A scattering microscope was developed to investigate single cells and biological microstructures by light scattering measurements. The spectrally resolved part of the setup and its validation are shown in detail. The analysis of light scattered by homogenous polystyrene spheres allows the determination of their diameters using Mie theory. The diameters of 150 single polystyrene spheres were determined by the spectrally resolved scattering microscope. In comparison, the same polystyrene suspension stock was investigated by a collimated transmission setup. Mean diameters and standard deviations of the size distribution were evaluated by both methods with a statistical error of less than 1nm. The systematic errors of both devices are in agreement within the measurement accuracy.

© 2011 OSA

1. A. Amelink, M. P. L. Bard, S. A. Burgers, and H. J. C. M. Sterenborg, “Single-scattering spectroscopy for the endoscopic analysis of particle size in superficial layers of turbid media,” Appl. Opt.42, 4095–4101 (2003). [CrossRef] [PubMed]
2. Y. Liu, X. Li, Y. L. Kim, and V. Backman, “Elastic backscattering spectroscopic microscopy,” Opt. Lett.30, 2445–2447 (2005). [CrossRef] [PubMed]
3. F. K. Forster, A. Kienle, R. Michels, and R. Hibst, “Phase function measurements on nonspherical scatterers using a two-axis goniometer,” J. Biomed. Opt.11, 024018 (2006). [CrossRef] [PubMed]
4. M. J. Berg, S. C. Hill, G. Videen, and K. P. Gurton, “Spatial filtering technique to image and measure two-dimensional near-forward scattering from single particles,” Opt. Express18, 9486–9495 (2010). [CrossRef] [PubMed]
5. P. Albella, J. M. Saiz, J. M. Sanz, F. González, and F. Moreno, “Nanoscopic surface inspection by analyzing the linear polarization degree of the scattered light,” Opt. Lett.34, 1906–1908 (2009). [CrossRef] [PubMed]
6. M. S. Patterson, B. Chance, and B. C. Wilson, “Time resolved reflectance and transmittance for the non-invasive measurement of tissue optical properties,” Appl. Opt.28, 2331–2336 (1989). [CrossRef] [PubMed]
7. T. Namita, Y. Kato, and K. Shimizu, “CT imaging of diffuse medium by time-resolved measurement of backscattered light,” Appl. Opt.48, D208–D217 (2009). [CrossRef] [PubMed]
8. J. R. Mourant, I. J. Bigio, D. A. Jack, T. M. Johnson, and H. D. Miller, “Measuring absorption coefficients in small volumes of highly scattering media: source-detector separations for which path lengths do not depend on scattering properties,” Appl. Opt.36, 5655–5661 (1997). [CrossRef] [PubMed]
9. A. Kienle, C. D’Andrea, F. Foschum, P. Taroni, and A. Pifferi, “Light propagation in dry and wet softwood,” Opt. Express16, 9895–9906 (2008). [CrossRef] [PubMed]
10. V. Backman, R. Gurjar, K. Badizadegan, I. Itzkan, R. Dasari, L. Perelman, and M. Feld, “Polarized light scattering spectroscopy for quantitative measurement of epithelial cellular structures in situ,” IEEE J. Sel. Top. Quantum Electron.5, 1019–1026 (1999). [CrossRef]
11. J. Allen, Y. Liu, Y. L. Kim, V. M. Turzhitsky, V. Backman, and G. A. Ameer, “Spectroscopic translation of cell-material interactions,” Biomaterials28, 162–174 (2007). [CrossRef]
12. L. B. Lovat, K. Johnson, G. D. Mackenzie, B. R. Clark, M. R. Novelli, S. Davies, M. O’Donovan, C. Selvasekar, S. M. Thorpe, D. Pickard, R. Fitzgerald, T. Fearn, I. Bigio, and S. G. Bown, “Elastic scattering spectroscopy accurately detects high grade dysplasia and cancer in Barrett’s oesophagus,” Gut55, 1078–1083 (2006). [CrossRef] [PubMed]
13. A. K. Popp, M. T. Valentine, P. D. Kaplan, and D. A. Weitz, “Microscopic origin of light scattering in tissue,” Appl. Opt.42, 2871–2880 (2003). [CrossRef] [PubMed]
14. K. Rebner, M. Schmitz, B. Boldrini, A. Kienle, D. Oelkrug, and R. W. Kessler, “Dark-field scattering microscopy for spectral characterization of polystyrene aggregates,” Opt. Express18, 3116–3127 (2010). [CrossRef] [PubMed]
15. H. Fang, L. Qiu, E. Vitkin, M. M. Zaman, C. Andersson, S. Salahuddin, L. M. Kimerer, P. B. Cipolloni, M. D. Modell, B. S. Turner, S. E. Keates, I. Bigio, I. Itzkan, S. D. Freedman, R. Bansil, E. B. Hanlon, and L. T. Perelman, “Confocal light absorption and scattering spectroscopic microscopy,” Appl. Opt.46, 1760–1769 (2007). [CrossRef] [PubMed]
