Abstract

This paper reports a light sheet fluorescence imaging flow cytometer for 3D sectioning of phytoplankton. The instrument developed has the inherent advantages of high cell counting throughput and high spatial resolution information derived from flow cytometry and light sheet microscopy. The throughput of the instrument is quantified by the sample volume flow rate of 0.5 μl/min with a spatial resolution as achieved by light sheet microscopy. Preliminary results from 3D morphology of the internal chlorophyll-a structure of two dinoflagellates species show promising application potentials of the method for phytoplankton taxonomy of selected species and species groups.

© 2013 OSA

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  1. Z. V. Finkel, J. Beardall, K. J. Flynn, A. Quigg, T. A. V. Rees, and J. A. Raven, “Phytoplankton in a changing world: cell size and elemental stoichiometry,” J. Plankton Res.32(1), 119–137 (2010).
    [CrossRef]
  2. L. Bonetta, “Flow cytometry smaller and better,” Nat. Methods2(10), 785–795 (2005).
    [CrossRef]
  3. D. A. Basiji, W. E. Ortyn, L. Liang, V. Venkatachalam, and P. Morrissey, “Cellular image analysis and imaging by flow cytometry,” Clin. Lab. Med.27(3), 653–670, viii (2007).
    [CrossRef] [PubMed]
  4. . F. Culverhouse, R. Williams, M. Benfield, P. R. Flood, A. F. Sell, M. G. Mazzocchi, I. Buttino, and M. Sieracki, “Automatic image analysis of plankton: future perspectives,” Mar. Ecol. Prog. Ser.312, 297–309 (2006).
    [CrossRef]
  5. R. Boistel, J. Swoger, U. Kržič, V. Fernandez, B. Gillet, and E. G. Reynaud, “The future of three-dimensional microscopic imaging in marine biology,” Mar. Ecol. (Berl.)32(4), 438–452 (2011).
    [CrossRef]
  6. J. A. Conchello and J. W. Lichtman, “Optical sectioning microscopy,” Nat. Methods2(12), 920–931 (2005).
    [CrossRef] [PubMed]
  7. E. G. Reynaud, U. Kržič, K. Greger, and E. H. K. Stelzer, “Light sheet-based fluorescence microscopy: more dimensions, more photons, and less photodamage,” HFSP J2(5), 266–275 (2008).
    [CrossRef] [PubMed]
  8. J. Huisken, J. Swoger, F. Del Bene, J. Wittbrodt, and E. H. K. Stelzer, “Optical sectioning deep inside live embryos by selective plane illumination microscopy,” Science305(5686), 1007–1009 (2004).
    [CrossRef] [PubMed]
  9. L. Silvestri, A. Bria, L. Sacconi, G. Iannello, and F. S. Pavone, “Confocal light sheet microscopy: micron-scale neuroanatomy of the entire mouse brain,” Opt. Express20(18), 20582–20598 (2012).
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    [CrossRef] [PubMed]
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  12. K. Khairy and P. J. Keller, “Reconstructing embryonic development,” Genesis49(7), 488–513 (2011).
    [CrossRef] [PubMed]
  13. S. Kumar, D. Wilding, M. B. Sikkel, A. R. Lyon, K. T. MacLeod, and C. Dunsby, “High-speed 2D and 3D fluorescence microscopy of cardiac myocytes,” Opt. Express19(15), 13839–13847 (2011).
    [CrossRef] [PubMed]
  14. K. Greger, J. Swoger, and E. H. K. Stelzer, “Basic building units and properties of a fluorescence single plane illumination microscope,” Rev. Sci. Instrum.78(2), 023705 (2007).
    [CrossRef] [PubMed]
  15. H. M. Shapiro, Practical flow cytometry (Wiley-Liss, 1993), Chapter. 4.
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    [CrossRef] [PubMed]

2012 (1)

2011 (4)

S. Kumar, D. Wilding, M. B. Sikkel, A. R. Lyon, K. T. MacLeod, and C. Dunsby, “High-speed 2D and 3D fluorescence microscopy of cardiac myocytes,” Opt. Express19(15), 13839–13847 (2011).
[CrossRef] [PubMed]

M. Weber and J. Huisken, “Light sheet microscopy for real-time developmental biology,” Curr. Opin. Genet. Dev.21(5), 566–572 (2011).
[CrossRef] [PubMed]

