Abstract

Hyperspectral imaging is a common technique in fluorescence microscopy to obtain the emission spectrum at each pixel of an image. However, methods to obtain spectral resolution based on diffraction gratings or integrated prisms work poorly when the sample is strongly scattering. We developed a microscope named the DIVER that collects the fluorescence emission over a very large angle. Since the fluorescence light after passing through the multiple scattering sample is not collimated, the use of grating or prisms strongly limits the amount of light that can be used with available hyperspectral devices. Here we show that 2 filters that accept uncollimated light over a large aperture are sufficient to calculate the spectral phasor rather than displaying the entire spectrum. Using the properties of the spectral phasors, we can resolve spectral components and perform the type of data analyses that are usually performed in hyperspectral image analysis.

© 2018 Optical Society of America under the terms of the OSA Open Access Publishing Agreement

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References

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  1. L. M. Andrews, M. R. Jones, M. A. Digman, and E. Gratton, “Detecting Pyronin Y labeled RNA transcripts in live cell microenvironments by phasor-FLIM analysis,” Methods Appl. Fluoresc. 1(1), 015001 (2013).
    [Crossref] [PubMed]
  2. F. Cutrale, A. Salih, and E. Gratton, “Spectral Phasor approach for fingerprinting of photo-activatable fluorescent proteins Dronpa, Kaede and KikGR,” Methods Appl. Fluoresc. 1(3), 035001 (2013).
    [Crossref] [PubMed]
  3. L. Malacrida, S. Astrada, A. Briva, M. Bollati-Fogolín, E. Gratton, and L. A. Bagatolli, “Spectral phasor analysis of LAURDAN fluorescence in live A549 lung cells to study the hydration and time evolution of intracellular lamellar body-like structures,” Biochim. Biophys. Acta 1858(11), 2625–2635 (2016).
    [Crossref] [PubMed]
  4. L. Malacrida, E. Gratton, and D. M. Jameson, “Model-free methods to study membrane environmental probes: a comparison of the spectral phasor and generalized polarization approaches,” Methods Appl. Fluoresc. 3(4), 047001 (2015).
    [Crossref]
  5. L. Malacrida, D. M. Jameson, and E. Gratton, “A multidimensional phasor approach reveals LAURDAN photophysics in NIH-3T3 cell membranes,” Sci. Rep. 7(1), 9215 (2017).
    [Crossref] [PubMed]
  6. S. Ranjit, A. Dvornikov, M. Levi, S. Furgeson, and E. Gratton, “Characterizing fibrosis in UUO mice model using multiparametric analysis of phasor distribution from FLIM images,” Biomed. Opt. Express 7(9), 3519–3530 (2016).
    [Crossref] [PubMed]
  7. F. Fereidouni, A. N. Bader, A. Colonna, and H. C. Gerritsen, “Phasor analysis of multiphoton spectral images distinguishes autofluorescence components of in vivo human skin,” J. Biophotonics 7(8), 589–596 (2014).
    [Crossref] [PubMed]
  8. F. Fereidouni, A. N. Bader, and H. C. Gerritsen, “Spectral phasor analysis allows rapid and reliable unmixing of fluorescence microscopy spectral images,” Opt. Express 20(12), 12729–12741 (2012).
    [Crossref] [PubMed]
  9. P. Wang, G. Turcatel, C. Arnesano, D. Warburton, S. E. Fraser, and F. Cutrale, “Fiber pattern removal and image reconstruction method for snapshot mosaic hyperspectral endoscopic images,” Biomed. Opt. Express 9(2), 780–790 (2018).
    [Crossref] [PubMed]
  10. 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).
    [Crossref] [PubMed]
  11. Z. Lavagnino, J. Dwight, A. Ustione, T. U. Nguyen, T. S. Tkaczyk, and D. W. Piston, “Snapshot Hyperspectral Light-Sheet Imaging of Signal Transduction in Live Pancreatic Islets,” Biophys. J. 111(2), 409–417 (2016).
    [Crossref] [PubMed]
  12. 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(20), 4833–4840 (2012).
    [Crossref] [PubMed]
  13. V. Crosignani, A. S. Dvornikov, and E. Gratton, “Enhancement of imaging depth in turbid media using a wide area detector,” J. Biophotonics 4(9), 592–599 (2011).
    [PubMed]
  14. L. Malacrida, E. Gratton, and D. M. Jameson, “Model-free methods to study membrane environmental probes: a comparison of the spectral phasor and generalized polarization approaches,” Methods Appl. Fluoresc. 3(4), 047001 (2015).
    [Crossref]

