Abstract

We have developed a new, high performance, hyperspectral microscope for biological and other applications. For each voxel within a three-dimensional specimen, the microscope simultaneously records the emission spectrum from 500  nm to 800   nm, with better than 3   nm spectral resolution. The microscope features a fully confocal design to ensure high spatial resolution and high quality optical sectioning. Optical throughput and detection efficiency are maximized through the use of a custom prism spectrometer and a backside thinned electron multiplying charge coupled device (EMCCD) array. A custom readout mode and synchronization scheme enable 512-point spectra to be recorded at a rate of 8300 spectra per second. In addition, the EMCCD readout mode eliminates curvature and keystone artifacts that often plague spectral imaging systems. The architecture of the new microscope is described in detail, and hyperspectral images from several specimens are presented.

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  26. If two (or more) emitting species occur with the same relative concentrations in every pixel of the image, the multivariate analysis will find a single pure component whose spectrum is an admixture of the spectra of the covarying species.
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2005

C. Buehler, K. H. Kim, U. Greuter, N. Schlumpf, and P. T. C. So, "Single-photon counting multicolor multiphoton fluorescence microscope," J. Fluoresc. 15, 41-51 (2005).
[CrossRef] [PubMed]

I. Pelletier, C. Pellerin, D. B. Chase, and J. F. Rabolt, "New developments in planar array infrared spectroscopy," Appl. Spectrosc. 59, 156-163 (2005).
[CrossRef] [PubMed]

2004

M. R. Keenan and P. G. Kotula, "Accounting for Poisson noise in the multivariate analysis of ToF-SIMS spectrum images," Surf. Interface Anal. 36, 203-212 (2004).
[CrossRef]

M. B. Sinclair, J. A. Timlin, D. M. Haaland, and M. Werner-Washburne, "Design, construction, characterization, and application of a hyperspectral microarray scanner," Appl. Opt. 43, 2079-2088 (2004).
[CrossRef] [PubMed]

R. A. Neville, L. X. Sun, and K. Staenz, "Detection of keystone in imaging spectrometer data," in Algorithms and Technologies for MultiSpectral, Hyperspectral, and Ultraspectral Imagery X, S. S. Shen and P. E. Lewis, eds., Proc. SPIE 5425, 208-217 (2004).
[CrossRef]

2003

V. Seyfreid, H. Birk, R. Storz, and H. Ulrich, "Advances in multispectral confocal imaging," in Confocal, Multiphoton, and Nonlinear Microscopic Imaging, T. Wilson, ed., Proc. SPIE 5139, 147-157 (2003).

D. M. Haaland, J. A. Timlin, M. B. Sinclair, and M. H. Van Benthem, M. J. Martinez, A. D. Aragon, and M. Werner-Washburne, "Multivariate curve resolution For hyperspectral image analysis:applications to microarray technology," in Spectral Imaging:Instrumentation, Applications, and Analysis, R. M. Levenson, G. H. Bearman, and A. Mahadevan-Jensen, eds., Proc. SPIE 4959, 55-66 (2003).
[CrossRef]

P. G. Kotula, M. R. Keenan, and J. R. Michael, "Automated analysis of SEM X-ray spectral images:a powerful new microanalysis tool," Microsc. Microanal. 9, 1-17 (2003).
[CrossRef] [PubMed]

C. D. Mackay, J. E. Baldwin, and R. N. Tubbs, "Noise free detectors in the visible and infrared:implications for the design of next-generation AO systems and large telescopes," in Future Giant Telescopes, J. R. P. Angel and R. Gilmozzi eds. Proc. SPIE 4840, 436-442 (2003).

2002

M. E. Dickinson, C. W. Waters, G. Bearman, R. Wolleschensky, S. Tille, and S. E. Fraser, "Sensitive imaging of spectrally overlapping fluorochromes using the LSM 510 Meta," in Multiphoton Microscopy in the Biomedical Sciences II, P. T. C. So, ed., Proc. SPIE 4620, 123-136 (2002).