K. Khairy and P. J. Keller, “Reconstructing embryonic development,” Genesis49(7), 488–513 (2011).
[CrossRef] [PubMed]

R. Boistel, J. Swoger, U. Kržič, V. Fernandez, B. Gillet, and E. G. Reynaud, “The future of three-dimensional microscopic imaging in marine biology,” Mar. Ecol. (Berl.)32(4), 438–452 (2011).
[CrossRef]

2010 (1)

Z. V. Finkel, J. Beardall, K. J. Flynn, A. Quigg, T. A. V. Rees, and J. A. Raven, “Phytoplankton in a changing world: cell size and elemental stoichiometry,” J. Plankton Res.32(1), 119–137 (2010).
[CrossRef]

2009 (1)

J. Huisken and D. Y. R. Stainier, “Selective plane illumination microscopy techniques in developmental biology,” Development136(12), 1963–1975 (2009).
[CrossRef] [PubMed]

2008 (1)

E. G. Reynaud, U. Kržič, K. Greger, and E. H. K. Stelzer, “Light sheet-based fluorescence microscopy: more dimensions, more photons, and less photodamage,” HFSP J2(5), 266–275 (2008).
[CrossRef] [PubMed]

2007 (3)

D. A. Basiji, W. E. Ortyn, L. Liang, V. Venkatachalam, and P. Morrissey, “Cellular image analysis and imaging by flow cytometry,” Clin. Lab. Med.27(3), 653–670, viii (2007).
[CrossRef] [PubMed]

K. Greger, J. Swoger, and E. H. K. Stelzer, “Basic building units and properties of a fluorescence single plane illumination microscope,” Rev. Sci. Instrum.78(2), 023705 (2007).
[CrossRef] [PubMed]

J. Huisken and D. Y. R. Stainier, “Even fluorescence excitation by multidirectional selective plane illumination microscopy (mSPIM),” Opt. Lett.32(17), 2608–2610 (2007).
[CrossRef] [PubMed]

2006 (2)

C. J. Engelbrecht and E. H. K. Stelzer, “Resolution enhancement in a light-sheet-based microscope (SPIM),” Opt. Lett.31(10), 1477–1479 (2006).
[CrossRef] [PubMed]

. F. Culverhouse, R. Williams, M. Benfield, P. R. Flood, A. F. Sell, M. G. Mazzocchi, I. Buttino, and M. Sieracki, “Automatic image analysis of plankton: future perspectives,” Mar. Ecol. Prog. Ser.312, 297–309 (2006).
[CrossRef]

2005 (2)

L. Bonetta, “Flow cytometry smaller and better,” Nat. Methods2(10), 785–795 (2005).
[CrossRef]

J. A. Conchello and J. W. Lichtman, “Optical sectioning microscopy,” Nat. Methods2(12), 920–931 (2005).
[CrossRef] [PubMed]

2004 (1)

J. Huisken, J. Swoger, F. Del Bene, J. Wittbrodt, and E. H. K. Stelzer, “Optical sectioning deep inside live embryos by selective plane illumination microscopy,” Science305(5686), 1007–1009 (2004).
[CrossRef] [PubMed]

2002 (1)

Azam, F.

Basiji, D. A.

D. A. Basiji, W. E. Ortyn, L. Liang, V. Venkatachalam, and P. Morrissey, “Cellular image analysis and imaging by flow cytometry,” Clin. Lab. Med.27(3), 653–670, viii (2007).
[CrossRef] [PubMed]

Beardall, J.

Z. V. Finkel, J. Beardall, K. J. Flynn, A. Quigg, T. A. V. Rees, and J. A. Raven, “Phytoplankton in a changing world: cell size and elemental stoichiometry,” J. Plankton Res.32(1), 119–137 (2010).
[CrossRef]

Benfield, M.

. F. Culverhouse, R. Williams, M. Benfield, P. R. Flood, A. F. Sell, M. G. Mazzocchi, I. Buttino, and M. Sieracki, “Automatic image analysis of plankton: future perspectives,” Mar. Ecol. Prog. Ser.312, 297–309 (2006).
[CrossRef]

Boistel, R.

R. Boistel, J. Swoger, U. Kržič, V. Fernandez, B. Gillet, and E. G. Reynaud, “The future of three-dimensional microscopic imaging in marine biology,” Mar. Ecol. (Berl.)32(4), 438–452 (2011).
[CrossRef]

Bonetta, L.