2018 (1)

2017 (2)

L. Malacrida, D. M. Jameson, and E. Gratton, “A multidimensional phasor approach reveals LAURDAN photophysics in NIH-3T3 cell membranes,” Sci. Rep. 7(1), 9215 (2017).
[Crossref] [PubMed]

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).
[Crossref] [PubMed]

2016 (3)

Z. Lavagnino, J. Dwight, A. Ustione, T. U. Nguyen, T. S. Tkaczyk, and D. W. Piston, “Snapshot Hyperspectral Light-Sheet Imaging of Signal Transduction in Live Pancreatic Islets,” Biophys. J. 111(2), 409–417 (2016).
[Crossref] [PubMed]

L. Malacrida, S. Astrada, A. Briva, M. Bollati-Fogolín, E. Gratton, and L. A. Bagatolli, “Spectral phasor analysis of LAURDAN fluorescence in live A549 lung cells to study the hydration and time evolution of intracellular lamellar body-like structures,” Biochim. Biophys. Acta 1858(11), 2625–2635 (2016).
[Crossref] [PubMed]

S. Ranjit, A. Dvornikov, M. Levi, S. Furgeson, and E. Gratton, “Characterizing fibrosis in UUO mice model using multiparametric analysis of phasor distribution from FLIM images,” Biomed. Opt. Express 7(9), 3519–3530 (2016).
[Crossref] [PubMed]

2015 (2)

L. Malacrida, E. Gratton, and D. M. Jameson, “Model-free methods to study membrane environmental probes: a comparison of the spectral phasor and generalized polarization approaches,” Methods Appl. Fluoresc. 3(4), 047001 (2015).
[Crossref]

L. Malacrida, E. Gratton, and D. M. Jameson, “Model-free methods to study membrane environmental probes: a comparison of the spectral phasor and generalized polarization approaches,” Methods Appl. Fluoresc. 3(4), 047001 (2015).
[Crossref]

2014 (1)

F. Fereidouni, A. N. Bader, A. Colonna, and H. C. Gerritsen, “Phasor analysis of multiphoton spectral images distinguishes autofluorescence components of in vivo human skin,” J. Biophotonics 7(8), 589–596 (2014).
[Crossref] [PubMed]

2013 (2)

L. M. Andrews, M. R. Jones, M. A. Digman, and E. Gratton, “Detecting Pyronin Y labeled RNA transcripts in live cell microenvironments by phasor-FLIM analysis,” Methods Appl. Fluoresc. 1(1), 015001 (2013).
[Crossref] [PubMed]

F. Cutrale, A. Salih, and E. Gratton, “Spectral Phasor approach for fingerprinting of photo-activatable fluorescent proteins Dronpa, Kaede and KikGR,” Methods Appl. Fluoresc. 1(3), 035001 (2013).
[Crossref] [PubMed]

2012 (2)

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(20), 4833–4840 (2012).
[Crossref] [PubMed]

F. Fereidouni, A. N. Bader, and H. C. Gerritsen, “Spectral phasor analysis allows rapid and reliable unmixing of fluorescence microscopy spectral images,” Opt. Express 20(12), 12729–12741 (2012).
[Crossref] [PubMed]

2011 (1)

V. Crosignani, A. S. Dvornikov, and E. Gratton, “Enhancement of imaging depth in turbid media using a wide area detector,” J. Biophotonics 4(9), 592–599 (2011).
[PubMed]

Andrews, L. M.

L. M. Andrews, M. R. Jones, M. A. Digman, and E. Gratton, “Detecting Pyronin Y labeled RNA transcripts in live cell microenvironments by phasor-FLIM analysis,” Methods Appl. Fluoresc. 1(1), 015001 (2013).
[Crossref] [PubMed]

Arnesano, C.

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).
[Crossref] [PubMed]

Astrada, S.

