Ultraviolet refractometry using field-based light scattering spectroscopy Citation

Accurate refractive index measurement in the deep ultraviolet (UV) range is important for the separate quantification of biomolecules such as proteins and DNA in biology. This task is demanding and has not been fully exploited so far. Here we report a new method of measuring refractive index using field-based light scattering spectroscopy, which is applicable to any wavelength range and suitable for both solutions and homogenous objects with well-defined shape such as microspheres. The angular scattering distribution of single microspheres immersed in homogeneous media is measured over the wavelength range 260 to 315 nm using quantitative phase microscopy. By least square fitting the observed scattering distribution with Mie scattering theory, the refractive index of either the sphere or the immersion medium can be determined provided that one is known a priori. Using this method, we have measured the refractive index dispersion of SiO2 spheres and bovine serum albumin (BSA) solutions in the deep UV region. Specific refractive index increments of BSA are also extracted. Typical accuracy of the present refractive index technique is ≤0.003. The precision of refractive index measurements is ≤0.002 and that of specific refractive index increment determination is ≤0.01 mL/g. ©2009 Optical Society of America OCIS codes: (120.3180) Interferometry; (180.0180) Microscopy; (170.3880) Medical and biological imaging. References and Links 1. A. Barty, K. A. Nugent, D. Paganin, and A. Roberts, "Quantitative optical phase microscopy," Opt. Lett. 23, 817-819 (1998). 2. B. Rappaz, P. Marquet, E. Cuche, Y. Emery, C. Depeursinge, and P. Magistretti, "Measurement of the integral refractive index and dynamic cell morphometry of living cells with digital holographic microscopy," Opt. Express 13, 9361-9373 (2005). 3. C. J. Mann, L. F. Yu, C. M. Lo, and M. K. Kim, "High-resolution quantitative phase-contrast microscopy by digital holography," Opt. Express 13, 8693-8698 (2005). 4. G. Popescu, T. Ikeda, R. R. Dasari, and M. S. Feld, "Diffraction phase microscopy for quantifying cell structure and dynamics," Opt. Lett. 31, 775-777 (2006). 5. W. Choi, C. Fang-Yen, K. Badizadegan, S. Oh, N. Lue, R. R. Dasari, and M. S. Feld, "Tomographic phase microscopy," Nat. Methods 4, 717-719 (2007). 6. Y. J. Sung, W. Choi, C. Fang-Yen, K. Badizadegan, R. R. Dasari, and M. S. Feld, "Optical diffraction tomography for high resolution live cell imaging," Opt. Express 17, 266-277 (2009). 7. F. Charriere, A. Marian, F. Montfort, J. Kuehn, T. Colomb, E. Cuche, P. Marquet, and C. Depeursinge, "Cell refractive index tomography by digital holographic microscopy," Opt. Lett. 31, 178-180 (2006). 8. B. Rappaz, F. Charriere, 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, 744-746 (2008). 9. 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, C538-C544 (2008). 10. Y. Park, M. Diez-Silva, G. Popescu, G. Lykotrafitis, W. Choi, M. S. Feld, and S. Suresh, "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). 11. R. Barer, "Refractometry and Interferometry of Living Cells," J. Opt. Soc. Am. 47, 545-556 (1957). 12. B. S. Chincholi, A. J. Havlik, and R. D. Vold, "Specific refractive index increments of polymer systems at four wavelengths," J. Chem. Eng. Data 19, 148-152 (1974). 13. 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, 567-569 (2007). 14. A. Pinchuk, "Optical constants and dielectric function of DNA's nucleotides in UV range," J. Quant. Spectrosc. Radiat. Transfer 85, 211-215 (2004). 15. M. Andersen, L. R. Painter, and S. Nir, "Dispersion-Equation and Polarizability of Bovine Serum-Albumin from Measurements of Refractive-Indexes," Biopolymers 13, 1261-1267 (1974). 16. M. Friebel and M. Meinke, "Determination of the complex refractive index of highly concentrated hemoglobin solutions using transmittance and reflectance measurements," J. Biomed. Opt. 10, 064019064015 (2005). 17. W. Choi, C. C. Yu, C. Fang-Yen, K. Badizadegan, R. R. Dasari, and M. S. Feld, "Field-based angleresolved light-scattering study of single live cells," Opt. Lett. 33, 1596-1598 (2008). 18. H. F. Ding, Z. Wang, F. Nguyen, S. A. Boppart, and G. Popescu, "Fourier Transform Light Scattering of Inhomogeneous and Dynamic Structures," Phys. Rev. Lett. 101, 238102 (2008). 19. A. Wax, C. Yang, R. R. Dasari, and M. S. Feld, "Measurement of angular distributions by use of lowcoherence interferometry for light-scattering spectroscopy," Opt. Lett. 26, 322-324 (2001). 20. 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). 21. 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). 22. C. Matzler, "Matlab functions for Mie scattering and absorption," IAP Research Report, No. 2002-08, June 2002. 23. M. Daimon and A. Masumura, "Measurement of the refractive index of distilled water from the nearinfrared region to the ultraviolet region," Appl. Opt. 46, 3811-3820 (2007). 24. C. M. Stoscheck and P. D. Murray, "Quantitation of protein," Methods Enzymol. 182, 50-68 (1990).