16. P. Huang, M. Hunter, and I. Georgakoudi, “Confocal light scattering spectroscopic imaging system for in situ tissue characterization,” Appl. Opt.48, 2595–2599 (2009). [CrossRef] [PubMed]
17. W. J. Cottrell, J. D. Wilson, and T. H. Foster, “Microscope enabling multimodality imaging, angle-resolved scattering, and scattering spectroscopy,” Opt. Lett.32, 2348–2350 (2007). [CrossRef] [PubMed]
18. Z. J. Smith and A. J. Berger, “Validation of an integrated Raman- and angular-scattering microscopy system on heterogeneous bead mixtures and single human immune cells,” Appl. Opt.48, D109–D120 (2009). [CrossRef] [PubMed]
19. R. Arimoto and J. Murray, “Orientation-dependent visibility of long thin objects in polarization-based microscopy,” Biophys. J.70, 2969–2980 (1996). [CrossRef] [PubMed]
20. H. K. Roy, Y. Liu, R. K. Wali, Y. L. Kim, A. K. Kromine, M. J. Goldberg, and V. Backman, “Four-dimensional elastic light-scattering fingerprints as preneoplastic markers in the rat model of colon carcinogenesis,” Gastroenterology126, 1071–1081 (2004). [CrossRef] [PubMed]
21. T. Rothe, M. Schmitz, and A. Kienle, “Angular resolved scattering microscopy,” in “Advanced Microscopy Techniques II,” (SPIE, 2011), 808613.
22. L. T. Perelman, V. Backman, M. Wallace, G. Zonios, R. Manoharan, A. Nusrat, S. Shields, M. Seiler, C. Lima, T. Hamano, I. Itzkan, J. Van Dam, J. M. Crawford, and M. S. Feld, “Observation of periodic fine structure in reflectance from biological tissue: A new technique for measuring nuclear size distribution,” Phys. Rev. Lett.80, 627–630 (1998). [CrossRef]
23. H. Fang, M. Ollero, E. Vitkin, L. Kimerer, P. Cipolloni, M. Zaman, S. Freedman, I. Bigio, I. Itzkan, E. Hanlon, and L. Perelman, “Noninvasive sizing of subcellular organelles with light scattering spectroscopy,” IEEE J Sel Top Quantum Electron9, 267–276 (2003). [CrossRef]
24. A. Kienle and R. Hibst, “Light guiding in biological tissue due to scattering,” Phys. Rev. Lett.97, 018104 (2006). [CrossRef] [PubMed]
25. A. Kienle, C. Wetzel, A. Bassi, D. Comelli, P. Taroni, and A. Pifferi, “Determination of the optical properties of anisotropic biological media using an isotropic diffusion model,” J. Biomed. Opt.12, 014026 (2007). [CrossRef] [PubMed]
26. G. Mie, “Beiträge zur Optik trüber Medien, speziell kolloidaler Metallösungen,” Ann. Phys.330, 377–445 (1908). [CrossRef]
27. C. F. Bohren and D. R. Huffman, Absorption and Scattering of Light by Small Particles (Wiley, 1983).
28. R. Michels, “Verständnis des mikroskopischen Ursprungs der Lichtstreuung in biologischem Gewebe,” doctoral dissertation, Ulm University (2010).
29. C. Tribastone and W. Peck, “Designing plastic optics: New applications emerging for optical glass substitutes,” in The Photonics Design and Applications Handbook, (Laurin Publishing, 1998), pp. H426–H433.
30. M. Schmitz, T. Rothe, and A. Kienle, “Comparison between spectral resolved scattering microscopy and collimated transmission measurements,” in “Advanced Microscopy Techniques II,” (SPIE, 2011), 808614.
31. M. Daimon and A. Masumura, “Measurement of the refractive index of distilled water from the near-infrared region to the ultraviolet region,” Appl. Opt.46, 3811–3820 (2007). [CrossRef] [PubMed]
32. X. Ma, J. Q. Lu, R. S. Brock, 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]
33. E. Collett, “Mueller-stokes matrix formulation of Fresnel’s equations,” Am. J. Phys.39, 517–528 (1971). [CrossRef]
34. M. Schmitz, R. Michels, and A. Kienle, “Darkfield scattering spectroscopic microscopy evaluation using polystyrene beads,” in “Clinical and Biomedical Spectroscopy,” (SPIE, 2009), 73681W.
35. A. D. Ward, M. Zhang, and O. Hunt, “Broadband Mie scattering from optically levitated aerosol droplets using a white LED,” Opt. Express16, 16390–16403 (2008). [CrossRef] [PubMed]
36. A. Graßmann and F. Peters, “Size measurement of very small spherical particles by Mie scattering imaging (MSI),” Part. Part. Syst. Character.21, 379–389 (2004). [CrossRef]
37. C. S. Mulvey, C. A. Sherwood, and I. J. Bigio, “Wavelength-dependent backscattering measurements for quantitative real-time monitoring of apoptosis in living cells,” J. Biomed. Opt.14, 064013 (2009). [CrossRef]

