Abstract

Structured-illumination microscopy delivers confocal-imaging capabilities and may be used for optical sectioning in bio-imaging applications. However, previous structured-illumination implementations are not capable of imaging molecular changes within highly scattering, biological samples in reflectance mode. Here, we present two advances which enable successful structured illumination reflectance microscopy to image molecular changes in epithelial tissue phantoms. First, we present the sine approximation algorithm to improve the ability to reconstruct the in-focus plane when the out-of-focus light is much greater in magnitude. We characterize the dependencies of this algorithm on phase step error, random noise and backscattered out-of-focus contributions. Second, we utilize a molecular-specific reflectance contrast agent based on gold nanoparticles to label disease-related biomarkers and increase the signal and signal-to-noise ratio (SNR) in structured illumination microscopy of biological tissue. Imaging results for multi-layer epithelial cell phantoms with optical properties characteristic of normal and cancerous tissue labeled with nanoparticles targeted against the epidermal growth factor receptor (EGFR) are presented. Structured illumination images reconstructed with the sine approximation algorithm compare favorably to those obtained with a standard confocal microscope; this new technique can be implemented in simple and small imaging platforms for future clinical studies.

© 2004 Optical Society of America

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References

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    [PubMed]

Acad. Radiol.

T. Collier, A. Lacy, A. Malpica, M. Follen, R. Richards-Kortum; ???Near Real Time Confocal Microscopy of Amelanotic Tissue: Detection of Dysplasia in Ex-Vivo Cervical Tissue,??? Acad. Radiol. 9, 504-512 (2002).
[CrossRef] [PubMed]

Cancer (Pila.)

T. Maruo, M. Yamasaki, C. A. Ladines-Llave, and M. Mochizuki, ???Immunohistochemical demonstration of elevated expression of epidermal growth factor receptor in the neoplastic changes of cervical squamous epithelium,??? Cancer (Pila.), 69, 1182-1187 (1992).
[CrossRef]

Cancer Research

Sokolov K, Follen M, Aaron J, Pavlova I, Malpica A, Lotan R, Richards-Kortum R; ???Real Time Vital Imaging of Pre-Cancer Using Anti-EGFR Antibodies Conjugated to Gold Nanoparticles,??? Cancer Research 63, 1999-2004 (2003).
[PubMed]

K. Sokolov, M. Follen, J. Aaron, I. Pavlova, A. Malpica, R. Lotan , R. Richards-Kortum: ???Real Time Vital Imaging of Pre-Cancer Using Anti-EGFR Antibodies Conjugated to Gold Nanoparticles,??? Cancer Research 63, 1999-2004 (2003).
[PubMed]

Gynecol. Oncol.

M. Wobus, R. Kuns, C. Wolf, L. C. Horn, U. Hoehler, I. Sheyn, B. A Werness, and L. S. Sherman: ???CD44 mediates constitutive type I receptor signaling in cervical carcinoma cells,??? Gynecol. Oncol. 83, 227-234 (2001).
[CrossRef] [PubMed]

J. Microsc.

M.A.A.Neil, A.Squire, R.Juškaitis, P.I.H.Bastiaens, T.Wilson, ???Wide-field optically sectioning fluorescence microscopy with laser illumination,??? J. Microsc. 197, 1-4 (2000).
[CrossRef] [PubMed]

J. of Biomed. Opt.

K. Sokolov, J. Galvan , A. Myakov , A. Lacy , R. Lotan, R. Richards-Kortum; ???Realistic Three Dimensional Epithelial Tissue Phantoms for Biomedical Optics,??? J. of Biomed. Opt. 7, 148-156 (2002).
[CrossRef]

Journal of Investigative Dermatology

M. Menaker, G. Flotte, T. Dwyer, PJ. Gonzalez: ???Confocal examination of nonmelanoma cancers in thick skin excisions to potentially guide mohs micrographic surgery without frozen histopathology,??? Journal of Investigative Dermatology 117, 1137-43 (2001).
[CrossRef] [PubMed]

Meas. Sci. Technol.

M.Dobosz, T.Usuda, and T.Kurosawa, ???Methods for the calibration of vibration pick-ups by laser interferometry: I. Theoretical analysis,??? Meas. Sci. Technol. 9, 232-239 (1998).
[CrossRef]

M.Dobosz, T.Usuda, and T.Kurosawa, ???Methods for the calibration of vibration pick-ups by laser interferometry: II. Experimental verification,??? Meas. Sci. Technol., 9, 240-249 (1998).
[CrossRef]

Melanoma Research

KJ. Busam, C. Charles, CM. Lohmann, A. Marghoob, M. Goldgeier, AC. Halpern: ???Detection of intraepidermal malignant melanoma in vivo by confocal scanning laser microscopy,??? Melanoma Research 12, 349-55 (2000).
[CrossRef]

Opt. Eng.

T. Tkaczyk, R. Józwicki, ???Full-field heterodyne interferometer for shape measurement: experimental characteristics of the system,??? Opt. Eng. 42, 2391???2399 (2003).
[CrossRef]

Opt. Express

Opt. Lett.

