Abstract

Digital holography offers a unique method for studying microscopic objects using quantitative measurements of the optical phase delays of transmitted light. The optical phase may be integrated across the object to produce an optical volume measurement, a parameter related to dry mass by a simple scaling factor. While digital holography is useful for comparing the properties of microscopic objects, especially cells, we show here that quantitative comparisons of optical phase can be influenced by the focal plane of the measurement. Although holographic images can be refocused digitally using Fresnel propagation, ambiguity can result if this aspect is not carefully controlled. We demonstrate that microscopic objects can be accurately profiled by employing a digital refocusing method to analyze phase profiles of polystyrene microspheres and red blood cells.

© 2015 Optical Society of America

Full Article  |  PDF Article
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References

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    [Crossref] [PubMed]
  3. N. T. Shaked, J. D. Finan, F. Guilak, and A. Wax, “Quantitative phase microscopy of articular chondrocyte dynamics by wide-field digital interferometry,” J. Biomed. Opt. 15(1), 010505 (2010).
    [Crossref] [PubMed]
  4. Z. Wang, L. Millet, M. Mir, H. Ding, S. Unarunotai, J. Rogers, M. U. Gillette, and G. Popescu, “Spatial light interference microscopy (SLIM),” Opt. Express 19(2), 1016–1026 (2011).
    [Crossref] [PubMed]
  5. P. Marquet, C. Depeursinge, and P. J. Magistretti, “Review of quantitative phase-digital holographic microscopy: promising novel imaging technique to resolve neuronal network activity and identify cellular biomarkers of psychiatric disorders,” Neurophotonics 1(2), 020901 (2014).
    [Crossref]
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    [Crossref] [PubMed]
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    [Crossref]
  14. I. N. Tang and H. R. Munkelwitz, “Simultaneous determination of refractive index and density of an evaporating aqueous solution droplet,” Aerosol Sci. Technol. 15(3), 201–207 (1991).
    [Crossref]
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    [Crossref]
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    [Crossref] [PubMed]
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  19. G. Levin, A. Kovalev, V. Minaev, and K. Sukhorukov, “Error in measuring dry cell mass with a computerized interference microscope,” Meas. Tech. 47(4), 412–416 (2004).
    [Crossref]
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2014 (1)

P. Marquet, C. Depeursinge, and P. J. Magistretti, “Review of quantitative phase-digital holographic microscopy: promising novel imaging technique to resolve neuronal network activity and identify cellular biomarkers of psychiatric disorders,” Neurophotonics 1(2), 020901 (2014).
[Crossref]

2012 (2)

K. G. Phillips, S. L. Jacques, and O. J. McCarty, “Measurement of single cell refractive index, dry mass, volume, and density using a transillumination microscope,” Phys. Rev. Lett. 109(11), 118105 (2012).
[Crossref] [PubMed]

M. Rinehart, Y. Zhu, and A. Wax, “Quantitative phase spectroscopy,” Biomed. Opt. Express 3(5), 958–965 (2012).
[Crossref] [PubMed]

2011 (3)

2010 (2)

N. T. Shaked, J. D. Finan, F. Guilak, and A. Wax, “Quantitative phase microscopy of articular chondrocyte dynamics by wide-field digital interferometry,” J. Biomed. Opt. 15(1), 010505 (2010).
[Crossref] [PubMed]

N. T. Shaked, J. D. Finan, F. Guilak, and A. Wax, “Quantitative phase microscopy of articular chondrocyte dynamics by wide-field digital interferometry,” J. Biomed. Opt. 15, 010505 (2010).

2008 (3)

G. Popescu, Y. Park, N. Lue, C. Best-Popescu, L. Deflores, R. R. Dasari, M. S. Feld, and K. Badizadegan, “Optical imaging of cell mass and growth dynamics,” Am. J. Physiol. Cell Physiol. 295(2), C538–C544 (2008).
[Crossref] [PubMed]

Y. Park, M. Diez-Silva, G. Popescu, G. Lykotrafitis, W. Choi, M. S. Feld, and S. Suresh, “Refractive index maps and membrane dynamics of human red blood cells parasitized by Plasmodium falciparum,” Proc. Natl. Acad. Sci. U.S.A. 105(37), 13730–13735 (2008).
[Crossref] [PubMed]

P. Langehanenberg, B. Kemper, D. Dirksen, and G. von Bally, “Autofocusing in digital holographic phase contrast microscopy on pure phase objects for live cell imaging,” Appl. Opt. 47(19), D176–D182 (2008).
[Crossref] [PubMed]

