Abstract

Imaging fluorescence in moving cells is fundamentally challenging because the exposure time is constrained by motion-blur, which limits the available signal. We report a method to image fluorescently labeled leukemia cells in fluid flow that has an effective exposure time of up to 50 times the motion-blur limit. Flowing cells are illuminated with a pseudo-random excitation pulse sequence, resulting in a motion-blur that can be computationally removed to produce near diffraction-limited images. This method enables observation of cellular organelles and their behavior in a fluid environment that resembles the vasculature.

© 2013 OSA

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  1. R. Yuste, “Fluorescence microscopy today,” Nat. Methods2(12), 902–904 (2005).
    [CrossRef] [PubMed]
  2. B. Huang, H. Babcock, and X. Zhuang, “Breaking the diffraction barrier: super-resolution imaging of cells,” Cell143(7), 1047–1058 (2010).
    [CrossRef] [PubMed]
  3. L. A. Sklar, Flow Cytometry for Biotechnology (Oxford, 2005).
  4. T. R. Jones, A. E. Carpenter, M. R. Lamprecht, J. Moffat, S. J. Silver, J. K. Grenier, A. B. Castoreno, U. S. Eggert, D. E. Root, P. Golland, and D. M. Sabatini, “Scoring diverse cellular morphologies in image-based screens with iterative feedback and machine learning,” Proc. Natl. Acad. Sci. U.S.A.106(6), 1826–1831 (2009).
    [CrossRef] [PubMed]
  5. D. A. Basiji and W. E. Ortyn, Imaging and analyzing parameters of small moving objects such as cells. Amnis Corporation, assignee. US Patent 6211955, 2000–03–29 (2001).
  6. R. D. Kamm, “Cellular fluid mechanics,” Annu. Rev. Fluid Mech.34(1), 211–232 (2002).
    [CrossRef] [PubMed]
  7. S. Suresh, “Biomechanics and biophysics of cancer cells,” Acta Biomater.3(4), 413–438 (2007).
    [CrossRef] [PubMed]
  8. J. W. Lichtman and J. A. Conchello, “Fluorescence Microscopy,” Nat. Methods2(12), 910–919 (2005).
    [CrossRef] [PubMed]
  9. J. Vermot, S. E. Fraser, and M. Liebling, “Fast fluorescence microscopy for imaging the dynamics of embryonic development,” HFSP J2(3), 143–155 (2008).
    [CrossRef] [PubMed]
  10. B. K. McKenna, J. G. Evans, M. C. Cheung, and D. J. Ehrlich, “A parallel microfluidic flow cytometer for high-content screening,” Nat. Methods8(5), 401–403 (2011).
    [CrossRef] [PubMed]
  11. E. Schonbrun, A. R. Abate, P. E. Steinvurzel, D. A. Weitz, and K. B. Crozier, “High-throughput fluorescence detection using an integrated zone-plate array,” Lab Chip10(7), 852–856 (2010).
    [CrossRef] [PubMed]
  12. E. Schonbrun, S. S. Gorthi, and D. Schaak, “Microfabricated multiple field of view imaging flow cytometry,” Lab Chip12(2), 268–273 (2011).
    [CrossRef] [PubMed]
  13. R. Raskar, A. Agrawal, and J. Tumblin, “Coded exposure photography: motion deblurring using fluttered shutter,” ACM SIGGRAPG25(3), 795–804 (2006).
    [CrossRef]
  14. P. Kiesel, M. Bassler, M. Beck, and N. Johnson, “Spatially modulated fluorescence emission from moving particles,” Appl. Phys. Lett.94(4), 041107 (2009).
    [CrossRef]
  15. R. C. Gonzalez, R. E. Woods, and S. L. Eddins, Digital Image Processing Using Matlab (Gatemark, 2009).
  16. R. A. Saleem, B. Knoblach, F. D. Mast, J. J. Smith, J. Boyle, C. M. Dobson, R. Long-O’Donnell, R. A. Rachubinski, and J. D. Aitchison, “Genome-wide analysis of signaling networks regulating fatty acid-induced gene expression and organelle biogenesis,” J. Cell Biol.181(2), 281–292 (2008).
    [CrossRef] [PubMed]

2011 (2)

