Abstract

We present a novel chromatic confocal microscope capable of volumetric reflectance imaging of microstructure in non-transparent tissue. Our design takes advantage of the chromatic aberration of aspheric lenses that are otherwise well corrected. Strong chromatic aberration, generated by multiple aspheres, longitudinally disperses supercontinuum light onto the sample. The backscattered light detected with a spectrometer is therefore wavelength encoded and each spectrum corresponds to a line image. This approach obviates the need for traditional axial mechanical scanning techniques that are difficult to implement for endoscopy and susceptible to motion artifact. A wavelength range of 590-775 nm yielded a >150 µm imaging depth with ~3 µm axial resolution. The system was further demonstrated by capturing volumetric images of buccal mucosa. We believe these represent the first microstructural images in non-transparent biological tissue using chromatic confocal microscopy that exhibit long imaging depth while maintaining acceptable resolution for resolving cell morphology. Miniaturization of this optical system could bring enhanced speed and accuracy to endomicroscopic in vivo volumetric imaging of epithelial tissue.

© 2013 OSA

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References

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2012 (1)

M. Vaishakh, “Optical sectioning in reciprocal fiber-optic based chromatic confocal microscope,” Optik (Stuttg.)123(16), 1450–1452 (2012).
[CrossRef]

2010 (2)

2008 (2)

I. Veilleux, J. A. Spencer, D. P. Biss, D. Cote, and C. P. Lin, “In vivo cell tracking with video rate multimodality laser scanning microscopy,” IEEE J. Quantum Electron.14(1), 10–18 (2008).
[CrossRef]

J. Garzón, T. Gharbi, and J. Meneses, “Real time determination of the optical thickness and topography of tissues by chromatic confocal microscopy,” J. Opt. A, Pure Appl. Opt.10(10), 104028 (2008).
[CrossRef]

2005 (2)

2004 (2)

2003 (1)

P. M. Lane, R. P. Elliott, and C. E. MacAulay, “Confocal microendoscopy with chromatic sectioning,” Proc. SPIE4959, 23–26 (2003).
[CrossRef]

2000 (1)

1999 (1)

1998 (1)

1996 (1)

1994 (2)

H. J. Tiziani and H. M. Uhde, “Three-dimensional image sensing by chromatic confocal microscopy,” Appl. Opt.33(10), 1838–1843 (1994).
[CrossRef] [PubMed]

M. Maly and A. Boyde, “Real-time stereoscopic confocal reflection microscopy using objective lenses with linear longitudinal chromatic dispersion,” Scanning16, 187–192 (1994).

1992 (1)

M. A. Browne, O. Akinyemi, and A. Boyde, “Confocal surface profiling utilizing chromatic aberration,” Scanning14(3), 145–153 (1992).
[CrossRef]

1987 (1)

Akinyemi, O.

M. A. Browne, O. Akinyemi, and A. Boyde, “Confocal surface profiling utilizing chromatic aberration,” Scanning14(3), 145–153 (1992).
[CrossRef]

Biss, D. P.

I. Veilleux, J. A. Spencer, D. P. Biss, D. Cote, and C. P. Lin, “In vivo cell tracking with video rate multimodality laser scanning microscopy,” IEEE J. Quantum Electron.14(1), 10–18 (2008).
[CrossRef]

Boudoux, C.

Bouma, B. E.

Boyde, A.

M. Maly and A. Boyde, “Real-time stereoscopic confocal reflection microscopy using objective lenses with linear longitudinal chromatic dispersion,” Scanning16, 187–192 (1994).

M. A. Browne, O. Akinyemi, and A. Boyde, “Confocal surface profiling utilizing chromatic aberration,” Scanning14(3), 145–153 (1992).
[CrossRef]

Browne, M. A.

M. A. Browne, O. Akinyemi, and A. Boyde, “Confocal surface profiling utilizing chromatic aberration,” Scanning14(3), 145–153 (1992).
[CrossRef]

Carlini, A. R.

Collier, T.

Cote, D.

