Abstract

Refractive index dispersion is an intrinsic optical property and a useful source of contrast in biological imaging studies. In this report, we present the first dispersion phase imaging of living eukaryotic cells. We have developed quantitative dispersion microscopy based on the principle of quantitative phase microscopy. The dual-wavelength quantitative phase microscope makes phase measurements at 310 nm and 400 nm wavelengths to quantify dispersion (refractive index increment ratio) of live cells. The measured dispersion of living HeLa cells is found to be around 1.088, which agrees well with that measured directly for protein solutions using total internal reflection. This technique, together with the dry mass and morphology measurements provided by quantitative phase microscopy, could prove to be a useful tool for distinguishing different types of biomaterials and studying spatial inhomogeneities of biological samples.

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References

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2010

2009

2008

B. Rappaz, F. Charrière, C. Depeursinge, P. J. Magistretti, and P. Marquet, “Simultaneous cell morphometry and refractive index measurement with dual-wavelength digital holographic microscopy and dye-enhanced dispersion of perfusion medium,” Opt. Lett. 33(7), 744–746 (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]

2007

B. J. Zeskind, C. D. Jordan, W. Timp, L. Trapani, G. Waller, V. Horodincu, D. J. Ehrlich, and P. Matsudaira, “Nucleic acid and protein mass mapping by live-cell deep-ultraviolet microscopy,” Nat. Methods 4(7), 567–569 (2007).
[CrossRef] [PubMed]

2006

Y. L. Jin, J. Y. Chen, L. Xu, and P. N. Wang, “Refractive index measurement for biomaterial samples by total internal reflection,” Phys. Med. Biol. 51(20), N371–N379 (2006).
[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]

2004

2003

W. R. Zipfel, R. M. Williams, and W. W. Webb, “Nonlinear magic: multiphoton microscopy in the biosciences,” Nat. Biotechnol. 21(11), 1369–1377 (2003).
[CrossRef] [PubMed]

2002

R. P. Sinha and D. P. Häder, “UV-induced DNA damage and repair: a review,” Photochem. Photobiol. Sci. 1(4), 225–236 (2002).
[CrossRef] [PubMed]

2001

1957

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]

Barer, R.

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]

Charrière, F.

Chen, J. Y.

Y. L. Jin, J. Y. Chen, L. Xu, and P. N. Wang, “Refractive index measurement for biomaterial samples by total internal reflection,” Phys. Med. Biol. 51(20), N371–N379 (2006).
[CrossRef] [PubMed]

Choi, W.

Dasari, R.

Dasari, R. R.

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.

Dorn, A.

Duker, J.

Ehrlich, D. J.

B. J. Zeskind, C. D. Jordan, W. Timp, L. Trapani, G. Waller, V. Horodincu, D. J. Ehrlich, and P. Matsudaira, “Nucleic acid and protein mass mapping by live-cell deep-ultraviolet microscopy,” Nat. Methods 4(7), 567–569 (2007).
[CrossRef] [PubMed]

Feld, M. S.

Fu, D.

Fujimoto, J.

Häder, D. P.

R. P. Sinha and D. P. Häder, “UV-induced DNA damage and repair: a review,” Photochem. Photobiol. Sci. 1(4), 225–236 (2002).
[CrossRef] [PubMed]

Horodincu, V.

B. J. Zeskind, C. D. Jordan, W. Timp, L. Trapani, G. Waller, V. Horodincu, D. J. Ehrlich, and P. Matsudaira, “Nucleic acid and protein mass mapping by live-cell deep-ultraviolet microscopy,” Nat. Methods 4(7), 567–569 (2007).
[CrossRef] [PubMed]

Ikeda, T.

Jin, Y. L.

Y. L. Jin, J. Y. Chen, L. Xu, and P. N. Wang, “Refractive index measurement for biomaterial samples by total internal reflection,” Phys. Med. Biol. 51(20), N371–N379 (2006).
[CrossRef] [PubMed]

Jordan, C. D.

B. J. Zeskind, C. D. Jordan, W. Timp, L. Trapani, G. Waller, V. Horodincu, D. J. Ehrlich, and P. Matsudaira, “Nucleic acid and protein mass mapping by live-cell deep-ultraviolet microscopy,” Nat. Methods 4(7), 567–569 (2007).
[CrossRef] [PubMed]

Ko, T.

Kowalczyk, A.

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]

Magistretti, P. J.

Marquet, P.

Matsudaira, P.

B. J. Zeskind, C. D. Jordan, W. Timp, L. Trapani, G. Waller, V. Horodincu, D. J. Ehrlich, and P. Matsudaira, “Nucleic acid and protein mass mapping by live-cell deep-ultraviolet microscopy,” Nat. Methods 4(7), 567–569 (2007).
[CrossRef] [PubMed]

Oh, S.

Park, Y.

Popescu, G.

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]

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]

Rappaz, B.

Sinha, R. P.

R. P. Sinha and D. P. Häder, “UV-induced DNA damage and repair: a review,” Photochem. Photobiol. Sci. 1(4), 225–236 (2002).
[CrossRef] [PubMed]

Srinivasan, V.

Sung, Y.

Timp, W.

B. J. Zeskind, C. D. Jordan, W. Timp, L. Trapani, G. Waller, V. Horodincu, D. J. Ehrlich, and P. Matsudaira, “Nucleic acid and protein mass mapping by live-cell deep-ultraviolet microscopy,” Nat. Methods 4(7), 567–569 (2007).
[CrossRef] [PubMed]

Trapani, L.