B. K. McKenna, J. G. Evans, M. C. Cheung, and D. J. Ehrlich, “A parallel microfluidic flow cytometer for high-content screening,” Nat. Methods8(5), 401–403 (2011).
[CrossRef] [PubMed]

E. Schonbrun, S. S. Gorthi, and D. Schaak, “Microfabricated multiple field of view imaging flow cytometry,” Lab Chip12(2), 268–273 (2011).
[CrossRef] [PubMed]

2010 (2)

E. Schonbrun, A. R. Abate, P. E. Steinvurzel, D. A. Weitz, and K. B. Crozier, “High-throughput fluorescence detection using an integrated zone-plate array,” Lab Chip10(7), 852–856 (2010).
[CrossRef] [PubMed]

B. Huang, H. Babcock, and X. Zhuang, “Breaking the diffraction barrier: super-resolution imaging of cells,” Cell143(7), 1047–1058 (2010).
[CrossRef] [PubMed]

2009 (2)

T. R. Jones, A. E. Carpenter, M. R. Lamprecht, J. Moffat, S. J. Silver, J. K. Grenier, A. B. Castoreno, U. S. Eggert, D. E. Root, P. Golland, and D. M. Sabatini, “Scoring diverse cellular morphologies in image-based screens with iterative feedback and machine learning,” Proc. Natl. Acad. Sci. U.S.A.106(6), 1826–1831 (2009).
[CrossRef] [PubMed]

P. Kiesel, M. Bassler, M. Beck, and N. Johnson, “Spatially modulated fluorescence emission from moving particles,” Appl. Phys. Lett.94(4), 041107 (2009).
[CrossRef]

2008 (2)

R. A. Saleem, B. Knoblach, F. D. Mast, J. J. Smith, J. Boyle, C. M. Dobson, R. Long-O’Donnell, R. A. Rachubinski, and J. D. Aitchison, “Genome-wide analysis of signaling networks regulating fatty acid-induced gene expression and organelle biogenesis,” J. Cell Biol.181(2), 281–292 (2008).
[CrossRef] [PubMed]

J. Vermot, S. E. Fraser, and M. Liebling, “Fast fluorescence microscopy for imaging the dynamics of embryonic development,” HFSP J2(3), 143–155 (2008).
[CrossRef] [PubMed]

2007 (1)

S. Suresh, “Biomechanics and biophysics of cancer cells,” Acta Biomater.3(4), 413–438 (2007).
[CrossRef] [PubMed]

2006 (1)

R. Raskar, A. Agrawal, and J. Tumblin, “Coded exposure photography: motion deblurring using fluttered shutter,” ACM SIGGRAPG25(3), 795–804 (2006).
[CrossRef]

2005 (2)

J. W. Lichtman and J. A. Conchello, “Fluorescence Microscopy,” Nat. Methods2(12), 910–919 (2005).
[CrossRef] [PubMed]

R. Yuste, “Fluorescence microscopy today,” Nat. Methods2(12), 902–904 (2005).
[CrossRef] [PubMed]

2002 (1)

R. D. Kamm, “Cellular fluid mechanics,” Annu. Rev. Fluid Mech.34(1), 211–232 (2002).
[CrossRef] [PubMed]

Abate, A. R.

E. Schonbrun, A. R. Abate, P. E. Steinvurzel, D. A. Weitz, and K. B. Crozier, “High-throughput fluorescence detection using an integrated zone-plate array,” Lab Chip10(7), 852–856 (2010).
[CrossRef] [PubMed]

Agrawal, A.

R. Raskar, A. Agrawal, and J. Tumblin, “Coded exposure photography: motion deblurring using fluttered shutter,” ACM SIGGRAPG25(3), 795–804 (2006).
[CrossRef]

Aitchison, J. D.

R. A. Saleem, B. Knoblach, F. D. Mast, J. J. Smith, J. Boyle, C. M. Dobson, R. Long-O’Donnell, R. A. Rachubinski, and J. D. Aitchison, “Genome-wide analysis of signaling networks regulating fatty acid-induced gene expression and organelle biogenesis,” J. Cell Biol.181(2), 281–292 (2008).
[CrossRef] [PubMed]

Babcock, H.

B. Huang, H. Babcock, and X. Zhuang, “Breaking the diffraction barrier: super-resolution imaging of cells,” Cell143(7), 1047–1058 (2010).
[CrossRef] [PubMed]

Bassler, M.

