Abstract

Traditional Normarski differential interference contrast (DIC) microscopy is a very powerful method for imaging nonstained biological samples. However, one of its major limitations is the nonquantitative nature of the imaging. To overcome this problem, we developed a quantitative DIC microscopy method based on off-axis sample self- interference. The digital holography algorithm is applied to obtain quantitative phase gradients in orthogonal directions, which leads to a quantitative phase image through a spiral integration of the phase gradients. This method is practically simple to implement on any standard microscope without stringent requirements on polarization optics. Optical sectioning can be obtained through enlarged illumination NA.

© 2010 Optical Society of America

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References

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2009 (1)

2008 (2)

S. V. King, A. Libertun, R. Piestun, C. J. Cogswell, and C. Preza, J. Biomed. Opt. 13, 024020 (2008).
[CrossRef] [PubMed]

M. Shribak, J. LaFountain, D. Biggs, and S. Inoue, J. Biomed. Opt. 13, 014011 (2008).
[CrossRef] [PubMed]

2007 (1)

2006 (1)

2005 (1)

2004 (1)

M. R. Arnison, K. G. Larkin, C. J. R. Sheppard, N. I. Smith, and C. J. Cogswell, J. Microsc. 214, 7 (2004).
[CrossRef] [PubMed]

1998 (2)

1973 (1)

Arnison, M. R.

M. R. Arnison, K. G. Larkin, C. J. R. Sheppard, N. I. Smith, and C. J. Cogswell, J. Microsc. 214, 7 (2004).
[CrossRef] [PubMed]

Badizadegan, K.

Barty, A.

Biggs, D.

M. Shribak, J. LaFountain, D. Biggs, and S. Inoue, J. Biomed. Opt. 13, 014011 (2008).
[CrossRef] [PubMed]

Bon, P.

Choi, W.

Cogswell, C. J.

S. V. King, A. Libertun, R. Piestun, C. J. Cogswell, and C. Preza, J. Biomed. Opt. 13, 024020 (2008).
[CrossRef] [PubMed]

M. R. Arnison, K. G. Larkin, C. J. R. Sheppard, N. I. Smith, and C. J. Cogswell, J. Microsc. 214, 7 (2004).
[CrossRef] [PubMed]

Cuche, E.

Dasari, R. R.

Depeursinge, C.

Emery, Y.

Feld, M. S.

Ikeda, T.

Inoue, S.

M. Shribak, J. LaFountain, D. Biggs, and S. Inoue, J. Biomed. Opt. 13, 014011 (2008).
[CrossRef] [PubMed]

King, S. V.

S. V. King, A. Libertun, R. Piestun, C. J. Cogswell, and C. Preza, J. Biomed. Opt. 13, 024020 (2008).
[CrossRef] [PubMed]

LaFountain, J.

M. Shribak, J. LaFountain, D. Biggs, and S. Inoue, J. Biomed. Opt. 13, 014011 (2008).
[CrossRef] [PubMed]

Larkin, K. G.

M. R. Arnison, K. G. Larkin, C. J. R. Sheppard, N. I. Smith, and C. J. Cogswell, J. Microsc. 214, 7 (2004).
[CrossRef] [PubMed]

Libertun, A.

S. V. King, A. Libertun, R. Piestun, C. J. Cogswell, and C. Preza, J. Biomed. Opt. 13, 024020 (2008).
[CrossRef] [PubMed]

Lue, N.

Magistretti, P.

Marquet, P.

Maucort, G.

Monneret, S.

Nugent, K. A.

Paganin, D.

Piestun, R.

S. V. King, A. Libertun, R. Piestun, C. J. Cogswell, and C. Preza, J. Biomed. Opt. 13, 024020 (2008).
[CrossRef] [PubMed]

Popescu, G.

Preza, C.

S. V. King, A. Libertun, R. Piestun, C. J. Cogswell, and C. Preza, J. Biomed. Opt. 13, 024020 (2008).
[CrossRef] [PubMed]

Rappaz, B.

Roberts, A.

Sheppard, C. J. R.

M. R. Arnison, K. G. Larkin, C. J. R. Sheppard, N. I. Smith, and C. J. Cogswell, J. Microsc. 214, 7 (2004).
[CrossRef] [PubMed]

Shribak, M.

M. Shribak, J. LaFountain, D. Biggs, and S. Inoue, J. Biomed. Opt. 13, 014011 (2008).
[CrossRef] [PubMed]

Smith, N. I.

M. R. Arnison, K. G. Larkin, C. J. R. Sheppard, N. I. Smith, and C. J. Cogswell, J. Microsc. 214, 7 (2004).
[CrossRef] [PubMed]

Wattellier, B.

Wyant, J. C.

Yamaguchi, I.

Zhang, T.

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Figures (3)

Fig. 1
Fig. 1

Schematic diagram of our off-axis DIC microscope setup. The inset shows the lateral shearing of the wavefront caused by the displacement of the grating from the image plane. IP1, IP2, image plane 1 and 2; G, grating; SF, spatial filter.

Fig. 2
Fig. 2

(a) Interferogram measured on the CCD (inset shows magnified fringes of the boxed area); (b) two- dimensional Fourier transform of the interferogram: the circles show the frequency component used for obtaining phase gradient images, while the arrows show the respective gradient directions; (c), (d) phase gradient images of the cell; (e) phase image of the cell obtained through spiral integration. Scale bar, 15 μm .

Fig. 3
Fig. 3

(a), (b) Bright-field images of a HeLa cell at different focal positions; (c), (d) phase gradient images of the cell under illumination NA of 0.05; (e), (f) phase gradient images of the cell under illumination NA of 0.8.

Equations (2)

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S = | A 0 ( x , y ) e i ( k 0 x + ϕ ( x , y ) ) + A 1 ( x + Δ x , y ) e i ( k 1 x + ϕ ( x + Δ x , y ) ) | 2 = A 0 ( x , y ) 2 + A 1 ( x , y ) 2 + 2 A 0 ( x , y ) A 1 ( x + Δ x , y ) cos [ ( k 1 k 0 ) x + ( ϕ ( x , y ) / x ) Δ x ] ,
Δ x sample d sin ( θ ) Mag = d λ L Mag ,

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