arrigoni, G. Turra, and A. Signoroni, “Hyperspectral image analysis for rapid and accurate discrimination of bacterial infections: A benchmark study,” Comput. Biol. Med. 88, 60–71 (2017).
[PubMed]

Artiga, M. S.

F. Cutrale, V. Trivedi, L. A. Trinh, C. L. Chiu, J. M. Choi, M. S. Artiga, and S. E. Fraser, “Hyperspectral phasor analysis enables multiplexed 5D in vivo imaging,” Nat. Methods 14(2), 149–152 (2017).
[PubMed]

Astrain, A.

N. Pujol, S. Cypowyj, K. Ziegler, A. Millet, A. Astrain, A. Goncharov, Y. Jin, A. D. Chisholm, and J. J. Ewbank, “Distinct innate immune responses to infection and wounding in the C. elegans epidermis,” Curr. Biol. 18(7), 481–489 (2008).
[PubMed]

Bar-Am, I.

Y. Garini, A. Gil, I. Bar-Am, D. Cabib, and N. Katzir, “Signal to noise analysis of multiple color fluorescence imaging microscopy,” Cytometry 35(3), 214–226 (1999).
[PubMed]

Bearman, G.

R. Lansford, G. Bearman, and S. E. Fraser, “Resolution of multiple green fluorescent protein color variants and dyes using two-photon microscopy and imaging spectroscopy,” J. Biomed. Opt. 6(3), 311–318 (2001).
[PubMed]

Bedard, N.

A. D. Elliott, L. Gao, A. Ustione, N. Bedard, R. Kester, D. W. Piston, and T. S. Tkaczyk, “Real-time hyperspectral fluorescence imaging of pancreatic β-cell dynamics with the image mapping spectrometer,” J. Cell Sci. 125(Pt 20), 4833–4840 (2012).
[PubMed]

Begue, N. J.

Bertani, F. R.

F. R. Bertani, E. Botti, L. Ferrari, V. Mussi, A. Costanzo, M. D’Alessandro, F. Cilloco, and S. Selci, “Label-free and non-invasive discrimination of HaCaT and melanoma cells in a co-culture model by hyperspectral confocal reflectance microscopy,” J. Biophotonics 9(6), 619–625 (2016).
[PubMed]

Bohndiek, S. E.

A. S. Luthman, S. Dumitru, I. Quiros-Gonzalez, J. Joseph, and S. E. Bohndiek, “Fluorescence hyperspectral imaging (fHSI) using a spectrally resolved detector array,” J. Biophotonics 10(6-7), 840–853 (2017).
[PubMed]

Botti, E.

F. R. Bertani, E. Botti, L. Ferrari, V. Mussi, A. Costanzo, M. D’Alessandro, F. Cilloco, and S. Selci, “Label-free and non-invasive discrimination of HaCaT and melanoma cells in a co-culture model by hyperspectral confocal reflectance microscopy,” J. Biophotonics 9(6), 619–625 (2016).
[PubMed]

Bouman, C. A.

Boyden, E. S.

R. Prevedel, Y. G. Yoon, M. Hoffmann, N. Pak, G. Wetzstein, S. Kato, T. Schrödel, R. Raskar, M. Zimmer, E. S. Boyden, and A. Vaziri, “Simultaneous whole-animal 3D imaging of neuronal activity using light-field microscopy,” Nat. Methods 11(7), 727–730 (2014).
[PubMed]

Brady, D. J.

Cabib, D.

Y. Garini, A. Gil, I. Bar-Am, D. Cabib, and N. Katzir, “Signal to noise analysis of multiple color fluorescence imaging microscopy,” Cytometry 35(3), 214–226 (1999).
[PubMed]

Carlsen, M. S.

Chisholm, A. D.

N. Pujol, S. Cypowyj, K. Ziegler, A. Millet, A. Astrain, A. Goncharov, Y. Jin, A. D. Chisholm, and J. J. Ewbank, “Distinct innate immune responses to infection and wounding in the C. elegans epidermis,” Curr. Biol. 18(7), 481–489 (2008).
[PubMed]

Chiu, C. L.

F. Cutrale, V. Trivedi, L. A. Trinh, C. L. Chiu, J. M. Choi, M. S. Artiga, and S. E. Fraser, “Hyperspectral phasor analysis enables multiplexed 5D in vivo imaging,” Nat. Methods 14(2), 149–152 (2017).
[PubMed]

Choi, J. M.

F. Cutrale, V. Trivedi, L. A. Trinh, C. L. Chiu, J. M. Choi, M. S. Artiga, and S. E. Fraser, “Hyperspectral phasor analysis enables multiplexed 5D in vivo imaging,” Nat. Methods 14(2), 149–152 (2017).
[PubMed]

Choi, K.

Cilloco, F.

F. R. Bertani, E. Botti, L. Ferrari, V. Mussi, A. Costanzo, M. D’Alessandro, F. Cilloco, and S. Selci, “Label-free and non-invasive discrimination of HaCaT and melanoma cells in a co-culture model by hyperspectral confocal reflectance microscopy,” J. Biophotonics 9(6), 619–625 (2016).
[PubMed]

Colvin, J. M.

