Abstract

Understanding the complexity of cellular biology often requires capturing and processing an enormous amount of data. In high-content drug screens, each cell is labeled with several different fluorescent markers and frequently thousands to millions of cells need to be analyzed in order to characterize biology’s intrinsic variability. In this work, we demonstrate a new microlens-based multispectral microscope designed to meet this throughput-intensive demand. We report multispectral image cubes of up to 1.30 gigapixels in the spatial domain, with up to 13 spectral samples per pixel, for a total image size of 16.8 billion spatial–spectral samples. To our knowledge, this is the largest multispectral microscopy dataset reported in the literature. Our system has highly reconfigurable spectral sampling and bandwidth settings, and we have demonstrated spectral unmixing of up to six fluorescent channels. This technology has the potential to speed up drug discovery by alleviating the imaging bottleneck in image-based assays.

© 2015 Optical Society of America

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References

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2014 (1)

2013 (4)

Q. Li, X. He, Y. Wang, H. Liu, D. Xu, and F. Guo, “Review of spectral imaging technology in biomedical engineering: achievements and challenges,” J. Biomed. Opt. 18, 100901 (2013).
[Crossref]

G. Di Caprio, D. Schaak, and E. Schonbrun, “Hyperspectral fluorescence microfluidic (HFM) microscopy,” Biomed. Opt. Express 4, 1486–1493 (2013).
[Crossref]

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A. Orth and K. Crozier, “Gigapixel fluorescence microscopy with a water immersion microlens array,” Opt. Express 21, 2361–2368 (2013).
[Crossref]

2012 (1)

2010 (3)

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, 14330–14344 (2010).
[Crossref]

F. Zanella, J. B. Lorens, and W. Link, “High content screening: seeing is believing,” Trends Biotechnol. 28, 237–245 (2010).
[Crossref]

M. Bickle, “The beautiful cell: high-content screening in drug discovery,” Anal. Bioanal. Chem. 398, 219–226 (2010).
[Crossref]

2009 (4)

J. Eid, A. Fehr, J. Gray, K. Luong, J. Lyle, G. Otto, P. Peluso, D. Rank, P. Baybayan, B. Bettman, A. Bibillo, K. Bjornson, B. Chaudhuri, F. Christians, R. Cicero, S. Clark, R. Dalal, A. deWinter, J. Dixon, M. Foquet, A. Gaertner, P. Hardenbol, C. Heiner, K. Hester, D. Holden, G. Kearns, X. Kong, R. Kuse, Y. Lacroix, S. Lin, P. Lundquist, C. Ma, P. Marks, M. Maxham, D. Murphy, I. Park, T. Pham, M. Phillips, J. Roy, R. Sebra, G. Shen, J. Sorenson, A. Tomaney, K. Travers, M. Trulson, J. Vieceli, J. Wegener, D. Wu, A. Yang, D. Zaccarin, P. Zhao, F. Zhong, J. Korlach, and S. Turner, “Real-time DNA sequencing from single polymerase molecules,” Science 323, 133–138 (2009).
[Crossref]

A. A. Wagadarikar, N. P. Pitsianis, X. Sun, and D. J. Brady, “Video rate spectral imaging using a coded aperture snapshot spectral imager,” Opt. Express 17, 6368–6388 (2009).
[Crossref]

E. Schonbrun, W. N. Ye, and K. B. Crozier, “Scanning microscopy using a short-focal-length Fresnel zone plate,” Opt. Lett. 34, 2228–2230 (2009).
[Crossref]

Y. Urano, D. Asanuma, Y. Hama, Y. Koyama, T. Barrett, M. Kamiya, T. Nagano, T. Watanabe, A. Hasegawa, P. L. Choyke, and H. Kobayashi, “Selective molecular imaging of viable cancer cells with pH-activatable fluorescence probes,” Nat. Med. 15, 104–109 (2009).
[Crossref]

2008 (3)

2007 (2)

M. E. Gehm, R. John, D. J. Brady, R. M. Willett, and T. J. Schulz, “Single-shot compressive spectral imaging with a dual-disperser architecture,” Opt. Express 15, 14013–14027 (2007).
[Crossref]

B. Kraus, M. Ziegler, and H. Wolff, “Linear fluorescence unmixing in cell biological research,” Mod. Res. Educ. Top. Microsc. 2, 863–873 (2007).