2006 (1)

2004 (2)

M. Liebling and M. Unser, “Autofocus for digital Fresnel holograms by use of a Fresnelet-sparsity criterion,” J. Opt. Soc. Am. A 21(12), 2424–2430 (2004).
[Crossref] [PubMed]

G. Levin, A. Kovalev, V. Minaev, and K. Sukhorukov, “Error in measuring dry cell mass with a computerized interference microscope,” Meas. Tech. 47(4), 412–416 (2004).
[Crossref]

2001 (1)

1998 (1)

V. Ball and J. J. Ramsden, “Buffer dependence of refractive index increments of protein solutions,” Biopolymers 46(7), 489–492 (1998).
[Crossref]

1991 (1)

I. N. Tang and H. R. Munkelwitz, “Simultaneous determination of refractive index and density of an evaporating aqueous solution droplet,” Aerosol Sci. Technol. 15(3), 201–207 (1991).
[Crossref]

1965 (1)

W. Heller, “Remarks on refractive index mixture rules,” J. Phys. Chem. 69(4), 1123–1129 (1965).
[Crossref]

1952 (2)

H. G. Davies and M. H. Wilkins, “Interference microscopy and mass determination,” Nature 169(4300), 541 (1952).
[Crossref] [PubMed]

R. Barer, “Interference microscopy and mass determination,” Nature 169(4296), 366–367 (1952).
[Crossref] [PubMed]

Badizadegan, K.

G. Popescu, Y. Park, N. Lue, C. Best-Popescu, L. Deflores, R. R. Dasari, M. S. Feld, and K. Badizadegan, “Optical imaging of cell mass and growth dynamics,” Am. J. Physiol. Cell Physiol. 295(2), C538–C544 (2008).
[Crossref] [PubMed]

Ball, V.

V. Ball and J. J. Ramsden, “Buffer dependence of refractive index increments of protein solutions,” Biopolymers 46(7), 489–492 (1998).
[Crossref]

Barer, R.

R. Barer, “Interference microscopy and mass determination,” Nature 169(4296), 366–367 (1952).
[Crossref] [PubMed]

Bashir, R.

M. Mir, Z. Wang, Z. Shen, M. Bednarz, R. Bashir, I. Golding, S. G. Prasanth, and G. Popescu, “Optical measurement of cycle-dependent cell growth,” Proc. Natl. Acad. Sci. U.S.A. 108(32), 13124–13129 (2011).
[Crossref] [PubMed]

Bednarz, M.

M. Mir, Z. Wang, Z. Shen, M. Bednarz, R. Bashir, I. Golding, S. G. Prasanth, and G. Popescu, “Optical measurement of cycle-dependent cell growth,” Proc. Natl. Acad. Sci. U.S.A. 108(32), 13124–13129 (2011).
[Crossref] [PubMed]

Best-Popescu, C.

G. Popescu, Y. Park, N. Lue, C. Best-Popescu, L. Deflores, R. R. Dasari, M. S. Feld, and K. Badizadegan, “Optical imaging of cell mass and growth dynamics,” Am. J. Physiol. Cell Physiol. 295(2), C538–C544 (2008).
[Crossref] [PubMed]

Callens, N.

Choi, W.

Y. Park, M. Diez-Silva, G. Popescu, G. Lykotrafitis, W. Choi, M. S. Feld, and S. Suresh, “Refractive index maps and membrane dynamics of human red blood cells parasitized by Plasmodium falciparum,” Proc. Natl. Acad. Sci. U.S.A. 105(37), 13730–13735 (2008).
[Crossref] [PubMed]

Dasari, R. R.

G. Popescu, Y. Park, N. Lue, C. Best-Popescu, L. Deflores, R. R. Dasari, M. S. Feld, and K. Badizadegan, “Optical imaging of cell mass and growth dynamics,” Am. J. Physiol. Cell Physiol. 295(2), C538–C544 (2008).
[Crossref] [PubMed]

Davies, H. G.

H. G. Davies and M. H. Wilkins, “Interference microscopy and mass determination,” Nature 169(4300), 541 (1952).
[Crossref] [PubMed]

De Nicola, S.

Deflores, L.

G. Popescu, Y. Park, N. Lue, C. Best-Popescu, L. Deflores, R. R. Dasari, M. S. Feld, and K. Badizadegan, “Optical imaging of cell mass and growth dynamics,” Am. J. Physiol. Cell Physiol. 295(2), C538–C544 (2008).
[Crossref] [PubMed]

Depeursinge, C.

