Abstract

A slightly-off-axis interferometry based Hilbert phase microscopy (HPM) method is developed to quantitatively obtain the phase distribution. Owing to its single-shot nature and details detection ability, HPM can be used to investigate rapid phenomena that take place in transparent structures such as biological cells. Moreover, the slightly-off-axis interferometry owns higher effective bandwidth and more sensitivity than traditional off-axis interferometry. The proposed method takes advantages of the above techniques to obtain the phase image of the red blood cells and compared with the traditional off-axis interferometry and phase retrieval algorithm based on the FFT. The experimental results show that the proposed method owns fine spatial details and real-time imaging ability. We are sure that the proposed method provides a breakthrough for real-time observing and quantitative analyzing of cells in vivo.

© 2011 OSA

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  1. N. T. Shaked, M. T. Rinehart, and A. Wax, “Dual-interference-channel quantitative-phase microscopy of live cell dynamics,” Opt. Lett. 34(6), 767–769 (2009).
    [CrossRef] [PubMed]
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    [CrossRef] [PubMed]
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    [CrossRef] [PubMed]
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    [CrossRef] [PubMed]
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    [CrossRef]
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    [CrossRef] [PubMed]
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    [CrossRef] [PubMed]
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    [CrossRef] [PubMed]

2010 (2)

K. Dillon and Y. Fainman, “Depth sectioning of attenuation,” J. Opt. Soc. Am. A 27(6), 1347–1354 (2010).
[CrossRef] [PubMed]

J. Jang, C. Y. Bae, J.-K. Park, and J. C. Ye, “Self-reference extended depth-of-field quantitative phase microscopy,” Proc. SPIE 7570, 757018, 757018-8 (2010).
[CrossRef]

2009 (5)

2007 (3)

2006 (2)

G. Popescu, T. Ikeda, R. R. Dasari, and M. S. Feld, “Diffraction phase microscopy for quantifying cell structure and dynamics,” Opt. Lett. 31(6), 775–777 (2006).
[CrossRef] [PubMed]

C. Uzoigwe, “The human erythrocyte has developed the biconcave disc shape to optimise the flow properties of the blood in the large vessels,” Med. Hypotheses 67(5), 1159–1163 (2006).
[CrossRef] [PubMed]

2005 (2)

2003 (1)

D. J. Stephens and V. J. Allan, “Light microscopy techniques for live cell imaging,” Science 300(5616), 82–86 (2003).
[CrossRef] [PubMed]

2002 (1)

R. Nafe and W. Schlote, “Methods for shape analysis of two-dimensional closed contours—a biologically important, but widely neglected field in histopathology,” Electron J. Pathol. 8, 022-02 (2002).

2000 (1)

1999 (2)

Y. Takaki, H. Kawai, and H. Ohzu, “Hybrid holographic microscopy free of conjugate and zero-order images,” Appl. Opt. 38(23), 4990–4996 (1999).
[CrossRef] [PubMed]

E. D. Taylor, C. Cates, M. E. Mauel, D. A. Maurer, D. Nadle, G. A. Navratil, and M. Shilov, “Nonstationary signal analysis of magnetic islands in plasmas,” Rev. Sci. Instrum. 70(12), 4545–4551 (1999).
[CrossRef]

1997 (2)

G. N. Vishnyakov and G. G. Levin, “Interferometric computed-microtomography of 3D phase objects,” Proc. SPIE 2984, 64–71 (1997).
[CrossRef]

I. Yamaguchi and T. Zhang, “Phase-shifting digital holography,” Opt. Lett. 22(16), 1268–1270 (1997).
[CrossRef] [PubMed]

1994 (1)

L. L. Wheeless, R. D. Robinson, O. P. Lapets, C. Cox, A. Rubio, M. Weintraub, and L. J. Benjamin, “Classification of red blood cells as normal, sickle, or other abnormal, using a single image analysis feature,” Cytometry 17(2), 159–166 (1994).
[CrossRef] [PubMed]

1962 (1)

Allan, V. J.

D. J. Stephens and V. J. Allan, “Light microscopy techniques for live cell imaging,” Science 300(5616), 82–86 (2003).
[CrossRef] [PubMed]

Badizadegan, K.

Bae, C. Y.