2002 (1)

2000 (1)

T. Scholzen and J. Gerdes, “The Ki-67 protein: from the known and the unknown,” J. Cell. Physiol. 182, 311–322 (2000).
[Crossref]

1994 (1)

Alterman, M.

M. Alterman, Y. Y. Schechner, and A. Weiss, “Multiplexed fluorescence unmixing,” in 2010 IEEE International Conference on Computational Photography (ICCP) (IEEE, 2010).

Asanuma, D.

Y. Urano, D. Asanuma, Y. Hama, Y. Koyama, T. Barrett, M. Kamiya, T. Nagano, T. Watanabe, A. Hasegawa, P. L. Choyke, and H. Kobayashi, “Selective molecular imaging of viable cancer cells with pH-activatable fluorescence probes,” Nat. Med. 15, 104–109 (2009).
[Crossref]

Barrett, T.

Y. Urano, D. Asanuma, Y. Hama, Y. Koyama, T. Barrett, M. Kamiya, T. Nagano, T. Watanabe, A. Hasegawa, P. L. Choyke, and H. Kobayashi, “Selective molecular imaging of viable cancer cells with pH-activatable fluorescence probes,” Nat. Med. 15, 104–109 (2009).
[Crossref]

Baybayan, P.

J. Eid, A. Fehr, J. Gray, K. Luong, J. Lyle, G. Otto, P. Peluso, D. Rank, P. Baybayan, B. Bettman, A. Bibillo, K. Bjornson, B. Chaudhuri, F. Christians, R. Cicero, S. Clark, R. Dalal, A. deWinter, J. Dixon, M. Foquet, A. Gaertner, P. Hardenbol, C. Heiner, K. Hester, D. Holden, G. Kearns, X. Kong, R. Kuse, Y. Lacroix, S. Lin, P. Lundquist, C. Ma, P. Marks, M. Maxham, D. Murphy, I. Park, T. Pham, M. Phillips, J. Roy, R. Sebra, G. Shen, J. Sorenson, A. Tomaney, K. Travers, M. Trulson, J. Vieceli, J. Wegener, D. Wu, A. Yang, D. Zaccarin, P. Zhao, F. Zhong, J. Korlach, and S. Turner, “Real-time DNA sequencing from single polymerase molecules,” Science 323, 133–138 (2009).
[Crossref]

Bettman, B.

J. Eid, A. Fehr, J. Gray, K. Luong, J. Lyle, G. Otto, P. Peluso, D. Rank, P. Baybayan, B. Bettman, A. Bibillo, K. Bjornson, B. Chaudhuri, F. Christians, R. Cicero, S. Clark, R. Dalal, A. deWinter, J. Dixon, M. Foquet, A. Gaertner, P. Hardenbol, C. Heiner, K. Hester, D. Holden, G. Kearns, X. Kong, R. Kuse, Y. Lacroix, S. Lin, P. Lundquist, C. Ma, P. Marks, M. Maxham, D. Murphy, I. Park, T. Pham, M. Phillips, J. Roy, R. Sebra, G. Shen, J. Sorenson, A. Tomaney, K. Travers, M. Trulson, J. Vieceli, J. Wegener, D. Wu, A. Yang, D. Zaccarin, P. Zhao, F. Zhong, J. Korlach, and S. Turner, “Real-time DNA sequencing from single polymerase molecules,” Science 323, 133–138 (2009).
[Crossref]

P. M. Lundquist, C. F. Zhong, P. Zhao, A. B. Tomaney, P. S. Peluso, J. Dixon, B. Bettman, Y. Lacroix, D. P. Kwo, E. McCullough, M. Maxham, K. Hester, P. McNitt, D. M. Grey, C. Henriquez, M. Foquet, S. W. Turner, and D. Zaccarin, “Parallel confocal detection of single molecules in real time,” Opt. Lett. 33, 1026–1028 (2008).
[Crossref]

Bibillo, A.

