Abstract

Existing darkfield illumination schemes are incompatible with many types of samples and/or procedures. We present a darkfield epi-illumination scheme which addresses these incompatibilities by providing illumination through the imaging objective. We validate the system performance using silver nanospheres in varying refractive index environments, characterize the intensity distribution of the darkfield illumination, and demonstrate system capabilities through a preliminary study of functionalized gold nanosphere interactions with cancer cells in culture. We observe a broadened scattering spectrum from unconjugated nanoparticles, as compared with anti-EGFR conjugated nanoparticles, upon incubation with cancer cells, and discuss the implications of this observation.

© 2006 Optical Society of America

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  1. S. Schultz, D. R. Smith, J. J. Mock, and D. A. Schultz, "Single-target molecule detection with nonbleaching multicolor optical immunolabels," PNAS 97, 996-1001 (2000).
    [CrossRef] [PubMed]
  2. I. El-Sayed, X. Huang, and M. El-Sayed, "Surface plasmon resonance scattering and absorption of anti-egfr antibody conjugated gold nanoparticles in cancer diagnostics: Applications in oral cancer," Nano Lett. 5, 829-834 (2005).
    [CrossRef] [PubMed]
  3. C. Loo, A. Lowery, N. Halas, J. West, and R. Drezek, "Immunotargeted nanoshells for integrated cancer imaging and therapy," Nano Lett. 5, 709-711 (2005).
    [CrossRef] [PubMed]
  4. C. Sonnichsen, B. Reinhard, J. Liphard, and A. Alivisatos, "A molecular ruler based on plasmon coupling of single gold and silver nanoparticles," Nat. Biotechnol. 23, 741-745 (2005).
    [CrossRef] [PubMed]
  5. A. Haes, W. Hall, L. Chang, W. Klein, and R. Van Duyne, "A localized surface plasmon resonance biosensor: First steps toward an assay for alzheimer's disease," Nano Lett. 4, 1029-1034 (2004).
    [CrossRef]
  6. N. Nath, and A. Chilkoti, "Label free colorimetric biosensing using nanoparticles," J. Fluoresc. 14, 377-389 (2004).
    [CrossRef] [PubMed]
  7. K. Sokolov, M. Follen, J. Aaron, I. Pavlova, A. Malpica, R. Lotan, and R. Richards-Kortum, "Real-time vital optical imaging of precancer using anti-epidermal growth factor receptor antibodies conjugated to gold nanoparticles," Cancer Res. 63, 1999-2004 (2003).
    [PubMed]
  8. A. Curry, G. Nusz, A. Chilkoti, and A. Wax, "Substrate effect on refractive index dependence of plasmon resonance for individual silver nanoparticles observed using darkfield micro-spectroscopy," Opt. Express 13, 2668-2677 (2005).
    [CrossRef] [PubMed]
  9. H. Haigler, J. F. Ash, S. J. Singer, and S. Cohen, "Visualization by fluorescence of binding and internalization of epidermal growth-factor in human carcinoma cells a-431," PNAS 75, 3317-3321 (1978).
    [CrossRef] [PubMed]

2005 (4)

I. El-Sayed, X. Huang, and M. El-Sayed, "Surface plasmon resonance scattering and absorption of anti-egfr antibody conjugated gold nanoparticles in cancer diagnostics: Applications in oral cancer," Nano Lett. 5, 829-834 (2005).
[CrossRef] [PubMed]

C. Loo, A. Lowery, N. Halas, J. West, and R. Drezek, "Immunotargeted nanoshells for integrated cancer imaging and therapy," Nano Lett. 5, 709-711 (2005).
[CrossRef] [PubMed]

C. Sonnichsen, B. Reinhard, J. Liphard, and A. Alivisatos, "A molecular ruler based on plasmon coupling of single gold and silver nanoparticles," Nat. Biotechnol. 23, 741-745 (2005).
[CrossRef] [PubMed]