C. L. Rink, M. M. Wernke, H. M. Powell, M. Tornero, S. C. Gnyawali, R. M. Schroeder, J. Y. Kim, J. A. Denune, A. W. Albury, G. M. Gordillo, J. M. Colvin, and C. K. Sen, “Standardized Approach to Quantitatively Measure Residual Limb Skin Health in Individuals with Lower Limb Amputation,” Adv. Wound Care (New Rochelle) 6(7), 225–232 (2017).
[PubMed]

Costanzo, A.

F. R. Bertani, E. Botti, L. Ferrari, V. Mussi, A. Costanzo, M. D’Alessandro, F. Cilloco, and S. Selci, “Label-free and non-invasive discrimination of HaCaT and melanoma cells in a co-culture model by hyperspectral confocal reflectance microscopy,” J. Biophotonics 9(6), 619–625 (2016).
[PubMed]

Couillault, C.

N. Pujol, O. Zugasti, D. Wong, C. Couillault, C. L. Kurz, H. Schulenburg, and J. J. Ewbank, “Anti-fungal innate immunity in C. elegans is enhanced by evolutionary diversification of antimicrobial peptides,” PLoS Pathog. 4(7), e1000105 (2008).
[PubMed]

Cull, C. F.

Cutrale, F.

F. Cutrale, V. Trivedi, L. A. Trinh, C. L. Chiu, J. M. Choi, M. S. Artiga, and S. E. Fraser, “Hyperspectral phasor analysis enables multiplexed 5D in vivo imaging,” Nat. Methods 14(2), 149–152 (2017).
[PubMed]

Cypowyj, S.

N. Pujol, S. Cypowyj, K. Ziegler, A. Millet, A. Astrain, A. Goncharov, Y. Jin, A. D. Chisholm, and J. J. Ewbank, “Distinct innate immune responses to infection and wounding in the C. elegans epidermis,” Curr. Biol. 18(7), 481–489 (2008).
[PubMed]

D’Alessandro, M.

F. R. Bertani, E. Botti, L. Ferrari, V. Mussi, A. Costanzo, M. D’Alessandro, F. Cilloco, and S. Selci, “Label-free and non-invasive discrimination of HaCaT and melanoma cells in a co-culture model by hyperspectral confocal reflectance microscopy,” J. Biophotonics 9(6), 619–625 (2016).
[PubMed]

Denune, J. A.

C. L. Rink, M. M. Wernke, H. M. Powell, M. Tornero, S. C. Gnyawali, R. M. Schroeder, J. Y. Kim, J. A. Denune, A. W. Albury, G. M. Gordillo, J. M. Colvin, and C. K. Sen, “Standardized Approach to Quantitatively Measure Residual Limb Skin Health in Individuals with Lower Limb Amputation,” Adv. Wound Care (New Rochelle) 6(7), 225–232 (2017).
[PubMed]

Doerge, C.

Drexhage, K. H.

G. A. Reynolds and K. H. Drexhage, “New coumarin dyes with rigidized structure for flashlamp-pumped dye lasers,” Opt. Commun. 13, 222–225 (1975).

Dumitru, S.

A. S. Luthman, S. Dumitru, I. Quiros-Gonzalez, J. Joseph, and S. E. Bohndiek, “Fluorescence hyperspectral imaging (fHSI) using a spectrally resolved detector array,” J. Biophotonics 10(6-7), 840–853 (2017).
[PubMed]

Dwight, J. G.

Elliott, A. D.

A. D. Elliott, L. Gao, A. Ustione, N. Bedard, R. Kester, D. W. Piston, and T. S. Tkaczyk, “Real-time hyperspectral fluorescence imaging of pancreatic β-cell dynamics with the image mapping spectrometer,” J. Cell Sci. 125(Pt 20), 4833–4840 (2012).
[PubMed]

Everly, R. M.

Ewbank, J. J.

N. Pujol, S. Cypowyj, K. Ziegler, A. Millet, A. Astrain, A. Goncharov, Y. Jin, A. D. Chisholm, and J. J. Ewbank, “Distinct innate immune responses to infection and wounding in the C. elegans epidermis,” Curr. Biol. 18(7), 481–489 (2008).
[PubMed]

N. Pujol, O. Zugasti, D. Wong, C. Couillault, C. L. Kurz, H. Schulenburg, and J. J. Ewbank, “Anti-fungal innate immunity in C. elegans is enhanced by evolutionary diversification of antimicrobial peptides,” PLoS Pathog. 4(7), e1000105 (2008).
[PubMed]

Ferrari, L.

F. R. Bertani, E. Botti, L. Ferrari, V. Mussi, A. Costanzo, M. D’Alessandro, F. Cilloco, and S. Selci, “Label-free and non-invasive discrimination of HaCaT and melanoma cells in a co-culture model by hyperspectral confocal reflectance microscopy,” J. Biophotonics 9(6), 619–625 (2016).
[PubMed]

Fraser, S. E.

F. Cutrale, V. Trivedi, L. A. Trinh, C. L. Chiu, J. M. Choi, M. S. Artiga, and S. E. Fraser, “Hyperspectral phasor analysis enables multiplexed 5D in vivo imaging,” Nat. Methods 14(2), 149–152 (2017).
[PubMed]

R. Lansford, G. Bearman, and S. E. Fraser, “Resolution of multiple green fluorescent protein color variants and dyes using two-photon microscopy and imaging spectroscopy,” J. Biomed. Opt. 6(3), 311–318 (2001).
[PubMed]

Gao, L.