J. Eid, A. Fehr, J. Gray, K. Luong, J. Lyle, G. Otto, P. Peluso, D. Rank, P. Baybayan, B. Bettman, A. Bibillo, K. Bjornson, B. Chaudhuri, F. Christians, R. Cicero, S. Clark, R. Dalal, A. deWinter, J. Dixon, M. Foquet, A. Gaertner, P. Hardenbol, C. Heiner, K. Hester, D. Holden, G. Kearns, X. Kong, R. Kuse, Y. Lacroix, S. Lin, P. Lundquist, C. Ma, P. Marks, M. Maxham, D. Murphy, I. Park, T. Pham, M. Phillips, J. Roy, R. Sebra, G. Shen, J. Sorenson, A. Tomaney, K. Travers, M. Trulson, J. Vieceli, J. Wegener, D. Wu, A. Yang, D. Zaccarin, P. Zhao, F. Zhong, J. Korlach, and S. Turner, “Real-time DNA sequencing from single polymerase molecules,” Science 323, 133–138 (2009).
[Crossref]

Bickle, M.

M. Bickle, “The beautiful cell: high-content screening in drug discovery,” Anal. Bioanal. Chem. 398, 219–226 (2010).
[Crossref]

Bjornson, K.

J. Eid, A. Fehr, J. Gray, K. Luong, J. Lyle, G. Otto, P. Peluso, D. Rank, P. Baybayan, B. Bettman, A. Bibillo, K. Bjornson, B. Chaudhuri, F. Christians, R. Cicero, S. Clark, R. Dalal, A. deWinter, J. Dixon, M. Foquet, A. Gaertner, P. Hardenbol, C. Heiner, K. Hester, D. Holden, G. Kearns, X. Kong, R. Kuse, Y. Lacroix, S. Lin, P. Lundquist, C. Ma, P. Marks, M. Maxham, D. Murphy, I. Park, T. Pham, M. Phillips, J. Roy, R. Sebra, G. Shen, J. Sorenson, A. Tomaney, K. Travers, M. Trulson, J. Vieceli, J. Wegener, D. Wu, A. Yang, D. Zaccarin, P. Zhao, F. Zhong, J. Korlach, and S. Turner, “Real-time DNA sequencing from single polymerase molecules,” Science 323, 133–138 (2009).
[Crossref]

Brady, D.

Brady, D. J.

Chaudhuri, B.

J. Eid, A. Fehr, J. Gray, K. Luong, J. Lyle, G. Otto, P. Peluso, D. Rank, P. Baybayan, B. Bettman, A. Bibillo, K. Bjornson, B. Chaudhuri, F. Christians, R. Cicero, S. Clark, R. Dalal, A. deWinter, J. Dixon, M. Foquet, A. Gaertner, P. Hardenbol, C. Heiner, K. Hester, D. Holden, G. Kearns, X. Kong, R. Kuse, Y. Lacroix, S. Lin, P. Lundquist, C. Ma, P. Marks, M. Maxham, D. Murphy, I. Park, T. Pham, M. Phillips, J. Roy, R. Sebra, G. Shen, J. Sorenson, A. Tomaney, K. Travers, M. Trulson, J. Vieceli, J. Wegener, D. Wu, A. Yang, D. Zaccarin, P. Zhao, F. Zhong, J. Korlach, and S. Turner, “Real-time DNA sequencing from single polymerase molecules,” Science 323, 133–138 (2009).
[Crossref]

Choyke, P. L.

Y. Urano, D. Asanuma, Y. Hama, Y. Koyama, T. Barrett, M. Kamiya, T. Nagano, T. Watanabe, A. Hasegawa, P. L. Choyke, and H. Kobayashi, “Selective molecular imaging of viable cancer cells with pH-activatable fluorescence probes,” Nat. Med. 15, 104–109 (2009).
[Crossref]

Christians, F.

J. Eid, A. Fehr, J. Gray, K. Luong, J. Lyle, G. Otto, P. Peluso, D. Rank, P. Baybayan, B. Bettman, A. Bibillo, K. Bjornson, B. Chaudhuri, F. Christians, R. Cicero, S. Clark, R. Dalal, A. deWinter, J. Dixon, M. Foquet, A. Gaertner, P. Hardenbol, C. Heiner, K. Hester, D. Holden, G. Kearns, X. Kong, R. Kuse, Y. Lacroix, S. Lin, P. Lundquist, C. Ma, P. Marks, M. Maxham, D. Murphy, I. Park, T. Pham, M. Phillips, J. Roy, R. Sebra, G. Shen, J. Sorenson, A. Tomaney, K. Travers, M. Trulson, J. Vieceli, J. Wegener, D. Wu, A. Yang, D. Zaccarin, P. Zhao, F. Zhong, J. Korlach, and S. Turner, “Real-time DNA sequencing from single polymerase molecules,” Science 323, 133–138 (2009).
[Crossref]

Cicero, R.