2001

M. Ikeuchi and S. Tabata, "Synechocystis sp PCC 6803: a useful tool in the study of the genetics of cyanobacteria," Photosynth. Res. 70, 73-83 (2001).
[CrossRef]

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

1999

M. J. Stimson, N. Haralampus-Grynaviski, and J. D. Simon, "A unique optical arrangement for obtaining spectrally resolved confocal images," Rev. Sci. Instrum. 70, 3351-3354 (1999).
[CrossRef]

1997

D. W. Warren, J. A. Hackwell, and D. J. Gutierrez, "Compact prism spectrograph based on aplanatic principles," Opt. Eng. 36, 1174-1182 (1997).
[CrossRef]

R. Bro and S. DeJong, "A fast non-negativity-constrained least squares algorithm," J. Chemometrics 11, 393-401 (1997).
[CrossRef]

1996

R. H. Webb, "Confocal optical microscopy," Rep. Prog. Phys. 59, 427-471 (1996).
[CrossRef]

1980

Aragon, A. D.

D. M. Haaland, J. A. Timlin, M. B. Sinclair, and M. H. Van Benthem, M. J. Martinez, A. D. Aragon, and M. Werner-Washburne, "Multivariate curve resolution For hyperspectral image analysis:applications to microarray technology," in Spectral Imaging:Instrumentation, Applications, and Analysis, R. M. Levenson, G. H. Bearman, and A. Mahadevan-Jensen, eds., Proc. SPIE 4959, 55-66 (2003).
[CrossRef]

Baldwin, J. E.

C. D. Mackay, J. E. Baldwin, and R. N. Tubbs, "Noise free detectors in the visible and infrared:implications for the design of next-generation AO systems and large telescopes," in Future Giant Telescopes, J. R. P. Angel and R. Gilmozzi eds. Proc. SPIE 4840, 436-442 (2003).

Bearman, G.

M. E. Dickinson, C. W. Waters, G. Bearman, R. Wolleschensky, S. Tille, and S. E. Fraser, "Sensitive imaging of spectrally overlapping fluorochromes using the LSM 510 Meta," in Multiphoton Microscopy in the Biomedical Sciences II, P. T. C. So, ed., Proc. SPIE 4620, 123-136 (2002).

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

Birk, H.

V. Seyfreid, H. Birk, R. Storz, and H. Ulrich, "Advances in multispectral confocal imaging," in Confocal, Multiphoton, and Nonlinear Microscopic Imaging, T. Wilson, ed., Proc. SPIE 5139, 147-157 (2003).

Bro, R.

R. Bro and S. DeJong, "A fast non-negativity-constrained least squares algorithm," J. Chemometrics 11, 393-401 (1997).
[CrossRef]

Buehler, C.

C. Buehler, K. H. Kim, U. Greuter, N. Schlumpf, and P. T. C. So, "Single-photon counting multicolor multiphoton fluorescence microscope," J. Fluoresc. 15, 41-51 (2005).
[CrossRef] [PubMed]

Chase, D. B.

DeJong, S.

R. Bro and S. DeJong, "A fast non-negativity-constrained least squares algorithm," J. Chemometrics 11, 393-401 (1997).
[CrossRef]

Dickinson, M. E.

M. E. Dickinson, C. W. Waters, G. Bearman, R. Wolleschensky, S. Tille, and S. E. Fraser, "Sensitive imaging of spectrally overlapping fluorochromes using the LSM 510 Meta," in Multiphoton Microscopy in the Biomedical Sciences II, P. T. C. So, ed., Proc. SPIE 4620, 123-136 (2002).

Easterling, R. G.

Fraser, S. E.

M. E. Dickinson, C. W. Waters, G. Bearman, R. Wolleschensky, S. Tille, and S. E. Fraser, "Sensitive imaging of spectrally overlapping fluorochromes using the LSM 510 Meta," in Multiphoton Microscopy in the Biomedical Sciences II, P. T. C. So, ed., Proc. SPIE 4620, 123-136 (2002).

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

Greuter, U.

C. Buehler, K. H. Kim, U. Greuter, N. Schlumpf, and P. T. C. So, "Single-photon counting multicolor multiphoton fluorescence microscope," J. Fluoresc. 15, 41-51 (2005).
[CrossRef] [PubMed]

Gutierrez, D. J.