L. Gao and L. V. Wang, “A review of snapshot multidimensional optical imaging: Measuring photon tags in parallel,” Phys. Rep. 616, 1–37 (2016).
[PubMed]

L. Gao and R. T. Smith, “Optical hyperspectral imaging in microscopy and spectroscopy - a review of data acquisition,” J. Biophotonics 8(6), 441–456 (2015).
[PubMed]

A. D. Elliott, L. Gao, A. Ustione, N. Bedard, R. Kester, D. W. Piston, and T. S. Tkaczyk, “Real-time hyperspectral fluorescence imaging of pancreatic β-cell dynamics with the image mapping spectrometer,” J. Cell Sci. 125(Pt 20), 4833–4840 (2012).
[PubMed]

L. Gao, R. T. Kester, N. Hagen, and T. S. Tkaczyk, “Snapshot Image Mapping Spectrometer (IMS) with high sampling density for hyperspectral microscopy,” Opt. Express 18(14), 14330–14344 (2010).
[PubMed]

Garini, Y.

Y. Garini, A. Gil, I. Bar-Am, D. Cabib, and N. Katzir, “Signal to noise analysis of multiple color fluorescence imaging microscopy,” Cytometry 35(3), 214–226 (1999).
[PubMed]

Gil, A.

Y. Garini, A. Gil, I. Bar-Am, D. Cabib, and N. Katzir, “Signal to noise analysis of multiple color fluorescence imaging microscopy,” Cytometry 35(3), 214–226 (1999).
[PubMed]

Gnyawali, S. C.

C. L. Rink, M. M. Wernke, H. M. Powell, M. Tornero, S. C. Gnyawali, R. M. Schroeder, J. Y. Kim, J. A. Denune, A. W. Albury, G. M. Gordillo, J. M. Colvin, and C. K. Sen, “Standardized Approach to Quantitatively Measure Residual Limb Skin Health in Individuals with Lower Limb Amputation,” Adv. Wound Care (New Rochelle) 6(7), 225–232 (2017).
[PubMed]

Goncharov, A.

N. Pujol, S. Cypowyj, K. Ziegler, A. Millet, A. Astrain, A. Goncharov, Y. Jin, A. D. Chisholm, and J. J. Ewbank, “Distinct innate immune responses to infection and wounding in the C. elegans epidermis,” Curr. Biol. 18(7), 481–489 (2008).
[PubMed]

Gordillo, G. M.

C. L. Rink, M. M. Wernke, H. M. Powell, M. Tornero, S. C. Gnyawali, R. M. Schroeder, J. Y. Kim, J. A. Denune, A. W. Albury, G. M. Gordillo, J. M. Colvin, and C. K. Sen, “Standardized Approach to Quantitatively Measure Residual Limb Skin Health in Individuals with Lower Limb Amputation,” Adv. Wound Care (New Rochelle) 6(7), 225–232 (2017).
[PubMed]

Haaland, D. M.

Hagen, N.

Hoffmann, M.

R. Prevedel, Y. G. Yoon, M. Hoffmann, N. Pak, G. Wetzstein, S. Kato, T. Schrödel, R. Raskar, M. Zimmer, E. S. Boyden, and A. Vaziri, “Simultaneous whole-animal 3D imaging of neuronal activity using light-field microscopy,” Nat. Methods 11(7), 727–730 (2014).
[PubMed]

Hoyt, C. C.

Jin, Y.

N. Pujol, S. Cypowyj, K. Ziegler, A. Millet, A. Astrain, A. Goncharov, Y. Jin, A. D. Chisholm, and J. J. Ewbank, “Distinct innate immune responses to infection and wounding in the C. elegans epidermis,” Curr. Biol. 18(7), 481–489 (2008).
[PubMed]

Jones, H. D.

Joseph, J.

A. S. Luthman, S. Dumitru, I. Quiros-Gonzalez, J. Joseph, and S. E. Bohndiek, “Fluorescence hyperspectral imaging (fHSI) using a spectrally resolved detector array,” J. Biophotonics 10(6-7), 840–853 (2017).
[PubMed]

Kasili, P. M.

P. M. Kasili and T. Vo-Dinh, “Hyperspectral imaging system using acousto-optic tunable filter for flow cytometry applications,” Cytometry A 69(8), 835–841 (2006).
[PubMed]

Kato, S.

R. Prevedel, Y. G. Yoon, M. Hoffmann, N. Pak, G. Wetzstein, S. Kato, T. Schrödel, R. Raskar, M. Zimmer, E. S. Boyden, and A. Vaziri, “Simultaneous whole-animal 3D imaging of neuronal activity using light-field microscopy,” Nat. Methods 11(7), 727–730 (2014).
[PubMed]

Katzir, N.

Y. Garini, A. Gil, I. Bar-Am, D. Cabib, and N. Katzir, “Signal to noise analysis of multiple color fluorescence imaging microscopy,” Cytometry 35(3), 214–226 (1999).
[PubMed]

Kester, R.