Techn. in Cancer Res. and Treat.

K. Sokolov, J. Aaron, B. Hsu, D. Nida, A. Gillenwater, M. Follen, C. MacAulay, K. Adler-Storthz, B. Korgel, M. Descour, R. Pasqualini, W. Arap, Wan Lam, R. Richards-Kortum; ???Optical Systems for In Vivo Molecular Imaging of Cancer,??? Techn. in Cancer Res. and Treat., 2, 491-504 (2003).

Other

ApoTome structured-illumination head by Zeiss Inc. at www.zeiss.com <a href= "http://www.zeiss.com/4125681F004CA025/Contents-Frame/286BA4D22B14DEE985256B4A007C3686">http://www.zeiss.com/4125681F004CA025/Contents-Frame/286BA4D22B14DEE985256B4A007C3686</a>.

OptiGrid structured-illumination system by Thales Optem at <a href= "http://www.thales-optem.com/optigrid.html">http://www.thales-optem.com/optigrid.html</a>.

Supplementary Material (2)

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Figures (10)

Fig. 1.
Fig. 1.

Layout of the typical structured illumination microscope system.

Fig. 2.
Fig. 2.

Imaging sequence at the CMOS/CCD detector for the structured-illumination technique. Five pixels are shown at the detector (D1–D5), and the illumination sequence is shown over three steps of the grating. The actual grating step size is ΔφS , and the start phase of the image sequence at a particular pixel is φ0 For example, the initial phase for pixel D1 is 0. The image is reconstructed using the assumed step size of Δφ.

Fig. 3.
Fig. 3.

(a) Topographical projection of the normalized reconstructed amplitude B as a function of the ratio of the real sampling step ΔφS to the assumed sampling step Δφ and phase φ0 at the pixel (b) Cross-sections for locations marked with blue lines in (a) and sampling steps ΔφS equal to 1.25Δφ and 1.50Δφ.

Fig. 4.
Fig. 4.

(a) Reconstructed, normalized amplitude B (blue), amplitude of residual grating BM (green), residual grating strength defined as the ratio of BA/BM (red), (b) BA/BM ratio for values of the step size within ±5% of the expected step size.

Fig. 5.
Fig. 5.

(a) Dependence of the reconstructed amplitude on the SNR and the normalized input amplitude and the number of images used in reconstruction. The input amplitude and SNR were varied in tandem, so that for input amplitude B equal to 1.0, the SNR was also set to 1.0. (b) Reconstructed amplitude as a function of SNR and input amplitude. Each curve showed in plot (a) was scaled to 0–1 range.

Fig. 6.
Fig. 6.

(a) Reconstruction results for 24 pixel test object. The SNR for gray level 3 was equal 0.02. Only 4 images were used for the reconstruction. The red columns represent the reconstructed amplitude, while green denote input data. (b) Magnified region marked with blue box in (a).

Fig. 7.
Fig. 7.

Images of phantoms containing SiHa cervical cancer cells labeled with anti-EGFR gold conjugates. The field of view is 54×54 µm2. The approximate depth of the imaged optical section is 15–20 µm below the phantom surface. Part (a) shows an inverted widefield reflectancemicroscope image. Part (b) shows a structured-illumination raw image. Part (c) shows a reconstructed optical-section image (animation - 1.9MB).

Fig. 8.
Fig. 8.

Image of phantom containing SiHa cervical cancer cells labeled with anti-EGFR gold conjugates reconstructed with classical structured illumination algorithm.

Fig. 9.
Fig. 9.

Images of phantom containing MDA-MB-435S cells labeled with anti-EGFR gold conjugates. The field of view is 54 µm×54 µm. The approximate depth of the optical section is 20 µm beneath the surface of the phantom. Part (a) shows a structured-illumination raw image. Part (b) shows a reconstructed optical-section image.

Fig. 10.
Fig. 10.

Reflectance confocal-microscope image of the phantom containing SiHa cells labeled with anti-EGFR gold conjugates. The field of view measures approximately 60 µm×60 µm. This field of view represents a segment of the full field of view of the confocal microscope chosen in size to match results obtained with structured-illumination (animation – 2.3MB).

Equations (7)

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S ( i , j , n ) = A ( i , j ) + B ( i , j ) { 0.5 + 0.5 cos [ φ 0 ( i , j ) + n Δ φ ] } ,
S ( n ) = A + 0.5 B + 0.5 B cos φ 0 cos ( n Δ φ ) 0.5 B sin φ 0 sin ( n Δ φ ) .
S ( n ) = b 0 + b 1 x 1 ( n ) + b 2 x 2 ( n ) ,
S = Xb + m ,
b ̂ = ( X T X ) 1 X T S .
B = ( b 1 2 + b 2 2 ) .
M ¯ neo M ¯ norm > ( σ neo + σ norm ) : 62.5 28.5 = 34.0 > ( 4.9 + 3.5 ) = 8.4 .

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