P. Marquet, C. Depeursinge, and P. J. Magistretti, “Review of quantitative phase-digital holographic microscopy: promising novel imaging technique to resolve neuronal network activity and identify cellular biomarkers of psychiatric disorders,” Neurophotonics 1(2), 020901 (2014).
[Crossref]

Diez-Silva, M.

Y. Park, M. Diez-Silva, G. Popescu, G. Lykotrafitis, W. Choi, M. S. Feld, and S. Suresh, “Refractive index maps and membrane dynamics of human red blood cells parasitized by Plasmodium falciparum,” Proc. Natl. Acad. Sci. U.S.A. 105(37), 13730–13735 (2008).
[Crossref] [PubMed]

Ding, H.

Dirksen, D.

Dubois, F.

Feld, M. S.

Y. Park, M. Diez-Silva, G. Popescu, G. Lykotrafitis, W. Choi, M. S. Feld, and S. Suresh, “Refractive index maps and membrane dynamics of human red blood cells parasitized by Plasmodium falciparum,” Proc. Natl. Acad. Sci. U.S.A. 105(37), 13730–13735 (2008).
[Crossref] [PubMed]

G. Popescu, Y. Park, N. Lue, C. Best-Popescu, L. Deflores, R. R. Dasari, M. S. Feld, and K. Badizadegan, “Optical imaging of cell mass and growth dynamics,” Am. J. Physiol. Cell Physiol. 295(2), C538–C544 (2008).
[Crossref] [PubMed]

Ferraro, P.

Finan, J. D.

N. T. Shaked, J. D. Finan, F. Guilak, and A. Wax, “Quantitative phase microscopy of articular chondrocyte dynamics by wide-field digital interferometry,” J. Biomed. Opt. 15, 010505 (2010).

N. T. Shaked, J. D. Finan, F. Guilak, and A. Wax, “Quantitative phase microscopy of articular chondrocyte dynamics by wide-field digital interferometry,” J. Biomed. Opt. 15(1), 010505 (2010).
[Crossref] [PubMed]

Finizio, A.

Gillette, M. U.

Golding, I.

M. Mir, Z. Wang, Z. Shen, M. Bednarz, R. Bashir, I. Golding, S. G. Prasanth, and G. Popescu, “Optical measurement of cycle-dependent cell growth,” Proc. Natl. Acad. Sci. U.S.A. 108(32), 13124–13129 (2011).
[Crossref] [PubMed]

Grilli, S.

Guilak, F.

N. T. Shaked, J. D. Finan, F. Guilak, and A. Wax, “Quantitative phase microscopy of articular chondrocyte dynamics by wide-field digital interferometry,” J. Biomed. Opt. 15(1), 010505 (2010).
[Crossref] [PubMed]

N. T. Shaked, J. D. Finan, F. Guilak, and A. Wax, “Quantitative phase microscopy of articular chondrocyte dynamics by wide-field digital interferometry,” J. Biomed. Opt. 15, 010505 (2010).

Heller, W.

W. Heller, “Remarks on refractive index mixture rules,” J. Phys. Chem. 69(4), 1123–1129 (1965).
[Crossref]

Jacques, S. L.

K. G. Phillips, S. L. Jacques, and O. J. McCarty, “Measurement of single cell refractive index, dry mass, volume, and density using a transillumination microscope,” Phys. Rev. Lett. 109(11), 118105 (2012).
[Crossref] [PubMed]

Kemper, B.

Kovalev, A.

G. Levin, A. Kovalev, V. Minaev, and K. Sukhorukov, “Error in measuring dry cell mass with a computerized interference microscope,” Meas. Tech. 47(4), 412–416 (2004).
[Crossref]

Langehanenberg, P.

Levin, G.

G. Levin, A. Kovalev, V. Minaev, and K. Sukhorukov, “Error in measuring dry cell mass with a computerized interference microscope,” Meas. Tech. 47(4), 412–416 (2004).
[Crossref]

Liebling, M.

Lue, N.

G. Popescu, Y. Park, N. Lue, C. Best-Popescu, L. Deflores, R. R. Dasari, M. S. Feld, and K. Badizadegan, “Optical imaging of cell mass and growth dynamics,” Am. J. Physiol. Cell Physiol. 295(2), C538–C544 (2008).
[Crossref] [PubMed]

Lykotrafitis, G.