A. D. Elliott, L. Gao, A. Ustione, N. Bedard, R. Kester, D. W. Piston, and T. S. Tkaczyk, “Real-time hyperspectral fluorescence imaging of pancreatic β-cell dynamics with the image mapping spectrometer,” J. Cell Sci. 125(Pt 20), 4833–4840 (2012).
[PubMed]

Kester, R. T.

Kim, J. Y.

C. L. Rink, M. M. Wernke, H. M. Powell, M. Tornero, S. C. Gnyawali, R. M. Schroeder, J. Y. Kim, J. A. Denune, A. W. Albury, G. M. Gordillo, J. M. Colvin, and C. K. Sen, “Standardized Approach to Quantitatively Measure Residual Limb Skin Health in Individuals with Lower Limb Amputation,” Adv. Wound Care (New Rochelle) 6(7), 225–232 (2017).
[PubMed]

Kurz, C. L.

N. Pujol, O. Zugasti, D. Wong, C. Couillault, C. L. Kurz, H. Schulenburg, and J. J. Ewbank, “Anti-fungal innate immunity in C. elegans is enhanced by evolutionary diversification of antimicrobial peptides,” PLoS Pathog. 4(7), e1000105 (2008).
[PubMed]

Lansford, R.

R. Lansford, G. Bearman, and S. E. Fraser, “Resolution of multiple green fluorescent protein color variants and dyes using two-photon microscopy and imaging spectroscopy,” J. Biomed. Opt. 6(3), 311–318 (2001).
[PubMed]

Luthman, A. S.

A. S. Luthman, S. Dumitru, I. Quiros-Gonzalez, J. Joseph, and S. E. Bohndiek, “Fluorescence hyperspectral imaging (fHSI) using a spectrally resolved detector array,” J. Biophotonics 10(6-7), 840–853 (2017).
[PubMed]

Millet, A.

N. Pujol, S. Cypowyj, K. Ziegler, A. Millet, A. Astrain, A. Goncharov, Y. Jin, A. D. Chisholm, and J. J. Ewbank, “Distinct innate immune responses to infection and wounding in the C. elegans epidermis,” Curr. Biol. 18(7), 481–489 (2008).
[PubMed]

Molodtsov, M. I.

T. Nöbauer, O. Skocek, A. J. Pernía-Andrade, L. Weilguny, F. M. Traub, M. I. Molodtsov, and A. Vaziri, “Video rate volumetric Ca2+ imaging across cortex using seeded iterative demixing (SID) microscopy,” Nat. Methods 14(8), 811–818 (2017).
[PubMed]

Morris, H. R.

Muir, R. D.

Mussi, V.

F. R. Bertani, E. Botti, L. Ferrari, V. Mussi, A. Costanzo, M. D’Alessandro, F. Cilloco, and S. Selci, “Label-free and non-invasive discrimination of HaCaT and melanoma cells in a co-culture model by hyperspectral confocal reflectance microscopy,” J. Biophotonics 9(6), 619–625 (2016).
[PubMed]

Newman, J. A.

Nöbauer, T.

T. Nöbauer, O. Skocek, A. J. Pernía-Andrade, L. Weilguny, F. M. Traub, M. I. Molodtsov, and A. Vaziri, “Video rate volumetric Ca2+ imaging across cortex using seeded iterative demixing (SID) microscopy,” Nat. Methods 14(8), 811–818 (2017).
[PubMed]

Oliver, T.

Pak, N.

R. Prevedel, Y. G. Yoon, M. Hoffmann, N. Pak, G. Wetzstein, S. Kato, T. Schrödel, R. Raskar, M. Zimmer, E. S. Boyden, and A. Vaziri, “Simultaneous whole-animal 3D imaging of neuronal activity using light-field microscopy,” Nat. Methods 11(7), 727–730 (2014).
[PubMed]

Patterson, G. H.

G. H. Patterson and D. W. Piston, “Photobleaching in two-photon excitation microscopy,” Biophys. J. 78(4), 2159–2162 (2000).
[PubMed]

Pernía-Andrade, A. J.

T. Nöbauer, O. Skocek, A. J. Pernía-Andrade, L. Weilguny, F. M. Traub, M. I. Molodtsov, and A. Vaziri, “Video rate volumetric Ca2+ imaging across cortex using seeded iterative demixing (SID) microscopy,” Nat. Methods 14(8), 811–818 (2017).
[PubMed]

Piston, D. W.

A. D. Elliott, L. Gao, A. Ustione, N. Bedard, R. Kester, D. W. Piston, and T. S. Tkaczyk, “Real-time hyperspectral fluorescence imaging of pancreatic β-cell dynamics with the image mapping spectrometer,” J. Cell Sci. 125(Pt 20), 4833–4840 (2012).
[PubMed]

G. H. Patterson and D. W. Piston, “Photobleaching in two-photon excitation microscopy,” Biophys. J. 78(4), 2159–2162 (2000).
[PubMed]

Powell, H. M.

C. L. Rink, M. M. Wernke, H. M. Powell, M. Tornero, S. C. Gnyawali, R. M. Schroeder, J. Y. Kim, J. A. Denune, A. W. Albury, G. M. Gordillo, J. M. Colvin, and C. K. Sen, “Standardized Approach to Quantitatively Measure Residual Limb Skin Health in Individuals with Lower Limb Amputation,” Adv. Wound Care (New Rochelle) 6(7), 225–232 (2017).
[PubMed]

Prevedel, R.