J. Eid, A. Fehr, J. Gray, K. Luong, J. Lyle, G. Otto, P. Peluso, D. Rank, P. Baybayan, B. Bettman, A. Bibillo, K. Bjornson, B. Chaudhuri, F. Christians, R. Cicero, S. Clark, R. Dalal, A. deWinter, J. Dixon, M. Foquet, A. Gaertner, P. Hardenbol, C. Heiner, K. Hester, D. Holden, G. Kearns, X. Kong, R. Kuse, Y. Lacroix, S. Lin, P. Lundquist, C. Ma, P. Marks, M. Maxham, D. Murphy, I. Park, T. Pham, M. Phillips, J. Roy, R. Sebra, G. Shen, J. Sorenson, A. Tomaney, K. Travers, M. Trulson, J. Vieceli, J. Wegener, D. Wu, A. Yang, D. Zaccarin, P. Zhao, F. Zhong, J. Korlach, and S. Turner, “Real-time DNA sequencing from single polymerase molecules,” Science 323, 133–138 (2009).
[Crossref]

Clark, S.

J. Eid, A. Fehr, J. Gray, K. Luong, J. Lyle, G. Otto, P. Peluso, D. Rank, P. Baybayan, B. Bettman, A. Bibillo, K. Bjornson, B. Chaudhuri, F. Christians, R. Cicero, S. Clark, R. Dalal, A. deWinter, J. Dixon, M. Foquet, A. Gaertner, P. Hardenbol, C. Heiner, K. Hester, D. Holden, G. Kearns, X. Kong, R. Kuse, Y. Lacroix, S. Lin, P. Lundquist, C. Ma, P. Marks, M. Maxham, D. Murphy, I. Park, T. Pham, M. Phillips, J. Roy, R. Sebra, G. Shen, J. Sorenson, A. Tomaney, K. Travers, M. Trulson, J. Vieceli, J. Wegener, D. Wu, A. Yang, D. Zaccarin, P. Zhao, F. Zhong, J. Korlach, and S. Turner, “Real-time DNA sequencing from single polymerase molecules,” Science 323, 133–138 (2009).
[Crossref]

Crozier, K.

Crozier, K. B.

Dalal, R.

J. Eid, A. Fehr, J. Gray, K. Luong, J. Lyle, G. Otto, P. Peluso, D. Rank, P. Baybayan, B. Bettman, A. Bibillo, K. Bjornson, B. Chaudhuri, F. Christians, R. Cicero, S. Clark, R. Dalal, A. deWinter, J. Dixon, M. Foquet, A. Gaertner, P. Hardenbol, C. Heiner, K. Hester, D. Holden, G. Kearns, X. Kong, R. Kuse, Y. Lacroix, S. Lin, P. Lundquist, C. Ma, P. Marks, M. Maxham, D. Murphy, I. Park, T. Pham, M. Phillips, J. Roy, R. Sebra, G. Shen, J. Sorenson, A. Tomaney, K. Travers, M. Trulson, J. Vieceli, J. Wegener, D. Wu, A. Yang, D. Zaccarin, P. Zhao, F. Zhong, J. Korlach, and S. Turner, “Real-time DNA sequencing from single polymerase molecules,” Science 323, 133–138 (2009).
[Crossref]

deWinter, A.

J. Eid, A. Fehr, J. Gray, K. Luong, J. Lyle, G. Otto, P. Peluso, D. Rank, P. Baybayan, B. Bettman, A. Bibillo, K. Bjornson, B. Chaudhuri, F. Christians, R. Cicero, S. Clark, R. Dalal, A. deWinter, J. Dixon, M. Foquet, A. Gaertner, P. Hardenbol, C. Heiner, K. Hester, D. Holden, G. Kearns, X. Kong, R. Kuse, Y. Lacroix, S. Lin, P. Lundquist, C. Ma, P. Marks, M. Maxham, D. Murphy, I. Park, T. Pham, M. Phillips, J. Roy, R. Sebra, G. Shen, J. Sorenson, A. Tomaney, K. Travers, M. Trulson, J. Vieceli, J. Wegener, D. Wu, A. Yang, D. Zaccarin, P. Zhao, F. Zhong, J. Korlach, and S. Turner, “Real-time DNA sequencing from single polymerase molecules,” Science 323, 133–138 (2009).
[Crossref]

Di Caprio, G.