P. Kiesel, M. Bassler, M. Beck, and N. Johnson, “Spatially modulated fluorescence emission from moving particles,” Appl. Phys. Lett.94(4), 041107 (2009).
[CrossRef]

Beck, M.

P. Kiesel, M. Bassler, M. Beck, and N. Johnson, “Spatially modulated fluorescence emission from moving particles,” Appl. Phys. Lett.94(4), 041107 (2009).
[CrossRef]

Boyle, J.

R. A. Saleem, B. Knoblach, F. D. Mast, J. J. Smith, J. Boyle, C. M. Dobson, R. Long-O’Donnell, R. A. Rachubinski, and J. D. Aitchison, “Genome-wide analysis of signaling networks regulating fatty acid-induced gene expression and organelle biogenesis,” J. Cell Biol.181(2), 281–292 (2008).
[CrossRef] [PubMed]

Carpenter, A. E.

T. R. Jones, A. E. Carpenter, M. R. Lamprecht, J. Moffat, S. J. Silver, J. K. Grenier, A. B. Castoreno, U. S. Eggert, D. E. Root, P. Golland, and D. M. Sabatini, “Scoring diverse cellular morphologies in image-based screens with iterative feedback and machine learning,” Proc. Natl. Acad. Sci. U.S.A.106(6), 1826–1831 (2009).
[CrossRef] [PubMed]

Castoreno, A. B.

T. R. Jones, A. E. Carpenter, M. R. Lamprecht, J. Moffat, S. J. Silver, J. K. Grenier, A. B. Castoreno, U. S. Eggert, D. E. Root, P. Golland, and D. M. Sabatini, “Scoring diverse cellular morphologies in image-based screens with iterative feedback and machine learning,” Proc. Natl. Acad. Sci. U.S.A.106(6), 1826–1831 (2009).
[CrossRef] [PubMed]

Cheung, M. C.

B. K. McKenna, J. G. Evans, M. C. Cheung, and D. J. Ehrlich, “A parallel microfluidic flow cytometer for high-content screening,” Nat. Methods8(5), 401–403 (2011).
[CrossRef] [PubMed]

Conchello, J. A.

J. W. Lichtman and J. A. Conchello, “Fluorescence Microscopy,” Nat. Methods2(12), 910–919 (2005).
[CrossRef] [PubMed]

Crozier, K. B.

E. Schonbrun, A. R. Abate, P. E. Steinvurzel, D. A. Weitz, and K. B. Crozier, “High-throughput fluorescence detection using an integrated zone-plate array,” Lab Chip10(7), 852–856 (2010).
[CrossRef] [PubMed]

Dobson, C. M.

R. A. Saleem, B. Knoblach, F. D. Mast, J. J. Smith, J. Boyle, C. M. Dobson, R. Long-O’Donnell, R. A. Rachubinski, and J. D. Aitchison, “Genome-wide analysis of signaling networks regulating fatty acid-induced gene expression and organelle biogenesis,” J. Cell Biol.181(2), 281–292 (2008).
[CrossRef] [PubMed]

Eggert, U. S.

T. R. Jones, A. E. Carpenter, M. R. Lamprecht, J. Moffat, S. J. Silver, J. K. Grenier, A. B. Castoreno, U. S. Eggert, D. E. Root, P. Golland, and D. M. Sabatini, “Scoring diverse cellular morphologies in image-based screens with iterative feedback and machine learning,” Proc. Natl. Acad. Sci. U.S.A.106(6), 1826–1831 (2009).
[CrossRef] [PubMed]

Ehrlich, D. J.

B. K. McKenna, J. G. Evans, M. C. Cheung, and D. J. Ehrlich, “A parallel microfluidic flow cytometer for high-content screening,” Nat. Methods8(5), 401–403 (2011).
[CrossRef] [PubMed]

Evans, J. G.

B. K. McKenna, J. G. Evans, M. C. Cheung, and D. J. Ehrlich, “A parallel microfluidic flow cytometer for high-content screening,” Nat. Methods8(5), 401–403 (2011).
[CrossRef] [PubMed]

Fraser, S. E.

J. Vermot, S. E. Fraser, and M. Liebling, “Fast fluorescence microscopy for imaging the dynamics of embryonic development,” HFSP J2(3), 143–155 (2008).
[CrossRef] [PubMed]

Golland, P.