Y. Park, M. Diez-Silva, G. Popescu, G. Lykotrafitis, W. Choi, M. S. Feld, and S. Suresh, “Refractive index maps and membrane dynamics of human red blood cells parasitized by Plasmodium falciparum,” Proc. Natl. Acad. Sci. U.S.A. 105(37), 13730–13735 (2008).
[Crossref] [PubMed]

Magistretti, P. J.

P. Marquet, C. Depeursinge, and P. J. Magistretti, “Review of quantitative phase-digital holographic microscopy: promising novel imaging technique to resolve neuronal network activity and identify cellular biomarkers of psychiatric disorders,” Neurophotonics 1(2), 020901 (2014).
[Crossref]

Marquet, P.

P. Marquet, C. Depeursinge, and P. J. Magistretti, “Review of quantitative phase-digital holographic microscopy: promising novel imaging technique to resolve neuronal network activity and identify cellular biomarkers of psychiatric disorders,” Neurophotonics 1(2), 020901 (2014).
[Crossref]

Mater, M.

McCarty, O. J.

K. G. Phillips, S. L. Jacques, and O. J. McCarty, “Measurement of single cell refractive index, dry mass, volume, and density using a transillumination microscope,” Phys. Rev. Lett. 109(11), 118105 (2012).
[Crossref] [PubMed]

Meucci, R.

Millet, L.

Minaev, V.

G. Levin, A. Kovalev, V. Minaev, and K. Sukhorukov, “Error in measuring dry cell mass with a computerized interference microscope,” Meas. Tech. 47(4), 412–416 (2004).
[Crossref]

Mir, M.

Z. Wang, L. Millet, M. Mir, H. Ding, S. Unarunotai, J. Rogers, M. U. Gillette, and G. Popescu, “Spatial light interference microscopy (SLIM),” Opt. Express 19(2), 1016–1026 (2011).
[Crossref] [PubMed]

M. Mir, Z. Wang, Z. Shen, M. Bednarz, R. Bashir, I. Golding, S. G. Prasanth, and G. Popescu, “Optical measurement of cycle-dependent cell growth,” Proc. Natl. Acad. Sci. U.S.A. 108(32), 13124–13129 (2011).
[Crossref] [PubMed]

Munkelwitz, H. R.

I. N. Tang and H. R. Munkelwitz, “Simultaneous determination of refractive index and density of an evaporating aqueous solution droplet,” Aerosol Sci. Technol. 15(3), 201–207 (1991).
[Crossref]

Ni, J.

Park, Y.

G. Popescu, Y. Park, N. Lue, C. Best-Popescu, L. Deflores, R. R. Dasari, M. S. Feld, and K. Badizadegan, “Optical imaging of cell mass and growth dynamics,” Am. J. Physiol. Cell Physiol. 295(2), C538–C544 (2008).
[Crossref] [PubMed]

Y. Park, M. Diez-Silva, G. Popescu, G. Lykotrafitis, W. Choi, M. S. Feld, and S. Suresh, “Refractive index maps and membrane dynamics of human red blood cells parasitized by Plasmodium falciparum,” Proc. Natl. Acad. Sci. U.S.A. 105(37), 13730–13735 (2008).
[Crossref] [PubMed]

Phillips, K. G.

K. G. Phillips, S. L. Jacques, and O. J. McCarty, “Measurement of single cell refractive index, dry mass, volume, and density using a transillumination microscope,” Phys. Rev. Lett. 109(11), 118105 (2012).
[Crossref] [PubMed]

Pierattini, G.

Popescu, G.

Z. Wang, L. Millet, M. Mir, H. Ding, S. Unarunotai, J. Rogers, M. U. Gillette, and G. Popescu, “Spatial light interference microscopy (SLIM),” Opt. Express 19(2), 1016–1026 (2011).
[Crossref] [PubMed]

M. Mir, Z. Wang, Z. Shen, M. Bednarz, R. Bashir, I. Golding, S. G. Prasanth, and G. Popescu, “Optical measurement of cycle-dependent cell growth,” Proc. Natl. Acad. Sci. U.S.A. 108(32), 13124–13129 (2011).
[Crossref] [PubMed]

Y. Park, M. Diez-Silva, G. Popescu, G. Lykotrafitis, W. Choi, M. S. Feld, and S. Suresh, “Refractive index maps and membrane dynamics of human red blood cells parasitized by Plasmodium falciparum,” Proc. Natl. Acad. Sci. U.S.A. 105(37), 13730–13735 (2008).
[Crossref] [PubMed]

G. Popescu, Y. Park, N. Lue, C. Best-Popescu, L. Deflores, R. R. Dasari, M. S. Feld, and K. Badizadegan, “Optical imaging of cell mass and growth dynamics,” Am. J. Physiol. Cell Physiol. 295(2), C538–C544 (2008).
[Crossref] [PubMed]

Prasanth, S. G.