Dixon, J.

J. Eid, A. Fehr, J. Gray, K. Luong, J. Lyle, G. Otto, P. Peluso, D. Rank, P. Baybayan, B. Bettman, A. Bibillo, K. Bjornson, B. Chaudhuri, F. Christians, R. Cicero, S. Clark, R. Dalal, A. deWinter, J. Dixon, M. Foquet, A. Gaertner, P. Hardenbol, C. Heiner, K. Hester, D. Holden, G. Kearns, X. Kong, R. Kuse, Y. Lacroix, S. Lin, P. Lundquist, C. Ma, P. Marks, M. Maxham, D. Murphy, I. Park, T. Pham, M. Phillips, J. Roy, R. Sebra, G. Shen, J. Sorenson, A. Tomaney, K. Travers, M. Trulson, J. Vieceli, J. Wegener, D. Wu, A. Yang, D. Zaccarin, P. Zhao, F. Zhong, J. Korlach, and S. Turner, “Real-time DNA sequencing from single polymerase molecules,” Science 323, 133–138 (2009).
[Crossref]

P. M. Lundquist, C. F. Zhong, P. Zhao, A. B. Tomaney, P. S. Peluso, J. Dixon, B. Bettman, Y. Lacroix, D. P. Kwo, E. McCullough, M. Maxham, K. Hester, P. McNitt, D. M. Grey, C. Henriquez, M. Foquet, S. W. Turner, and D. Zaccarin, “Parallel confocal detection of single molecules in real time,” Opt. Lett. 33, 1026–1028 (2008).
[Crossref]

Eid, J.

J. Eid, A. Fehr, J. Gray, K. Luong, J. Lyle, G. Otto, P. Peluso, D. Rank, P. Baybayan, B. Bettman, A. Bibillo, K. Bjornson, B. Chaudhuri, F. Christians, R. Cicero, S. Clark, R. Dalal, A. deWinter, J. Dixon, M. Foquet, A. Gaertner, P. Hardenbol, C. Heiner, K. Hester, D. Holden, G. Kearns, X. Kong, R. Kuse, Y. Lacroix, S. Lin, P. Lundquist, C. Ma, P. Marks, M. Maxham, D. Murphy, I. Park, T. Pham, M. Phillips, J. Roy, R. Sebra, G. Shen, J. Sorenson, A. Tomaney, K. Travers, M. Trulson, J. Vieceli, J. Wegener, D. Wu, A. Yang, D. Zaccarin, P. Zhao, F. Zhong, J. Korlach, and S. Turner, “Real-time DNA sequencing from single polymerase molecules,” Science 323, 133–138 (2009).
[Crossref]

Fehr, A.

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Lacroix, Y.

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Lundquist, P. M.

Luong, K.

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P. M. Lundquist, C. F. Zhong, P. Zhao, A. B. Tomaney, P. S. Peluso, J. Dixon, B. Bettman, Y. Lacroix, D. P. Kwo, E. McCullough, M. Maxham, K. Hester, P. McNitt, D. M. Grey, C. Henriquez, M. Foquet, S. W. Turner, and D. Zaccarin, “Parallel confocal detection of single molecules in real time,” Opt. Lett. 33, 1026–1028 (2008).
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Supplementary Material (2)

NameDescription
» Visualization 1: AVI (350 KB)      Complete spectral stack.
» Visualization 2: MOV (166 KB)      Animation showing all six channels.