L. Malacrida, S. Astrada, A. Briva, M. Bollati-Fogolín, E. Gratton, and L. A. Bagatolli, “Spectral phasor analysis of LAURDAN fluorescence in live A549 lung cells to study the hydration and time evolution of intracellular lamellar body-like structures,” Biochim. Biophys. Acta 1858(11), 2625–2635 (2016).
[Crossref] [PubMed]

Bader, A. N.

F. Fereidouni, A. N. Bader, A. Colonna, and H. C. Gerritsen, “Phasor analysis of multiphoton spectral images distinguishes autofluorescence components of in vivo human skin,” J. Biophotonics 7(8), 589–596 (2014).
[Crossref] [PubMed]

F. Fereidouni, A. N. Bader, and H. C. Gerritsen, “Spectral phasor analysis allows rapid and reliable unmixing of fluorescence microscopy spectral images,” Opt. Express 20(12), 12729–12741 (2012).
[Crossref] [PubMed]

Bagatolli, L. A.

L. Malacrida, S. Astrada, A. Briva, M. Bollati-Fogolín, E. Gratton, and L. A. Bagatolli, “Spectral phasor analysis of LAURDAN fluorescence in live A549 lung cells to study the hydration and time evolution of intracellular lamellar body-like structures,” Biochim. Biophys. Acta 1858(11), 2625–2635 (2016).
[Crossref] [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(20), 4833–4840 (2012).
[Crossref] [PubMed]

Bollati-Fogolín, M.

L. Malacrida, S. Astrada, A. Briva, M. Bollati-Fogolín, E. Gratton, and L. A. Bagatolli, “Spectral phasor analysis of LAURDAN fluorescence in live A549 lung cells to study the hydration and time evolution of intracellular lamellar body-like structures,” Biochim. Biophys. Acta 1858(11), 2625–2635 (2016).
[Crossref] [PubMed]

Briva, A.

L. Malacrida, S. Astrada, A. Briva, M. Bollati-Fogolín, E. Gratton, and L. A. Bagatolli, “Spectral phasor analysis of LAURDAN fluorescence in live A549 lung cells to study the hydration and time evolution of intracellular lamellar body-like structures,” Biochim. Biophys. Acta 1858(11), 2625–2635 (2016).
[Crossref] [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).
[Crossref] [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).
[Crossref] [PubMed]

Colonna, A.

F. Fereidouni, A. N. Bader, A. Colonna, and H. C. Gerritsen, “Phasor analysis of multiphoton spectral images distinguishes autofluorescence components of in vivo human skin,” J. Biophotonics 7(8), 589–596 (2014).
[Crossref] [PubMed]

Crosignani, V.

V. Crosignani, A. S. Dvornikov, and E. Gratton, “Enhancement of imaging depth in turbid media using a wide area detector,” J. Biophotonics 4(9), 592–599 (2011).
[PubMed]

Cutrale, F.

P. Wang, G. Turcatel, C. Arnesano, D. Warburton, S. E. Fraser, and F. Cutrale, “Fiber pattern removal and image reconstruction method for snapshot mosaic hyperspectral endoscopic images,” Biomed. Opt. Express 9(2), 780–790 (2018).
[Crossref] [PubMed]

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).
[Crossref] [PubMed]

F. Cutrale, A. Salih, and E. Gratton, “Spectral Phasor approach for fingerprinting of photo-activatable fluorescent proteins Dronpa, Kaede and KikGR,” Methods Appl. Fluoresc. 1(3), 035001 (2013).
[Crossref] [PubMed]

Digman, M. A.

L. M. Andrews, M. R. Jones, M. A. Digman, and E. Gratton, “Detecting Pyronin Y labeled RNA transcripts in live cell microenvironments by phasor-FLIM analysis,” Methods Appl. Fluoresc. 1(1), 015001 (2013).
[Crossref] [PubMed]

Dvornikov, A.

Dvornikov, A. S.

V. Crosignani, A. S. Dvornikov, and E. Gratton, “Enhancement of imaging depth in turbid media using a wide area detector,” J. Biophotonics 4(9), 592–599 (2011).
[PubMed]

Dwight, J.

