Abstract

We describe the implementation of quantitative Differential Interference Contrast (DIC) Microscopy using a spatial light modulator (SLM) as a flexible Fourier filter in the optical path. The experimental arrangement allows for the all-electronic acquisition of multiple phase shifted DIC-images at video rates which are analyzed to yield the optical path length variation of the sample. The resolution of the technique is analyzed by retrieving the phase profiles of polystyrene spheres in immersion oil, and the method is then applied for quantitative imaging of biological samples. By reprogramming the diffractive structure displayed at the SLM it is possible to record the whole set of phase shifted DIC images simultaneously in different areas of the same camera chip. This allows for quantitative snap-shot imaging of a sample, which has applications for the investigation of dynamic processes.

© 2010 Optical Society of America

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    [CrossRef] [PubMed]
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    [CrossRef] [PubMed]
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    [CrossRef]
  29. N. Ghosh, P. Buddhiwant, A. Uppal, S. K. Majumder, H. S. Patel, and P. K. Gupta, “Simultaneous determination of size and refractive index of red blood cells by light scattering measurements,” Appl. Phys. Lett. 88, 084101 (2006).
    [CrossRef]
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    [CrossRef] [PubMed]
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2010 (2)

C. Maurer, S. Khan, S. Fassl, S. Bernet, and M. Ritsch-Marte, “Depth of field multiplexing in microscopy,” Opt. Express 3, 3023–3034 (2010).
[CrossRef]

H. Ding, and G. Popescu, “Instantaneous spatial light interference microscopy,” Opt. Express 18, 1569–1575 (2010).
[CrossRef] [PubMed]

2009 (5)

2008 (5)

M. Shribak, J. LaFountain, D. Biggs, and S. Inoué, “Orientation-independent differential interference contrast microscopy and its combination with an orientation independent polarization system,” J. Biomed. Opt. 13, 014011 (2008).
[CrossRef] [PubMed]

A. Grjasnow, A. Wuttig, and R. Riesenberg, “Phase resolving microscopy by multi-plane diffraction detection,” J. Microsc. 231(1), 115–123 (2008).
[CrossRef] [PubMed]

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

B. Kemper, and G. von Bally, “Digital holographic microscopy for life cell applications and technical inspection,” Appl. Opt. 47, A52–A61 (2008).
[CrossRef] [PubMed]

O. von Hofsten, M. Bertilson, and U. Vogt, “Theoretical development of a high-resolution differential-interference contrast optic for x-ray microscopy,” Opt. Express 16, 1132–1141 (2008).
[CrossRef] [PubMed]

2006 (5)

2005 (1)

2004 (2)

M. R. Arnison, K. G. Larkin, C. J. R. Sheppard, N. I. Smith, and C. J. Cogswell, “Linear phase imaging using differential interference contrast microscopy,” J. Microsc. 214, 7–12 (2004).
[CrossRef] [PubMed]

A. Y. M. Ng, C. W. See, and M. G. Somekh, “Quantitative optical microscope with enhanced resolution using a pixilated liquid crystal spatial light modulator,” J. Microsc. 214(3), 334–340 (2004).
[CrossRef] [PubMed]

2003 (2)

X. Ma, J. Q. Lu, R. S. Scott, K. M. Jacobs, P. Yang, and X.-H. Hu, “Determination of complex refractive index of polystyrene microspheres from 370 to 1610 nm,” Phys. Med. Biol. 48, 4165–4172 (2003).
[CrossRef]

E. Di Fabrizio, D. Cojoc, S. Cabrini, B. Kaulich, J. Susini, P. Facci, and T. Wilhein, “Diffractive optical elements for differential interference contrast x-ray microscopy,” Opt. Express 11, 2278–2288 (2003).
[CrossRef] [PubMed]

2000 (1)

1999 (1)

1994 (1)

1990 (1)

1972 (1)

R. W. Gerchberg, and W. O. Saxton, “A practical algorithm for the determination of phase from image and diffraction plane pictures,” Optik (Stuttg.) 35, 237–246 (1972).

1955 (1)

G. Nomarski, “Microinterferometrie differentiel a ondes polarisees,” J. Phys. Radium 16, 9S–11S (1955).

Alferi, D.

Arnison, M. R.

M. R. Arnison, K. G. Larkin, C. J. R. Sheppard, N. I. Smith, and C. J. Cogswell, “Linear phase imaging using differential interference contrast microscopy,” J. Microsc. 214, 7–12 (2004).
[CrossRef] [PubMed]

Bernet, S.