Am. J. Phys.

• E. Collett, “Mueller-stokes matrix formulation of Fresnel’s equations,” Am. J. Phys.39, 517–528 (1971). [CrossRef]

Ann. Phys.

• G. Mie, “Beiträge zur Optik trüber Medien, speziell kolloidaler Metallösungen,” Ann. Phys.330, 377–445 (1908). [CrossRef]

Appl. Opt.

• H. Fang, L. Qiu, E. Vitkin, M. M. Zaman, C. Andersson, S. Salahuddin, L. M. Kimerer, P. B. Cipolloni, M. D. Modell, B. S. Turner, S. E. Keates, I. Bigio, I. Itzkan, S. D. Freedman, R. Bansil, E. B. Hanlon, and L. T. Perelman, “Confocal light absorption and scattering spectroscopic microscopy,” Appl. Opt.46, 1760–1769 (2007). [CrossRef] [PubMed]
• P. Huang, M. Hunter, and I. Georgakoudi, “Confocal light scattering spectroscopic imaging system for in situ tissue characterization,” Appl. Opt.48, 2595–2599 (2009). [CrossRef] [PubMed]
• M. Daimon and A. Masumura, “Measurement of the refractive index of distilled water from the near-infrared region to the ultraviolet region,” Appl. Opt.46, 3811–3820 (2007). [CrossRef] [PubMed]
• A. Amelink, M. P. L. Bard, S. A. Burgers, and H. J. C. M. Sterenborg, “Single-scattering spectroscopy for the endoscopic analysis of particle size in superficial layers of turbid media,” Appl. Opt.42, 4095–4101 (2003). [CrossRef] [PubMed]
• M. S. Patterson, B. Chance, and B. C. Wilson, “Time resolved reflectance and transmittance for the non-invasive measurement of tissue optical properties,” Appl. Opt.28, 2331–2336 (1989). [CrossRef] [PubMed]
• T. Namita, Y. Kato, and K. Shimizu, “CT imaging of diffuse medium by time-resolved measurement of backscattered light,” Appl. Opt.48, D208–D217 (2009). [CrossRef] [PubMed]
• J. R. Mourant, I. J. Bigio, D. A. Jack, T. M. Johnson, and H. D. Miller, “Measuring absorption coefficients in small volumes of highly scattering media: source-detector separations for which path lengths do not depend on scattering properties,” Appl. Opt.36, 5655–5661 (1997). [CrossRef] [PubMed]
• A. K. Popp, M. T. Valentine, P. D. Kaplan, and D. A. Weitz, “Microscopic origin of light scattering in tissue,” Appl. Opt.42, 2871–2880 (2003). [CrossRef] [PubMed]
• Z. J. Smith and A. J. Berger, “Validation of an integrated Raman- and angular-scattering microscopy system on heterogeneous bead mixtures and single human immune cells,” Appl. Opt.48, D109–D120 (2009). [CrossRef] [PubMed]

