Abstract

We present a fiber optic confocal reflectance microscope (FCRM) which can be used to image epithelial tissue with sub-cellular resolution in vivo. Confocal images of normal and abnormal appearing cervical tissue were obtained in vivo from eighteen patients undergoing colposcopic examination of the cervix; biopsy specimens were taken from imaged sites. The measured lateral and axial resolutions of the system were 1.6 µm and 3 µm, respectively. Morphologic features, including nuclear size and nuclear-to-cytoplasmic ratio, were extracted from confocal images obtained at various depths beneath the epithelial surface. Image features extracted from confocal images compared well with features extracted from confocal images obtained in vitro and from previous histopathologic studies. This study shows that fiber optic confocal reflectance microscopy can be used to visualize the morphology of cervical epithelium in vivo.

© 2003 Optical Society of America

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References

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Acad. Radiol. (1)

T. Collier, A. Lacy, A. Malpica, M. Follen, and 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]

Am. J. Obstet. Gynecol. (1)

R. Drezek, T. Collier, C. Brookner, A. Malpica, R. Lotan, and R. Richards-Kortum, �??Laser scanning confocal microscopy of cervical tissue before and after application of acetic acid,�?? Am. J. Obstet. Gynecol. 182, 1135-1139 (2000).
[CrossRef] [PubMed]

Appl. Opt. (4)

EUROGIN 2003 (1)

W. McLaren, J. Tan, and M. Quinn, �??Detection of cervical neoplasia using non-invasive fibre-optic confocal microscopy,�?? 5th international multidisciplinary congress EUROGIN 2003, 213-217, Paris, France (April 13-16, 2003).

IEEE J. Sel. Top. Quantum Electron. (1)

T. Collier, D. Arifler, A. Malpica, M. Follen, and R. Richards-Kortum, �??Determination of epithelial tissue scattering coefficient using confocal microscopy,�?? IEEE J. Sel. Top. Quantum Electron. 9, 307-313 (2003).
[CrossRef]

IEEE trans. biomed. eng. (1)

K. B. Sung, C. Liang, M. Descour, T. Collier, M. Follen, and R. Richards-Kortum, �??Fiber optic confocal reflectance microscope with miniature objective for in vivo imaging of human tissues,�?? IEEE trans. biomed. eng. 49, 1168-1172 (2002).
[CrossRef] [PubMed]

J. Am. Acad. Dermatol. (1)

M. K. Silverman, F. M. Golomb, A. W. Kopf, C. M. Grin-Jorgensen, K. A. Vossaert, J. P. Doyle, and M. J. Levenstein, �??Verification of a formula for determination of preexcision surgical margins from fixed-tissue melanoma specimens,�?? J. Am. Acad. Dermatol. 27, 214�??219 (1992).
[CrossRef] [PubMed]

J. Biomed. Opt. (1)

D. Arifler, M. Guillaud, A. Carraro, A. Malpica, M. Follen, and R. Richards-Kortum, �??Light scattering from normal and dysplastic cervical cells at different epithelial depths: finite-difference time-domain modeling with a perfectly matched layer boundary condition,�?? J. Biomed. Opt. 8, 484-494 (2003).
[CrossRef] [PubMed]

J. Cosmet. Sci. (1)

P. Corcuff, C. Chaussepied, G. Madry, and C. Hadjur, �??Skin optics revisited by in vivo confocal microscopy: melanin and sun exposure,�?? J. Cosmet. Sci. 52, 91-102 (2001).
[PubMed]

J. Microsc. (1)

K. B. Sung, C. Liang, M. Descour, M. Follen, A. Malpica, and R. Richards-Kortum, �??Near real time in vivo fiber optic confocal microscopy: sub-cellular structure resolved,�?? J. Microsc. 207, 137-145 (2002).
[CrossRef] [PubMed]

J. Opt. Soc. Am. A (1)

Laryngoscope (1)

W. M. White, M. Rajadhyaksha, S. Gonzalez, R. L. Fabian, and R. R. Anderson, �??Noninvasive imaging of human oral mucosa in vivo by confocal reflectance microscopy,�?? Laryngoscope 109, 1709-1717 (1999).
[CrossRef] [PubMed]

Opt. Lett. (1)

Physiological Measurement (1)

D. C. Walker, B. J. Brown, A. D. Blackett, J. Tidy and R. H. Smallwood, �??A study of the morphological parameters of cervical squamous epithelium,�?? Physiological Measurement 24, 121-135 (2003).
[CrossRef] [PubMed]

Other (1)

Mauna Kea Technologies, Paris, France. <a href="http://www.maunakeatech.com">http://www.maunakeatech.com</a>

Supplementary Material (2)

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Figures (8)

Fig. 1.
Fig. 1.

Schematic diagram of the fiber optic confocal reflectance microscope.

Fig. 2.
Fig. 2.

Photograph of the imaging probe, showing the fiber optic bundle (F), Teflon tube (T) and the brass tube (B).

Fig. 3.
Fig. 3.

The optical sectioning capability of the system is illustrated by imaging a planar reflective grating at different positions along the optical axis. Average intensity over a 16×36 µm area from thirty images of the grating is normalized and plotted. The full width at half of the maximum magnitude is 3 µm.

Fig. 4.
Fig. 4.

(2.41 MB) Video of normal cervical epithelium. The depth of the image plane increases from the superficial layer of epithelial cells to the basement membrane. The field of view is 250×240 µm. (7.03 MB version).

Fig. 5.
Fig. 5.

(a) and (c) Confocal images of normal cervical epithelium taken from two patients. (e) Confocal image of hyperkeratotic and atrophic cervical epithelium from another patient. Photographs in (b), (d), and (f) show the corresponding H&E sections of biopsy specimens taken from the imaged tissue sites. The scale bars are 20 µm in the confocal images and 50 µm in the H&E sections.

Fig. 6.
Fig. 6.

(a) Confocal image of cervical epithelium diagnosed as CIN 1. (c) Confocal image of CIN 3 cervical epithelium from another patient. Photographs in (b) and (d) show the corresponding H&E sections of biopsy specimens taken from the imaged tissue sites. The scale bars are 20 µm in the confocal images and 50 µm in the H&E sections.

Fig. 7.
Fig. 7.

Scatter plot of measured average nuclear area and nuclear-cytoplasmic ratio (N/C) from this in vivo study (open triangles) and a previous in vitro study reported by Collier et al. (black diamonds) [1]

Fig. 8.
Fig. 8.

Comparison of (a) average nuclear diameter (µm) and (b) nuclear-cytoplasmic ratio (N/C) between this study and previous studies by Collier [1] and Walker [15].

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