Abstract

We demonstrate high-throughput label-free single-cell image cytometry and image-based classification of Euglena gracilis (a microalgal species) under different culture conditions. We perform it with our high-throughput optofluidic image cytometer composed of a time-stretch microscope with 780-nm resolution and 75-MHz line rate, and an inertial-focusing microfluidic device. By analyzing a large number of single-cell images from the image cytometer, we identify differences in morphological and intracellular phenotypes between E. gracilis cell groups and statistically classify them under various culture conditions including nitrogen deficiency for lipid induction. Our method holds promise for real-time evaluation of culture techniques for E. gracilis and possibly other microalgae in a non-invasive manner.

© 2016 Optical Society of America

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Corrections

21 June 2016: A correction was made to the abstract.

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References

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  1. L. Christenson and R. Sims, “Production and harvesting of microalgae for wastewater treatment, biofuels, and bioproducts,” Biotechnol. Adv. 29(6), 686–702 (2011).
    [Crossref] [PubMed]
  2. D. O. Hessen, H. J. De Lange, and E. Van Donk, “UV-induced changes in phytoplankton cells and its effects on grazers,” Freshw. Biol. 38(3), 513–524 (1997).
    [Crossref]
  3. M. Cramer and J. Myers, “Growth and photosynthetic characteristics of Euglena gracilis,” Arch. Mikrobiol. 17(4), 384–402 (1952).
    [Crossref]
  4. D. R. Georgianna and S. P. Mayfield, “Exploiting diversity and synthetic biology for the production of algal biofuels,” Nature 488(7411), 329–335 (2012).
    [Crossref] [PubMed]
  5. K. Goda, K. K. Tsia, and B. Jalali, “Serial time-encoded amplified imaging for real-time observation of fast dynamic phenomena,” Nature 458(7242), 1145–1149 (2009).
    [Crossref] [PubMed]
  6. K. Goda and B. Jalali, “Dispersive Fourier transformation for fast continuous single-shot measurements,” Nat. Photonics 7(2), 102–112 (2013).
    [Crossref]
  7. C. Lei, B. Guo, Z. Cheng, and K. Goda, “Optical time-stretch imaging: Principles and applications,” Applied Physics Reviews 3(1), 011102 (2016).
    [Crossref]
  8. C. L. Chen, A. Mahjoubfar, and B. Jalali, “Optical data compression in time stretch imaging,” PLoS One 10(4), e0125106 (2015).
    [Crossref] [PubMed]
  9. J. Xu, X. Wei, L. Yu, C. Zhang, J. Xu, K. K. Y. Wong, and K. K. Tsia, “High-performance multi-megahertz optical coherence tomography based on amplified optical time-stretch,” Biomed. Opt. Express 6(4), 1340–1350 (2015).
    [Crossref] [PubMed]
  10. A. K. Lau, H. C. Shum, K. K. Wong, and K. K. Tsia, “Optofluidic time-stretch imaging - an emerging tool for high-throughput imaging flow cytometry,” Lab Chip 16(10), 1743–1756 (2016).
    [Crossref] [PubMed]
  11. H. Chen, C. Lei, F. Xing, Z. Weng, M. Chen, S. Yang, and S. Xie, “Multiwavelength time-stretch imaging system,” Opt. Lett. 39(7), 2202–2205 (2014).
    [Crossref] [PubMed]
  12. C. Lei, H. Chen, F. Xing, M. Chen, S. Yang, and S. Xie, “Time-stretch high-speed microscopic imaging system based on temporally and spectrally shaped amplified spontaneous emission,” Opt. Lett. 40(6), 946–949 (2015).
    [Crossref] [PubMed]
  13. Q. Guo, H. Chen, Z. Weng, M. Chen, S. Yang, and S. Xie, “Fast time-lens-based line-scan single-pixel camera with multi-wavelength source,” Biomed. Opt. Express 6(9), 3610–3617 (2015).
    [Crossref] [PubMed]
  14. M. Ugawa, C. Lei, T. Nozawa, T. Ideguchi, D. Di Carlo, S. Ota, Y. Ozeki, and K. Goda, “High-throughput optofluidic particle profiling with morphological and chemical specificity,” Opt. Lett. 40(20), 4803–4806 (2015).
    [Crossref] [PubMed]
  15. A. J. Chung, D. R. Gossett, and D. Di Carlo, “Three dimensional, sheathless, and high-throughput microparticle inertial focusing through geometry-induced secondary flows,” Small 9(5), 685–690 (2013).
    [Crossref] [PubMed]
  16. L. Golan and D. Yelin, “Flow cytometry using spectrally encoded confocal microscopy,” Opt. Lett. 35(13), 2218–2220 (2010).
    [Crossref] [PubMed]
  17. D. Yelin, I. Rizvi, W. M. White, J. T. Motz, T. Hasan, B. E. Bouma, and G. J. Tearney, “Three-dimensional miniature endoscopy,” Nature 443(7113), 765 (2006).
    [Crossref] [PubMed]
  18. Y. N. Xia and G. M. Whitesides, “Soft lithography,” Angew. Chem. Int. Ed. Engl. 37(5), 550–575 (1998).
    [Crossref]
  19. R. A. Andersen, J. A. Berges, P. J. Harrison, and M. M. Watanabe, Recipes for Freshwater and Seawater Media in Algal Culturing Techniques (Academic Press, 2005), 429–538.