R. Prevedel, Y. G. Yoon, M. Hoffmann, N. Pak, G. Wetzstein, S. Kato, T. Schrödel, R. Raskar, M. Zimmer, E. S. Boyden, and A. Vaziri, “Simultaneous whole-animal 3D imaging of neuronal activity using light-field microscopy,” Nat. Methods 11(7), 727–730 (2014).
[PubMed]

Pujol, N.

N. Pujol, S. Cypowyj, K. Ziegler, A. Millet, A. Astrain, A. Goncharov, Y. Jin, A. D. Chisholm, and J. J. Ewbank, “Distinct innate immune responses to infection and wounding in the C. elegans epidermis,” Curr. Biol. 18(7), 481–489 (2008).
[PubMed]

N. Pujol, O. Zugasti, D. Wong, C. Couillault, C. L. Kurz, H. Schulenburg, and J. J. Ewbank, “Anti-fungal innate immunity in C. elegans is enhanced by evolutionary diversification of antimicrobial peptides,” PLoS Pathog. 4(7), e1000105 (2008).
[PubMed]

Quiros-Gonzalez, I.

A. S. Luthman, S. Dumitru, I. Quiros-Gonzalez, J. Joseph, and S. E. Bohndiek, “Fluorescence hyperspectral imaging (fHSI) using a spectrally resolved detector array,” J. Biophotonics 10(6-7), 840–853 (2017).
[PubMed]

Raskar, R.

R. Prevedel, Y. G. Yoon, M. Hoffmann, N. Pak, G. Wetzstein, S. Kato, T. Schrödel, R. Raskar, M. Zimmer, E. S. Boyden, and A. Vaziri, “Simultaneous whole-animal 3D imaging of neuronal activity using light-field microscopy,” Nat. Methods 11(7), 727–730 (2014).
[PubMed]

Reynolds, G. A.

G. A. Reynolds and K. H. Drexhage, “New coumarin dyes with rigidized structure for flashlamp-pumped dye lasers,” Opt. Commun. 13, 222–225 (1975).

Rink, C. L.

C. L. Rink, M. M. Wernke, H. M. Powell, M. Tornero, S. C. Gnyawali, R. M. Schroeder, J. Y. Kim, J. A. Denune, A. W. Albury, G. M. Gordillo, J. M. Colvin, and C. K. Sen, “Standardized Approach to Quantitatively Measure Residual Limb Skin Health in Individuals with Lower Limb Amputation,” Adv. Wound Care (New Rochelle) 6(7), 225–232 (2017).
[PubMed]

Schrödel, T.

R. Prevedel, Y. G. Yoon, M. Hoffmann, N. Pak, G. Wetzstein, S. Kato, T. Schrödel, R. Raskar, M. Zimmer, E. S. Boyden, and A. Vaziri, “Simultaneous whole-animal 3D imaging of neuronal activity using light-field microscopy,” Nat. Methods 11(7), 727–730 (2014).
[PubMed]

Schroeder, R. M.

C. L. Rink, M. M. Wernke, H. M. Powell, M. Tornero, S. C. Gnyawali, R. M. Schroeder, J. Y. Kim, J. A. Denune, A. W. Albury, G. M. Gordillo, J. M. Colvin, and C. K. Sen, “Standardized Approach to Quantitatively Measure Residual Limb Skin Health in Individuals with Lower Limb Amputation,” Adv. Wound Care (New Rochelle) 6(7), 225–232 (2017).
[PubMed]

Schulenburg, H.

N. Pujol, O. Zugasti, D. Wong, C. Couillault, C. L. Kurz, H. Schulenburg, and J. J. Ewbank, “Anti-fungal innate immunity in C. elegans is enhanced by evolutionary diversification of antimicrobial peptides,” PLoS Pathog. 4(7), e1000105 (2008).
[PubMed]

Selci, S.

F. R. Bertani, E. Botti, L. Ferrari, V. Mussi, A. Costanzo, M. D’Alessandro, F. Cilloco, and S. Selci, “Label-free and non-invasive discrimination of HaCaT and melanoma cells in a co-culture model by hyperspectral confocal reflectance microscopy,” J. Biophotonics 9(6), 619–625 (2016).
[PubMed]

Sen, C. K.

C. L. Rink, M. M. Wernke, H. M. Powell, M. Tornero, S. C. Gnyawali, R. M. Schroeder, J. Y. Kim, J. A. Denune, A. W. Albury, G. M. Gordillo, J. M. Colvin, and C. K. Sen, “Standardized Approach to Quantitatively Measure Residual Limb Skin Health in Individuals with Lower Limb Amputation,” Adv. Wound Care (New Rochelle) 6(7), 225–232 (2017).
[PubMed]

Signoroni, A.

S. Arrigoni, G. Turra, and A. Signoroni, “Hyperspectral image analysis for rapid and accurate discrimination of bacterial infections: A benchmark study,” Comput. Biol. Med. 88, 60–71 (2017).
[PubMed]

Simpson, G. J.

