Abstract

We present a study of the three-dimensional structure of cancer cells using dual-wavelength phase-imaging digital holographic microscopy. Phase imaging of objects with optical height variation greater than the wavelength of light is ambiguous and causes phase wrapping. By comparing two phase images recorded at different wavelengths, the images can be accurately unwrapped. The unwrapping method is computationally fast and straightforward, and it can process complex topologies. Additionally, the limitations on the total optical height are significantly relaxed. This new methodology is widely applicable to other phase-imaging techniques as well as in applications beyond optical microscopy.

© 2011 Optical Society of America

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References

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  1. D. C. Ghiglia and M. D. Pritt, Two-Dimensional Phase Unwrapping: Theory, Algorithms, and Software (Wiley, 1998).
  2. A. Khmaladze, A. Restrepo-Martínez, M. K. Kim, R. Castañeda, and A. Blandón, Appl. Opt. 47, 3203 (2008).
    [CrossRef] [PubMed]
  3. U. Schnars and W. Jüptner, Digital Holography: Digital Hologram Recording, Numerical Reconstruction, and Related Techniques (Springer, 2004).
  4. J. Gass, A. Dakoff, and M. K. Kim, Opt. Lett. 28, 1141 (2003).
    [CrossRef] [PubMed]
  5. C. Mann, P. Bingham, V. Paquit, and K. Tobin, Opt. Express 16, 9753 (2008).
    [CrossRef] [PubMed]
  6. H. Hendargo, M. Zhao, N. Shepherd, and J. Izatt, Opt. Express 17, 5039 (2009).
    [CrossRef] [PubMed]
  7. A. Khmaladze, M. K. Kim, and C.-M. Lo, Opt. Express 16, 10900 (2008).
    [CrossRef] [PubMed]

2009 (1)

2008 (3)

2003 (1)

Bingham, P.

Blandón, A.

Castañeda, R.

Dakoff, A.

Gass, J.

Ghiglia, D. C.

D. C. Ghiglia and M. D. Pritt, Two-Dimensional Phase Unwrapping: Theory, Algorithms, and Software (Wiley, 1998).

Hendargo, H.

Izatt, J.

Jüptner, W.

U. Schnars and W. Jüptner, Digital Holography: Digital Hologram Recording, Numerical Reconstruction, and Related Techniques (Springer, 2004).

Khmaladze, A.

Kim, M. K.

Lo, C.-M.

Mann, C.

Paquit, V.

Pritt, M. D.

D. C. Ghiglia and M. D. Pritt, Two-Dimensional Phase Unwrapping: Theory, Algorithms, and Software (Wiley, 1998).

Restrepo-Martínez, A.

Schnars, U.

U. Schnars and W. Jüptner, Digital Holography: Digital Hologram Recording, Numerical Reconstruction, and Related Techniques (Springer, 2004).

Shepherd, N.

Tobin, K.

Zhao, M.

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Figures (3)

Fig. 1
Fig. 1

Linear regression phase unwrapping: (a)  m 1 ( m 2 ) via Eq. (6) and (b)  r ( m 2 ) —the square of the difference between m 1 and the nearest integer. In this example, the wavelengths are 675 and 635 nm , and r ( m 2 ) is minimal at m 2 = 16 .

Fig. 2
Fig. 2

Digital holographic imaging of KB cell: (a) phase image at 675 nm , (b) phase image at 635 nm , (c) dual-wavelength unwrapped phase image, (d) 3D pseudocolor rendering of (c). Images are 150 × 150 pixels, 60 μm × 60 μm .

Fig. 3
Fig. 3

Digital holographic imaging of two ovarian cancer cells (marked with circles on all four images) with the substrate at an angle: phase images at (a) 532 and (b)  633 nm , (c) dual-wavelength unwrapped phase image, (d) 3D pseudocolor rendering of (c). The images are 256 × 256 pixels, 78 μm × 78 μm , and m 2 was set to 12.

Equations (8)

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h ( x , y ) = λ 2 π φ ( x , y ) .
h ( x , y ) = λ 2 π φ ( x , y ) ( n n 0 ) ,
h ( x , y ) = λ 1 2 π φ 1 ( x , y ) + λ 1 m 1 ( x , y ) = λ 2 2 π φ 2 ( x , y ) + λ 2 m 2 ( x , y ) ,
φ 1 ( x , y ) 2 π + m 1 ( x , y ) = h ( x , y ) λ 1 , φ 2 ( x , y ) 2 π + m 2 ( x , y ) = h ( x , y ) λ 2 .
h ( x , y ) = λ 2 λ 1 λ 2 λ 1 · [ φ 1 ( x , y ) φ 2 ( x , y ) 2 π + m 1 ( x , y ) m 2 ( x , y ) ] .
ϕ 1 ( x , y ) ϕ 2 ( x , y ) 2 π + m 1 ( x , y ) m 2 ( x , y )
m 1 ( x , y ) = λ 2 λ 1 m 2 ( x , y ) + 1 2 π ( λ 2 λ 1 φ 2 ( x , y ) φ 1 ( x , y ) ) .
m 1 ( x , y ) = round ( λ 2 λ 1 m 2 ( x , y ) + 1 2 π ( λ 2 λ 1 φ 2 ( x , y ) φ 1 ( x , y ) ) ) ,

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