2016 (2)

A. K. Lau, H. C. Shum, K. K. Wong, and K. K. Tsia, “Optofluidic time-stretch imaging - an emerging tool for high-throughput imaging flow cytometry,” Lab Chip 16(10), 1743–1756 (2016).
[Crossref] [PubMed]

C. Lei, B. Guo, Z. Cheng, and K. Goda, “Optical time-stretch imaging: Principles and applications,” Applied Physics Reviews 3(1), 011102 (2016).
[Crossref]

2015 (5)

2014 (1)

2013 (2)

K. Goda and B. Jalali, “Dispersive Fourier transformation for fast continuous single-shot measurements,” Nat. Photonics 7(2), 102–112 (2013).
[Crossref]

A. J. Chung, D. R. Gossett, and D. Di Carlo, “Three dimensional, sheathless, and high-throughput microparticle inertial focusing through geometry-induced secondary flows,” Small 9(5), 685–690 (2013).
[Crossref] [PubMed]

2012 (1)

D. R. Georgianna and S. P. Mayfield, “Exploiting diversity and synthetic biology for the production of algal biofuels,” Nature 488(7411), 329–335 (2012).
[Crossref] [PubMed]

2011 (1)

L. Christenson and R. Sims, “Production and harvesting of microalgae for wastewater treatment, biofuels, and bioproducts,” Biotechnol. Adv. 29(6), 686–702 (2011).
[Crossref] [PubMed]

2010 (1)

2009 (1)

K. Goda, K. K. Tsia, and B. Jalali, “Serial time-encoded amplified imaging for real-time observation of fast dynamic phenomena,” Nature 458(7242), 1145–1149 (2009).
[Crossref] [PubMed]

2006 (1)

D. Yelin, I. Rizvi, W. M. White, J. T. Motz, T. Hasan, B. E. Bouma, and G. J. Tearney, “Three-dimensional miniature endoscopy,” Nature 443(7113), 765 (2006).
[Crossref] [PubMed]

1998 (1)

Y. N. Xia and G. M. Whitesides, “Soft lithography,” Angew. Chem. Int. Ed. Engl. 37(5), 550–575 (1998).
[Crossref]

1997 (1)

D. O. Hessen, H. J. De Lange, and E. Van Donk, “UV-induced changes in phytoplankton cells and its effects on grazers,” Freshw. Biol. 38(3), 513–524 (1997).
[Crossref]

1952 (1)

M. Cramer and J. Myers, “Growth and photosynthetic characteristics of Euglena gracilis,” Arch. Mikrobiol. 17(4), 384–402 (1952).
[Crossref]

Bouma, B. E.

D. Yelin, I. Rizvi, W. M. White, J. T. Motz, T. Hasan, B. E. Bouma, and G. J. Tearney, “Three-dimensional miniature endoscopy,” Nature 443(7113), 765 (2006).
[Crossref] [PubMed]

Chen, C. L.

C. L. Chen, A. Mahjoubfar, and B. Jalali, “Optical data compression in time stretch imaging,” PLoS One 10(4), e0125106 (2015).
[Crossref] [PubMed]

Chen, H.

Chen, M.

Cheng, Z.

C. Lei, B. Guo, Z. Cheng, and K. Goda, “Optical time-stretch imaging: Principles and applications,” Applied Physics Reviews 3(1), 011102 (2016).
[Crossref]

Christenson, L.

L. Christenson and R. Sims, “Production and harvesting of microalgae for wastewater treatment, biofuels, and bioproducts,” Biotechnol. Adv. 29(6), 686–702 (2011).
[Crossref] [PubMed]

Chung, A. J.