M. Mir, Z. Wang, Z. Shen, M. Bednarz, R. Bashir, I. Golding, S. G. Prasanth, and G. Popescu, “Optical measurement of cycle-dependent cell growth,” Proc. Natl. Acad. Sci. U.S.A. 108(32), 13124–13129 (2011).
[Crossref] [PubMed]

Ramsden, J. J.

V. Ball and J. J. Ramsden, “Buffer dependence of refractive index increments of protein solutions,” Biopolymers 46(7), 489–492 (1998).
[Crossref]

Rinehart, M.

Rogers, J.

Schockaert, C.

Shaked, N. T.

N. T. Shaked, J. D. Finan, F. Guilak, and A. Wax, “Quantitative phase microscopy of articular chondrocyte dynamics by wide-field digital interferometry,” J. Biomed. Opt. 15(1), 010505 (2010).
[Crossref] [PubMed]

N. T. Shaked, J. D. Finan, F. Guilak, and A. Wax, “Quantitative phase microscopy of articular chondrocyte dynamics by wide-field digital interferometry,” J. Biomed. Opt. 15, 010505 (2010).

Shen, Z.

M. Mir, Z. Wang, Z. Shen, M. Bednarz, R. Bashir, I. Golding, S. G. Prasanth, and G. Popescu, “Optical measurement of cycle-dependent cell growth,” Proc. Natl. Acad. Sci. U.S.A. 108(32), 13124–13129 (2011).
[Crossref] [PubMed]

Sukhorukov, K.

G. Levin, A. Kovalev, V. Minaev, and K. Sukhorukov, “Error in measuring dry cell mass with a computerized interference microscope,” Meas. Tech. 47(4), 412–416 (2004).
[Crossref]

Suresh, S.

Y. Park, M. Diez-Silva, G. Popescu, G. Lykotrafitis, W. Choi, M. S. Feld, and S. Suresh, “Refractive index maps and membrane dynamics of human red blood cells parasitized by Plasmodium falciparum,” Proc. Natl. Acad. Sci. U.S.A. 105(37), 13730–13735 (2008).
[Crossref] [PubMed]

Tang, I. N.

I. N. Tang and H. R. Munkelwitz, “Simultaneous determination of refractive index and density of an evaporating aqueous solution droplet,” Aerosol Sci. Technol. 15(3), 201–207 (1991).
[Crossref]

Unarunotai, S.

Unser, M.

von Bally, G.

Wang, Z.

Z. Wang, L. Millet, M. Mir, H. Ding, S. Unarunotai, J. Rogers, M. U. Gillette, and G. Popescu, “Spatial light interference microscopy (SLIM),” Opt. Express 19(2), 1016–1026 (2011).
[Crossref] [PubMed]

M. Mir, Z. Wang, Z. Shen, M. Bednarz, R. Bashir, I. Golding, S. G. Prasanth, and G. Popescu, “Optical measurement of cycle-dependent cell growth,” Proc. Natl. Acad. Sci. U.S.A. 108(32), 13124–13129 (2011).
[Crossref] [PubMed]

Wax, A.

M. Rinehart, Y. Zhu, and A. Wax, “Quantitative phase spectroscopy,” Biomed. Opt. Express 3(5), 958–965 (2012).
[Crossref] [PubMed]

N. T. Shaked, J. D. Finan, F. Guilak, and A. Wax, “Quantitative phase microscopy of articular chondrocyte dynamics by wide-field digital interferometry,” J. Biomed. Opt. 15, 010505 (2010).

N. T. Shaked, J. D. Finan, F. Guilak, and A. Wax, “Quantitative phase microscopy of articular chondrocyte dynamics by wide-field digital interferometry,” J. Biomed. Opt. 15(1), 010505 (2010).
[Crossref] [PubMed]

Wilkins, M. H.

H. G. Davies and M. H. Wilkins, “Interference microscopy and mass determination,” Nature 169(4300), 541 (1952).
[Crossref] [PubMed]

Xu, L.

Yourassowsky, C.

Zhu, Y.

