Abstract

Quantitative phase spectroscopy is presented as a novel method of measuring the wavelength-dependent refractive index of microscopic volumes. Light from a broadband source is filtered to an ~5 nm bandwidth and rapidly tuned across the visible spectrum in 1 nm increments by an acousto-optic tunable filter (AOTF). Quantitative phase images of semitransparent samples are recovered at each wavelength using off-axis interferometry and are processed to recover relative and absolute dispersion measurements. We demonstrate the utility of this approach by (i) spectrally averaging phase images to reduce coherent noise, (ii) measuring absorptive and dispersive features in microspheres, and (iii) quantifying bulk hemoglobin concentrations by absolute refractive index measurements. Considerations of using low coherence illumination and the extension of spectral techniques in quantitative phase measurements are discussed.

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2011

2010

2009

2008

2006

M. A. Choma, A. K. Ellerbee, S. Yazdanfar, and J. A. Izatt, “Doppler flow imaging of cytoplasmic streaming using spectral domain phase microscopy,” J. Biomed. Opt.11(2), 024014 (2006).
[CrossRef] [PubMed]

1990

P. Schiebener, J. Straub, J. M. H. Levelt Sengers, and J. S. Gallagher, “Refractive index of water and steam as function of wavelength, temperature and density,” J. Phys. Chem. Ref. Data19(3), 677–717 (1990).
[CrossRef]

1971

1957

Ahrenkiel, R. K.

Barer, R.

Charrière, F.

Choi, W.

Choma, M. A.

M. A. Choma, A. K. Ellerbee, S. Yazdanfar, and J. A. Izatt, “Doppler flow imaging of cytoplasmic streaming using spectral domain phase microscopy,” J. Biomed. Opt.11(2), 024014 (2006).
[CrossRef] [PubMed]

Chowdhury, S.

Clark, R. L.

Dasari, R.

Dasari, R. R.

Depeursinge, C.

Douplik, A.

O. Zhernovaya, O. Sydoruk, V. Tuchin, and A. Douplik, “The refractive index of human hemoglobin in the visible range,” Phys. Med. Biol.56(13), 4013–4021 (2011).
[CrossRef] [PubMed]

Drake, T. K.

M. T. Rinehart, T. K. Drake, F. E. Robles, L. C. Rohan, D. Katz, and A. Wax, “Time-resolved imaging refractometry of microbicidal films using quantitative phase microscopy,” J. Biomed. Opt.16(12), 120510 (2011).
[CrossRef] [PubMed]

Ellerbee, A. K.

M. A. Choma, A. K. Ellerbee, S. Yazdanfar, and J. A. Izatt, “Doppler flow imaging of cytoplasmic streaming using spectral domain phase microscopy,” J. Biomed. Opt.11(2), 024014 (2006).
[CrossRef] [PubMed]

Emery, Y.

Feld, M.

Feld, M. S.

Fu, D.

Gallagher, J. S.

P. Schiebener, J. Straub, J. M. H. Levelt Sengers, and J. S. Gallagher, “Refractive index of water and steam as function of wavelength, temperature and density,” J. Phys. Chem. Ref. Data19(3), 677–717 (1990).
[CrossRef]

Izatt, J. A.

M. A. Choma, A. K. Ellerbee, S. Yazdanfar, and J. A. Izatt, “Doppler flow imaging of cytoplasmic streaming using spectral domain phase microscopy,” J. Biomed. Opt.11(2), 024014 (2006).
[CrossRef] [PubMed]

Jenness, N. J.

Katz, D.

M. T. Rinehart, T. K. Drake, F. E. Robles, L. C. Rohan, D. Katz, and A. Wax, “Time-resolved imaging refractometry of microbicidal films using quantitative phase microscopy,” J. Biomed. Opt.16(12), 120510 (2011).
[CrossRef] [PubMed]

Levelt Sengers, J. M. H.

P. Schiebener, J. Straub, J. M. H. Levelt Sengers, and J. S. Gallagher, “Refractive index of water and steam as function of wavelength, temperature and density,” J. Phys. Chem. Ref. Data19(3), 677–717 (1990).
[CrossRef]

Magistretti, P. J.

Marquet, P.

Monemhaghdoust, Z.

Montfort, F.

Moser, C.

Park, Y.

Rappaz, B.

Rinehart, M. T.

Robles, F. E.

Rohan, L. C.

M. T. Rinehart, T. K. Drake, F. E. Robles, L. C. Rohan, D. Katz, and A. Wax, “Time-resolved imaging refractometry of microbicidal films using quantitative phase microscopy,” J. Biomed. Opt.16(12), 120510 (2011).
[CrossRef] [PubMed]

Satterwhite, L. L.

Schiebener, P.

P. Schiebener, J. Straub, J. M. H. Levelt Sengers, and J. S. Gallagher, “Refractive index of water and steam as function of wavelength, temperature and density,” J. Phys. Chem. Ref. Data19(3), 677–717 (1990).
[CrossRef]

Shaked, N. T.

Straub, J.

P. Schiebener, J. Straub, J. M. H. Levelt Sengers, and J. S. Gallagher, “Refractive index of water and steam as function of wavelength, temperature and density,” J. Phys. Chem. Ref. Data19(3), 677–717 (1990).
[CrossRef]

Sung, Y.