T. R. Jones, A. E. Carpenter, M. R. Lamprecht, J. Moffat, S. J. Silver, J. K. Grenier, A. B. Castoreno, U. S. Eggert, D. E. Root, P. Golland, and D. M. Sabatini, “Scoring diverse cellular morphologies in image-based screens with iterative feedback and machine learning,” Proc. Natl. Acad. Sci. U.S.A.106(6), 1826–1831 (2009).
[CrossRef] [PubMed]

Gorthi, S. S.

E. Schonbrun, S. S. Gorthi, and D. Schaak, “Microfabricated multiple field of view imaging flow cytometry,” Lab Chip12(2), 268–273 (2011).
[CrossRef] [PubMed]

Grenier, J. K.

T. R. Jones, A. E. Carpenter, M. R. Lamprecht, J. Moffat, S. J. Silver, J. K. Grenier, A. B. Castoreno, U. S. Eggert, D. E. Root, P. Golland, and D. M. Sabatini, “Scoring diverse cellular morphologies in image-based screens with iterative feedback and machine learning,” Proc. Natl. Acad. Sci. U.S.A.106(6), 1826–1831 (2009).
[CrossRef] [PubMed]

Huang, B.

B. Huang, H. Babcock, and X. Zhuang, “Breaking the diffraction barrier: super-resolution imaging of cells,” Cell143(7), 1047–1058 (2010).
[CrossRef] [PubMed]

Johnson, N.

P. Kiesel, M. Bassler, M. Beck, and N. Johnson, “Spatially modulated fluorescence emission from moving particles,” Appl. Phys. Lett.94(4), 041107 (2009).
[CrossRef]

Jones, T. R.

T. R. Jones, A. E. Carpenter, M. R. Lamprecht, J. Moffat, S. J. Silver, J. K. Grenier, A. B. Castoreno, U. S. Eggert, D. E. Root, P. Golland, and D. M. Sabatini, “Scoring diverse cellular morphologies in image-based screens with iterative feedback and machine learning,” Proc. Natl. Acad. Sci. U.S.A.106(6), 1826–1831 (2009).
[CrossRef] [PubMed]

Kamm, R. D.

R. D. Kamm, “Cellular fluid mechanics,” Annu. Rev. Fluid Mech.34(1), 211–232 (2002).
[CrossRef] [PubMed]

Kiesel, P.

P. Kiesel, M. Bassler, M. Beck, and N. Johnson, “Spatially modulated fluorescence emission from moving particles,” Appl. Phys. Lett.94(4), 041107 (2009).
[CrossRef]

Knoblach, B.

R. A. Saleem, B. Knoblach, F. D. Mast, J. J. Smith, J. Boyle, C. M. Dobson, R. Long-O’Donnell, R. A. Rachubinski, and J. D. Aitchison, “Genome-wide analysis of signaling networks regulating fatty acid-induced gene expression and organelle biogenesis,” J. Cell Biol.181(2), 281–292 (2008).
[CrossRef] [PubMed]

Lamprecht, M. R.

T. R. Jones, A. E. Carpenter, M. R. Lamprecht, J. Moffat, S. J. Silver, J. K. Grenier, A. B. Castoreno, U. S. Eggert, D. E. Root, P. Golland, and D. M. Sabatini, “Scoring diverse cellular morphologies in image-based screens with iterative feedback and machine learning,” Proc. Natl. Acad. Sci. U.S.A.106(6), 1826–1831 (2009).
[CrossRef] [PubMed]

Lichtman, J. W.

J. W. Lichtman and J. A. Conchello, “Fluorescence Microscopy,” Nat. Methods2(12), 910–919 (2005).
[CrossRef] [PubMed]

Liebling, M.

J. Vermot, S. E. Fraser, and M. Liebling, “Fast fluorescence microscopy for imaging the dynamics of embryonic development,” HFSP J2(3), 143–155 (2008).
[CrossRef] [PubMed]

Long-O’Donnell, R.

R. A. Saleem, B. Knoblach, F. D. Mast, J. J. Smith, J. Boyle, C. M. Dobson, R. Long-O’Donnell, R. A. Rachubinski, and J. D. Aitchison, “Genome-wide analysis of signaling networks regulating fatty acid-induced gene expression and organelle biogenesis,” J. Cell Biol.181(2), 281–292 (2008).
[CrossRef] [PubMed]

Mast, F. D.