Biomaterials

• J. Allen, Y. Liu, Y. L. Kim, V. M. Turzhitsky, V. Backman, and G. A. Ameer, “Spectroscopic translation of cell-material interactions,” Biomaterials28, 162–174 (2007). [CrossRef]

Biophys. J.

• R. Arimoto and J. Murray, “Orientation-dependent visibility of long thin objects in polarization-based microscopy,” Biophys. J.70, 2969–2980 (1996). [CrossRef] [PubMed]

Gastroenterology

• H. K. Roy, Y. Liu, R. K. Wali, Y. L. Kim, A. K. Kromine, M. J. Goldberg, and V. Backman, “Four-dimensional elastic light-scattering fingerprints as preneoplastic markers in the rat model of colon carcinogenesis,” Gastroenterology126, 1071–1081 (2004). [CrossRef] [PubMed]

Gut

• L. B. Lovat, K. Johnson, G. D. Mackenzie, B. R. Clark, M. R. Novelli, S. Davies, M. O’Donovan, C. Selvasekar, S. M. Thorpe, D. Pickard, R. Fitzgerald, T. Fearn, I. Bigio, and S. G. Bown, “Elastic scattering spectroscopy accurately detects high grade dysplasia and cancer in Barrett’s oesophagus,” Gut55, 1078–1083 (2006). [CrossRef] [PubMed]

IEEE J Sel Top Quantum Electron

• H. Fang, M. Ollero, E. Vitkin, L. Kimerer, P. Cipolloni, M. Zaman, S. Freedman, I. Bigio, I. Itzkan, E. Hanlon, and L. Perelman, “Noninvasive sizing of subcellular organelles with light scattering spectroscopy,” IEEE J Sel Top Quantum Electron9, 267–276 (2003). [CrossRef]

IEEE J. Sel. Top. Quantum Electron.

• V. Backman, R. Gurjar, K. Badizadegan, I. Itzkan, R. Dasari, L. Perelman, and M. Feld, “Polarized light scattering spectroscopy for quantitative measurement of epithelial cellular structures in situ,” IEEE J. Sel. Top. Quantum Electron.5, 1019–1026 (1999). [CrossRef]

J. Biomed. Opt.

• F. K. Forster, A. Kienle, R. Michels, and R. Hibst, “Phase function measurements on nonspherical scatterers using a two-axis goniometer,” J. Biomed. Opt.11, 024018 (2006). [CrossRef] [PubMed]
• A. Kienle, C. Wetzel, A. Bassi, D. Comelli, P. Taroni, and A. Pifferi, “Determination of the optical properties of anisotropic biological media using an isotropic diffusion model,” J. Biomed. Opt.12, 014026 (2007). [CrossRef] [PubMed]
• C. S. Mulvey, C. A. Sherwood, and I. J. Bigio, “Wavelength-dependent backscattering measurements for quantitative real-time monitoring of apoptosis in living cells,” J. Biomed. Opt.14, 064013 (2009). [CrossRef]

