Abstract

Various staining techniques are commonly used in biomedical research to investigate cellular morphology. By inducing absorption of light, staining dyes change the intracellular refractive index due to the Kramers–Kronig relationship. We present a method for creating 2D maps of real and imaginary refractive indices of stained biological cells using their thickness and absorptance. We validate our technique on dyed polystyrene microspheres and quantify the alteration in refractive index of stained biological cells. We reveal that specific staining of individual organelles can increase their scattering cross-section by orders of magnitudes, implying a major impact in the field of biophotonics.

© 2012 Optical Society of America

Full Article  |  PDF Article

References

  • View by:
  • |
  • |
  • |

  1. E. D. P. De Robertis, F. A. Saez, and E. M. F. De Robertis, Cell Biology, 6th ed. (Saunders, 1975).
  2. E. S. Cibas and B. S. Ducatman, Cytology: Diagnostic Principles and Clinical Correlates (Saunders, 1996).
  3. J. D. Wilson, W. J. Cottrell, and T. H. Foster, J. Biomed. Opt. 12, 014010 (2007).
    [CrossRef]
  4. R. d. L. Kronig, J. Opt. Soc. Am. 12, 547 (1926).
    [CrossRef]
  5. H. A. Kramers, Atti del Congresso Internazionale dei Fisici 2, 545 (1927).
    [CrossRef]
  6. S. Uttam, R. K. Bista, D. J. Hartman, R. E. Brand, and Y. Liu, J. Biomed. Opt. 16, 116013 (2011).
    [CrossRef]
  7. V. Lucarini, Kramers-Kronig Relations in Optical Materials Research (Springer, 2005).
  8. H. C. v. d. Hulst, Light Scattering by Small Particles (Wiley, 1957).
  9. S. A. Prahl, “Inverse adding-doubling software,” http://omlc.ogi.edu/software/iad/index.html .
  10. J. W. Pickering, S. A. Prahl, N. Vanwieringen, J. F. Beek, H. J. C. M. Sterenborg, and M. J. C. Vangemert, Appl. Opt. 32, 399 (1993).
    [CrossRef]
  11. S. A. Prahl, M. J. C. Vangemert, and A. J. Welch, Appl. Opt. 32, 559 (1993).
    [CrossRef]
  12. C. Maetzler, “MATLAB functions for Mie scattering and absorption,” Research Report 2002-08 (Bern, 2002).
  13. D. J. Cook, in Cellular Pathology, 2nd ed. (Scion, 2006), pp. 67–104.
  14. G. C. Crossmon, Stain Technol. 24, 241 (1949).
    [CrossRef]
  15. H. G. Davies, M. H. F. Wilkins, J. Chayen, and L. F. Lacour, Q. J. Microsc. Sci. 95, 271 (1954).
  16. J. M. Schmitt and G. Kumar, Appl. Opt. 37, 2788 (1998).
    [CrossRef]
  17. G. W. Kattawar and G. N. Plass, Appl. Opt. 6, 1377(1967).
    [CrossRef]
  18. N. N. Boustany, S. A. Boppart, and V. Backman, Annu. Rev. Biomed. Eng. 12, 285 (2010).
    [CrossRef]
  19. H. Subramanian, P. Pradhan, Y. Liu, I. R. Capoglu, J. D. Rogers, H. K. Roy, R. E. Brand, and V. Backman, Opt. Lett. 34, 518 (2009).
    [CrossRef]

2011 (1)

S. Uttam, R. K. Bista, D. J. Hartman, R. E. Brand, and Y. Liu, J. Biomed. Opt. 16, 116013 (2011).
[CrossRef]

2010 (1)

N. N. Boustany, S. A. Boppart, and V. Backman, Annu. Rev. Biomed. Eng. 12, 285 (2010).
[CrossRef]

2009 (1)

2007 (1)

J. D. Wilson, W. J. Cottrell, and T. H. Foster, J. Biomed. Opt. 12, 014010 (2007).
[CrossRef]

1998 (1)

1993 (2)

1967 (1)

1954 (1)

H. G. Davies, M. H. F. Wilkins, J. Chayen, and L. F. Lacour, Q. J. Microsc. Sci. 95, 271 (1954).

1949 (1)

G. C. Crossmon, Stain Technol. 24, 241 (1949).
[CrossRef]

1927 (1)

H. A. Kramers, Atti del Congresso Internazionale dei Fisici 2, 545 (1927).
[CrossRef]

1926 (1)

Backman, V.