A. Curry, G. Nusz, A. Chilkoti, and A. Wax, "Substrate effect on refractive index dependence of plasmon resonance for individual silver nanoparticles observed using darkfield micro-spectroscopy," Opt. Express 13, 2668-2677 (2005).
[CrossRef] [PubMed]

2004 (2)

A. Haes, W. Hall, L. Chang, W. Klein, and R. Van Duyne, "A localized surface plasmon resonance biosensor: First steps toward an assay for alzheimer's disease," Nano Lett. 4, 1029-1034 (2004).
[CrossRef]

N. Nath, and A. Chilkoti, "Label free colorimetric biosensing using nanoparticles," J. Fluoresc. 14, 377-389 (2004).
[CrossRef] [PubMed]

2003 (1)

K. Sokolov, M. Follen, J. Aaron, I. Pavlova, A. Malpica, R. Lotan, and R. Richards-Kortum, "Real-time vital optical imaging of precancer using anti-epidermal growth factor receptor antibodies conjugated to gold nanoparticles," Cancer Res. 63, 1999-2004 (2003).
[PubMed]

2000 (1)

S. Schultz, D. R. Smith, J. J. Mock, and D. A. Schultz, "Single-target molecule detection with nonbleaching multicolor optical immunolabels," PNAS 97, 996-1001 (2000).
[CrossRef] [PubMed]

1978 (1)

H. Haigler, J. F. Ash, S. J. Singer, and S. Cohen, "Visualization by fluorescence of binding and internalization of epidermal growth-factor in human carcinoma cells a-431," PNAS 75, 3317-3321 (1978).
[CrossRef] [PubMed]

Aaron, J.

K. Sokolov, M. Follen, J. Aaron, I. Pavlova, A. Malpica, R. Lotan, and R. Richards-Kortum, "Real-time vital optical imaging of precancer using anti-epidermal growth factor receptor antibodies conjugated to gold nanoparticles," Cancer Res. 63, 1999-2004 (2003).
[PubMed]

Alivisatos, A.

C. Sonnichsen, B. Reinhard, J. Liphard, and A. Alivisatos, "A molecular ruler based on plasmon coupling of single gold and silver nanoparticles," Nat. Biotechnol. 23, 741-745 (2005).
[CrossRef] [PubMed]

Ash, J. F.

H. Haigler, J. F. Ash, S. J. Singer, and S. Cohen, "Visualization by fluorescence of binding and internalization of epidermal growth-factor in human carcinoma cells a-431," PNAS 75, 3317-3321 (1978).
[CrossRef] [PubMed]

Chang, L.

A. Haes, W. Hall, L. Chang, W. Klein, and R. Van Duyne, "A localized surface plasmon resonance biosensor: First steps toward an assay for alzheimer's disease," Nano Lett. 4, 1029-1034 (2004).
[CrossRef]

Chilkoti, A.

Cohen, S.

H. Haigler, J. F. Ash, S. J. Singer, and S. Cohen, "Visualization by fluorescence of binding and internalization of epidermal growth-factor in human carcinoma cells a-431," PNAS 75, 3317-3321 (1978).
[CrossRef] [PubMed]

Curry, A.

Drezek, R.

C. Loo, A. Lowery, N. Halas, J. West, and R. Drezek, "Immunotargeted nanoshells for integrated cancer imaging and therapy," Nano Lett. 5, 709-711 (2005).
[CrossRef] [PubMed]

El-Sayed, I.

I. El-Sayed, X. Huang, and M. El-Sayed, "Surface plasmon resonance scattering and absorption of anti-egfr antibody conjugated gold nanoparticles in cancer diagnostics: Applications in oral cancer," Nano Lett. 5, 829-834 (2005).
[CrossRef] [PubMed]

El-Sayed, M.