C. Maurer, S. Khan, S. Fassl, S. Bernet, and M. Ritsch-Marte, “Depth of field multiplexing in microscopy,” Opt. Express 3, 3023–3034 (2010).
[CrossRef]

T. J. McIntyre, C. Maurer, S. Bernet, and M. Ritsch-Marte, “Differential interference contrast imaging using a spatial light modulator,” Opt. Lett. 34, 2988–2990 (2009).
[CrossRef] [PubMed]

C. Maurer, S. Bernet, and M. Ritsch-Marte, “Refining common path interferometry with a spiral-phase Fourier filter,” J. Opt. A, Pure Appl. Opt. 11(8), 094023 (2009).
[CrossRef]

S. Bernet, A. Jesacher, S. Fürhapter, C. Maurer, and M. Ritsch-Marte, “Quantitative imaging of complex samples by spiral phase contrast microscopy,” Opt. Express 14, 3792–3805 (2006).
[CrossRef] [PubMed]

Bertilson, M.

Biggs, D.

M. Shribak, J. LaFountain, D. Biggs, and S. Inoué, “Orientation-independent differential interference contrast microscopy and its combination with an orientation independent polarization system,” J. Biomed. Opt. 13, 014011 (2008).
[CrossRef] [PubMed]

Brophy, C. P.

Buddhiwant, P.

N. Ghosh, P. Buddhiwant, A. Uppal, S. K. Majumder, H. S. Patel, and P. K. Gupta, “Simultaneous determination of size and refractive index of red blood cells by light scattering measurements,” Appl. Phys. Lett. 88, 084101 (2006).
[CrossRef]

Cabrini, S.

Charrière, F.

Choi, W.

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

Cogswell, C. J.

M. R. Arnison, K. G. Larkin, C. J. R. Sheppard, N. I. Smith, and C. J. Cogswell, “Linear phase imaging using differential interference contrast microscopy,” J. Microsc. 214, 7–12 (2004).
[CrossRef] [PubMed]

Cojoc, D.

Colomb, T.

Conchello, J. A.

Cottrell, D. M.

Dasari, R. R.

Davis, J. A.

Depeursinge, C.

Di Fabrizio, E.

Diez-Silva, M.

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

Ding, H.

Facci, P.

Fassl, S.

C. Maurer, S. Khan, S. Fassl, S. Bernet, and M. Ritsch-Marte, “Depth of field multiplexing in microscopy,” Opt. Express 3, 3023–3034 (2010).
[CrossRef]

Feld, M. S.

Y. Park, M. Diez-Silva, G. Popescu, G. Lykotrafitis, W. Choi, and M. S. Feld, “Refractive index maps and membrane dynamics of human red blood cells parasitized by Plasmodium falciparum,” Proc. Natl. Acad. Sci. U.S.A. 105, 13730–13735 (2008).
[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, 1165–1167 (2005).
[CrossRef] [PubMed]

Ferraro, P.

Finizio, A.

Fürhapter, S.

Gerchberg, R. W.

R. W. Gerchberg, and W. O. Saxton, “A practical algorithm for the determination of phase from image and diffraction plane pictures,” Optik (Stuttg.) 35, 237–246 (1972).

Ghosh, N.

N. Ghosh, P. Buddhiwant, A. Uppal, S. K. Majumder, H. S. Patel, and P. K. Gupta, “Simultaneous determination of size and refractive index of red blood cells by light scattering measurements,” Appl. Phys. Lett. 88, 084101 (2006).
[CrossRef]

Grjasnow, A.

A. Grjasnow, A. Wuttig, and R. Riesenberg, “Phase resolving microscopy by multi-plane diffraction detection,” J. Microsc. 231(1), 115–123 (2008).
[CrossRef] [PubMed]

Gupta, P. K.

N. Ghosh, P. Buddhiwant, A. Uppal, S. K. Majumder, H. S. Patel, and P. K. Gupta, “Simultaneous determination of size and refractive index of red blood cells by light scattering measurements,” Appl. Phys. Lett. 88, 084101 (2006).
[CrossRef]

Heger, T. J.

Heise, B.

Hu, X.-H.

X. Ma, J. Q. Lu, R. S. Scott, K. M. Jacobs, P. Yang, and X.-H. Hu, “Determination of complex refractive index of polystyrene microspheres from 370 to 1610 nm,” Phys. Med. Biol. 48, 4165–4172 (2003).
[CrossRef]

Ikeda, T.

Inoué, S.

M. Shribak, J. LaFountain, D. Biggs, and S. Inoué, “Orientation-independent differential interference contrast microscopy and its combination with an orientation independent polarization system,” J. Biomed. Opt. 13, 014011 (2008).
[CrossRef] [PubMed]

M. Shribak, and S. Inoué, “Orientation-independent differential interference contrast microscopy,” Appl. Opt. 45, 460–469 (2006).
[CrossRef] [PubMed]

Ivanov, C. D.