Z. Lavagnino, J. Dwight, A. Ustione, T. U. Nguyen, T. S. Tkaczyk, and D. W. Piston, “Snapshot Hyperspectral Light-Sheet Imaging of Signal Transduction in Live Pancreatic Islets,” Biophys. J. 111(2), 409–417 (2016).
[Crossref] [PubMed]

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(20), 4833–4840 (2012).
[Crossref] [PubMed]

Fereidouni, F.

F. Fereidouni, A. N. Bader, A. Colonna, and H. C. Gerritsen, “Phasor analysis of multiphoton spectral images distinguishes autofluorescence components of in vivo human skin,” J. Biophotonics 7(8), 589–596 (2014).
[Crossref] [PubMed]

F. Fereidouni, A. N. Bader, and H. C. Gerritsen, “Spectral phasor analysis allows rapid and reliable unmixing of fluorescence microscopy spectral images,” Opt. Express 20(12), 12729–12741 (2012).
[Crossref] [PubMed]

Fraser, S. E.

P. Wang, G. Turcatel, C. Arnesano, D. Warburton, S. E. Fraser, and F. Cutrale, “Fiber pattern removal and image reconstruction method for snapshot mosaic hyperspectral endoscopic images,” Biomed. Opt. Express 9(2), 780–790 (2018).
[Crossref] [PubMed]

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).
[Crossref] [PubMed]

Furgeson, S.

Gao, L.

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(20), 4833–4840 (2012).
[Crossref] [PubMed]

Gerritsen, H. C.

F. Fereidouni, A. N. Bader, A. Colonna, and H. C. Gerritsen, “Phasor analysis of multiphoton spectral images distinguishes autofluorescence components of in vivo human skin,” J. Biophotonics 7(8), 589–596 (2014).
[Crossref] [PubMed]

F. Fereidouni, A. N. Bader, and H. C. Gerritsen, “Spectral phasor analysis allows rapid and reliable unmixing of fluorescence microscopy spectral images,” Opt. Express 20(12), 12729–12741 (2012).
[Crossref] [PubMed]

Gratton, E.

L. Malacrida, D. M. Jameson, and E. Gratton, “A multidimensional phasor approach reveals LAURDAN photophysics in NIH-3T3 cell membranes,” Sci. Rep. 7(1), 9215 (2017).
[Crossref] [PubMed]

L. Malacrida, S. Astrada, A. Briva, M. Bollati-Fogolín, E. Gratton, and L. A. Bagatolli, “Spectral phasor analysis of LAURDAN fluorescence in live A549 lung cells to study the hydration and time evolution of intracellular lamellar body-like structures,” Biochim. Biophys. Acta 1858(11), 2625–2635 (2016).
[Crossref] [PubMed]

S. Ranjit, A. Dvornikov, M. Levi, S. Furgeson, and E. Gratton, “Characterizing fibrosis in UUO mice model using multiparametric analysis of phasor distribution from FLIM images,” Biomed. Opt. Express 7(9), 3519–3530 (2016).
[Crossref] [PubMed]

L. Malacrida, E. Gratton, and D. M. Jameson, “Model-free methods to study membrane environmental probes: a comparison of the spectral phasor and generalized polarization approaches,” Methods Appl. Fluoresc. 3(4), 047001 (2015).
[Crossref]

L. Malacrida, E. Gratton, and D. M. Jameson, “Model-free methods to study membrane environmental probes: a comparison of the spectral phasor and generalized polarization approaches,” Methods Appl. Fluoresc. 3(4), 047001 (2015).
[Crossref]

L. M. Andrews, M. R. Jones, M. A. Digman, and E. Gratton, “Detecting Pyronin Y labeled RNA transcripts in live cell microenvironments by phasor-FLIM analysis,” Methods Appl. Fluoresc. 1(1), 015001 (2013).
[Crossref] [PubMed]

F. Cutrale, A. Salih, and E. Gratton, “Spectral Phasor approach for fingerprinting of photo-activatable fluorescent proteins Dronpa, Kaede and KikGR,” Methods Appl. Fluoresc. 1(3), 035001 (2013).
[Crossref] [PubMed]

V. Crosignani, A. S. Dvornikov, and E. Gratton, “Enhancement of imaging depth in turbid media using a wide area detector,” J. Biophotonics 4(9), 592–599 (2011).
[PubMed]

Jameson, D. M.