R. A. Saleem, B. Knoblach, F. D. Mast, J. J. Smith, J. Boyle, C. M. Dobson, R. Long-O’Donnell, R. A. Rachubinski, and J. D. Aitchison, “Genome-wide analysis of signaling networks regulating fatty acid-induced gene expression and organelle biogenesis,” J. Cell Biol.181(2), 281–292 (2008).
[CrossRef] [PubMed]

McKenna, B. K.

B. K. McKenna, J. G. Evans, M. C. Cheung, and D. J. Ehrlich, “A parallel microfluidic flow cytometer for high-content screening,” Nat. Methods8(5), 401–403 (2011).
[CrossRef] [PubMed]

Moffat, J.

T. R. Jones, A. E. Carpenter, M. R. Lamprecht, J. Moffat, S. J. Silver, J. K. Grenier, A. B. Castoreno, U. S. Eggert, D. E. Root, P. Golland, and D. M. Sabatini, “Scoring diverse cellular morphologies in image-based screens with iterative feedback and machine learning,” Proc. Natl. Acad. Sci. U.S.A.106(6), 1826–1831 (2009).
[CrossRef] [PubMed]

Rachubinski, R. A.

R. A. Saleem, B. Knoblach, F. D. Mast, J. J. Smith, J. Boyle, C. M. Dobson, R. Long-O’Donnell, R. A. Rachubinski, and J. D. Aitchison, “Genome-wide analysis of signaling networks regulating fatty acid-induced gene expression and organelle biogenesis,” J. Cell Biol.181(2), 281–292 (2008).
[CrossRef] [PubMed]

Raskar, R.

R. Raskar, A. Agrawal, and J. Tumblin, “Coded exposure photography: motion deblurring using fluttered shutter,” ACM SIGGRAPG25(3), 795–804 (2006).
[CrossRef]

Root, D. E.

T. R. Jones, A. E. Carpenter, M. R. Lamprecht, J. Moffat, S. J. Silver, J. K. Grenier, A. B. Castoreno, U. S. Eggert, D. E. Root, P. Golland, and D. M. Sabatini, “Scoring diverse cellular morphologies in image-based screens with iterative feedback and machine learning,” Proc. Natl. Acad. Sci. U.S.A.106(6), 1826–1831 (2009).
[CrossRef] [PubMed]

Sabatini, D. M.

T. R. Jones, A. E. Carpenter, M. R. Lamprecht, J. Moffat, S. J. Silver, J. K. Grenier, A. B. Castoreno, U. S. Eggert, D. E. Root, P. Golland, and D. M. Sabatini, “Scoring diverse cellular morphologies in image-based screens with iterative feedback and machine learning,” Proc. Natl. Acad. Sci. U.S.A.106(6), 1826–1831 (2009).
[CrossRef] [PubMed]

Saleem, R. A.

R. A. Saleem, B. Knoblach, F. D. Mast, J. J. Smith, J. Boyle, C. M. Dobson, R. Long-O’Donnell, R. A. Rachubinski, and J. D. Aitchison, “Genome-wide analysis of signaling networks regulating fatty acid-induced gene expression and organelle biogenesis,” J. Cell Biol.181(2), 281–292 (2008).
[CrossRef] [PubMed]

Schaak, D.

E. Schonbrun, S. S. Gorthi, and D. Schaak, “Microfabricated multiple field of view imaging flow cytometry,” Lab Chip12(2), 268–273 (2011).
[CrossRef] [PubMed]

Schonbrun, E.

E. Schonbrun, S. S. Gorthi, and D. Schaak, “Microfabricated multiple field of view imaging flow cytometry,” Lab Chip12(2), 268–273 (2011).
[CrossRef] [PubMed]

E. Schonbrun, A. R. Abate, P. E. Steinvurzel, D. A. Weitz, and K. B. Crozier, “High-throughput fluorescence detection using an integrated zone-plate array,” Lab Chip10(7), 852–856 (2010).
[CrossRef] [PubMed]

Silver, S. J.