J. Jang, C. Y. Bae, J.-K. Park, and J. C. Ye, “Self-reference extended depth-of-field quantitative phase microscopy,” Proc. SPIE 7570, 757018, 757018-8 (2010).
[CrossRef]

Benjamin, L. J.

L. L. Wheeless, R. D. Robinson, O. P. Lapets, C. Cox, A. Rubio, M. Weintraub, and L. J. Benjamin, “Classification of red blood cells as normal, sickle, or other abnormal, using a single image analysis feature,” Cytometry 17(2), 159–166 (1994).
[CrossRef] [PubMed]

Bewersdorf, J.

Cates, C.

E. D. Taylor, C. Cates, M. E. Mauel, D. A. Maurer, D. Nadle, G. A. Navratil, and M. Shilov, “Nonstationary signal analysis of magnetic islands in plasmas,” Rev. Sci. Instrum. 70(12), 4545–4551 (1999).
[CrossRef]

Choi, W.

Colomb, T.

Cox, C.

L. L. Wheeless, R. D. Robinson, O. P. Lapets, C. Cox, A. Rubio, M. Weintraub, and L. J. Benjamin, “Classification of red blood cells as normal, sickle, or other abnormal, using a single image analysis feature,” Cytometry 17(2), 159–166 (1994).
[CrossRef] [PubMed]

Cuche, E.

Dasari, R. R.

Y. Sung, W. Choi, C. Fang-Yen, K. Badizadegan, R. R. Dasari, and M. S. Feld, “Optical diffraction tomography for high resolution live cell imaging,” Opt. Express 17(1), 266–277 (2009).
[CrossRef] [PubMed]

Z. Yaqoob, W. Choi, S. Oh, N. Lue, Y. Park, C. Fang-Yen, R. R. Dasari, K. Badizadegan, and M. S. Feld, “Improved phase sensitivity in spectral domain phase microscopy using line-field illumination and self phase-referencing,” Opt. Express 17(13), 10681–10687 (2009).
[CrossRef] [PubMed]

C. Fang-Yen, S. Oh, Y. Park, W. Choi, S. Song, H. S. Seung, R. R. Dasari, and M. S. Feld, “Imaging voltage-dependent cell motions with heterodyne Mach-Zehnder phase microscopy,” Opt. Lett. 32(11), 1572–1574 (2007).
[CrossRef] [PubMed]

N. Lue, J. Bewersdorf, M. D. Lessard, K. Badizadegan, R. R. Dasari, M. S. Feld, and G. Popescu, “Tissue refractometry using Hilbert phase microscopy,” Opt. Lett. 32(24), 3522–3524 (2007).
[CrossRef] [PubMed]

W. Choi, C. Fang-Yen, K. Badizadegan, S. Oh, N. Lue, R. R. Dasari, and M. S. Feld, “Tomographic phase microscopy,” Nat. Methods 4(9), 717–719 (2007).
[CrossRef] [PubMed]

G. Popescu, T. Ikeda, R. R. Dasari, and M. S. Feld, “Diffraction phase microscopy for quantifying cell structure and dynamics,” Opt. Lett. 31(6), 775–777 (2006).
[CrossRef] [PubMed]

T. Ikeda, G. Popescu, R. R. Dasari, and M. S. Feld, “Hilbert phase microscopy for investigating fast dynamics in transparent systems,” Opt. Lett. 30(10), 1165–1167 (2005).
[CrossRef] [PubMed]

Depeursinge, C.

Dillon, K.

Emery, Y.

Fainman, Y.

Fang-Yen, C.

Feld, M. S.

Y. Sung, W. Choi, C. Fang-Yen, K. Badizadegan, R. R. Dasari, and M. S. Feld, “Optical diffraction tomography for high resolution live cell imaging,” Opt. Express 17(1), 266–277 (2009).
[CrossRef] [PubMed]

Z. Yaqoob, W. Choi, S. Oh, N. Lue, Y. Park, C. Fang-Yen, R. R. Dasari, K. Badizadegan, and M. S. Feld, “Improved phase sensitivity in spectral domain phase microscopy using line-field illumination and self phase-referencing,” Opt. Express 17(13), 10681–10687 (2009).
[CrossRef] [PubMed]