L. Malacrida, D. M. Jameson, and E. Gratton, “A multidimensional phasor approach reveals LAURDAN photophysics in NIH-3T3 cell membranes,” Sci. Rep. 7(1), 9215 (2017).
[Crossref] [PubMed]

L. Malacrida, E. Gratton, and D. M. Jameson, “Model-free methods to study membrane environmental probes: a comparison of the spectral phasor and generalized polarization approaches,” Methods Appl. Fluoresc. 3(4), 047001 (2015).
[Crossref]

L. Malacrida, E. Gratton, and D. M. Jameson, “Model-free methods to study membrane environmental probes: a comparison of the spectral phasor and generalized polarization approaches,” Methods Appl. Fluoresc. 3(4), 047001 (2015).
[Crossref]

Jones, M. R.

L. M. Andrews, M. R. Jones, M. A. Digman, and E. Gratton, “Detecting Pyronin Y labeled RNA transcripts in live cell microenvironments by phasor-FLIM analysis,” Methods Appl. Fluoresc. 1(1), 015001 (2013).
[Crossref] [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(20), 4833–4840 (2012).
[Crossref] [PubMed]

Lavagnino, Z.

Z. Lavagnino, J. Dwight, A. Ustione, T. U. Nguyen, T. S. Tkaczyk, and D. W. Piston, “Snapshot Hyperspectral Light-Sheet Imaging of Signal Transduction in Live Pancreatic Islets,” Biophys. J. 111(2), 409–417 (2016).
[Crossref] [PubMed]

Levi, M.

Malacrida, L.

L. Malacrida, D. M. Jameson, and E. Gratton, “A multidimensional phasor approach reveals LAURDAN photophysics in NIH-3T3 cell membranes,” Sci. Rep. 7(1), 9215 (2017).
[Crossref] [PubMed]

L. Malacrida, S. Astrada, A. Briva, M. Bollati-Fogolín, E. Gratton, and L. A. Bagatolli, “Spectral phasor analysis of LAURDAN fluorescence in live A549 lung cells to study the hydration and time evolution of intracellular lamellar body-like structures,” Biochim. Biophys. Acta 1858(11), 2625–2635 (2016).
[Crossref] [PubMed]

L. Malacrida, E. Gratton, and D. M. Jameson, “Model-free methods to study membrane environmental probes: a comparison of the spectral phasor and generalized polarization approaches,” Methods Appl. Fluoresc. 3(4), 047001 (2015).
[Crossref]

L. Malacrida, E. Gratton, and D. M. Jameson, “Model-free methods to study membrane environmental probes: a comparison of the spectral phasor and generalized polarization approaches,” Methods Appl. Fluoresc. 3(4), 047001 (2015).
[Crossref]

Nguyen, T. U.

Z. Lavagnino, J. Dwight, A. Ustione, T. U. Nguyen, T. S. Tkaczyk, and D. W. Piston, “Snapshot Hyperspectral Light-Sheet Imaging of Signal Transduction in Live Pancreatic Islets,” Biophys. J. 111(2), 409–417 (2016).
[Crossref] [PubMed]

Piston, D. W.

Z. Lavagnino, J. Dwight, A. Ustione, T. U. Nguyen, T. S. Tkaczyk, and D. W. Piston, “Snapshot Hyperspectral Light-Sheet Imaging of Signal Transduction in Live Pancreatic Islets,” Biophys. J. 111(2), 409–417 (2016).
[Crossref] [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(20), 4833–4840 (2012).
[Crossref] [PubMed]

Ranjit, S.

Salih, A.

F. Cutrale, A. Salih, and E. Gratton, “Spectral Phasor approach for fingerprinting of photo-activatable fluorescent proteins Dronpa, Kaede and KikGR,” Methods Appl. Fluoresc. 1(3), 035001 (2013).
[Crossref] [PubMed]

Tkaczyk, T. S.

Z. Lavagnino, J. Dwight, A. Ustione, T. U. Nguyen, T. S. Tkaczyk, and D. W. Piston, “Snapshot Hyperspectral Light-Sheet Imaging of Signal Transduction in Live Pancreatic Islets,” Biophys. J. 111(2), 409–417 (2016).
[Crossref] [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(20), 4833–4840 (2012).
[Crossref] [PubMed]

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).
[Crossref] [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).
[Crossref] [PubMed]

Turcatel, G.