T. R. Jones, A. E. Carpenter, M. R. Lamprecht, J. Moffat, S. J. Silver, J. K. Grenier, A. B. Castoreno, U. S. Eggert, D. E. Root, P. Golland, and D. M. Sabatini, “Scoring diverse cellular morphologies in image-based screens with iterative feedback and machine learning,” Proc. Natl. Acad. Sci. U.S.A.106(6), 1826–1831 (2009).
[CrossRef] [PubMed]

Smith, J. J.

R. A. Saleem, B. Knoblach, F. D. Mast, J. J. Smith, J. Boyle, C. M. Dobson, R. Long-O’Donnell, R. A. Rachubinski, and J. D. Aitchison, “Genome-wide analysis of signaling networks regulating fatty acid-induced gene expression and organelle biogenesis,” J. Cell Biol.181(2), 281–292 (2008).
[CrossRef] [PubMed]

Steinvurzel, P. E.

E. Schonbrun, A. R. Abate, P. E. Steinvurzel, D. A. Weitz, and K. B. Crozier, “High-throughput fluorescence detection using an integrated zone-plate array,” Lab Chip10(7), 852–856 (2010).
[CrossRef] [PubMed]

Suresh, S.

S. Suresh, “Biomechanics and biophysics of cancer cells,” Acta Biomater.3(4), 413–438 (2007).
[CrossRef] [PubMed]

Tumblin, J.

R. Raskar, A. Agrawal, and J. Tumblin, “Coded exposure photography: motion deblurring using fluttered shutter,” ACM SIGGRAPG25(3), 795–804 (2006).
[CrossRef]

Vermot, J.

J. Vermot, S. E. Fraser, and M. Liebling, “Fast fluorescence microscopy for imaging the dynamics of embryonic development,” HFSP J2(3), 143–155 (2008).
[CrossRef] [PubMed]

Weitz, D. A.

E. Schonbrun, A. R. Abate, P. E. Steinvurzel, D. A. Weitz, and K. B. Crozier, “High-throughput fluorescence detection using an integrated zone-plate array,” Lab Chip10(7), 852–856 (2010).
[CrossRef] [PubMed]

Yuste, R.

R. Yuste, “Fluorescence microscopy today,” Nat. Methods2(12), 902–904 (2005).
[CrossRef] [PubMed]

Zhuang, X.

B. Huang, H. Babcock, and X. Zhuang, “Breaking the diffraction barrier: super-resolution imaging of cells,” Cell143(7), 1047–1058 (2010).
[CrossRef] [PubMed]

ACM SIGGRAPG (1)

R. Raskar, A. Agrawal, and J. Tumblin, “Coded exposure photography: motion deblurring using fluttered shutter,” ACM SIGGRAPG25(3), 795–804 (2006).
[CrossRef]

Acta Biomater. (1)

S. Suresh, “Biomechanics and biophysics of cancer cells,” Acta Biomater.3(4), 413–438 (2007).
[CrossRef] [PubMed]

Annu. Rev. Fluid Mech. (1)

R. D. Kamm, “Cellular fluid mechanics,” Annu. Rev. Fluid Mech.34(1), 211–232 (2002).
[CrossRef] [PubMed]

Appl. Phys. Lett. (1)

P. Kiesel, M. Bassler, M. Beck, and N. Johnson, “Spatially modulated fluorescence emission from moving particles,” Appl. Phys. Lett.94(4), 041107 (2009).
[CrossRef]

Cell (1)

B. Huang, H. Babcock, and X. Zhuang, “Breaking the diffraction barrier: super-resolution imaging of cells,” Cell143(7), 1047–1058 (2010).
[CrossRef] [PubMed]

HFSP J (1)

J. Vermot, S. E. Fraser, and M. Liebling, “Fast fluorescence microscopy for imaging the dynamics of embryonic development,” HFSP J2(3), 143–155 (2008).
[CrossRef] [PubMed]

J. Cell Biol. (1)

R. A. Saleem, B. Knoblach, F. D. Mast, J. J. Smith, J. Boyle, C. M. Dobson, R. Long-O’Donnell, R. A. Rachubinski, and J. D. Aitchison, “Genome-wide analysis of signaling networks regulating fatty acid-induced gene expression and organelle biogenesis,” J. Cell Biol.181(2), 281–292 (2008).
[CrossRef] [PubMed]

Lab Chip (2)

E. Schonbrun, A. R. Abate, P. E. Steinvurzel, D. A. Weitz, and K. B. Crozier, “High-throughput fluorescence detection using an integrated zone-plate array,” Lab Chip10(7), 852–856 (2010).
[CrossRef] [PubMed]