N. Lue, J. Bewersdorf, M. D. Lessard, K. Badizadegan, R. R. Dasari, M. S. Feld, and G. Popescu, “Tissue refractometry using Hilbert phase microscopy,” Opt. Lett. 32(24), 3522–3524 (2007).
[CrossRef] [PubMed]

C. Fang-Yen, S. Oh, Y. Park, W. Choi, S. Song, H. S. Seung, R. R. Dasari, and M. S. Feld, “Imaging voltage-dependent cell motions with heterodyne Mach-Zehnder phase microscopy,” Opt. Lett. 32(11), 1572–1574 (2007).
[CrossRef] [PubMed]

W. Choi, C. Fang-Yen, K. Badizadegan, S. Oh, N. Lue, R. R. Dasari, and M. S. Feld, “Tomographic phase microscopy,” Nat. Methods 4(9), 717–719 (2007).
[CrossRef] [PubMed]

G. Popescu, T. Ikeda, R. R. Dasari, and M. S. Feld, “Diffraction phase microscopy for quantifying cell structure and dynamics,” Opt. Lett. 31(6), 775–777 (2006).
[CrossRef] [PubMed]

T. Ikeda, G. Popescu, R. R. Dasari, and M. S. Feld, “Hilbert phase microscopy for investigating fast dynamics in transparent systems,” Opt. Lett. 30(10), 1165–1167 (2005).
[CrossRef] [PubMed]

Ikeda, T.

Jang, J.

J. Jang, C. Y. Bae, J.-K. Park, and J. C. Ye, “Self-reference extended depth-of-field quantitative phase microscopy,” Proc. SPIE 7570, 757018, 757018-8 (2010).
[CrossRef]

Kawai, H.

Lapets, O. P.

L. L. Wheeless, R. D. Robinson, O. P. Lapets, C. Cox, A. Rubio, M. Weintraub, and L. J. Benjamin, “Classification of red blood cells as normal, sickle, or other abnormal, using a single image analysis feature,” Cytometry 17(2), 159–166 (1994).
[CrossRef] [PubMed]

Leith, E. N.

Lessard, M. D.

Levin, G. G.

G. N. Vishnyakov and G. G. Levin, “Interferometric computed-microtomography of 3D phase objects,” Proc. SPIE 2984, 64–71 (1997).
[CrossRef]

Lue, N.

Magistretti, P. J.

Marquet, P.

Mauel, M. E.

E. D. Taylor, C. Cates, M. E. Mauel, D. A. Maurer, D. Nadle, G. A. Navratil, and M. Shilov, “Nonstationary signal analysis of magnetic islands in plasmas,” Rev. Sci. Instrum. 70(12), 4545–4551 (1999).
[CrossRef]

Maurer, D. A.

E. D. Taylor, C. Cates, M. E. Mauel, D. A. Maurer, D. Nadle, G. A. Navratil, and M. Shilov, “Nonstationary signal analysis of magnetic islands in plasmas,” Rev. Sci. Instrum. 70(12), 4545–4551 (1999).
[CrossRef]

Mir, M.

Nadle, D.

E. D. Taylor, C. Cates, M. E. Mauel, D. A. Maurer, D. Nadle, G. A. Navratil, and M. Shilov, “Nonstationary signal analysis of magnetic islands in plasmas,” Rev. Sci. Instrum. 70(12), 4545–4551 (1999).
[CrossRef]

Nafe, R.

R. Nafe and W. Schlote, “Methods for shape analysis of two-dimensional closed contours—a biologically important, but widely neglected field in histopathology,” Electron J. Pathol. 8, 022-02 (2002).

Navratil, G. A.

E. D. Taylor, C. Cates, M. E. Mauel, D. A. Maurer, D. Nadle, G. A. Navratil, and M. Shilov, “Nonstationary signal analysis of magnetic islands in plasmas,” Rev. Sci. Instrum. 70(12), 4545–4551 (1999).
[CrossRef]

Oh, S.

Ohzu, H.

Park, J.-K.

J. Jang, C. Y. Bae, J.-K. Park, and J. C. Ye, “Self-reference extended depth-of-field quantitative phase microscopy,” Proc. SPIE 7570, 757018, 757018-8 (2010).
[CrossRef]

Park, Y.

Popescu, G.

Rappaz, B.

Rinehart, M. T.

Robinson, R. D.