Sinclair, M. B.

Skocek, O.

T. Nöbauer, O. Skocek, A. J. Pernía-Andrade, L. Weilguny, F. M. Traub, M. I. Molodtsov, and A. Vaziri, “Video rate volumetric Ca2+ imaging across cortex using seeded iterative demixing (SID) microscopy,” Nat. Methods 14(8), 811–818 (2017).
[PubMed]

Smith, R. T.

L. Gao and R. T. Smith, “Optical hyperspectral imaging in microscopy and spectroscopy - a review of data acquisition,” J. Biophotonics 8(6), 441–456 (2015).
[PubMed]

Sreehari, S.

Sullivan, S. Z.

Timlin, J. A.

Tkaczyk, T. S.

Tornero, M.

C. L. Rink, M. M. Wernke, H. M. Powell, M. Tornero, S. C. Gnyawali, R. M. Schroeder, J. Y. Kim, J. A. Denune, A. W. Albury, G. M. Gordillo, J. M. Colvin, and C. K. Sen, “Standardized Approach to Quantitatively Measure Residual Limb Skin Health in Individuals with Lower Limb Amputation,” Adv. Wound Care (New Rochelle) 6(7), 225–232 (2017).
[PubMed]

Traub, F. M.

T. Nöbauer, O. Skocek, A. J. Pernía-Andrade, L. Weilguny, F. M. Traub, M. I. Molodtsov, and A. Vaziri, “Video rate volumetric Ca2+ imaging across cortex using seeded iterative demixing (SID) microscopy,” Nat. Methods 14(8), 811–818 (2017).
[PubMed]

Treado, P. J.

Trinh, L. A.

F. Cutrale, V. Trivedi, L. A. Trinh, C. L. Chiu, J. M. Choi, M. S. Artiga, and S. E. Fraser, “Hyperspectral phasor analysis enables multiplexed 5D in vivo imaging,” Nat. Methods 14(2), 149–152 (2017).
[PubMed]

Trivedi, V.

F. Cutrale, V. Trivedi, L. A. Trinh, C. L. Chiu, J. M. Choi, M. S. Artiga, and S. E. Fraser, “Hyperspectral phasor analysis enables multiplexed 5D in vivo imaging,” Nat. Methods 14(2), 149–152 (2017).
[PubMed]

Turra, G.

S. Arrigoni, G. Turra, and A. Signoroni, “Hyperspectral image analysis for rapid and accurate discrimination of bacterial infections: A benchmark study,” Comput. Biol. Med. 88, 60–71 (2017).
[PubMed]

Ustione, A.

A. D. Elliott, L. Gao, A. Ustione, N. Bedard, R. Kester, D. W. Piston, and T. S. Tkaczyk, “Real-time hyperspectral fluorescence imaging of pancreatic β-cell dynamics with the image mapping spectrometer,” J. Cell Sci. 125(Pt 20), 4833–4840 (2012).
[PubMed]

Vaziri, A.

T. Nöbauer, O. Skocek, A. J. Pernía-Andrade, L. Weilguny, F. M. Traub, M. I. Molodtsov, and A. Vaziri, “Video rate volumetric Ca2+ imaging across cortex using seeded iterative demixing (SID) microscopy,” Nat. Methods 14(8), 811–818 (2017).
[PubMed]

R. Prevedel, Y. G. Yoon, M. Hoffmann, N. Pak, G. Wetzstein, S. Kato, T. Schrödel, R. Raskar, M. Zimmer, E. S. Boyden, and A. Vaziri, “Simultaneous whole-animal 3D imaging of neuronal activity using light-field microscopy,” Nat. Methods 11(7), 727–730 (2014).
[PubMed]

Vo-Dinh, T.

P. M. Kasili and T. Vo-Dinh, “Hyperspectral imaging system using acousto-optic tunable filter for flow cytometry applications,” Cytometry A 69(8), 835–841 (2006).
[PubMed]

Wang, L. V.

L. Gao and L. V. Wang, “A review of snapshot multidimensional optical imaging: Measuring photon tags in parallel,” Phys. Rep. 616, 1–37 (2016).
[PubMed]

Weilguny, L.

T. Nöbauer, O. Skocek, A. J. Pernía-Andrade, L. Weilguny, F. M. Traub, M. I. Molodtsov, and A. Vaziri, “Video rate volumetric Ca2+ imaging across cortex using seeded iterative demixing (SID) microscopy,” Nat. Methods 14(8), 811–818 (2017).
[PubMed]

Wernke, M. M.

C. L. Rink, M. M. Wernke, H. M. Powell, M. Tornero, S. C. Gnyawali, R. M. Schroeder, J. Y. Kim, J. A. Denune, A. W. Albury, G. M. Gordillo, J. M. Colvin, and C. K. Sen, “Standardized Approach to Quantitatively Measure Residual Limb Skin Health in Individuals with Lower Limb Amputation,” Adv. Wound Care (New Rochelle) 6(7), 225–232 (2017).
[PubMed]

Wetzstein, G.

R. Prevedel, Y. G. Yoon, M. Hoffmann, N. Pak, G. Wetzstein, S. Kato, T. Schrödel, R. Raskar, M. Zimmer, E. S. Boyden, and A. Vaziri, “Simultaneous whole-animal 3D imaging of neuronal activity using light-field microscopy,” Nat. Methods 11(7), 727–730 (2014).
[PubMed]

Wong, D.