I. El-Sayed, X. Huang, and M. El-Sayed, "Surface plasmon resonance scattering and absorption of anti-egfr antibody conjugated gold nanoparticles in cancer diagnostics: Applications in oral cancer," Nano Lett. 5, 829-834 (2005).
[CrossRef] [PubMed]

Follen, M.

K. Sokolov, M. Follen, J. Aaron, I. Pavlova, A. Malpica, R. Lotan, and R. Richards-Kortum, "Real-time vital optical imaging of precancer using anti-epidermal growth factor receptor antibodies conjugated to gold nanoparticles," Cancer Res. 63, 1999-2004 (2003).
[PubMed]

Haes, A.

A. Haes, W. Hall, L. Chang, W. Klein, and R. Van Duyne, "A localized surface plasmon resonance biosensor: First steps toward an assay for alzheimer's disease," Nano Lett. 4, 1029-1034 (2004).
[CrossRef]

Haigler, H.

H. Haigler, J. F. Ash, S. J. Singer, and S. Cohen, "Visualization by fluorescence of binding and internalization of epidermal growth-factor in human carcinoma cells a-431," PNAS 75, 3317-3321 (1978).
[CrossRef] [PubMed]

Halas, N.

C. Loo, A. Lowery, N. Halas, J. West, and R. Drezek, "Immunotargeted nanoshells for integrated cancer imaging and therapy," Nano Lett. 5, 709-711 (2005).
[CrossRef] [PubMed]

Hall, W.

A. Haes, W. Hall, L. Chang, W. Klein, and R. Van Duyne, "A localized surface plasmon resonance biosensor: First steps toward an assay for alzheimer's disease," Nano Lett. 4, 1029-1034 (2004).
[CrossRef]

Huang, X.

I. El-Sayed, X. Huang, and M. El-Sayed, "Surface plasmon resonance scattering and absorption of anti-egfr antibody conjugated gold nanoparticles in cancer diagnostics: Applications in oral cancer," Nano Lett. 5, 829-834 (2005).
[CrossRef] [PubMed]

Klein, W.

A. Haes, W. Hall, L. Chang, W. Klein, and R. Van Duyne, "A localized surface plasmon resonance biosensor: First steps toward an assay for alzheimer's disease," Nano Lett. 4, 1029-1034 (2004).
[CrossRef]

Liphard, J.

C. Sonnichsen, B. Reinhard, J. Liphard, and A. Alivisatos, "A molecular ruler based on plasmon coupling of single gold and silver nanoparticles," Nat. Biotechnol. 23, 741-745 (2005).
[CrossRef] [PubMed]

Loo, C.

C. Loo, A. Lowery, N. Halas, J. West, and R. Drezek, "Immunotargeted nanoshells for integrated cancer imaging and therapy," Nano Lett. 5, 709-711 (2005).
[CrossRef] [PubMed]

Lotan, R.

K. Sokolov, M. Follen, J. Aaron, I. Pavlova, A. Malpica, R. Lotan, and R. Richards-Kortum, "Real-time vital optical imaging of precancer using anti-epidermal growth factor receptor antibodies conjugated to gold nanoparticles," Cancer Res. 63, 1999-2004 (2003).
[PubMed]

Lowery, A.

C. Loo, A. Lowery, N. Halas, J. West, and R. Drezek, "Immunotargeted nanoshells for integrated cancer imaging and therapy," Nano Lett. 5, 709-711 (2005).
[CrossRef] [PubMed]

Malpica, A.

K. Sokolov, M. Follen, J. Aaron, I. Pavlova, A. Malpica, R. Lotan, and R. Richards-Kortum, "Real-time vital optical imaging of precancer using anti-epidermal growth factor receptor antibodies conjugated to gold nanoparticles," Cancer Res. 63, 1999-2004 (2003).
[PubMed]

Mock, J. J.

S. Schultz, D. R. Smith, J. J. Mock, and D. A. Schultz, "Single-target molecule detection with nonbleaching multicolor optical immunolabels," PNAS 97, 996-1001 (2000).
[CrossRef] [PubMed]

Nath, N.