Aerosol Sci. Technol. (1)

I. N. Tang and H. R. Munkelwitz, “Simultaneous determination of refractive index and density of an evaporating aqueous solution droplet,” Aerosol Sci. Technol. 15(3), 201–207 (1991).
[Crossref]

Am. J. Physiol. Cell Physiol. (1)

G. Popescu, Y. Park, N. Lue, C. Best-Popescu, L. Deflores, R. R. Dasari, M. S. Feld, and K. Badizadegan, “Optical imaging of cell mass and growth dynamics,” Am. J. Physiol. Cell Physiol. 295(2), C538–C544 (2008).
[Crossref] [PubMed]

Appl. Opt. (1)

Biomed. Opt. Express (1)

Biopolymers (1)

V. Ball and J. J. Ramsden, “Buffer dependence of refractive index increments of protein solutions,” Biopolymers 46(7), 489–492 (1998).
[Crossref]

J. Biomed. Opt. (2)

N. T. Shaked, J. D. Finan, F. Guilak, and A. Wax, “Quantitative phase microscopy of articular chondrocyte dynamics by wide-field digital interferometry,” J. Biomed. Opt. 15, 010505 (2010).

N. T. Shaked, J. D. Finan, F. Guilak, and A. Wax, “Quantitative phase microscopy of articular chondrocyte dynamics by wide-field digital interferometry,” J. Biomed. Opt. 15(1), 010505 (2010).
[Crossref] [PubMed]

J. Opt. Soc. Am. A (1)

J. Phys. Chem. (1)

W. Heller, “Remarks on refractive index mixture rules,” J. Phys. Chem. 69(4), 1123–1129 (1965).
[Crossref]

Meas. Tech. (1)

G. Levin, A. Kovalev, V. Minaev, and K. Sukhorukov, “Error in measuring dry cell mass with a computerized interference microscope,” Meas. Tech. 47(4), 412–416 (2004).
[Crossref]

Nature (2)

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K. G. Phillips, S. L. Jacques, and O. J. McCarty, “Measurement of single cell refractive index, dry mass, volume, and density using a transillumination microscope,” Phys. Rev. Lett. 109(11), 118105 (2012).
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Supplementary Material (1)

» Media 1: MOV (1349 KB)     

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Figures (5)

Fig. 1
Fig. 1

Schematic of experimental imaging system. Low coherence light is split into a reference arm and an input to the sample. Retroreflectors (RR) enables pathlength matching between the two arms. Matched 40x objectives image the sample and reference fields onto the CMOS detector. The reference arm is offset from the optical axis to enable off-axis digital holography. Sample images are shown as insets.

Fig. 2
Fig. 2

Digital refocusing of a single microsphere image. (a) xz-slice of amplitude focus; (b) representative phase images of microsphere at multiple propagation distances; (c) amplitude variance as a function of propagation distance, minimum variance location indicated with arrow; (d) measured microsphere volume vs. focal distance, red line indicates change in volume measurement with defocus distance, dashed lines and blue regions indicate the actual microsphere population distribution, ± σ, 2σ, 3σ.

Fig. 3
Fig. 3

Volume prediction plots for four microsphere populations. Left: blue dots and bars indicate population mean and standard deviation. Right: Individual measurements for 7-μm diameter microspheres. Red circles indicate measurements of hand-focused images, blue circles indicate digitally-refocused measurements. Dashed lines and blue regions indicate the actual microsphere population distribution, ± σ, 2σ, 3σ. Metrics are reported as means ± standard deviations.

Fig. 4
Fig. 4

Digital refocusing of a single red blood cell image and corresponding optical volume measurements. Top: Amplitude and phase of manually-defocused holograms of the same cell, followed by digitally-refocused phase images. Bottom-Left: Amplitude variance metric of holograms A-G. Bottom-Right: Computed OV of RBC from manually-focused phase images (black) and digitally-refocused phase images (blue). OV reported as mean ± standard deviation.

Fig. 5
Fig. 5

Digital refocusing of a single red blood cell image subjected to high flow rates. Top-Left: Optical volume of RBC with flow on and off (rates indicated on plot). Blue data points are OV calculations from raw images and red data points are from digitally refocused images. Images of cells at points A-E on plot are shown at the right with corresponding time indicated. Bottom-Left: Defocus at each time point as determined by the output of the digital refocusing algorithm (Media 1).

Equations (2)

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DM=( x,y Δϕ(x,y)dxdy )/α,
OV= x,y ΔOPL(x,y)dxdy = x,y Δn(x,y)h(x,y)dxdy .

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