A. J. Chung, D. R. Gossett, and D. Di Carlo, “Three dimensional, sheathless, and high-throughput microparticle inertial focusing through geometry-induced secondary flows,” Small 9(5), 685–690 (2013).
[Crossref] [PubMed]

Cramer, M.

M. Cramer and J. Myers, “Growth and photosynthetic characteristics of Euglena gracilis,” Arch. Mikrobiol. 17(4), 384–402 (1952).
[Crossref]

De Lange, H. J.

D. O. Hessen, H. J. De Lange, and E. Van Donk, “UV-induced changes in phytoplankton cells and its effects on grazers,” Freshw. Biol. 38(3), 513–524 (1997).
[Crossref]

Di Carlo, D.

M. Ugawa, C. Lei, T. Nozawa, T. Ideguchi, D. Di Carlo, S. Ota, Y. Ozeki, and K. Goda, “High-throughput optofluidic particle profiling with morphological and chemical specificity,” Opt. Lett. 40(20), 4803–4806 (2015).
[Crossref] [PubMed]

A. J. Chung, D. R. Gossett, and D. Di Carlo, “Three dimensional, sheathless, and high-throughput microparticle inertial focusing through geometry-induced secondary flows,” Small 9(5), 685–690 (2013).
[Crossref] [PubMed]

Georgianna, D. R.

D. R. Georgianna and S. P. Mayfield, “Exploiting diversity and synthetic biology for the production of algal biofuels,” Nature 488(7411), 329–335 (2012).
[Crossref] [PubMed]

Goda, K.

C. Lei, B. Guo, Z. Cheng, and K. Goda, “Optical time-stretch imaging: Principles and applications,” Applied Physics Reviews 3(1), 011102 (2016).
[Crossref]

M. Ugawa, C. Lei, T. Nozawa, T. Ideguchi, D. Di Carlo, S. Ota, Y. Ozeki, and K. Goda, “High-throughput optofluidic particle profiling with morphological and chemical specificity,” Opt. Lett. 40(20), 4803–4806 (2015).
[Crossref] [PubMed]

K. Goda and B. Jalali, “Dispersive Fourier transformation for fast continuous single-shot measurements,” Nat. Photonics 7(2), 102–112 (2013).
[Crossref]

K. Goda, K. K. Tsia, and B. Jalali, “Serial time-encoded amplified imaging for real-time observation of fast dynamic phenomena,” Nature 458(7242), 1145–1149 (2009).
[Crossref] [PubMed]

Golan, L.

Gossett, D. R.

A. J. Chung, D. R. Gossett, and D. Di Carlo, “Three dimensional, sheathless, and high-throughput microparticle inertial focusing through geometry-induced secondary flows,” Small 9(5), 685–690 (2013).
[Crossref] [PubMed]

Guo, B.

C. Lei, B. Guo, Z. Cheng, and K. Goda, “Optical time-stretch imaging: Principles and applications,” Applied Physics Reviews 3(1), 011102 (2016).
[Crossref]

Guo, Q.

Hasan, T.

D. Yelin, I. Rizvi, W. M. White, J. T. Motz, T. Hasan, B. E. Bouma, and G. J. Tearney, “Three-dimensional miniature endoscopy,” Nature 443(7113), 765 (2006).
[Crossref] [PubMed]

Hessen, D. O.

D. O. Hessen, H. J. De Lange, and E. Van Donk, “UV-induced changes in phytoplankton cells and its effects on grazers,” Freshw. Biol. 38(3), 513–524 (1997).
[Crossref]

Ideguchi, T.

Jalali, B.

C. L. Chen, A. Mahjoubfar, and B. Jalali, “Optical data compression in time stretch imaging,” PLoS One 10(4), e0125106 (2015).
[Crossref] [PubMed]

K. Goda and B. Jalali, “Dispersive Fourier transformation for fast continuous single-shot measurements,” Nat. Photonics 7(2), 102–112 (2013).
[Crossref]

K. Goda, K. K. Tsia, and B. Jalali, “Serial time-encoded amplified imaging for real-time observation of fast dynamic phenomena,” Nature 458(7242), 1145–1149 (2009).
[Crossref] [PubMed]

Lau, A. K.

A. K. Lau, H. C. Shum, K. K. Wong, and K. K. Tsia, “Optofluidic time-stretch imaging - an emerging tool for high-throughput imaging flow cytometry,” Lab Chip 16(10), 1743–1756 (2016).
[Crossref] [PubMed]

Lei, C.

Mahjoubfar, A.