Ustione, A.

Z. Lavagnino, J. Dwight, A. Ustione, T. U. Nguyen, T. S. Tkaczyk, and D. W. Piston, “Snapshot Hyperspectral Light-Sheet Imaging of Signal Transduction in Live Pancreatic Islets,” Biophys. J. 111(2), 409–417 (2016).
[Crossref] [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(20), 4833–4840 (2012).
[Crossref] [PubMed]

Wang, P.

Warburton, D.

Biochim. Biophys. Acta (1)

L. Malacrida, S. Astrada, A. Briva, M. Bollati-Fogolín, E. Gratton, and L. A. Bagatolli, “Spectral phasor analysis of LAURDAN fluorescence in live A549 lung cells to study the hydration and time evolution of intracellular lamellar body-like structures,” Biochim. Biophys. Acta 1858(11), 2625–2635 (2016).
[Crossref] [PubMed]

Biomed. Opt. Express (2)

Biophys. J. (1)

Z. Lavagnino, J. Dwight, A. Ustione, T. U. Nguyen, T. S. Tkaczyk, and D. W. Piston, “Snapshot Hyperspectral Light-Sheet Imaging of Signal Transduction in Live Pancreatic Islets,” Biophys. J. 111(2), 409–417 (2016).
[Crossref] [PubMed]

J. Biophotonics (2)

V. Crosignani, A. S. Dvornikov, and E. Gratton, “Enhancement of imaging depth in turbid media using a wide area detector,” J. Biophotonics 4(9), 592–599 (2011).
[PubMed]

F. Fereidouni, A. N. Bader, A. Colonna, and H. C. Gerritsen, “Phasor analysis of multiphoton spectral images distinguishes autofluorescence components of in vivo human skin,” J. Biophotonics 7(8), 589–596 (2014).
[Crossref] [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(20), 4833–4840 (2012).
[Crossref] [PubMed]

Methods Appl. Fluoresc. (4)

L. Malacrida, E. Gratton, and D. M. Jameson, “Model-free methods to study membrane environmental probes: a comparison of the spectral phasor and generalized polarization approaches,” Methods Appl. Fluoresc. 3(4), 047001 (2015).
[Crossref]

L. Malacrida, E. Gratton, and D. M. Jameson, “Model-free methods to study membrane environmental probes: a comparison of the spectral phasor and generalized polarization approaches,” Methods Appl. Fluoresc. 3(4), 047001 (2015).
[Crossref]

L. M. Andrews, M. R. Jones, M. A. Digman, and E. Gratton, “Detecting Pyronin Y labeled RNA transcripts in live cell microenvironments by phasor-FLIM analysis,” Methods Appl. Fluoresc. 1(1), 015001 (2013).
[Crossref] [PubMed]

F. Cutrale, A. Salih, and E. Gratton, “Spectral Phasor approach for fingerprinting of photo-activatable fluorescent proteins Dronpa, Kaede and KikGR,” Methods Appl. Fluoresc. 1(3), 035001 (2013).
[Crossref] [PubMed]

Nat. Methods (1)

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).
[Crossref] [PubMed]

Opt. Express (1)

Sci. Rep. (1)

L. Malacrida, D. M. Jameson, and E. Gratton, “A multidimensional phasor approach reveals LAURDAN photophysics in NIH-3T3 cell membranes,” Sci. Rep. 7(1), 9215 (2017).
[Crossref] [PubMed]

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Figures (8)