Jacobs, K. M.

X. Ma, J. Q. Lu, R. S. Scott, K. M. Jacobs, P. Yang, and X.-H. Hu, “Determination of complex refractive index of polystyrene microspheres from 370 to 1610 nm,” Phys. Med. Biol. 48, 4165–4172 (2003).
[CrossRef]

Jesacher, A.

Kaulich, B.

Kemper, B.

Khan, S.

C. Maurer, S. Khan, S. Fassl, S. Bernet, and M. Ritsch-Marte, “Depth of field multiplexing in microscopy,” Opt. Express 3, 3023–3034 (2010).
[CrossRef]

Kühn, J.

LaFountain, J.

M. Shribak, J. LaFountain, D. Biggs, and S. Inoué, “Orientation-independent differential interference contrast microscopy and its combination with an orientation independent polarization system,” 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, “Linear phase imaging using differential interference contrast microscopy,” J. Microsc. 214, 7–12 (2004).
[CrossRef] [PubMed]

Lu, J. Q.

X. Ma, J. Q. Lu, R. S. Scott, K. M. Jacobs, P. Yang, and X.-H. Hu, “Determination of complex refractive index of polystyrene microspheres from 370 to 1610 nm,” Phys. Med. Biol. 48, 4165–4172 (2003).
[CrossRef]

Lykotrafitis, G.

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

Ma, X.

X. Ma, J. Q. Lu, R. S. Scott, K. M. Jacobs, P. Yang, and X.-H. Hu, “Determination of complex refractive index of polystyrene microspheres from 370 to 1610 nm,” Phys. Med. Biol. 48, 4165–4172 (2003).
[CrossRef]

Majumder, S. K.

N. Ghosh, P. Buddhiwant, A. Uppal, S. K. Majumder, H. S. Patel, and P. K. Gupta, “Simultaneous determination of size and refractive index of red blood cells by light scattering measurements,” Appl. Phys. Lett. 88, 084101 (2006).
[CrossRef]

Marquet, P.

Maurer, C.

C. Maurer, S. Khan, S. Fassl, S. Bernet, and M. Ritsch-Marte, “Depth of field multiplexing in microscopy,” Opt. Express 3, 3023–3034 (2010).
[CrossRef]

T. J. McIntyre, C. Maurer, S. Bernet, and M. Ritsch-Marte, “Differential interference contrast imaging using a spatial light modulator,” Opt. Lett. 34, 2988–2990 (2009).
[CrossRef] [PubMed]

C. Maurer, S. Bernet, and M. Ritsch-Marte, “Refining common path interferometry with a spiral-phase Fourier filter,” J. Opt. A, Pure Appl. Opt. 11(8), 094023 (2009).
[CrossRef]

S. Bernet, A. Jesacher, S. Fürhapter, C. Maurer, and M. Ritsch-Marte, “Quantitative imaging of complex samples by spiral phase contrast microscopy,” Opt. Express 14, 3792–3805 (2006).
[CrossRef] [PubMed]

McIntyre, T. J.

Mitchell, E. A. D.

Montfort, F.

Moratal, C.

Ng, A. Y. M.

A. Y. M. Ng, C. W. See, and M. G. Somekh, “Quantitative optical microscope with enhanced resolution using a pixilated liquid crystal spatial light modulator,” J. Microsc. 214(3), 334–340 (2004).
[CrossRef] [PubMed]

Nicola, S. D.

Nikolov, I. D.

Nomarski, G.

G. Nomarski, “Microinterferometrie differentiel a ondes polarisees,” J. Phys. Radium 16, 9S–11S (1955).

Park, Y.

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

Patel, H. S.

N. Ghosh, P. Buddhiwant, A. Uppal, S. K. Majumder, H. S. Patel, and P. K. Gupta, “Simultaneous determination of size and refractive index of red blood cells by light scattering measurements,” Appl. Phys. Lett. 88, 084101 (2006).
[CrossRef]

Pavillon, N.

Petrocellis, L. D.

Pierattini, G.

Popescu, G.

H. Ding, and G. Popescu, “Instantaneous spatial light interference microscopy,” Opt. Express 18, 1569–1575 (2010).
[CrossRef] [PubMed]

Y. Park, M. Diez-Silva, G. Popescu, G. Lykotrafitis, W. Choi, and M. S. Feld, “Refractive index maps and membrane dynamics of human red blood cells parasitized by Plasmodium falciparum,” Proc. Natl. Acad. Sci. U.S.A. 105, 13730–13735 (2008).
[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, 1165–1167 (2005).
[CrossRef] [PubMed]

Preza, C.

