Abstract

Spectrally encoded confocal microscopy (SECM) is a technique that allows confocal microscopy to be performed through the confines of a narrow diameter optical fiber probe. We present a novel scheme for performing SECM in which a rapid wavelength swept source is used. The system allows large field of view images to be acquired at rates up to 30 frames/second. Images of resolution targets and tissue specimens acquired ex vivo demonstrate high lateral (1.4 μm) and axial (6 μm) resolution. Imaging of human skin was performed in vivo at depths of up to 350 μm, allowing cellular and sub-cellular details to be visualized in real time.

© 2005 Optical Society of America

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References

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Appl. Opt.

Endoscopy

M. Sakashita, H. Inoue, H. Kashida, J. Tanaka, J.Y. Cho, H. Satodate, E. Hidaka, T. Yoshida, N. Fukami, Y. Tamegai, A. Shiokawa, and S. Kudo, "Virtual histology of colorectal lesions using laserscanning confocal microscopy," Endoscopy 35, 1033-1038 (2003).
[CrossRef] [PubMed]

Gastroenterology

R. Kiesslich, J. Burg, M. Vieth, J. Gnaendiger, M. Enders, P. Delaney, A. Polglase, W. McLaren, D. Janell, S. Thomas, B. Nafe, P.R. Galle, and M.F. Neurath, "Confocal laser endoscopy for diagnosing intraepithelial neoplasias and colorectal cancer in vivo," Gastroenterology 127, 706-713 (2004).
[CrossRef] [PubMed]

J. Invest. Dermatol.

L.D. Swindle, S.G. Thomas, M. Freeman, and P.M. Delaney, "View of normal human skin in vivo as observed using fluorescent fiber-optic confocal microscopic imaging," J. Invest. Dermatol. 121, 706- 712 (2003).
[CrossRef] [PubMed]

M. Rajadhyaksha, M. Grossman, D. Esterowitz, R.H. Webb, and R.R. Anderson, "In vivo confocal scanning laser microscopy of human skin: Melanin provides strong contrast," J. Invest. Dermatol. 104, 946-952 (1995).
[CrossRef] [PubMed]

J. Opt. Soc. Am. A

Opt. Express

Opt. Lett.

Optics Commun.

J. Knittel, L. Schnieder, G. Buess, B. Messerschmidt, and T. Possner, "Endoscope-compatible confocal microscope using a gradient index-lens system," Optics Commun. 188, 267-273 (2001).
[CrossRef]

Other

T. Wilson, Confocal microscopy (Academic Press, San Diego, Calif., 1990).

T.R. Corle and G.S. Kino, Confocal scanning confocal microscopy and related imaging systems (Academic Press, San Diego, Calif., 1996)

Supplementary Material (1)

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Figures (5)

Fig. 1.
Fig. 1.

SECM system schematic. The rapidly wavelength-swept laser source includes a semiconductor optical amplifier (SOA) producing broadband spontaneous emission, which is coupled to the grating (Gr1)-based polygon filter through an optical circulator (C1). Two 90/10 fiber-optic couplers bring filtered light to the imaging and the synchronization arms. In the imaging arm, light remitted from the sample is directed to a photodiode (D1) via another circulator (C2). The electrical output from D1 is digitized to form the image. In the synchronization arm, a small portion of the light is coupled to a static filter, comprising a grating (Gr2) and mirror (M). A single wavelength of light is transmitted by the static filter to a detector (D2), via an additional circulator (C3). The detected signal from the synchronization arm serves as a trigger for the acquisition of each spectral line. Polarization controllers (OOO) are installed before polarization sensitive components (SOA and gratings) to maximize throughput.

Fig. 2.
Fig. 2.

Imaging arm configuration. A galvanometer mounted mirror (M) provides slow-axis scanning of collimated light into and out of the plane of the Fig. Fast-axis scanning results as wavelength-swept light diffracts from the high groove density transmission grating (Gr). A 0.9 NA water immersion microscope objective produces a focused spot on the sample. A telecentric telescope images the slow scan pivot onto the back pupil of the objective.

Fig. 3.
Fig. 3.

US Air Force resolution target. The smallest elements on this target (Group 7 Element 6) are 2.2 microns wide. The field of view is 440 microns × 400 microns. The wavelength encoded axis is the horizontal axis.

Fig. 4.
Fig. 4.

Skeletal muscle. (a, b) Images of freshly excised tissue acquired at 8 fps with SECM. In both images, the large muscle fibers are well delineated. In (a), a weak solution of acetic acid was applied to enhance the nucleic contrast. Nuclei appear as bright elongated structures in the periphery of the muscle fibers (solid arrows), which is consistent with the H&E stained histological appearance of muscle fibers and nuclei (c). SECM resolves muscular striations [rectangle in (b)] as evidenced by the enlarged portion of the SECM image (d). The striations are comparable to those seen in the histology section [rectangle in (c)], enlarged in (e) for better visualization. The scale bar is 100 microns.

Fig. 5.
Fig. 5.

SECM images of human skin, obtained from the ventral forearm in vivo and in real time. This “en face” image of the skin was obtained at 8 frames per second and shows the characteristic reticular pattern of keratinocytes within the epidermis. The scale bar is 100 microns. (Movie: 2.45 kb)

Equations (4)

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FOV δx = Δ λ δλ ,
FOV = 2 f tan ( Δ θ 2 ) ,
Δ θ = m cos θ L . Λ Δ λ ,
δ λ G = λ 0 Λ mD

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