L. Bonetta, “Flow cytometry smaller and better,” Nat. Methods2(10), 785–795 (2005).
[CrossRef]

Bria, A.

Buttino, I.

. F. Culverhouse, R. Williams, M. Benfield, P. R. Flood, A. F. Sell, M. G. Mazzocchi, I. Buttino, and M. Sieracki, “Automatic image analysis of plankton: future perspectives,” Mar. Ecol. Prog. Ser.312, 297–309 (2006).
[CrossRef]

Conchello, J. A.

J. A. Conchello and J. W. Lichtman, “Optical sectioning microscopy,” Nat. Methods2(12), 920–931 (2005).
[CrossRef] [PubMed]

Culverhouse, . F.

. F. Culverhouse, R. Williams, M. Benfield, P. R. Flood, A. F. Sell, M. G. Mazzocchi, I. Buttino, and M. Sieracki, “Automatic image analysis of plankton: future perspectives,” Mar. Ecol. Prog. Ser.312, 297–309 (2006).
[CrossRef]

Del Bene, F.

J. Huisken, J. Swoger, F. Del Bene, J. Wittbrodt, and E. H. K. Stelzer, “Optical sectioning deep inside live embryos by selective plane illumination microscopy,” Science305(5686), 1007–1009 (2004).
[CrossRef] [PubMed]

Dunsby, C.

Engelbrecht, C. J.

Fernandez, V.

R. Boistel, J. Swoger, U. Kržič, V. Fernandez, B. Gillet, and E. G. Reynaud, “The future of three-dimensional microscopic imaging in marine biology,” Mar. Ecol. (Berl.)32(4), 438–452 (2011).
[CrossRef]

Finkel, Z. V.

Z. V. Finkel, J. Beardall, K. J. Flynn, A. Quigg, T. A. V. Rees, and J. A. Raven, “Phytoplankton in a changing world: cell size and elemental stoichiometry,” J. Plankton Res.32(1), 119–137 (2010).
[CrossRef]

Flood, P. R.

. F. Culverhouse, R. Williams, M. Benfield, P. R. Flood, A. F. Sell, M. G. Mazzocchi, I. Buttino, and M. Sieracki, “Automatic image analysis of plankton: future perspectives,” Mar. Ecol. Prog. Ser.312, 297–309 (2006).
[CrossRef]

Flynn, K. J.

Z. V. Finkel, J. Beardall, K. J. Flynn, A. Quigg, T. A. V. Rees, and J. A. Raven, “Phytoplankton in a changing world: cell size and elemental stoichiometry,” J. Plankton Res.32(1), 119–137 (2010).
[CrossRef]

Fuchs, E.

Gillet, B.

R. Boistel, J. Swoger, U. Kržič, V. Fernandez, B. Gillet, and E. G. Reynaud, “The future of three-dimensional microscopic imaging in marine biology,” Mar. Ecol. (Berl.)32(4), 438–452 (2011).
[CrossRef]

Greger, K.

E. G. Reynaud, U. Kržič, K. Greger, and E. H. K. Stelzer, “Light sheet-based fluorescence microscopy: more dimensions, more photons, and less photodamage,” HFSP J2(5), 266–275 (2008).
[CrossRef] [PubMed]

K. Greger, J. Swoger, and E. H. K. Stelzer, “Basic building units and properties of a fluorescence single plane illumination microscope,” Rev. Sci. Instrum.78(2), 023705 (2007).
[CrossRef] [PubMed]

Huisken, J.

M. Weber and J. Huisken, “Light sheet microscopy for real-time developmental biology,” Curr. Opin. Genet. Dev.21(5), 566–572 (2011).
[CrossRef] [PubMed]

J. Huisken and D. Y. R. Stainier, “Selective plane illumination microscopy techniques in developmental biology,” Development136(12), 1963–1975 (2009).
[CrossRef] [PubMed]

J. Huisken and D. Y. R. Stainier, “Even fluorescence excitation by multidirectional selective plane illumination microscopy (mSPIM),” Opt. Lett.32(17), 2608–2610 (2007).
[CrossRef] [PubMed]

J. Huisken, J. Swoger, F. Del Bene, J. Wittbrodt, and E. H. K. Stelzer, “Optical sectioning deep inside live embryos by selective plane illumination microscopy,” Science305(5686), 1007–1009 (2004).
[CrossRef] [PubMed]

Iannello, G.