Rappaz, B.

Riesenberg, R.

A. Grjasnow, A. Wuttig, and R. Riesenberg, “Phase resolving microscopy by multi-plane diffraction detection,” J. Microsc. 231(1), 115–123 (2008).
[CrossRef] [PubMed]

Rinehart, M. T.

Ritsch-Marte, M.

C. Maurer, S. Khan, S. Fassl, S. Bernet, and M. Ritsch-Marte, “Depth of field multiplexing in microscopy,” Opt. Express 3, 3023–3034 (2010).
[CrossRef]

T. J. McIntyre, C. Maurer, S. Bernet, and M. Ritsch-Marte, “Differential interference contrast imaging using a spatial light modulator,” Opt. Lett. 34, 2988–2990 (2009).
[CrossRef] [PubMed]

C. Maurer, S. Bernet, and M. Ritsch-Marte, “Refining common path interferometry with a spiral-phase Fourier filter,” J. Opt. A, Pure Appl. Opt. 11(8), 094023 (2009).
[CrossRef]

S. Bernet, A. Jesacher, S. Fürhapter, C. Maurer, and M. Ritsch-Marte, “Quantitative imaging of complex samples by spiral phase contrast microscopy,” Opt. Express 14, 3792–3805 (2006).
[CrossRef] [PubMed]

Saxton, W. O.

R. W. Gerchberg, and W. O. Saxton, “A practical algorithm for the determination of phase from image and diffraction plane pictures,” Optik (Stuttg.) 35, 237–246 (1972).

Scott, R. S.

X. Ma, J. Q. Lu, R. S. Scott, K. M. Jacobs, P. Yang, and X.-H. Hu, “Determination of complex refractive index of polystyrene microspheres from 370 to 1610 nm,” Phys. Med. Biol. 48, 4165–4172 (2003).
[CrossRef]

See, C. W.

A. Y. M. Ng, C. W. See, and M. G. Somekh, “Quantitative optical microscope with enhanced resolution using a pixilated liquid crystal spatial light modulator,” J. Microsc. 214(3), 334–340 (2004).
[CrossRef] [PubMed]

Shaked, N. T.

Sheppard, C. J. R.

M. R. Arnison, K. G. Larkin, C. J. R. Sheppard, N. I. Smith, and C. J. Cogswell, “Linear phase imaging using differential interference contrast microscopy,” J. Microsc. 214, 7–12 (2004).
[CrossRef] [PubMed]

Shribak, M.

M. Shribak, J. LaFountain, D. Biggs, and S. Inoué, “Orientation-independent differential interference contrast microscopy and its combination with an orientation independent polarization system,” J. Biomed. Opt. 13, 014011 (2008).
[CrossRef] [PubMed]

M. Shribak, and S. Inoué, “Orientation-independent differential interference contrast microscopy,” Appl. Opt. 45, 460–469 (2006).
[CrossRef] [PubMed]

Smith, N. I.

M. R. Arnison, K. G. Larkin, C. J. R. Sheppard, N. I. Smith, and C. J. Cogswell, “Linear phase imaging using differential interference contrast microscopy,” J. Microsc. 214, 7–12 (2004).
[CrossRef] [PubMed]

Snyder, D. L.

Somekh, M. G.

A. Y. M. Ng, C. W. See, and M. G. Somekh, “Quantitative optical microscope with enhanced resolution using a pixilated liquid crystal spatial light modulator,” J. Microsc. 214(3), 334–340 (2004).
[CrossRef] [PubMed]

Stifter, D.

Susini, J.

Uppal, A.

N. Ghosh, P. Buddhiwant, A. Uppal, S. K. Majumder, H. S. Patel, and P. K. Gupta, “Simultaneous determination of size and refractive index of red blood cells by light scattering measurements,” Appl. Phys. Lett. 88, 084101 (2006).
[CrossRef]

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Figures (10)

Fig. 1.
Fig. 1.

Experimental setup. The beam path and all used lenses are shown in the image. For a detailed explanation see the text.

Fig. 2.
Fig. 2.

DIC images and intensity plots of a polystyrene bead for a π/2 phase difference illustrating the effect of illumination spatial coherence. (a) High level of coherence (NA 0.01); (b) Medium coherence (NA 0.38); (c) Low coherence (NA 0.75).

Fig. 3.
Fig. 3.