Beek, J. F.

Bista, R. K.

S. Uttam, R. K. Bista, D. J. Hartman, R. E. Brand, and Y. Liu, J. Biomed. Opt. 16, 116013 (2011).
[CrossRef]

Boppart, S. A.

N. N. Boustany, S. A. Boppart, and V. Backman, Annu. Rev. Biomed. Eng. 12, 285 (2010).
[CrossRef]

Boustany, N. N.

N. N. Boustany, S. A. Boppart, and V. Backman, Annu. Rev. Biomed. Eng. 12, 285 (2010).
[CrossRef]

Brand, R. E.

Capoglu, I. R.

Chayen, J.

H. G. Davies, M. H. F. Wilkins, J. Chayen, and L. F. Lacour, Q. J. Microsc. Sci. 95, 271 (1954).

Cibas, E. S.

E. S. Cibas and B. S. Ducatman, Cytology: Diagnostic Principles and Clinical Correlates (Saunders, 1996).

Cook, D. J.

D. J. Cook, in Cellular Pathology, 2nd ed. (Scion, 2006), pp. 67–104.

Cottrell, W. J.

J. D. Wilson, W. J. Cottrell, and T. H. Foster, J. Biomed. Opt. 12, 014010 (2007).
[CrossRef]

Crossmon, G. C.

G. C. Crossmon, Stain Technol. 24, 241 (1949).
[CrossRef]

Davies, H. G.

H. G. Davies, M. H. F. Wilkins, J. Chayen, and L. F. Lacour, Q. J. Microsc. Sci. 95, 271 (1954).

De Robertis, E. D. P.

E. D. P. De Robertis, F. A. Saez, and E. M. F. De Robertis, Cell Biology, 6th ed. (Saunders, 1975).

De Robertis, E. M. F.

E. D. P. De Robertis, F. A. Saez, and E. M. F. De Robertis, Cell Biology, 6th ed. (Saunders, 1975).

Ducatman, B. S.

E. S. Cibas and B. S. Ducatman, Cytology: Diagnostic Principles and Clinical Correlates (Saunders, 1996).

Foster, T. H.

J. D. Wilson, W. J. Cottrell, and T. H. Foster, J. Biomed. Opt. 12, 014010 (2007).
[CrossRef]

Hartman, D. J.

S. Uttam, R. K. Bista, D. J. Hartman, R. E. Brand, and Y. Liu, J. Biomed. Opt. 16, 116013 (2011).
[CrossRef]

Hulst, H. C. v. d.

H. C. v. d. Hulst, Light Scattering by Small Particles (Wiley, 1957).

Kattawar, G. W.

Kramers, H. A.

H. A. Kramers, Atti del Congresso Internazionale dei Fisici 2, 545 (1927).
[CrossRef]

Kronig, R. d. L.

Kumar, G.

Lacour, L. F.

H. G. Davies, M. H. F. Wilkins, J. Chayen, and L. F. Lacour, Q. J. Microsc. Sci. 95, 271 (1954).

Liu, Y.

Lucarini, V.

V. Lucarini, Kramers-Kronig Relations in Optical Materials Research (Springer, 2005).

Maetzler, C.

C. Maetzler, “MATLAB functions for Mie scattering and absorption,” Research Report 2002-08 (Bern, 2002).

Pickering, J. W.

Plass, G. N.

Pradhan, P.

Prahl, S. A.

Rogers, J. D.

Roy, H. K.

Saez, F. A.

E. D. P. De Robertis, F. A. Saez, and E. M. F. De Robertis, Cell Biology, 6th ed. (Saunders, 1975).

Schmitt, J. M.

Sterenborg, H. J. C. M.

Subramanian, H.

Uttam, S.

S. Uttam, R. K. Bista, D. J. Hartman, R. E. Brand, and Y. Liu, J. Biomed. Opt. 16, 116013 (2011).
[CrossRef]

Vangemert, M. J. C.