Jaffe, J. S.

Keller, P. J.

K. Khairy and P. J. Keller, “Reconstructing embryonic development,” Genesis49(7), 488–513 (2011).
[CrossRef] [PubMed]

Khairy, K.

K. Khairy and P. J. Keller, “Reconstructing embryonic development,” Genesis49(7), 488–513 (2011).
[CrossRef] [PubMed]

Kržic, U.

R. Boistel, J. Swoger, U. Kržič, V. Fernandez, B. Gillet, and E. G. Reynaud, “The future of three-dimensional microscopic imaging in marine biology,” Mar. Ecol. (Berl.)32(4), 438–452 (2011).
[CrossRef]

E. G. Reynaud, U. Kržič, K. Greger, and E. H. K. Stelzer, “Light sheet-based fluorescence microscopy: more dimensions, more photons, and less photodamage,” HFSP J2(5), 266–275 (2008).
[CrossRef] [PubMed]

Kumar, S.

Liang, L.

D. A. Basiji, W. E. Ortyn, L. Liang, V. Venkatachalam, and P. Morrissey, “Cellular image analysis and imaging by flow cytometry,” Clin. Lab. Med.27(3), 653–670, viii (2007).
[CrossRef] [PubMed]

Lichtman, J. W.

J. A. Conchello and J. W. Lichtman, “Optical sectioning microscopy,” Nat. Methods2(12), 920–931 (2005).
[CrossRef] [PubMed]

Long, R. A.

Lyon, A. R.

MacLeod, K. T.

Mazzocchi, M. G.

. F. Culverhouse, R. Williams, M. Benfield, P. R. Flood, A. F. Sell, M. G. Mazzocchi, I. Buttino, and M. Sieracki, “Automatic image analysis of plankton: future perspectives,” Mar. Ecol. Prog. Ser.312, 297–309 (2006).
[CrossRef]

Morrissey, P.

D. A. Basiji, W. E. Ortyn, L. Liang, V. Venkatachalam, and P. Morrissey, “Cellular image analysis and imaging by flow cytometry,” Clin. Lab. Med.27(3), 653–670, viii (2007).
[CrossRef] [PubMed]

Ortyn, W. E.

D. A. Basiji, W. E. Ortyn, L. Liang, V. Venkatachalam, and P. Morrissey, “Cellular image analysis and imaging by flow cytometry,” Clin. Lab. Med.27(3), 653–670, viii (2007).
[CrossRef] [PubMed]

Pavone, F. S.

Quigg, A.

Z. V. Finkel, J. Beardall, K. J. Flynn, A. Quigg, T. A. V. Rees, and J. A. Raven, “Phytoplankton in a changing world: cell size and elemental stoichiometry,” J. Plankton Res.32(1), 119–137 (2010).
[CrossRef]

Raven, J. A.

Z. V. Finkel, J. Beardall, K. J. Flynn, A. Quigg, T. A. V. Rees, and J. A. Raven, “Phytoplankton in a changing world: cell size and elemental stoichiometry,” J. Plankton Res.32(1), 119–137 (2010).
[CrossRef]

Rees, T. A. V.

Z. V. Finkel, J. Beardall, K. J. Flynn, A. Quigg, T. A. V. Rees, and J. A. Raven, “Phytoplankton in a changing world: cell size and elemental stoichiometry,” J. Plankton Res.32(1), 119–137 (2010).
[CrossRef]

Reynaud, E. G.

R. Boistel, J. Swoger, U. Kržič, V. Fernandez, B. Gillet, and E. G. Reynaud, “The future of three-dimensional microscopic imaging in marine biology,” Mar. Ecol. (Berl.)32(4), 438–452 (2011).
[CrossRef]

E. G. Reynaud, U. Kržič, K. Greger, and E. H. K. Stelzer, “Light sheet-based fluorescence microscopy: more dimensions, more photons, and less photodamage,” HFSP J2(5), 266–275 (2008).
[CrossRef] [PubMed]

Sacconi, L.

Sell, A. F.

. F. Culverhouse, R. Williams, M. Benfield, P. R. Flood, A. F. Sell, M. G. Mazzocchi, I. Buttino, and M. Sieracki, “Automatic image analysis of plankton: future perspectives,” Mar. Ecol. Prog. Ser.312, 297–309 (2006).
[CrossRef]

Sieracki, M.