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Figures (6)

Fig. 1.
Fig. 1. (a) Optical system layout. Dashed box below is the LED used for spectral resolution characterization. CAM, camera; N, notch and long-pass filters; SLR, single-lens reflex lens; WP, wedge prism; EM, fluorescence emission; DM, dichroic mirror; RGB, red, green, and blue laser beams; MLA, microlens array; S, sample; LED, white-light-emitting diode ring array. (b) Average of 100 frames recorded by the camera. Each microlens aperture image is replicated in a line along the prism dispersion axis. Scale bar is 1 mm. Inset: Detail of six spectra as recorded by the camera. Arrows indicate pixels corresponding to 511 and 679 nm. (c) Fluorescence emission exits the wedge prism (wedge angle φ = 3.85 ° and 7.68° for 2° and 4° deviation wedges, respectively) at a wavelength-dependent angle. The angular spread θ determines the spectrum size at the image sensor. (d) Micrograph of the microlens array. Space between microlens columns leaves space for emission spectra. Scale bar is half the column pitch (300 μm).
Fig. 2.
Fig. 2. (a) Fluorescence emission spectra of Dragon Green (blue curve) and Sky Blue (red curve) beads as measured by a commercial spectrometer. The emission peaks are separated by 166 nm. (b) Spectra of the same beads, measured by the multispectral microlens microscope. The emission peaks are separated by 8 pixels. The spectral axis in (b) is obtained by using the conversion 1 pixel = 21 nm . Note that the dip in intensity at 532 and 658 nm is due to the notch filters. (c) Spectral impulse at 473 nm. The blue curve is a Gaussian fit to the data (black dots). Pixel numbers 12 and 13 receive little signal and are not shown. The spectral FWHM at 473 nm is 1.56 pixels = 32.7 nm . Inset: Raw camera frame during spectral impulse measurement at 473 nm, with false blue color. The distance between microlens columns is 13.25 pixels.
Fig. 3.
Fig. 3. (a) A whole-well image of a HeLa cell culture. The original dataset has 11 spectral samples, which are reduced to 3 for display purposes. Signals from the spectral channels centered at 521, 612, and 673 nm are displayed as red, green, and blue, respectively. The full-resolution image is available online [25]. Scale bar: 1 mm. (b) High-magnification view of the boxed region in (a). Cell nuclei, labeled with DAPI and AlexaFluor 488, appear pink. The cell body, labeled with a whole cell stain, is blue, and subcellular fibrous actin filaments (AlexaFluor 555 Phalloidin) are visible in green. Scale bar: 50 μm. A complete spectral stack is shown in Visualization 1. (c) Raw spectral data for three representative points in the image, I–III, as indicated by the white arrows in (b). AF488, AlexaFluor 488; AF555, AlexaFluor 555; WCS, whole cell stain.
Fig. 4.
Fig. 4. (a) Large FOV image comprised of nine wells, each containing up to six distinct fluorescent beads. For display purposes, the 13 spectral bins of this data set are downsampled to 3 (RGB). (b), (c) High-magnification views of the regions labeled b and c, respectively, in (a). (b) contains five different color beads; (c) contains six different color beads. (d) Isolated fluorescent beads of each type, at 2 × magnification compared to (b) and (c). Scale bar: 25 μm.
Fig. 5.
Fig. 5. (a) Fluorescence spectra of each of the six bead types, as measured by a commercial spectrometer, (b) fluorescence spectra of each bead type as identified by the user-assisted unmixing process. (c) Top: False-colored composite image of all six unmixed channels (Ch 1–6). The color of each channel is the same as its corresponding spectral curve in (a) and (b). Bottom: The region inside of the dashed box is shown for each channel in false color. Channels are ordered from left to right by ascending peak emission wavelength. An animation showing all six channels is shown in Visualization 2. Scale bar: 50 μm. (d) Unmixing separation matrix. Each column describes the amount of signal from a bead type that is unmixed into a given channel (Ch 1–6). Each row describes the amount of signal in a channel that originated from a given bead type. Percentages on the right axis indicate the percentage of the channel that originated from the correct bead type. Percentages on the bottom indicate the percentage of bead signal that was unmixed into the correct channel. Perfect unmixing would produce a diagonal matrix and 100% for all percentages.
Fig. 6.
Fig. 6. (a) Estimated spectra for each of the four fluorophore species in the HeLa cells sample. AF488, AlexaFluor 488; AF555, AlexaFluor 555; WCS, whole cell stain. (b1)–(b4) Single channel images of the boxed region in (c) for each fluorophore, as indicated. Note that the cell nucleus in region I contains a high AlexaFluor 488 (labels Ki67 protein) signal, whereas the nucleus in region II does not, indicative of a cell that has stopped dividing. (c) RGB image containing three of the four demixed channels. Red, AlexaFluor 488; blue, DAPI; green, AlexaFluor 555. Whole cell stain not included for clarity. Scale bar: 50 μm.

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