I. Veilleux, J. A. Spencer, D. P. Biss, D. Cote, and C. P. Lin, “In vivo cell tracking with video rate multimodality laser scanning microscopy,” IEEE J. Quantum Electron.14(1), 10–18 (2008).
[CrossRef]

de Pradier, B.

Donaldson, L.

Dunn, A. K.

Elliott, R. P.

P. M. Lane, R. P. Elliott, and C. E. MacAulay, “Confocal microendoscopy with chromatic sectioning,” Proc. SPIE4959, 23–26 (2003).
[CrossRef]

Follen, M.

Garzón, J.

J. Garzón, T. Gharbi, and J. Meneses, “Real time determination of the optical thickness and topography of tissues by chromatic confocal microscopy,” J. Opt. A, Pure Appl. Opt.10(10), 104028 (2008).
[CrossRef]

Gharbi, T.

J. Garzón, T. Gharbi, and J. Meneses, “Real time determination of the optical thickness and topography of tissues by chromatic confocal microscopy,” J. Opt. A, Pure Appl. Opt.10(10), 104028 (2008).
[CrossRef]

Gmitro, A. F.

Gopalan, V.

Hopkins, M. F.

Iftimia, N. V.

Lane, P. M.

P. M. Lane, R. P. Elliott, and C. E. MacAulay, “Confocal microendoscopy with chromatic sectioning,” Proc. SPIE4959, 23–26 (2003).
[CrossRef]

Li, H. F.

Li, P.

Lin, C. P.

I. Veilleux, J. A. Spencer, D. P. Biss, D. Cote, and C. P. Lin, “In vivo cell tracking with video rate multimodality laser scanning microscopy,” IEEE J. Quantum Electron.14(1), 10–18 (2008).
[CrossRef]

Liu, Z. W.

MacAulay, C. E.

P. M. Lane, R. P. Elliott, and C. E. MacAulay, “Confocal microendoscopy with chromatic sectioning,” Proc. SPIE4959, 23–26 (2003).
[CrossRef]

Malpica, A.

Maly, M.

M. Maly and A. Boyde, “Real-time stereoscopic confocal reflection microscopy using objective lenses with linear longitudinal chromatic dispersion,” Scanning16, 187–192 (1994).

Meneses, J.

J. Garzón, T. Gharbi, and J. Meneses, “Real time determination of the optical thickness and topography of tissues by chromatic confocal microscopy,” J. Opt. A, Pure Appl. Opt.10(10), 104028 (2008).
[CrossRef]

Miks, A.

J. Novak and A. Miks, “Hyperchromats with linear dependence of longitudinal chromatic aberration on wavelength,” Optik (Stuttg.)116(4), 165–168 (2005).
[CrossRef]

Montigny, E. D.

Morneau, D.

Novak, J.

J. Novak and A. Miks, “Hyperchromats with linear dependence of longitudinal chromatic aberration on wavelength,” Optik (Stuttg.)116(4), 165–168 (2005).
[CrossRef]

Oh, W. Y.

Richards-Kortum, R.

Rouse, A. R.

Ruprecht, A. K.

Sabharwal, Y. S.

Shen, P.

Shi, K. B.

Shishkov, M.

Smithpeter, C.

Smithpeter, C. L.

Spencer, J. A.

I. Veilleux, J. A. Spencer, D. P. Biss, D. Cote, and C. P. Lin, “In vivo cell tracking with video rate multimodality laser scanning microscopy,” IEEE J. Quantum Electron.14(1), 10–18 (2008).
[CrossRef]

Strupler, M.

Sung, K. B.

Tearney, G. J.

Tiziani, H. J.

Uhde, H. M.

Vaishakh, M.

M. Vaishakh, “Optical sectioning in reciprocal fiber-optic based chromatic confocal microscope,” Optik (Stuttg.)123(16), 1450–1452 (2012).
[CrossRef]

Veilleux, I.

I. Veilleux, J. A. Spencer, D. P. Biss, D. Cote, and C. P. Lin, “In vivo cell tracking with video rate multimodality laser scanning microscopy,” IEEE J. Quantum Electron.14(1), 10–18 (2008).
[CrossRef]

Welch, A. J.

White, W. M.

Wiesendanger, T. F.

Wilson, T.