Vanwieringen, N.

Welch, A. J.

Wilkins, M. H. F.

H. G. Davies, M. H. F. Wilkins, J. Chayen, and L. F. Lacour, Q. J. Microsc. Sci. 95, 271 (1954).

Wilson, J. D.

J. D. Wilson, W. J. Cottrell, and T. H. Foster, J. Biomed. Opt. 12, 014010 (2007).
[CrossRef]

Annu. Rev. Biomed. Eng. (1)

N. N. Boustany, S. A. Boppart, and V. Backman, Annu. Rev. Biomed. Eng. 12, 285 (2010).
[CrossRef]

Appl. Opt. (4)

Atti del Congresso Internazionale dei Fisici (1)

H. A. Kramers, Atti del Congresso Internazionale dei Fisici 2, 545 (1927).
[CrossRef]

J. Biomed. Opt. (2)

S. Uttam, R. K. Bista, D. J. Hartman, R. E. Brand, and Y. Liu, J. Biomed. Opt. 16, 116013 (2011).
[CrossRef]

J. D. Wilson, W. J. Cottrell, and T. H. Foster, J. Biomed. Opt. 12, 014010 (2007).
[CrossRef]

J. Opt. Soc. Am. (1)

Opt. Lett. (1)

Q. J. Microsc. Sci. (1)

H. G. Davies, M. H. F. Wilkins, J. Chayen, and L. F. Lacour, Q. J. Microsc. Sci. 95, 271 (1954).

Stain Technol. (1)

G. C. Crossmon, Stain Technol. 24, 241 (1949).
[CrossRef]

Other (7)

E. D. P. De Robertis, F. A. Saez, and E. M. F. De Robertis, Cell Biology, 6th ed. (Saunders, 1975).

E. S. Cibas and B. S. Ducatman, Cytology: Diagnostic Principles and Clinical Correlates (Saunders, 1996).

V. Lucarini, Kramers-Kronig Relations in Optical Materials Research (Springer, 2005).

H. C. v. d. Hulst, Light Scattering by Small Particles (Wiley, 1957).

S. A. Prahl, “Inverse adding-doubling software,” http://omlc.ogi.edu/software/iad/index.html .

C. Maetzler, “MATLAB functions for Mie scattering and absorption,” Research Report 2002-08 (Bern, 2002).

D. J. Cook, in Cellular Pathology, 2nd ed. (Scion, 2006), pp. 67–104.

Cited By

OSA participates in CrossRef's Cited-By Linking service. Citing articles from OSA journals and other participating publishers are listed here.

Alert me when this article is cited.


Figures (3)

Fig. 1.
Fig. 1.

Schematic of the SM and sample design: reflected-light Kohler illumination brightfield microscope (60× objective lens; N.A. 0.4) projects sample image through a grating spectrometer on a CCD camera. A stage under the spectrometer spatially scans the sample image. (a) dyed microsphere phantom; (b) stained biological cell.

Fig. 2.
Fig. 2.

(a) n of dyed polystyrene microspheres and (b) their reduced scattering cross-section. IS-IAD data appears in red and is based on post-processing of the SM measurements in blue. Standard errors in corresponding shaded colors.

Fig. 3.
Fig. 3.

(a) shows the measured cell height and absorptance at 600 nm. (b) illustrates the n and n for 3 areas (black corresponds to unstained); letters H and E point to the absorption peaks of hematoxylin and eosin correspondingly. (c) illustrates the spatial distribution of n and n at 600 nm within the cell. (d) Size-dependent absorption efficiency (Qabs) and amplification of scattering efficiency (Qsca) (refractive index of surrounding medium assumed to be 1.52).

Equations (4)

Equations on this page are rendered with MathJax. Learn more.

I(x,y,λ)=I0(x,y,λ0)exp{(4π/λ)0L(x,y)n(x,y,z,λ)dz},
n=χdye/2n,n=n01+(χdye+n2)/n02,
χdye(k)=(2/π)P0kχdye(k)/((k2k2)dk,χdye(k)=(2k/π)P0χdye(k)/(k2k2)dk,
Im{n2(λ)nmed2(λ)n2(λ)+2nmed2(λ)}=λ8πaQabs(λ),

Metrics