N. Pujol, O. Zugasti, D. Wong, C. Couillault, C. L. Kurz, H. Schulenburg, and J. J. Ewbank, “Anti-fungal innate immunity in C. elegans is enhanced by evolutionary diversification of antimicrobial peptides,” PLoS Pathog. 4(7), e1000105 (2008).
[PubMed]

Yoon, Y. G.

R. Prevedel, Y. G. Yoon, M. Hoffmann, N. Pak, G. Wetzstein, S. Kato, T. Schrödel, R. Raskar, M. Zimmer, E. S. Boyden, and A. Vaziri, “Simultaneous whole-animal 3D imaging of neuronal activity using light-field microscopy,” Nat. Methods 11(7), 727–730 (2014).
[PubMed]

Ziegler, K.

N. Pujol, S. Cypowyj, K. Ziegler, A. Millet, A. Astrain, A. Goncharov, Y. Jin, A. D. Chisholm, and J. J. Ewbank, “Distinct innate immune responses to infection and wounding in the C. elegans epidermis,” Curr. Biol. 18(7), 481–489 (2008).
[PubMed]

Zimmer, M.

R. Prevedel, Y. G. Yoon, M. Hoffmann, N. Pak, G. Wetzstein, S. Kato, T. Schrödel, R. Raskar, M. Zimmer, E. S. Boyden, and A. Vaziri, “Simultaneous whole-animal 3D imaging of neuronal activity using light-field microscopy,” Nat. Methods 11(7), 727–730 (2014).
[PubMed]

Zugasti, O.

N. Pujol, O. Zugasti, D. Wong, C. Couillault, C. L. Kurz, H. Schulenburg, and J. J. Ewbank, “Anti-fungal innate immunity in C. elegans is enhanced by evolutionary diversification of antimicrobial peptides,” PLoS Pathog. 4(7), e1000105 (2008).
[PubMed]

Adv. Wound Care (New Rochelle) (1)

C. L. Rink, M. M. Wernke, H. M. Powell, M. Tornero, S. C. Gnyawali, R. M. Schroeder, J. Y. Kim, J. A. Denune, A. W. Albury, G. M. Gordillo, J. M. Colvin, and C. K. Sen, “Standardized Approach to Quantitatively Measure Residual Limb Skin Health in Individuals with Lower Limb Amputation,” Adv. Wound Care (New Rochelle) 6(7), 225–232 (2017).
[PubMed]

Appl. Opt. (2)

Appl. Spectrosc. (1)

Biomed. Opt. Express (1)

Biophys. J. (1)

G. H. Patterson and D. W. Piston, “Photobleaching in two-photon excitation microscopy,” Biophys. J. 78(4), 2159–2162 (2000).
[PubMed]

Comput. Biol. Med. (1)

S. Arrigoni, G. Turra, and A. Signoroni, “Hyperspectral image analysis for rapid and accurate discrimination of bacterial infections: A benchmark study,” Comput. Biol. Med. 88, 60–71 (2017).
[PubMed]

Curr. Biol. (1)

N. Pujol, S. Cypowyj, K. Ziegler, A. Millet, A. Astrain, A. Goncharov, Y. Jin, A. D. Chisholm, and J. J. Ewbank, “Distinct innate immune responses to infection and wounding in the C. elegans epidermis,” Curr. Biol. 18(7), 481–489 (2008).
[PubMed]

Cytometry (1)

Y. Garini, A. Gil, I. Bar-Am, D. Cabib, and N. Katzir, “Signal to noise analysis of multiple color fluorescence imaging microscopy,” Cytometry 35(3), 214–226 (1999).
[PubMed]

Cytometry A (1)

P. M. Kasili and T. Vo-Dinh, “Hyperspectral imaging system using acousto-optic tunable filter for flow cytometry applications,” Cytometry A 69(8), 835–841 (2006).
[PubMed]

J. Biomed. Opt. (1)

R. Lansford, G. Bearman, and S. E. Fraser, “Resolution of multiple green fluorescent protein color variants and dyes using two-photon microscopy and imaging spectroscopy,” J. Biomed. Opt. 6(3), 311–318 (2001).
[PubMed]

J. Biophotonics (3)

F. R. Bertani, E. Botti, L. Ferrari, V. Mussi, A. Costanzo, M. D’Alessandro, F. Cilloco, and S. Selci, “Label-free and non-invasive discrimination of HaCaT and melanoma cells in a co-culture model by hyperspectral confocal reflectance microscopy,” J. Biophotonics 9(6), 619–625 (2016).
[PubMed]

L. Gao and R. T. Smith, “Optical hyperspectral imaging in microscopy and spectroscopy - a review of data acquisition,” J. Biophotonics 8(6), 441–456 (2015).
[PubMed]

A. S. Luthman, S. Dumitru, I. Quiros-Gonzalez, J. Joseph, and S. E. Bohndiek, “Fluorescence hyperspectral imaging (fHSI) using a spectrally resolved detector array,” J. Biophotonics 10(6-7), 840–853 (2017).
[PubMed]