C. L. Chen, A. Mahjoubfar, and B. Jalali, “Optical data compression in time stretch imaging,” PLoS One 10(4), e0125106 (2015).
[Crossref] [PubMed]

Mayfield, S. P.

D. R. Georgianna and S. P. Mayfield, “Exploiting diversity and synthetic biology for the production of algal biofuels,” Nature 488(7411), 329–335 (2012).
[Crossref] [PubMed]

Motz, J. T.

D. Yelin, I. Rizvi, W. M. White, J. T. Motz, T. Hasan, B. E. Bouma, and G. J. Tearney, “Three-dimensional miniature endoscopy,” Nature 443(7113), 765 (2006).
[Crossref] [PubMed]

Myers, J.

M. Cramer and J. Myers, “Growth and photosynthetic characteristics of Euglena gracilis,” Arch. Mikrobiol. 17(4), 384–402 (1952).
[Crossref]

Nozawa, T.

Ota, S.

Ozeki, Y.

Rizvi, I.

D. Yelin, I. Rizvi, W. M. White, J. T. Motz, T. Hasan, B. E. Bouma, and G. J. Tearney, “Three-dimensional miniature endoscopy,” Nature 443(7113), 765 (2006).
[Crossref] [PubMed]

Shum, H. C.

A. K. Lau, H. C. Shum, K. K. Wong, and K. K. Tsia, “Optofluidic time-stretch imaging - an emerging tool for high-throughput imaging flow cytometry,” Lab Chip 16(10), 1743–1756 (2016).
[Crossref] [PubMed]

Sims, R.

L. Christenson and R. Sims, “Production and harvesting of microalgae for wastewater treatment, biofuels, and bioproducts,” Biotechnol. Adv. 29(6), 686–702 (2011).
[Crossref] [PubMed]

Tearney, G. J.

D. Yelin, I. Rizvi, W. M. White, J. T. Motz, T. Hasan, B. E. Bouma, and G. J. Tearney, “Three-dimensional miniature endoscopy,” Nature 443(7113), 765 (2006).
[Crossref] [PubMed]

Tsia, K. K.

A. K. Lau, H. C. Shum, K. K. Wong, and K. K. Tsia, “Optofluidic time-stretch imaging - an emerging tool for high-throughput imaging flow cytometry,” Lab Chip 16(10), 1743–1756 (2016).
[Crossref] [PubMed]

J. Xu, X. Wei, L. Yu, C. Zhang, J. Xu, K. K. Y. Wong, and K. K. Tsia, “High-performance multi-megahertz optical coherence tomography based on amplified optical time-stretch,” Biomed. Opt. Express 6(4), 1340–1350 (2015).
[Crossref] [PubMed]

K. Goda, K. K. Tsia, and B. Jalali, “Serial time-encoded amplified imaging for real-time observation of fast dynamic phenomena,” Nature 458(7242), 1145–1149 (2009).
[Crossref] [PubMed]

Ugawa, M.

Van Donk, E.

D. O. Hessen, H. J. De Lange, and E. Van Donk, “UV-induced changes in phytoplankton cells and its effects on grazers,” Freshw. Biol. 38(3), 513–524 (1997).
[Crossref]

Wei, X.

Weng, Z.

White, W. M.

D. Yelin, I. Rizvi, W. M. White, J. T. Motz, T. Hasan, B. E. Bouma, and G. J. Tearney, “Three-dimensional miniature endoscopy,” Nature 443(7113), 765 (2006).
[Crossref] [PubMed]

Whitesides, G. M.

Y. N. Xia and G. M. Whitesides, “Soft lithography,” Angew. Chem. Int. Ed. Engl. 37(5), 550–575 (1998).
[Crossref]

Wong, K. K.

A. K. Lau, H. C. Shum, K. K. Wong, and K. K. Tsia, “Optofluidic time-stretch imaging - an emerging tool for high-throughput imaging flow cytometry,” Lab Chip 16(10), 1743–1756 (2016).
[Crossref] [PubMed]

Wong, K. K. Y.

Xia, Y. N.

Y. N. Xia and G. M. Whitesides, “Soft lithography,” Angew. Chem. Int. Ed. Engl. 37(5), 550–575 (1998).
[Crossref]

Xie, S.

Xing, F.

Xu, J.

Yang, S.

Yelin, D.