D. W. Warren, J. A. Hackwell, and D. J. Gutierrez, "Compact prism spectrograph based on aplanatic principles," Opt. Eng. 36, 1174-1182 (1997).
[CrossRef]

Haaland, D. M.

M. B. Sinclair, J. A. Timlin, D. M. Haaland, and M. Werner-Washburne, "Design, construction, characterization, and application of a hyperspectral microarray scanner," Appl. Opt. 43, 2079-2088 (2004).
[CrossRef] [PubMed]

D. M. Haaland, J. A. Timlin, M. B. Sinclair, and M. H. Van Benthem, M. J. Martinez, A. D. Aragon, and M. Werner-Washburne, "Multivariate curve resolution For hyperspectral image analysis:applications to microarray technology," in Spectral Imaging:Instrumentation, Applications, and Analysis, R. M. Levenson, G. H. Bearman, and A. Mahadevan-Jensen, eds., Proc. SPIE 4959, 55-66 (2003).
[CrossRef]

D. M. Haaland and R. G. Easterling, "Improved sensitivity of infrared spectroscopy by the application of least squares methods," Appl. Spectrosc. 34, 539-548 (1980).
[CrossRef]

Hackwell, J. A.

D. W. Warren, J. A. Hackwell, and D. J. Gutierrez, "Compact prism spectrograph based on aplanatic principles," Opt. Eng. 36, 1174-1182 (1997).
[CrossRef]

Haralampus-Grynaviski, N.

M. J. Stimson, N. Haralampus-Grynaviski, and J. D. Simon, "A unique optical arrangement for obtaining spectrally resolved confocal images," Rev. Sci. Instrum. 70, 3351-3354 (1999).
[CrossRef]

Ikeuchi, M.

M. Ikeuchi and S. Tabata, "Synechocystis sp PCC 6803: a useful tool in the study of the genetics of cyanobacteria," Photosynth. Res. 70, 73-83 (2001).
[CrossRef]

Keenan, M. R.

M. R. Keenan and P. G. Kotula, "Accounting for Poisson noise in the multivariate analysis of ToF-SIMS spectrum images," Surf. Interface Anal. 36, 203-212 (2004).
[CrossRef]

P. G. Kotula, M. R. Keenan, and J. R. Michael, "Automated analysis of SEM X-ray spectral images:a powerful new microanalysis tool," Microsc. Microanal. 9, 1-17 (2003).
[CrossRef] [PubMed]

Kim, K. H.

C. Buehler, K. H. Kim, U. Greuter, N. Schlumpf, and P. T. C. So, "Single-photon counting multicolor multiphoton fluorescence microscope," J. Fluoresc. 15, 41-51 (2005).
[CrossRef] [PubMed]

Kotula, P. G.

M. R. Keenan and P. G. Kotula, "Accounting for Poisson noise in the multivariate analysis of ToF-SIMS spectrum images," Surf. Interface Anal. 36, 203-212 (2004).
[CrossRef]

P. G. Kotula, M. R. Keenan, and J. R. Michael, "Automated analysis of SEM X-ray spectral images:a powerful new microanalysis tool," Microsc. Microanal. 9, 1-17 (2003).
[CrossRef] [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," J. Biomed. Opt. 6, 311-318 (2001).
[CrossRef] [PubMed]

Mackay, C. D.

C. D. Mackay, J. E. Baldwin, and R. N. Tubbs, "Noise free detectors in the visible and infrared:implications for the design of next-generation AO systems and large telescopes," in Future Giant Telescopes, J. R. P. Angel and R. Gilmozzi eds. Proc. SPIE 4840, 436-442 (2003).

Martinez, M. J.

D. M. Haaland, J. A. Timlin, M. B. Sinclair, and M. H. Van Benthem, M. J. Martinez, A. D. Aragon, and M. Werner-Washburne, "Multivariate curve resolution For hyperspectral image analysis:applications to microarray technology," in Spectral Imaging:Instrumentation, Applications, and Analysis, R. M. Levenson, G. H. Bearman, and A. Mahadevan-Jensen, eds., Proc. SPIE 4959, 55-66 (2003).
[CrossRef]

Michael, J. R.