B. J. Zeskind, C. D. Jordan, W. Timp, L. Trapani, G. Waller, V. Horodincu, D. J. Ehrlich, and P. Matsudaira, “Nucleic acid and protein mass mapping by live-cell deep-ultraviolet microscopy,” Nat. Methods 4(7), 567–569 (2007).
[CrossRef] [PubMed]

Waller, G.

B. J. Zeskind, C. D. Jordan, W. Timp, L. Trapani, G. Waller, V. Horodincu, D. J. Ehrlich, and P. Matsudaira, “Nucleic acid and protein mass mapping by live-cell deep-ultraviolet microscopy,” Nat. Methods 4(7), 567–569 (2007).
[CrossRef] [PubMed]

Wang, P. N.

Y. L. Jin, J. Y. Chen, L. Xu, and P. N. Wang, “Refractive index measurement for biomaterial samples by total internal reflection,” Phys. Med. Biol. 51(20), N371–N379 (2006).
[CrossRef] [PubMed]

Wax, A.

Webb, W. W.

W. R. Zipfel, R. M. Williams, and W. W. Webb, “Nonlinear magic: multiphoton microscopy in the biosciences,” Nat. Biotechnol. 21(11), 1369–1377 (2003).
[CrossRef] [PubMed]

Williams, R. M.

W. R. Zipfel, R. M. Williams, and W. W. Webb, “Nonlinear magic: multiphoton microscopy in the biosciences,” Nat. Biotechnol. 21(11), 1369–1377 (2003).
[CrossRef] [PubMed]

Wojtkowski, M.

Xu, L.

Y. L. Jin, J. Y. Chen, L. Xu, and P. N. Wang, “Refractive index measurement for biomaterial samples by total internal reflection,” Phys. Med. Biol. 51(20), N371–N379 (2006).
[CrossRef] [PubMed]

Yamauchi, T.

Yang, C. H.

Yaqoob, Z.

Zeskind, B. J.

B. J. Zeskind, C. D. Jordan, W. Timp, L. Trapani, G. Waller, V. Horodincu, D. J. Ehrlich, and P. Matsudaira, “Nucleic acid and protein mass mapping by live-cell deep-ultraviolet microscopy,” Nat. Methods 4(7), 567–569 (2007).
[CrossRef] [PubMed]

Zipfel, W. R.

W. R. Zipfel, R. M. Williams, and W. W. Webb, “Nonlinear magic: multiphoton microscopy in the biosciences,” Nat. Biotechnol. 21(11), 1369–1377 (2003).
[CrossRef] [PubMed]

Am. J. Physiol. Cell Physiol.

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]

J. Opt. Soc. Am.

Nat. Biotechnol.

W. R. Zipfel, R. M. Williams, and W. W. Webb, “Nonlinear magic: multiphoton microscopy in the biosciences,” Nat. Biotechnol. 21(11), 1369–1377 (2003).
[CrossRef] [PubMed]

Nat. Methods

B. J. Zeskind, C. D. Jordan, W. Timp, L. Trapani, G. Waller, V. Horodincu, D. J. Ehrlich, and P. Matsudaira, “Nucleic acid and protein mass mapping by live-cell deep-ultraviolet microscopy,” Nat. Methods 4(7), 567–569 (2007).
[CrossRef] [PubMed]

Opt. Express

Opt. Lett.

Photochem. Photobiol. Sci.

R. P. Sinha and D. P. Häder, “UV-induced DNA damage and repair: a review,” Photochem. Photobiol. Sci. 1(4), 225–236 (2002).
[CrossRef] [PubMed]

Phys. Med. Biol.

Y. L. Jin, J. Y. Chen, L. Xu, and P. N. Wang, “Refractive index measurement for biomaterial samples by total internal reflection,” Phys. Med. Biol. 51(20), N371–N379 (2006).
[CrossRef] [PubMed]

Other

B. Alberts, A. Johnson, J. Lewis, M. Raff, K. Roberts, and P. Walter, Molecular Biology of the Cell (Garland Science, New York, 2002).

T. Vo-Dinh, Biomedical Photonics Handbook (CRC Press, 2003).

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Figures (4)

Fig. 1
Fig. 1

Schematic diagram of dual wavelength NUV quantitative phase microscope. RegA: Ti: sapphire regenerative amplifier; OPA: optical parametric amplifier; OBJ: objective; G: Ronchi grating; SPF: spatial filter (see the insert).

Fig. 2
Fig. 2

Refractive index measured at two wavelengths for (a) protein solutions and (b) DNA solutions versus concentrations and their corresponding linear fits to obtain specific refractive index increments, α.

Fig. 3
Fig. 3

Optical path length image of HeLa cells at (a) 310 nm and (b) 400 nm; (c) ratio image of magnification corrected OPL images at 310 nm and 400 nm; (d) composite image of corrected OPL images using red and green channels for 310 nm and 400 nm images, respectively. The number indicates the auto-segmented cell number and the gray dashed line depicts the borders of each cell. Scale bar: 20 μm.

Fig. 4
Fig. 4

Scatter and box plots of the (a) dry mass (b) projected area (c) dispersion of 54 HeLa cells. The box indicates 25%-75% range and whiskers indicates 5%-95% range.

Equations (3)

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O P L ( x , y ) = n α n C n d ( x , y ) = ϕ ( x , y ) λ 2 π
x , y O P L ( x , y ) d x d y = x , y n α n C n d ( x , y ) d x d y = n α n M n
R = O P L ( x , y ) 310 n m d x d y O P L ( x , y ) 400 n m d x d y

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