J. Cell Sci. (1)

A. D. Elliott, L. Gao, A. Ustione, N. Bedard, R. Kester, D. W. Piston, and T. S. Tkaczyk, “Real-time hyperspectral fluorescence imaging of pancreatic β-cell dynamics with the image mapping spectrometer,” J. Cell Sci. 125(Pt 20), 4833–4840 (2012).
[PubMed]

Nat. Methods (3)

F. Cutrale, V. Trivedi, L. A. Trinh, C. L. Chiu, J. M. Choi, M. S. Artiga, and S. E. Fraser, “Hyperspectral phasor analysis enables multiplexed 5D in vivo imaging,” Nat. Methods 14(2), 149–152 (2017).
[PubMed]

T. Nöbauer, O. Skocek, A. J. Pernía-Andrade, L. Weilguny, F. M. Traub, M. I. Molodtsov, and A. Vaziri, “Video rate volumetric Ca2+ imaging across cortex using seeded iterative demixing (SID) microscopy,” Nat. Methods 14(8), 811–818 (2017).
[PubMed]

R. Prevedel, Y. G. Yoon, M. Hoffmann, N. Pak, G. Wetzstein, S. Kato, T. Schrödel, R. Raskar, M. Zimmer, E. S. Boyden, and A. Vaziri, “Simultaneous whole-animal 3D imaging of neuronal activity using light-field microscopy,” Nat. Methods 11(7), 727–730 (2014).
[PubMed]

Opt. Commun. (1)

G. A. Reynolds and K. H. Drexhage, “New coumarin dyes with rigidized structure for flashlamp-pumped dye lasers,” Opt. Commun. 13, 222–225 (1975).

Opt. Express (2)

Phys. Rep. (1)

L. Gao and L. V. Wang, “A review of snapshot multidimensional optical imaging: Measuring photon tags in parallel,” Phys. Rep. 616, 1–37 (2016).
[PubMed]

PLoS Pathog. (1)

N. Pujol, O. Zugasti, D. Wong, C. Couillault, C. L. Kurz, H. Schulenburg, and J. J. Ewbank, “Anti-fungal innate immunity in C. elegans is enhanced by evolutionary diversification of antimicrobial peptides,” PLoS Pathog. 4(7), e1000105 (2008).
[PubMed]

Supplementary Material (2)

NameDescription
» Visualization 1       Heterogeneous mixtures of fluorescent dye droplets
» Visualization 2       In vivo imaging of genetically modified C. elegans.

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Figures (6)

Fig. 1
Fig. 1 Overview of spatial-spectral multiplexing. (A) Instrument schematic. (B) Illustration of spectra projected on the PMT array from different pixels in one image plane. (C) Spectral reconstruction from a single channel image for two components with different spectra.
Fig. 2
Fig. 2 (A) Beam scanning path (not the beam path). (B) The projection of spectrum of a narrow band emission on the PMT array. (C) Spectral window calibration with doubling crystal.
Fig. 3
Fig. 3 Flowchart for the iterative classification algorithm.
Fig. 4
Fig. 4 (A) Integrated total fluorescence of a dye mixture (5μM fluorescein in water and 50 μM coumarin 6 in chloroform). (B) Classification under the assumption of two components (a in green and b in blue). Insert: recovered spectra of component a and b. (C) Classification under the assumption of three components (a in green, b in blue and c in red). Insert: recovered spectra for component a, b and c. (D) Overlay of recovered spectrum of component a with the fluorescein emission spectrum from a benchtop fluorimeter, and the difference between them. (E) Overlay of recovered spectrum of component b with the coumarin 6 emission spectrum, and the difference between them. Both plotted spectra were averaged over all the spatial positions of the same classified component. (F) Spectrum of the third component (red dotted line) fitted as a linear combination (black solid line) of the first two components.
Fig. 5
Fig. 5 Dye mixture with three fluorophores: fluorescein (green) in water, coumarin 6 (blue) and eFluor 450 (violet) in chloroform separately (also see Visualization 1). Target droplet with coumarin 6 in chloroform (circled in red) was tracked from its first appearance in frame 55, set as 0 s (A) and final misclassification in frame 386 with during time 21.8 s (B). (C) Spectra recovered from the target droplet at 0 s (green solid line) and 21.8 s (purple dash line). (D) Photobleaching curve of coumarin 6 in the target droplet (blue dot) with the exponential fit (red line).
Fig. 6
Fig. 6 Two-photon fluorescence image of gene coded C. elegans analyzed without knowing the emission spectra of the fluorophores as a priori at (A) frame 33 and (B) frame 104. Pseudo-color based on different components recovered from custom classification algorithm with green for component 1 and red for component 2 (also see Visualization 2). (C) Recovered spectra for component 1 (green) and 2 (red) for both frame 33 (solid lines) and frame 104 (dash lines). Both plotted spectra were averaged over all the spatial positions of the same classified component. (D) Fluorescence image of gene coded C. elegans with conventional fluorescence microscope under 460 - 490 nm excitation.

Equations (3)

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s j meas = s j 0 (λ)dλ l=L/2 L/2 s j+l 0 δλ
s meas = s 0 rect( L )
cos( θ )= s ref s x,y | s ref || s x,y |

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