P. G. Kotula, M. R. Keenan, and J. R. Michael, "Automated analysis of SEM X-ray spectral images:a powerful new microanalysis tool," Microsc. Microanal. 9, 1-17 (2003).
[CrossRef] [PubMed]

Neville, R. A.

R. A. Neville, L. X. Sun, and K. Staenz, "Detection of keystone in imaging spectrometer data," in Algorithms and Technologies for MultiSpectral, Hyperspectral, and Ultraspectral Imagery X, S. S. Shen and P. E. Lewis, eds., Proc. SPIE 5425, 208-217 (2004).
[CrossRef]

Pellerin, C.

Pelletier, I.

Rabolt, J. F.

Schlumpf, N.

C. Buehler, K. H. Kim, U. Greuter, N. Schlumpf, and P. T. C. So, "Single-photon counting multicolor multiphoton fluorescence microscope," J. Fluoresc. 15, 41-51 (2005).
[CrossRef] [PubMed]

Seyfreid, V.

V. Seyfreid, H. Birk, R. Storz, and H. Ulrich, "Advances in multispectral confocal imaging," in Confocal, Multiphoton, and Nonlinear Microscopic Imaging, T. Wilson, ed., Proc. SPIE 5139, 147-157 (2003).

Simon, J. D.

M. J. Stimson, N. Haralampus-Grynaviski, and J. D. Simon, "A unique optical arrangement for obtaining spectrally resolved confocal images," Rev. Sci. Instrum. 70, 3351-3354 (1999).
[CrossRef]

Sinclair, M. B.

M. B. Sinclair, J. A. Timlin, D. M. Haaland, and M. Werner-Washburne, "Design, construction, characterization, and application of a hyperspectral microarray scanner," Appl. Opt. 43, 2079-2088 (2004).
[CrossRef] [PubMed]

D. M. Haaland, J. A. Timlin, M. B. Sinclair, and M. H. Van Benthem, M. J. Martinez, A. D. Aragon, and M. Werner-Washburne, "Multivariate curve resolution For hyperspectral image analysis:applications to microarray technology," in Spectral Imaging:Instrumentation, Applications, and Analysis, R. M. Levenson, G. H. Bearman, and A. Mahadevan-Jensen, eds., Proc. SPIE 4959, 55-66 (2003).
[CrossRef]

So, P. T. C.

C. Buehler, K. H. Kim, U. Greuter, N. Schlumpf, and P. T. C. So, "Single-photon counting multicolor multiphoton fluorescence microscope," J. Fluoresc. 15, 41-51 (2005).
[CrossRef] [PubMed]

Staenz, K.

R. A. Neville, L. X. Sun, and K. Staenz, "Detection of keystone in imaging spectrometer data," in Algorithms and Technologies for MultiSpectral, Hyperspectral, and Ultraspectral Imagery X, S. S. Shen and P. E. Lewis, eds., Proc. SPIE 5425, 208-217 (2004).
[CrossRef]

Stimson, M. J.

M. J. Stimson, N. Haralampus-Grynaviski, and J. D. Simon, "A unique optical arrangement for obtaining spectrally resolved confocal images," Rev. Sci. Instrum. 70, 3351-3354 (1999).
[CrossRef]

Storz, R.

V. Seyfreid, H. Birk, R. Storz, and H. Ulrich, "Advances in multispectral confocal imaging," in Confocal, Multiphoton, and Nonlinear Microscopic Imaging, T. Wilson, ed., Proc. SPIE 5139, 147-157 (2003).

Sun, L. X.

R. A. Neville, L. X. Sun, and K. Staenz, "Detection of keystone in imaging spectrometer data," in Algorithms and Technologies for MultiSpectral, Hyperspectral, and Ultraspectral Imagery X, S. S. Shen and P. E. Lewis, eds., Proc. SPIE 5425, 208-217 (2004).
[CrossRef]

Tabata, S.

M. Ikeuchi and S. Tabata, "Synechocystis sp PCC 6803: a useful tool in the study of the genetics of cyanobacteria," Photosynth. Res. 70, 73-83 (2001).
[CrossRef]

Tille, S.