Xu, Q. A.

Yang, C. A.

Yin, S. Z.

Yun, S. H.

Appl. Opt. (4)

IEEE J. Quantum Electron. (1)

I. Veilleux, J. A. Spencer, D. P. Biss, D. Cote, and C. P. Lin, “In vivo cell tracking with video rate multimodality laser scanning microscopy,” IEEE J. Quantum Electron.14(1), 10–18 (2008).
[CrossRef]

J. Opt. A, Pure Appl. Opt. (1)

J. Garzón, T. Gharbi, and J. Meneses, “Real time determination of the optical thickness and topography of tissues by chromatic confocal microscopy,” J. Opt. A, Pure Appl. Opt.10(10), 104028 (2008).
[CrossRef]

Opt. Express (4)

Opt. Lett. (3)

Optik (Stuttg.) (2)

J. Novak and A. Miks, “Hyperchromats with linear dependence of longitudinal chromatic aberration on wavelength,” Optik (Stuttg.)116(4), 165–168 (2005).
[CrossRef]

M. Vaishakh, “Optical sectioning in reciprocal fiber-optic based chromatic confocal microscope,” Optik (Stuttg.)123(16), 1450–1452 (2012).
[CrossRef]

Proc. SPIE (1)

P. M. Lane, R. P. Elliott, and C. E. MacAulay, “Confocal microendoscopy with chromatic sectioning,” Proc. SPIE4959, 23–26 (2003).
[CrossRef]

Scanning (2)

M. Maly and A. Boyde, “Real-time stereoscopic confocal reflection microscopy using objective lenses with linear longitudinal chromatic dispersion,” Scanning16, 187–192 (1994).

M. A. Browne, O. Akinyemi, and A. Boyde, “Confocal surface profiling utilizing chromatic aberration,” Scanning14(3), 145–153 (1992).
[CrossRef]

Other (2)

ANSI, “American National Standard for Safe Use of Lasers” (Laser Institute of America, 2007).

J. B. Pawley, ed., Handbook of Biological Confocal Microscopy, 3rd ed. (Springer, 2006), p. 985.

Supplementary Material (2)

» Media 1: AVI (6466 KB)     
» Media 2: AVI (9060 KB)     

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Figures (4)

Fig. 1
Fig. 1

(a) Schematic of the chromatic confocal microscope. PCF: photonic crystal fiber supercontinuum source; CL: collimating lens; BS: beam splitter; L1, L2, L3, and L4: aspheric lenses; OL: objective lens; L5: detection lens; F1: detection fiber. Inset shows a generic chromatic focal shift for any beam focus within the lens system. (b) Reference spectrum of the supercontinuum source measured by the spectrometer through the optical system.

Fig. 2
Fig. 2

(a) The chromatic shift shows the relative focus for each wavelength. The total shift is 157 µm over a 185 nm range. (b) Four axial PSFs are shown at 10, 50, 90, and 130 µm depth at wavelengths of 595, 635, 675, and 725 nm, respectively. The peak of each PSF corresponds to a single wavelength which is converted to relative depth using a calibration curve. The FWHM from left to right is 3.2, 2.9, 3.1, and 3.1 µm.

Fig. 3
Fig. 3

(a) CCM video (Media 1) of porcine buccal mucosa compared with (b) an image of the same tissue from the Lucid Vivascope confocal microscope.

Fig. 4
Fig. 4

(a) CCM video (Media 2) of the basal layer of porcine buccal mucosa. (b) The same cell morphology can be seen in an image taken with the Lucid Vivascope confocal microscope.

Equations (8)

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I(u)= 0 v p | 0 1 P(ρ)exp(ju ρ 2 ) J 0 (vρ)ρdρ | 2 vdv
u= 2π λ N A 2 n z
v= 2π λ NAr
PSF(λ)=oPSF(λ)sPSF(λ)
z(λ)=2213 λ 2 +3850λ1498.045
SNR= MPE p x E QFR ( MPE p x E QFR+ n d 2 + n r 2 ) 1/2
MPE=1.1A ( p x t p ) 0.25 [ J ]
SNR=MPE QFR p x E n r

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