L. L. Wheeless, R. D. Robinson, O. P. Lapets, C. Cox, A. Rubio, M. Weintraub, and L. J. Benjamin, “Classification of red blood cells as normal, sickle, or other abnormal, using a single image analysis feature,” Cytometry 17(2), 159–166 (1994).
[CrossRef] [PubMed]

Rubio, A.

L. L. Wheeless, R. D. Robinson, O. P. Lapets, C. Cox, A. Rubio, M. Weintraub, and L. J. Benjamin, “Classification of red blood cells as normal, sickle, or other abnormal, using a single image analysis feature,” Cytometry 17(2), 159–166 (1994).
[CrossRef] [PubMed]

Schlote, W.

R. Nafe and W. Schlote, “Methods for shape analysis of two-dimensional closed contours—a biologically important, but widely neglected field in histopathology,” Electron J. Pathol. 8, 022-02 (2002).

Seung, H. S.

Shaked, N. T.

Shilov, M.

E. D. Taylor, C. Cates, M. E. Mauel, D. A. Maurer, D. Nadle, G. A. Navratil, and M. Shilov, “Nonstationary signal analysis of magnetic islands in plasmas,” Rev. Sci. Instrum. 70(12), 4545–4551 (1999).
[CrossRef]

Song, S.

Stephens, D. J.

D. J. Stephens and V. J. Allan, “Light microscopy techniques for live cell imaging,” Science 300(5616), 82–86 (2003).
[CrossRef] [PubMed]

Sung, Y.

Takaki, Y.

Tangella, K.

Taylor, E. D.

E. D. Taylor, C. Cates, M. E. Mauel, D. A. Maurer, D. Nadle, G. A. Navratil, and M. Shilov, “Nonstationary signal analysis of magnetic islands in plasmas,” Rev. Sci. Instrum. 70(12), 4545–4551 (1999).
[CrossRef]

Upatnieks, J.

Uzoigwe, C.

C. Uzoigwe, “The human erythrocyte has developed the biconcave disc shape to optimise the flow properties of the blood in the large vessels,” Med. Hypotheses 67(5), 1159–1163 (2006).
[CrossRef] [PubMed]

Vishnyakov, G. N.

G. N. Vishnyakov and G. G. Levin, “Interferometric computed-microtomography of 3D phase objects,” Proc. SPIE 2984, 64–71 (1997).
[CrossRef]

Wang, Z.

Wax, A.

Weintraub, M.

L. L. Wheeless, R. D. Robinson, O. P. Lapets, C. Cox, A. Rubio, M. Weintraub, and L. J. Benjamin, “Classification of red blood cells as normal, sickle, or other abnormal, using a single image analysis feature,” Cytometry 17(2), 159–166 (1994).
[CrossRef] [PubMed]

Wheeless, L. L.

L. L. Wheeless, R. D. Robinson, O. P. Lapets, C. Cox, A. Rubio, M. Weintraub, and L. J. Benjamin, “Classification of red blood cells as normal, sickle, or other abnormal, using a single image analysis feature,” Cytometry 17(2), 159–166 (1994).
[CrossRef] [PubMed]

Yamaguchi, I.

Yaqoob, Z.

Ye, J. C.

J. Jang, C. Y. Bae, J.-K. Park, and J. C. Ye, “Self-reference extended depth-of-field quantitative phase microscopy,” Proc. SPIE 7570, 757018, 757018-8 (2010).
[CrossRef]

Zhang, T.

Zhu, Y.

Appl. Opt. (2)

Cytometry (1)

L. L. Wheeless, R. D. Robinson, O. P. Lapets, C. Cox, A. Rubio, M. Weintraub, and L. J. Benjamin, “Classification of red blood cells as normal, sickle, or other abnormal, using a single image analysis feature,” Cytometry 17(2), 159–166 (1994).
[CrossRef] [PubMed]

Electron J. Pathol. (1)

R. Nafe and W. Schlote, “Methods for shape analysis of two-dimensional closed contours—a biologically important, but widely neglected field in histopathology,” Electron J. Pathol. 8, 022-02 (2002).