Sydoruk, O.

O. Zhernovaya, O. Sydoruk, V. Tuchin, and A. Douplik, “The refractive index of human hemoglobin in the visible range,” Phys. Med. Biol.56(13), 4013–4021 (2011).
[CrossRef] [PubMed]

Tuchin, V.

O. Zhernovaya, O. Sydoruk, V. Tuchin, and A. Douplik, “The refractive index of human hemoglobin in the visible range,” Phys. Med. Biol.56(13), 4013–4021 (2011).
[CrossRef] [PubMed]

Wax, A.

Yamauchi, T.

Yaqoob, Z.

Yazdanfar, S.

M. A. Choma, A. K. Ellerbee, S. Yazdanfar, and J. A. Izatt, “Doppler flow imaging of cytoplasmic streaming using spectral domain phase microscopy,” J. Biomed. Opt.11(2), 024014 (2006).
[CrossRef] [PubMed]

Zhernovaya, O.

O. Zhernovaya, O. Sydoruk, V. Tuchin, and A. Douplik, “The refractive index of human hemoglobin in the visible range,” Phys. Med. Biol.56(13), 4013–4021 (2011).
[CrossRef] [PubMed]

Zhu, Y.

Biomed. Opt. Express

J. Biomed. Opt.

M. T. Rinehart, T. K. Drake, F. E. Robles, L. C. Rohan, D. Katz, and A. Wax, “Time-resolved imaging refractometry of microbicidal films using quantitative phase microscopy,” J. Biomed. Opt.16(12), 120510 (2011).
[CrossRef] [PubMed]

M. A. Choma, A. K. Ellerbee, S. Yazdanfar, and J. A. Izatt, “Doppler flow imaging of cytoplasmic streaming using spectral domain phase microscopy,” J. Biomed. Opt.11(2), 024014 (2006).
[CrossRef] [PubMed]

J. Opt. Soc. Am.

J. Phys. Chem. Ref. Data

P. Schiebener, J. Straub, J. M. H. Levelt Sengers, and J. S. Gallagher, “Refractive index of water and steam as function of wavelength, temperature and density,” J. Phys. Chem. Ref. Data19(3), 677–717 (1990).
[CrossRef]

Opt. Express

Opt. Lett.

Phys. Med. Biol.

O. Zhernovaya, O. Sydoruk, V. Tuchin, and A. Douplik, “The refractive index of human hemoglobin in the visible range,” Phys. Med. Biol.56(13), 4013–4021 (2011).
[CrossRef] [PubMed]

Other

Life Technologies, “FluoSpheres®,” http://probes.invitrogen.com/media/spectra/data/8809h2o.txt .

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Figures (4)

Fig. 1
Fig. 1

Simulation of the effects of low coherence illumination on off-axis QPM carrier frequency (λ0 = 500 nm): (A-D) Amplitude of complex envelope (term 3 of Eq. (2) of the interference cross-term. (E-H) High-frequency spatial modulation (term 2 of Eq. (2) cross-sections from dotted lines in corresponding (A-D). (A,E) δz = 0 μm, δλ = 1 nm; some roll-off of amplitude is seen. (B,F) δz = 0 μm, δλ = 5 nm; amplitude roll-off obscures signal at edges of field of view. (C,G) δz = 20 μm, δλ = 5 nm; peak of coherence envelope moves across field of view due to path length offset. (D,H) δz = 0 μm, δλ = 22 nm, ; large bandwidth severely limits field of view. Note that the angle between the sample and reference beams, and hence the spatial carrier frequency, are fixed in all of these figures. Lateral scale bars: 50 µm.

Fig. 2
Fig. 2

Phase images of a transparent PDMS phase object, demonstrating a reduction of coherent noise by spectral averaging. Letters have a nominal 90nm thickness. (A,B) Full field of view (~200x200µm) with reduced SNR at the edges; 50µm scale bars. (D,E) zoomed in view with insets corresponding to plots at the dotted lines; 20µm scale bars. (A,D) imaged with λ0 = 603 nm, δλ = 5.4 nm; (B,E) computed by averaging 220 phase maps across 500–720 nm, λ 0 =603 nm . (C,F) Simulation of a phase image acquired with δλ = 220 nm; note the area over which high-SNR phase information is significantly reduced.

Fig. 3
Fig. 3

(A) Phase images of a fluorescent 10μm polystyrene microsphere, λ 0 =594 nm ; scale bar is 5 μm. (B) Phase profile taken from dashed line (A). (C) Published absorption peak of fluorescent microsphere [10]. (D) Theoretical and measured absolute refractive index spectra of fluorescent and non-fluorescent microspheres, calculated from the points circled in (B).

Fig. 4
Fig. 4

(A) Phase image of PDMS chamber for bulk fluid RI spectroscopy, scale bar 25 μm. (B) Measured RI dispersion spectra for serial dilutions of oxy-hemoglobin and PBS. (C) Refractive index of measured fluids as a function of hemoglobin concentration. Blue diamonds correspond to values measured by QPS; Red triangles to values measured by a commercial refractometer. λ0 = 589 nm; fit trend line: n = 1.3362 + 0.1496C, R2 = 0.9962.

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