E. Schonbrun, S. S. Gorthi, and D. Schaak, “Microfabricated multiple field of view imaging flow cytometry,” Lab Chip12(2), 268–273 (2011).
[CrossRef] [PubMed]

Nat. Methods (3)

B. K. McKenna, J. G. Evans, M. C. Cheung, and D. J. Ehrlich, “A parallel microfluidic flow cytometer for high-content screening,” Nat. Methods8(5), 401–403 (2011).
[CrossRef] [PubMed]

R. Yuste, “Fluorescence microscopy today,” Nat. Methods2(12), 902–904 (2005).
[CrossRef] [PubMed]

J. W. Lichtman and J. A. Conchello, “Fluorescence Microscopy,” Nat. Methods2(12), 910–919 (2005).
[CrossRef] [PubMed]

Proc. Natl. Acad. Sci. U.S.A. (1)

T. R. Jones, A. E. Carpenter, M. R. Lamprecht, J. Moffat, S. J. Silver, J. K. Grenier, A. B. Castoreno, U. S. Eggert, D. E. Root, P. Golland, and D. M. Sabatini, “Scoring diverse cellular morphologies in image-based screens with iterative feedback and machine learning,” Proc. Natl. Acad. Sci. U.S.A.106(6), 1826–1831 (2009).
[CrossRef] [PubMed]

Other (3)

D. A. Basiji and W. E. Ortyn, Imaging and analyzing parameters of small moving objects such as cells. Amnis Corporation, assignee. US Patent 6211955, 2000–03–29 (2001).

L. A. Sklar, Flow Cytometry for Biotechnology (Oxford, 2005).

R. C. Gonzalez, R. E. Woods, and S. L. Eddins, Digital Image Processing Using Matlab (Gatemark, 2009).

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Figures (6)

Fig. 1
Fig. 1

Stationary images of a chronic myeloid leukemia (K562) cell. (a) Brightfield image. (b,c) Fluorescence images of the cell labeled with the nucleic acid stain Syto16. The exposure time in (c) is sixteen times greater than in (b), similar to the number of pulses in the following coded exposure measurements. The grayscale bar is in units of photoelectron counts. Scale bars are 5 μm.

Fig. 2
Fig. 2

Coded excitation fluorescence microscope. (a) Custom designed chopper wheel that modulates the fluorescence excitation with a pseudo-random code. (b) Cells travel through a microfluidic device and the fluorescence emission is imaged by a microscope (0.65 NA, 40 × ) and recorded onto a camera. (c) Blur encoded images are captured by the camera and computationally decoded to produce near diffraction-limited images.

Fig. 3
Fig. 3

Numerical reconstruction. (a) Shows the first step involving scaling the pseudo-random sequence by an estimate of the velocity to transform from time to space. (b) Convolution of the spatial code by the microscope point spread function (PSF) to produce the estimated system PSF. (c) Deconvolution of the collected image by the estimated system PSF. The peak signal value is found inside the red box and the deconvolution artifacts are found along the horizontal blue line.

Fig. 4
Fig. 4

Motion-blur decoding. (a) The pseudo-random 32 letter, 18 pulse code. (b-e) Images of 2 μm beads for decreasing illumination intensity. Recorded motion-blur images are shown in the first column, decoded images in the second column, and comparison of line profiles along the marked regions in the third column, respectively. The blue curve (D) is the deconvolved cross-section and the red curve (SP) is the single pulse cross-section. Scale bars are 2 μm.

Fig. 5
Fig. 5

Imaging of the nuclei of chronic myeloid leukemia cells at a range of velocities. The motion-blur encoded images are shown on the left and the decoded reconstructions are shown on the right. The motion-blur limited velocity due to the effective exposure is 1.9 mm/s, while cells are traveling at 27, 52, 80, and 103 mm/s in (a-d), respectively.

Fig. 6
Fig. 6

Imaging of green fluorescent protein expression in the peroxisomes of CML cells. (a,b) Show a motion-blur encoded and decoded image of cell peroxisomes. c) Shows a vertical (blue) and horizontal (red) cross-section of the fluorescent intensity through two pairs of closely spaced peroxisomes outlined in b). (d,e) Show decoded images of two other cells. Scale bars are 5 μm.

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