L. Golan and D. Yelin, “Flow cytometry using spectrally encoded confocal microscopy,” Opt. Lett. 35(13), 2218–2220 (2010).
[Crossref] [PubMed]

D. Yelin, I. Rizvi, W. M. White, J. T. Motz, T. Hasan, B. E. Bouma, and G. J. Tearney, “Three-dimensional miniature endoscopy,” Nature 443(7113), 765 (2006).
[Crossref] [PubMed]

Yu, L.

Zhang, C.

Angew. Chem. Int. Ed. Engl. (1)

Y. N. Xia and G. M. Whitesides, “Soft lithography,” Angew. Chem. Int. Ed. Engl. 37(5), 550–575 (1998).
[Crossref]

Applied Physics Reviews (1)

C. Lei, B. Guo, Z. Cheng, and K. Goda, “Optical time-stretch imaging: Principles and applications,” Applied Physics Reviews 3(1), 011102 (2016).
[Crossref]

Arch. Mikrobiol. (1)

M. Cramer and J. Myers, “Growth and photosynthetic characteristics of Euglena gracilis,” Arch. Mikrobiol. 17(4), 384–402 (1952).
[Crossref]

Biomed. Opt. Express (2)

Biotechnol. Adv. (1)

L. Christenson and R. Sims, “Production and harvesting of microalgae for wastewater treatment, biofuels, and bioproducts,” Biotechnol. Adv. 29(6), 686–702 (2011).
[Crossref] [PubMed]

Freshw. Biol. (1)

D. O. Hessen, H. J. De Lange, and E. Van Donk, “UV-induced changes in phytoplankton cells and its effects on grazers,” Freshw. Biol. 38(3), 513–524 (1997).
[Crossref]

Lab Chip (1)

A. K. Lau, H. C. Shum, K. K. Wong, and K. K. Tsia, “Optofluidic time-stretch imaging - an emerging tool for high-throughput imaging flow cytometry,” Lab Chip 16(10), 1743–1756 (2016).
[Crossref] [PubMed]

Nat. Photonics (1)

K. Goda and B. Jalali, “Dispersive Fourier transformation for fast continuous single-shot measurements,” Nat. Photonics 7(2), 102–112 (2013).
[Crossref]

Nature (3)

D. Yelin, I. Rizvi, W. M. White, J. T. Motz, T. Hasan, B. E. Bouma, and G. J. Tearney, “Three-dimensional miniature endoscopy,” Nature 443(7113), 765 (2006).
[Crossref] [PubMed]

D. R. Georgianna and S. P. Mayfield, “Exploiting diversity and synthetic biology for the production of algal biofuels,” Nature 488(7411), 329–335 (2012).
[Crossref] [PubMed]

K. Goda, K. K. Tsia, and B. Jalali, “Serial time-encoded amplified imaging for real-time observation of fast dynamic phenomena,” Nature 458(7242), 1145–1149 (2009).
[Crossref] [PubMed]

Opt. Lett. (4)

PLoS One (1)

C. L. Chen, A. Mahjoubfar, and B. Jalali, “Optical data compression in time stretch imaging,” PLoS One 10(4), e0125106 (2015).
[Crossref] [PubMed]

Small (1)

A. J. Chung, D. R. Gossett, and D. Di Carlo, “Three dimensional, sheathless, and high-throughput microparticle inertial focusing through geometry-induced secondary flows,” Small 9(5), 685–690 (2013).
[Crossref] [PubMed]

Other (1)

R. A. Andersen, J. A. Berges, P. J. Harrison, and M. M. Watanabe, Recipes for Freshwater and Seawater Media in Algal Culturing Techniques (Academic Press, 2005), 429–538.

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Figures (5)

Fig. 1
Fig. 1 Schematic of the high-throughput optofluidic image cytometer.

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Fig. 2
Fig. 2 Comparison of E. gracilis images taken by our image cytometer (left), CCD-based differential interference contrast microscope (middle), and CCD-based fluorescence microscope with Nile Red lipid staining (right).

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Fig. 3
Fig. 3 Images of E. gracilis cells under the three culture conditions taken by our image cytometer.

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Fig. 4
Fig. 4 Analysis of the images of the E. gracilis cells under the three culture conditions (N = 527 per culture group).

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Fig. 5
Fig. 5 Results of the LDA analysis and Student’s t-test to show the statistical differences between lipid-induced cells and two other cell groups.

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Equations (3)

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O D F = [ O ( 1 ) , O ( 2 ) , … , O ( n ) ] ,
a v e O D F = 1 n ⋅ ∑ j = 1 n O ( j ) .
s t d O D F = 1 n ⋅ ∑ j = 1 n [ O ( j ) − a v e O D F ] 2 .

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