. F. Culverhouse, R. Williams, M. Benfield, P. R. Flood, A. F. Sell, M. G. Mazzocchi, I. Buttino, and M. Sieracki, “Automatic image analysis of plankton: future perspectives,” Mar. Ecol. Prog. Ser.312, 297–309 (2006).
[CrossRef]

Sikkel, M. B.

Silvestri, L.

Stainier, D. Y. R.

J. Huisken and D. Y. R. Stainier, “Selective plane illumination microscopy techniques in developmental biology,” Development136(12), 1963–1975 (2009).
[CrossRef] [PubMed]

J. Huisken and D. Y. R. Stainier, “Even fluorescence excitation by multidirectional selective plane illumination microscopy (mSPIM),” Opt. Lett.32(17), 2608–2610 (2007).
[CrossRef] [PubMed]

Stelzer, E. H. K.

E. G. Reynaud, U. Kržič, K. Greger, and E. H. K. Stelzer, “Light sheet-based fluorescence microscopy: more dimensions, more photons, and less photodamage,” HFSP J2(5), 266–275 (2008).
[CrossRef] [PubMed]

K. Greger, J. Swoger, and E. H. K. Stelzer, “Basic building units and properties of a fluorescence single plane illumination microscope,” Rev. Sci. Instrum.78(2), 023705 (2007).
[CrossRef] [PubMed]

C. J. Engelbrecht and E. H. K. Stelzer, “Resolution enhancement in a light-sheet-based microscope (SPIM),” Opt. Lett.31(10), 1477–1479 (2006).
[CrossRef] [PubMed]

J. Huisken, J. Swoger, F. Del Bene, J. Wittbrodt, and E. H. K. Stelzer, “Optical sectioning deep inside live embryos by selective plane illumination microscopy,” Science305(5686), 1007–1009 (2004).
[CrossRef] [PubMed]

Swoger, J.

R. Boistel, J. Swoger, U. Kržič, V. Fernandez, B. Gillet, and E. G. Reynaud, “The future of three-dimensional microscopic imaging in marine biology,” Mar. Ecol. (Berl.)32(4), 438–452 (2011).
[CrossRef]

K. Greger, J. Swoger, and E. H. K. Stelzer, “Basic building units and properties of a fluorescence single plane illumination microscope,” Rev. Sci. Instrum.78(2), 023705 (2007).
[CrossRef] [PubMed]

J. Huisken, J. Swoger, F. Del Bene, J. Wittbrodt, and E. H. K. Stelzer, “Optical sectioning deep inside live embryos by selective plane illumination microscopy,” Science305(5686), 1007–1009 (2004).
[CrossRef] [PubMed]

Venkatachalam, V.

D. A. Basiji, W. E. Ortyn, L. Liang, V. Venkatachalam, and P. Morrissey, “Cellular image analysis and imaging by flow cytometry,” Clin. Lab. Med.27(3), 653–670, viii (2007).
[CrossRef] [PubMed]

Weber, M.

M. Weber and J. Huisken, “Light sheet microscopy for real-time developmental biology,” Curr. Opin. Genet. Dev.21(5), 566–572 (2011).
[CrossRef] [PubMed]

Wilding, D.

Williams, R.

. F. Culverhouse, R. Williams, M. Benfield, P. R. Flood, A. F. Sell, M. G. Mazzocchi, I. Buttino, and M. Sieracki, “Automatic image analysis of plankton: future perspectives,” Mar. Ecol. Prog. Ser.312, 297–309 (2006).
[CrossRef]

Wittbrodt, J.