N. Nath, and A. Chilkoti, "Label free colorimetric biosensing using nanoparticles," J. Fluoresc. 14, 377-389 (2004).
[CrossRef] [PubMed]

Nusz, G.

Pavlova, I.

K. Sokolov, M. Follen, J. Aaron, I. Pavlova, A. Malpica, R. Lotan, and R. Richards-Kortum, "Real-time vital optical imaging of precancer using anti-epidermal growth factor receptor antibodies conjugated to gold nanoparticles," Cancer Res. 63, 1999-2004 (2003).
[PubMed]

Reinhard, B.

C. Sonnichsen, B. Reinhard, J. Liphard, and A. Alivisatos, "A molecular ruler based on plasmon coupling of single gold and silver nanoparticles," Nat. Biotechnol. 23, 741-745 (2005).
[CrossRef] [PubMed]

Richards-Kortum, R.

K. Sokolov, M. Follen, J. Aaron, I. Pavlova, A. Malpica, R. Lotan, and R. Richards-Kortum, "Real-time vital optical imaging of precancer using anti-epidermal growth factor receptor antibodies conjugated to gold nanoparticles," Cancer Res. 63, 1999-2004 (2003).
[PubMed]

Schultz, D. A.

S. Schultz, D. R. Smith, J. J. Mock, and D. A. Schultz, "Single-target molecule detection with nonbleaching multicolor optical immunolabels," PNAS 97, 996-1001 (2000).
[CrossRef] [PubMed]

Schultz, S.

S. Schultz, D. R. Smith, J. J. Mock, and D. A. Schultz, "Single-target molecule detection with nonbleaching multicolor optical immunolabels," PNAS 97, 996-1001 (2000).
[CrossRef] [PubMed]

Singer, S. J.

H. Haigler, J. F. Ash, S. J. Singer, and S. Cohen, "Visualization by fluorescence of binding and internalization of epidermal growth-factor in human carcinoma cells a-431," PNAS 75, 3317-3321 (1978).
[CrossRef] [PubMed]

Smith, D. R.

S. Schultz, D. R. Smith, J. J. Mock, and D. A. Schultz, "Single-target molecule detection with nonbleaching multicolor optical immunolabels," PNAS 97, 996-1001 (2000).
[CrossRef] [PubMed]

Sokolov, K.

K. Sokolov, M. Follen, J. Aaron, I. Pavlova, A. Malpica, R. Lotan, and R. Richards-Kortum, "Real-time vital optical imaging of precancer using anti-epidermal growth factor receptor antibodies conjugated to gold nanoparticles," Cancer Res. 63, 1999-2004 (2003).
[PubMed]

Sonnichsen, C.

C. Sonnichsen, B. Reinhard, J. Liphard, and A. Alivisatos, "A molecular ruler based on plasmon coupling of single gold and silver nanoparticles," Nat. Biotechnol. 23, 741-745 (2005).
[CrossRef] [PubMed]

Van Duyne, R.

A. Haes, W. Hall, L. Chang, W. Klein, and R. Van Duyne, "A localized surface plasmon resonance biosensor: First steps toward an assay for alzheimer's disease," Nano Lett. 4, 1029-1034 (2004).
[CrossRef]

Wax, A.

West, J.

C. Loo, A. Lowery, N. Halas, J. West, and R. Drezek, "Immunotargeted nanoshells for integrated cancer imaging and therapy," Nano Lett. 5, 709-711 (2005).
[CrossRef] [PubMed]

Cancer Res. (1)

K. Sokolov, M. Follen, J. Aaron, I. Pavlova, A. Malpica, R. Lotan, and R. Richards-Kortum, "Real-time vital optical imaging of precancer using anti-epidermal growth factor receptor antibodies conjugated to gold nanoparticles," Cancer Res. 63, 1999-2004 (2003).
[PubMed]