Opt. Express

• M. J. Berg, S. C. Hill, G. Videen, and K. P. Gurton, “Spatial filtering technique to image and measure two-dimensional near-forward scattering from single particles,” Opt. Express18, 9486–9495 (2010). [CrossRef] [PubMed]
• A. Kienle, C. D’Andrea, F. Foschum, P. Taroni, and A. Pifferi, “Light propagation in dry and wet softwood,” Opt. Express16, 9895–9906 (2008). [CrossRef] [PubMed]
• K. Rebner, M. Schmitz, B. Boldrini, A. Kienle, D. Oelkrug, and R. W. Kessler, “Dark-field scattering microscopy for spectral characterization of polystyrene aggregates,” Opt. Express18, 3116–3127 (2010). [CrossRef] [PubMed]
• A. D. Ward, M. Zhang, and O. Hunt, “Broadband Mie scattering from optically levitated aerosol droplets using a white LED,” Opt. Express16, 16390–16403 (2008). [CrossRef] [PubMed]

Opt. Lett.

• W. J. Cottrell, J. D. Wilson, and T. H. Foster, “Microscope enabling multimodality imaging, angle-resolved scattering, and scattering spectroscopy,” Opt. Lett.32, 2348–2350 (2007). [CrossRef] [PubMed]
• P. Albella, J. M. Saiz, J. M. Sanz, F. González, and F. Moreno, “Nanoscopic surface inspection by analyzing the linear polarization degree of the scattered light,” Opt. Lett.34, 1906–1908 (2009). [CrossRef] [PubMed]
• Y. Liu, X. Li, Y. L. Kim, and V. Backman, “Elastic backscattering spectroscopic microscopy,” Opt. Lett.30, 2445–2447 (2005). [CrossRef] [PubMed]

Part. Part. Syst. Character.

• A. Graßmann and F. Peters, “Size measurement of very small spherical particles by Mie scattering imaging (MSI),” Part. Part. Syst. Character.21, 379–389 (2004). [CrossRef]

Phys. Med. Biol.

• X. Ma, J. Q. Lu, R. S. Brock, 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]

Phys. Rev. Lett.

• L. T. Perelman, V. Backman, M. Wallace, G. Zonios, R. Manoharan, A. Nusrat, S. Shields, M. Seiler, C. Lima, T. Hamano, I. Itzkan, J. Van Dam, J. M. Crawford, and M. S. Feld, “Observation of periodic fine structure in reflectance from biological tissue: A new technique for measuring nuclear size distribution,” Phys. Rev. Lett.80, 627–630 (1998). [CrossRef]
• A. Kienle and R. Hibst, “Light guiding in biological tissue due to scattering,” Phys. Rev. Lett.97, 018104 (2006). [CrossRef] [PubMed]

The Photonics Design and Applications Handbook

• C. Tribastone and W. Peck, “Designing plastic optics: New applications emerging for optical glass substitutes,” in The Photonics Design and Applications Handbook, (Laurin Publishing, 1998), pp. H426–H433.

Other

• M. Schmitz, T. Rothe, and A. Kienle, “Comparison between spectral resolved scattering microscopy and collimated transmission measurements,” in “Advanced Microscopy Techniques II,” (SPIE, 2011), 808614.
• M. Schmitz, R. Michels, and A. Kienle, “Darkfield scattering spectroscopic microscopy evaluation using polystyrene beads,” in “Clinical and Biomedical Spectroscopy,” (SPIE, 2009), 73681W.
• C. F. Bohren and D. R. Huffman, Absorption and Scattering of Light by Small Particles (Wiley, 1983).
• R. Michels, “Verständnis des mikroskopischen Ursprungs der Lichtstreuung in biologischem Gewebe,” doctoral dissertation, Ulm University (2010).
• T. Rothe, M. Schmitz, and A. Kienle, “Angular resolved scattering microscopy,” in “Advanced Microscopy Techniques II,” (SPIE, 2011), 808613.

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