J. Huisken, J. Swoger, F. Del Bene, J. Wittbrodt, and E. H. K. Stelzer, “Optical sectioning deep inside live embryos by selective plane illumination microscopy,” Science305(5686), 1007–1009 (2004).
[CrossRef] [PubMed]

Clin. Lab. Med. (1)

D. A. Basiji, W. E. Ortyn, L. Liang, V. Venkatachalam, and P. Morrissey, “Cellular image analysis and imaging by flow cytometry,” Clin. Lab. Med.27(3), 653–670, viii (2007).
[CrossRef] [PubMed]

Curr. Opin. Genet. Dev. (1)

M. Weber and J. Huisken, “Light sheet microscopy for real-time developmental biology,” Curr. Opin. Genet. Dev.21(5), 566–572 (2011).
[CrossRef] [PubMed]

Development (1)

J. Huisken and D. Y. R. Stainier, “Selective plane illumination microscopy techniques in developmental biology,” Development136(12), 1963–1975 (2009).
[CrossRef] [PubMed]

Genesis (1)

K. Khairy and P. J. Keller, “Reconstructing embryonic development,” Genesis49(7), 488–513 (2011).
[CrossRef] [PubMed]

HFSP J (1)

E. G. Reynaud, U. Kržič, K. Greger, and E. H. K. Stelzer, “Light sheet-based fluorescence microscopy: more dimensions, more photons, and less photodamage,” HFSP J2(5), 266–275 (2008).
[CrossRef] [PubMed]

J. Plankton Res. (1)

Z. V. Finkel, J. Beardall, K. J. Flynn, A. Quigg, T. A. V. Rees, and J. A. Raven, “Phytoplankton in a changing world: cell size and elemental stoichiometry,” J. Plankton Res.32(1), 119–137 (2010).
[CrossRef]

Mar. Ecol. (Berl.) (1)

R. Boistel, J. Swoger, U. Kržič, V. Fernandez, B. Gillet, and E. G. Reynaud, “The future of three-dimensional microscopic imaging in marine biology,” Mar. Ecol. (Berl.)32(4), 438–452 (2011).
[CrossRef]

Mar. Ecol. Prog. Ser. (1)

. F. Culverhouse, R. Williams, M. Benfield, P. R. Flood, A. F. Sell, M. G. Mazzocchi, I. Buttino, and M. Sieracki, “Automatic image analysis of plankton: future perspectives,” Mar. Ecol. Prog. Ser.312, 297–309 (2006).
[CrossRef]

Nat. Methods (2)

L. Bonetta, “Flow cytometry smaller and better,” Nat. Methods2(10), 785–795 (2005).
[CrossRef]

J. A. Conchello and J. W. Lichtman, “Optical sectioning microscopy,” Nat. Methods2(12), 920–931 (2005).
[CrossRef] [PubMed]

Opt. Express (3)

Opt. Lett. (2)

Rev. Sci. Instrum. (1)

K. Greger, J. Swoger, and E. H. K. Stelzer, “Basic building units and properties of a fluorescence single plane illumination microscope,” Rev. Sci. Instrum.78(2), 023705 (2007).
[CrossRef] [PubMed]

Science (1)

J. Huisken, J. Swoger, F. Del Bene, J. Wittbrodt, and E. H. K. Stelzer, “Optical sectioning deep inside live embryos by selective plane illumination microscopy,” Science305(5686), 1007–1009 (2004).
[CrossRef] [PubMed]

Other (1)

H. M. Shapiro, Practical flow cytometry (Wiley-Liss, 1993), Chapter. 4.

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Figures (5)

Fig. 1
Fig. 1

Schematic setup of the light sheet based 3D imaging flow cytometer.

Fig. 2
Fig. 2

(a) Laser scattering image of the sample core, 580 × 580 pixels, scale bar: 20 μm, exposure time: 500 ms, (b) Timing chart of the trigger and camera synchronization control.

Fig. 3
Fig. 3

Characteristics of the light sheet generated. (a) Image of the light sheet, taken with a 5 × /0.2 lens from the side with the flowing of chlorophyll solution. (b) Intensity profile of the light sheet, FWHM is 5.39 ± 0.13 μm.

Fig. 4
Fig. 4

Experimentally measured PSFs. (a) Lateral PSF measured by fluorescence imaging: the FWHM is 0.81 ± 0.07 μm. (b) Axial PSF measured by laser scattering imaging: the FWHM is 1.42 ± 0.15 μm.

Fig. 5
Fig. 5

Images of Gambierdiscus sp. and Procentrum sp. captured with the 3D imaging flow cytometer. (a) Maximum projection of the stack of 30 images of Procentrum sp. ; (b) A single image out of the 30 planes; (c) and (d) are maximum projection of two different Gambierdiscus sp. cells. (c) has a stack of 35 images and (d) has a stack of 38 images; (e) Projections of the same stack used in Fig. 5(d) along lateral direction. Scale bars are 10 μm.

Metrics