Raw DIC images at a shear of 0.126 µm. (a)–(c) Horizontal (x) shear with phase differences of 0, 2π/3 and 4π/3 respectively. (d)–(f) Vertical (y) shear with phase differences of 0, 2π/3 and 4π/3 respectively.

Fig. 4.
Fig. 4.

Measured phase gradients and optical path lengths for a polystyrene bead in immersion oil. (a) Phase gradient for a shear in the horizontal (x) direction; (b) Phase gradient for a shear in the vertical (y) direction; (c) Recovered two-dimensional optical path length difference; (d) Cross-sectional optical path length difference from left to right through the center of the bead shown in (c).

Fig. 5.
Fig. 5.

A plot of measured values and the fit corresponding to Eq. (18).

Fig. 6.
Fig. 6.

Recovered thickness for a polystyrene bead in immersion oil. (a) Three-dimensional representation. (b) A cross-section compared to the shape for a perfect sphere with the measured radius.

Fig. 7.
Fig. 7.

SLM-DIC imaging of blood cells in a buffer solution. (a)–(c) Sample raw images with a horizontal shear at a relative phase of 0, 2π/3 and 4π/3 respectively. (d) The recovered phase gradient from these images, Δϕx. (e) A colour map of the optical path length obtained from the x and y gradients. (f) A cross-section of the optical path length through one of the blood cells.

Fig. 8.
Fig. 8.

Blood cell thickness determined using the current SLM-DIC measurements and a literature value for the refractive index difference between the cell and the buffer solution of 0.06. (a) Three-dimensional representation; (b) Cross-section through the center of the blood cell.

Fig. 9.
Fig. 9.

SLM-DIC measurements of chromosomes. (a) A sample DIC image with a phase difference of 4π/3. (b) The recovered horizontal phase gradient Δϕx. (c) The optical path length difference obtained from the gradients.

Fig. 10.
Fig. 10.

Quantitative calculation of the phase with a single snapshot. (a) A single camera image showing the raw images with annotations giving the varying shear directions and relative phases. (b) Gradient field in the horizontal (x) direction. (c) Gradient field in the the vertical (y) direction. (d) Calculated phase distribution. (e) The profile along the line shown in (d).

Tables (3)

Tables Icon

Table 1. Experimental Parameters

Tables Icon

Table 2. Measured and calculated values for the bead DIC measurements with a shear of 0.126 µm

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Table 3. Measured refractive index difference between the polystyrene bead and the immersion oil and bead radius obtained from the measurements with various shears

Equations (18)

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E ( x , y ) = A ( x , y ) cos [ k z ω t + ϕ ( x , y ) ] ,
I = E 1 + E 2 2 = A 1 2 + A 2 2 2 + A 1 A 2 cos [ ϕ 1 ϕ 2 ] + I INC ,
I A = A 1 2 + A 2 2 2 + A 1 A 2 cos [ Δ ϕ ] + I INC
I B = A 1 2 A 2 2 2 + A 1 A 2 cos [ Δ ϕ 2 π 3 ] + I INC
I C = A 1 2 A 2 2 2 + A 1 A 2 cos [ Δ ϕ 4 π 3 ] + I INC .
Δ ϕ ( x , y ) = tan 1 { I A sin ( 0 ) + I B sin ( 2 π 3 ) + I C sin ( 4 π 3 ) I A cos ( 0 ) + I B cos ( 2 π 3 ) + I C cos ( 4 π 3 ) } .
ϕ ( x , y ) = 2 π ( n n s ) h ( x , y ) λ = 2 π Λ ( x , y ) λ ,
Δ ϕ x ( x , y ) = 2 π λ [ Λ ( x , y ) Λ ( x + Δ x r , y ) ] .
Λ ( x , y ) x λ 2 π Δ ϕ x ( x , y ) Δ x r ,
Λ ( x , y ) y λ 2 π Δ ϕ y ( x , y ) Δ y r .
θ = sin 1 { λ a } .
x = L tan ( θ ) = L tan ( sin 1 { λ a } ) L λ a
Δ x r = x 2 x 1 = L ( λ a 2 λ a 1 ) .
L = a 1 X 0 1 λ .
Δ x r = X 0 1 ( a 1 a 2 1 ) .
h ( x , y ) = 2 r 2 ( x x 0 ) 2 ( y y 0 ) 2 ,
ϕ ( x , y ) = 4 π ( n n s ) r 2 ( x x 0 ) 2 ( y y 0 ) 2 λ .
ϕ 2 ( x , y ) = 16 π 2 ( n n s ) 2 r 2 λ 2 16 π 2 ( n n s ) 2 [ ( x x 0 ) 2 + ( y y 0 ) 2 ] λ 2

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