Fig. 1
Fig. 1 Cos-sin filters transmission. A) The filters transmission used to obtain the first harmonic of the spectral bandwidth. Outside the region 400nm 600nm the transmission is zero with the addition of bandpass filters. B) The output of the filters is normalized and shifted to give the range of the cosine and sine function. C) The phasor representation of the G and S function obtained with these filters. Note that the polar phasor plot of the filters deviates from the perfect cosine and sine functions (should be a circle).
Fig. 2
Fig. 2 Correction for the non-ideal response of the filters. A) Correction factors for the phase and amplitude of the filter obtained by comparing the measured response with the ideal response of the cos-sin functions. The phase measured at a given wavelength is compared with the theoretical value (red arrow). For the theoretical value of the phase, the correction factor of the modulation is read from the graph. B) After the correction is applied, the filters have the ideal response. C) The phasor representation of the filter after correction is now a circle, as it should be for a perfect (cos-sin) filter combination.
Fig. 3
Fig. 3 Experimental setup. The detailed description of the DIVER microscope can be found in [13]. For the demonstration, we show the microscope objective, a container with fluorescence solutions (Sample), a transparent block or scattering block that we insert between the sample and the detector and the position of the Cyan (cos) and Green (sin) filters. The filters were inserted in a rotating filter wheel placed inside the sealed chamber filled with the index matching fluid. Matching refractive index in the optical path from a sample to the detector allows minimizing photon losses due to reflections. For cos-sin filters calibration we used collimated light in the 400-600 nm range, which was generated by passing tungsten lamp light through the monochromator (in this case the objective lens and the sample were removed). For two-photon imaging, the Spectra-Physics InSight DS + femtosecond laser coupled with the scanning system was used as an excitation light source.
Fig. 4
Fig. 4 Calibration of the spectral phasor in the DIVER microscope for a transparent and scattering sample. The expected correlation is perfect for the transparent media and has a very slight deviation due to scattering for the strongly scattering sample.
Fig. 5
Fig. 5 Measurement of the spectral phasor of 6 dyes. The dyes spectral phasor position recovered using a hyperspectral device (Zeiss LSM 710 with 32 channels spectral detector) and the spectral phasor position recovered by the cos-sin filter method. A) Coumarin 1 /EtOH, 445nm; B) ECFP /buffer, 477nm; C) Coumarin 1/ MeOH, 504nm; D) Rhodamine 110 /water, 520nm; E) Rhodamine 6G/EtOH, 552nm, F) Rhodamine B /water, 576nm.
Fig. 6
Fig. 6 Spectral phasors of 3 dyes measured with and without the scattering slab. A) Coumarin 1 /EtOH, 445nm; B) Coumarin 1/MeOH, 504nm; C) Rhodamine 6G/EtOH, 552nm. The presence of the scattering slab gives the same spectral phasor as with the transparent slab.
Fig. 7
Fig. 7 Linear combination of molecular species. Measurement of spectral phasor for a mixture of Coumarin 1 and Rhodamine 6G ethanol solutions. Concentrations of pure dye solutions were adjusted to yield approximately the same fluorescence signal under two-photon excitation at 740nm. Mixtures containing 0, 25, 50, 75 and 100% of pure dye solution we used for measurements. A) Linear combination of phasor positions obtained using the cos-sin filters and B) linear combination using the lifetime phasor approach. According to phasor linear combination law, phasors of mixed solutions should be positioned on the line connecting phasors of pure solutions. This is demonstrated for both spectral (A) and lifetime (B) phasors measured for dyes mixtures.
Fig. 8
Fig. 8 Comparison of spectral phasor and hyperspectral images of a convallaria sample. A) spectral phasor image obtained with the cos-sin filter in the DIVER microscope. B) Hyperspectral image of the same sample obtained with the wavelength detector of the Zeiss 710 Confocal microscope equipped with the 32-spectral detector. The sample was excited at 740nm using two photon excitation in both cases.

Equations (8)

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G= λ I(λ)*cos(n*2π*λ/Δλ)/ λ I(λ)
S= λ I(λ)*sin(n*2π*λ/Δλ)/ λ I(λ)
F (λ) norm =2*( F cos (λ) F cosMIN )/( F cosMAX F cosMIN )1
I cos =( λ F cos (λ)*I(λ))/ λ I(λ)
G=2( I cos F cosMIN )/( F cosMAX F cosMIN )1
G 1 = λ I 1 (λ)* I 1cos (λ)/I(λ)
G 2 = λ I 2 (λ)* I 1cos (λ)/I(λ)
G= λ I(λ) I cos (λ) λ I(λ) = λ f 1 I 1cos (λ) λ I(λ) + λ f 2 I 2cos (λ) λ I(λ) = f 1 G 1 +( 1 f 1 ) G 2