M. E. Dickinson, C. W. Waters, G. Bearman, R. Wolleschensky, S. Tille, and S. E. Fraser, "Sensitive imaging of spectrally overlapping fluorochromes using the LSM 510 Meta," in Multiphoton Microscopy in the Biomedical Sciences II, P. T. C. So, ed., Proc. SPIE 4620, 123-136 (2002).

Timlin, J. A.

M. B. Sinclair, J. A. Timlin, D. M. Haaland, and M. Werner-Washburne, "Design, construction, characterization, and application of a hyperspectral microarray scanner," Appl. Opt. 43, 2079-2088 (2004).
[CrossRef] [PubMed]

D. M. Haaland, J. A. Timlin, M. B. Sinclair, and M. H. Van Benthem, M. J. Martinez, A. D. Aragon, and M. Werner-Washburne, "Multivariate curve resolution For hyperspectral image analysis:applications to microarray technology," in Spectral Imaging:Instrumentation, Applications, and Analysis, R. M. Levenson, G. H. Bearman, and A. Mahadevan-Jensen, eds., Proc. SPIE 4959, 55-66 (2003).
[CrossRef]

Tubbs, R. N.

C. D. Mackay, J. E. Baldwin, and R. N. Tubbs, "Noise free detectors in the visible and infrared:implications for the design of next-generation AO systems and large telescopes," in Future Giant Telescopes, J. R. P. Angel and R. Gilmozzi eds. Proc. SPIE 4840, 436-442 (2003).

Ulrich, H.

V. Seyfreid, H. Birk, R. Storz, and H. Ulrich, "Advances in multispectral confocal imaging," in Confocal, Multiphoton, and Nonlinear Microscopic Imaging, T. Wilson, ed., Proc. SPIE 5139, 147-157 (2003).

Van Benthem, M. H.

D. M. Haaland, J. A. Timlin, M. B. Sinclair, and M. H. Van Benthem, M. J. Martinez, A. D. Aragon, and M. Werner-Washburne, "Multivariate curve resolution For hyperspectral image analysis:applications to microarray technology," in Spectral Imaging:Instrumentation, Applications, and Analysis, R. M. Levenson, G. H. Bearman, and A. Mahadevan-Jensen, eds., Proc. SPIE 4959, 55-66 (2003).
[CrossRef]

Warren, D. W.

D. W. Warren, J. A. Hackwell, and D. J. Gutierrez, "Compact prism spectrograph based on aplanatic principles," Opt. Eng. 36, 1174-1182 (1997).
[CrossRef]

Waters, C. W.

M. E. Dickinson, C. W. Waters, G. Bearman, R. Wolleschensky, S. Tille, and S. E. Fraser, "Sensitive imaging of spectrally overlapping fluorochromes using the LSM 510 Meta," in Multiphoton Microscopy in the Biomedical Sciences II, P. T. C. So, ed., Proc. SPIE 4620, 123-136 (2002).

Webb, R. H.

R. H. Webb, "Confocal optical microscopy," Rep. Prog. Phys. 59, 427-471 (1996).
[CrossRef]

Werner-Washburne, M.

M. B. Sinclair, J. A. Timlin, D. M. Haaland, and M. Werner-Washburne, "Design, construction, characterization, and application of a hyperspectral microarray scanner," Appl. Opt. 43, 2079-2088 (2004).
[CrossRef] [PubMed]

D. M. Haaland, J. A. Timlin, M. B. Sinclair, and M. H. Van Benthem, M. J. Martinez, A. D. Aragon, and M. Werner-Washburne, "Multivariate curve resolution For hyperspectral image analysis:applications to microarray technology," in Spectral Imaging:Instrumentation, Applications, and Analysis, R. M. Levenson, G. H. Bearman, and A. Mahadevan-Jensen, eds., Proc. SPIE 4959, 55-66 (2003).
[CrossRef]

Wolleschensky, R.

M. E. Dickinson, C. W. Waters, G. Bearman, R. Wolleschensky, S. Tille, and S. E. Fraser, "Sensitive imaging of spectrally overlapping fluorochromes using the LSM 510 Meta," in Multiphoton Microscopy in the Biomedical Sciences II, P. T. C. So, ed., Proc. SPIE 4620, 123-136 (2002).

Appl. Opt.

Appl. Spectrosc.