J. Fluoresc. (1)

N. Nath, and A. Chilkoti, "Label free colorimetric biosensing using nanoparticles," J. Fluoresc. 14, 377-389 (2004).
[CrossRef] [PubMed]

Nano Lett. (3)

I. El-Sayed, X. Huang, and M. El-Sayed, "Surface plasmon resonance scattering and absorption of anti-egfr antibody conjugated gold nanoparticles in cancer diagnostics: Applications in oral cancer," Nano Lett. 5, 829-834 (2005).
[CrossRef] [PubMed]

C. Loo, A. Lowery, N. Halas, J. West, and R. Drezek, "Immunotargeted nanoshells for integrated cancer imaging and therapy," Nano Lett. 5, 709-711 (2005).
[CrossRef] [PubMed]

A. Haes, W. Hall, L. Chang, W. Klein, and R. Van Duyne, "A localized surface plasmon resonance biosensor: First steps toward an assay for alzheimer's disease," Nano Lett. 4, 1029-1034 (2004).
[CrossRef]

Nat. Biotechnol. (1)

C. Sonnichsen, B. Reinhard, J. Liphard, and A. Alivisatos, "A molecular ruler based on plasmon coupling of single gold and silver nanoparticles," Nat. Biotechnol. 23, 741-745 (2005).
[CrossRef] [PubMed]

Opt. Express (1)

PNAS (2)

H. Haigler, J. F. Ash, S. J. Singer, and S. Cohen, "Visualization by fluorescence of binding and internalization of epidermal growth-factor in human carcinoma cells a-431," PNAS 75, 3317-3321 (1978).
[CrossRef] [PubMed]

S. Schultz, D. R. Smith, J. J. Mock, and D. A. Schultz, "Single-target molecule detection with nonbleaching multicolor optical immunolabels," PNAS 97, 996-1001 (2000).
[CrossRef] [PubMed]

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Figures (6)

Fig. 1.
Fig. 1.

Epi-illumination darkfield scheme showing the incident, reflected, and scattered light paths in relation to the microscope objective and darkfield illumination optics.

Fig. 2.
Fig. 2.

Darkfield images of 80 nm Ag NPs in: (a) air, (b) water, and (c) index-matching oil environments. (d) Resonance spectra for the same single NP in each refractive index environment. (e) Experimental versus predicted SPR peak positions as a function of refractive index environment for a weighting factor of 0.58.

Fig. 3.
Fig. 3.

(a) Map of darkfield illumination intensity, and (b) plot of illumination intensity variation along line of spectral acquisition.

Fig. 4.
Fig. 4.

Darkfield images of (a) cells incubated in pure media [1s acquisition time], (b) cell incubated in media with non-conjugated NPs [0.4s acquisition time], and (c) cells incubated with anti-EGFR conjugated NPs [0.4s acquisition time]. Note the increased scattering intensity due to the NPs and the more distinct boundaries for the cells incubated with anti-EGFR NPs, which are expected to localize preferentially on cell surfaces where EGFR is expressed.

Fig. 5.
Fig. 5.

Demonstration of dual-mode imaging with epi-illuminated darkfield for registration of NPs within cells. Each image is a single acquisition of the microscope FOV, under different illumination conditions, and not an overlay of images.

Fig. 6.
Fig. 6.

Spectra of cells acquired with microspectroscopy system. (a) The spectrum from a single site in a cell incubated with non-conjugated NPs is complex, and the resonance peak is not apparent. (b) The resonance peak becomes more apparent when the spectra from a single acquisition are averaged. (c) The peak is clearly seen when 6 acquisitions from the same cell are averaged. (d) The ramp in scattering intensity toward longer wavelengths is present in cells without NPs and may therefore be subtracted from the spectra of cells with NPs to isolate the scattering spectrum due to the NPs. The results of this baseline correction are displayed for (e) a cell incubated with non-conjugated NPs and (f) a cell incubated with conjugated NPs.

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