J. Opt. Soc. Am. (1)

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

Med. Hypotheses (1)

C. Uzoigwe, “The human erythrocyte has developed the biconcave disc shape to optimise the flow properties of the blood in the large vessels,” Med. Hypotheses 67(5), 1159–1163 (2006).
[CrossRef] [PubMed]

Nat. Methods (1)

W. Choi, C. Fang-Yen, K. Badizadegan, S. Oh, N. Lue, R. R. Dasari, and M. S. Feld, “Tomographic phase microscopy,” Nat. Methods 4(9), 717–719 (2007).
[CrossRef] [PubMed]

Opt. Express (4)

Opt. Lett. (7)

Proc. SPIE (2)

G. N. Vishnyakov and G. G. Levin, “Interferometric computed-microtomography of 3D phase objects,” Proc. SPIE 2984, 64–71 (1997).
[CrossRef]

J. Jang, C. Y. Bae, J.-K. Park, and J. C. Ye, “Self-reference extended depth-of-field quantitative phase microscopy,” Proc. SPIE 7570, 757018, 757018-8 (2010).
[CrossRef]

Rev. Sci. Instrum. (1)

E. D. Taylor, C. Cates, M. E. Mauel, D. A. Maurer, D. Nadle, G. A. Navratil, and M. Shilov, “Nonstationary signal analysis of magnetic islands in plasmas,” Rev. Sci. Instrum. 70(12), 4545–4551 (1999).
[CrossRef]

Science (1)

D. J. Stephens and V. J. Allan, “Light microscopy techniques for live cell imaging,” Science 300(5616), 82–86 (2003).
[CrossRef] [PubMed]

Other (2)

S. C. Pei and J. J. Ding, “The generalized radial Hilbert transform and its applications to 2-D edge detection (Any direction or specified directions),” in Proceedings of the IEEE International Conference on Acoustics, (Institute of Electrical and Electronics Engineers, Hong Kong, 2003), pp. 357–360.

“Red blood cell,” http://en.wikipedia.org/wiki/Red_blood_cell .

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Figures (6)

Fig. 1
Fig. 1

Experimental setup for slightly-off-axis interference. SPF = Spatial filter (beam expander with a confocally-positioned pinhole), BS1, BS2 = Beam splitters; M1, M2 = Mirrors; MO1, MO2 = Micro objectives; S = Sample.

Fig. 2
Fig. 2

Interferograms of RBCs (7–8 μm in diameter). (a) Off-axis interferogram. (b) slightly-off-axis interferogram. White horizontal scale bars in (a, b) represent 1 μm.

Fig. 3
Fig. 3

The first group of quantitative phase images of 7–8 μm red blood cells. (a-d) Final unwrapped phase maps obtained by method 1 to method 4, respectively. Black horizontal scale bars in (a–d) represent 1 μm. The common color bar indicates phase in radians.

Fig. 5
Fig. 5

The third group of quantitative phase images of 7–8 μm red blood cells. (a-d) Final unwrapped phase maps obtained by method 1 to method 4, respectively. Black horizontal scale bars in (a–d) represent 1 μm. It is noted that the first two methods process the same interferogram; the same with the last two methods. (e-h) Vertical views associated with (a-d).The color bars indicate phase in radians. (i-l) Phase profile fluctuations from the dotted line in that crosses the cell in (a-d).

Fig. 4
Fig. 4

The second group of quantitative phase images of 7–8 μm red blood cells. (a-d) Final unwrapped phase maps obtained by method 1 to method 4, respectively. Black horizontal scale bars in (a–d) represent 1 μm. The common color bar indicates phase in radians.

Fig. 6
Fig. 6

Corresponding histograms of the phase shifts in Figs. 5 (a-d). The red dashed line is a division, and the left of it represents NaCl solution information while the right part shows cell information.

Tables (2)

Tables Icon

Table 1 Extracted Image Analysis Features for Each Feature Based on the Consensus Data Set

Tables Icon

Table 2 Calculated effective data from the phase histogram

Equations (4)

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I ( x ) = I R + I S + 2 [ I R I S ( x ) ] 1 / 2 cos [ q x + ϕ ( x ) ]
z ( x ) = 1 2 u ( x ) + 1 2 j H T u ( x ) , Φ ( x ) = tan 1 { Im [ z ( x ) ] / Re [ z ( x ) ] } ,
Form factor = 4  × π   × Area Perimeter 2  
S N = 1 N i = 1 N ( ϕ i ϕ ¯ ) 2

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