J. Biomed. Opt.

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

J. Chemometrics

R. Bro and S. DeJong, "A fast non-negativity-constrained least squares algorithm," J. Chemometrics 11, 393-401 (1997).
[CrossRef]

J. Fluoresc.

C. Buehler, K. H. Kim, U. Greuter, N. Schlumpf, and P. T. C. So, "Single-photon counting multicolor multiphoton fluorescence microscope," J. Fluoresc. 15, 41-51 (2005).
[CrossRef] [PubMed]

Microsc. Microanal.

P. G. Kotula, M. R. Keenan, and J. R. Michael, "Automated analysis of SEM X-ray spectral images:a powerful new microanalysis tool," Microsc. Microanal. 9, 1-17 (2003).
[CrossRef] [PubMed]

Opt. Eng.

D. W. Warren, J. A. Hackwell, and D. J. Gutierrez, "Compact prism spectrograph based on aplanatic principles," Opt. Eng. 36, 1174-1182 (1997).
[CrossRef]

Photosynth. Res.

M. Ikeuchi and S. Tabata, "Synechocystis sp PCC 6803: a useful tool in the study of the genetics of cyanobacteria," Photosynth. Res. 70, 73-83 (2001).
[CrossRef]

Proc. SPIE

R. A. Neville, L. X. Sun, and K. Staenz, "Detection of keystone in imaging spectrometer data," in Algorithms and Technologies for MultiSpectral, Hyperspectral, and Ultraspectral Imagery X, S. S. Shen and P. E. Lewis, eds., Proc. SPIE 5425, 208-217 (2004).
[CrossRef]

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If two (or more) emitting species occur with the same relative concentrations in every pixel of the image, the multivariate analysis will find a single pure component whose spectrum is an admixture of the spectra of the covarying species.

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Figures (9)

Fig. 1
Fig. 1

Schematic diagram of the layout of the hyperspectral confocal microscope.

Fig. 2
Fig. 2

Optical raytrace of the aplanatic prism spectrometer.

Fig. 3
Fig. 3

Diagram showing the scan geometry for the hyperspectral confocal microscope. A galvanometer driven mirror scans the focused laser spot in the y direction, while a translation stage moves the specimen in the x direction.

Fig. 4
Fig. 4

Schematic illustration of the EMCCD readout mode developed for the hyperspectral confocal microscope. The EMCCD array is illuminated within a small vertical range immediately adjacent to the frame transfer region of the detector. The photocharge generated during a given exposure interval is sequentially shifted down with successive trigger pulses until it is shifted into the serial register for readout.

Fig. 5
Fig. 5

(a) Small portion of a hyperspectral image stack, containing the image of a 0.17 μm diameter fluorescent microsphere. The pixels of this image were recorded at 0.12 μm intervals in the x and y directions. (b) The emission spectrum of the microsphere, as recorded in the brightest pixel in the hyperspectral data set. (c) A lateral intensity profile through the center of the microsphere, obtained from the in-focus frame of the three-dimensional image stack. (d) An axial intensity profile through the center of the microsphere, obtained from successive frames of the three-dimensional image stack.

Fig. 6
Fig. 6

(a) Sequence of gray-scale images derived from a hyperspectral image stack obtained using 2.5 μm diameter fluorescent microspheres. The frames were recorded with 0.3 μm increments in the axial direction and show the optical sectioning performance of the hyperspectral confocal microscope. (b) A single-pixel emission spectrum corresponding to one of the brighter pixels of the hyperspectral data set.

Fig. 7
Fig. 7

Hyperspectral image of 2.3 μm diameter silica microspheres labeled on their surface with four different fluorescent compounds (one type of tag per microsphere). The raw emission spectra of the five compounds are shown in the inset, with the arrows showing the microsphere from which each spectrum was obtained.

Fig. 8
Fig. 8

False color confocal image of a group of Synechocystis sp. PC 6803 cells. This image was constructed using the MCR-derived concentration maps of the four contributing fluorescent photosynthetic pigments. The insets show two raw single-pixel spectra along with the contributions of the pure-component spectra to the overall raw spectra.

Fig. 9
Fig. 9

Pure-component spectra obtained from MCR analysis of the Synechocystis data, normalized to unit peak height.

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