Abstract

This paper presents the use and characterization of an electrically focus tunable lens to perform axial scanning in a confocal microscope. Lateral and axial resolution are characterized over a >250 µm axial scan range. Confocal microscopy using optical axial scanning is demonstrated in epithelial tissue and compared to traditional stage scanning. By enabling rapid axial scanning, minimizing motion artifacts, and reducing mechanical complexity, this technique has potential to enhance in vivo three-dimensional imaging in confocal endomicroscopy.

© 2014 Optical Society of America

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References

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  1. J. M. Jabbour, M. A. Saldua, J. N. Bixler, and K. C. Maitland, “Confocal endomicroscopy: instrumentation and medical applications,” Ann. Biomed. Eng. 40(2), 378–397 (2012).
    [Crossref] [PubMed]
  2. J. M. Jabbour, S. Cheng, B. H. Malik, R. Cuenca, J. A. Jo, J. Wright, Y. S. Cheng, and K. C. Maitland, “Fluorescence lifetime imaging and reflectance confocal microscopy for multiscale imaging of oral precancer,” J. Biomed. Opt. 18(4), 046012 (2013).
    [Crossref] [PubMed]
  3. A. A. Tanbakuchi, A. R. Rouse, J. A. Udovich, K. D. Hatch, and A. F. Gmitro, “Clinical confocal microlaparoscope for real-time in vivo optical biopsies,” J. Biomed. Opt. 14(4), 044030 (2009).
    [Crossref] [PubMed]
  4. 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(10), 1168–1172 (2002).
    [Crossref] [PubMed]
  5. C. Olsovsky, R. Shelton, O. Carrasco-Zevallos, B. E. Applegate, and K. C. Maitland, “Chromatic confocal microscopy for multi-depth imaging of epithelial tissue,” Biomed. Opt. Express 4(5), 732–740 (2013).
    [Crossref] [PubMed]
  6. P. M. Lane, R. P. Elliott, and C. E. MacAulay, “Confocal microendoscopy with chromatic sectioning,” Proc. SPIE 4959, 23–26 (2003).
    [Crossref]
  7. A. J. Thompson, C. Paterson, M. A. Neil, C. Dunsby, and P. M. French, “Adaptive phase compensation for ultracompact laser scanning endomicroscopy,” Opt. Lett. 36(9), 1707–1709 (2011).
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  9. S. Kumar, D. Wilding, M. B. Sikkel, A. R. Lyon, K. T. MacLeod, and C. Dunsby, “High-speed 2D and 3D fluorescence microscopy of cardiac myocytes,” Opt. Express 19(15), 13839–13847 (2011).
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  10. Q. Wu, S. Guo, Y. Ma, F. Gao, C. Yang, M. Yang, X. Yu, X. Zhang, R. A. Rupp, and J. Xu, “Optical refocusing three-dimensional wide-field fluorescence lifetime imaging microscopy,” Opt. Express 20(2), 960–965 (2012).
    [Crossref] [PubMed]
  11. L. Yang, A. Mac Raighne, E. M. McCabe, L. A. Dunbar, and T. Scharf, “Confocal microscopy using variable-focal-length microlenses and an optical fiber bundle,” Appl. Opt. 44(28), 5928–5936 (2005).
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  12. B. F. Grewe, F. F. Voigt, M. van ’t Hoff, and F. Helmchen, “Fast two-layer two-photon imaging of neuronal cell populations using an electrically tunable lens,” Biomed. Opt. Express 2(7), 2035–2046 (2011).
    [Crossref] [PubMed]
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  14. K.-S. Lee, P. Vanderwall, and J. P. Rolland, “Two-photon microscopy with dynamic focusing objective using a liquid lens,” Proc. SPIE 7569, 756923 (2010).
    [Crossref]
  15. F. O. Fahrbach, F. F. Voigt, B. Schmid, F. Helmchen, and J. Huisken, “Rapid 3D light-sheet microscopy with a tunable lens,” Opt. Express 21(18), 21010–21026 (2013).
    [Crossref] [PubMed]
  16. H. S. Chen and Y. H. Lin, “An endoscopic system adopting a liquid crystal lens with an electrically tunable depth-of-field,” Opt. Express 21(15), 18079–18088 (2013).
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  17. N. Savidis, G. Peyman, N. Peyghambarian, and J. Schwiegerling, “Nonmechanical zoom system through pressure-controlled tunable fluidic lenses,” Appl. Opt. 52(12), 2858–2865 (2013).
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  18. D. G. Ouzounov, D. R. Rivera, W. W. Webb, J. Bentley, and C. Xu, “Miniature varifocal objective lens for endomicroscopy,” Opt. Lett. 38(16), 3103–3106 (2013).
    [Crossref] [PubMed]
  19. R. A. Drezek, T. Collier, C. K. Brookner, A. Malpica, R. Lotan, R. R. Richards-Kortum, and M. Follen, “Laser scanning confocal microscopy of cervical tissue before and after application of acetic acid,” Am. J. Obstet. Gynecol. 182(5), 1135–1139 (2000).
    [Crossref] [PubMed]

2013 (6)

2012 (2)

Q. Wu, S. Guo, Y. Ma, F. Gao, C. Yang, M. Yang, X. Yu, X. Zhang, R. A. Rupp, and J. Xu, “Optical refocusing three-dimensional wide-field fluorescence lifetime imaging microscopy,” Opt. Express 20(2), 960–965 (2012).
[Crossref] [PubMed]

J. M. Jabbour, M. A. Saldua, J. N. Bixler, and K. C. Maitland, “Confocal endomicroscopy: instrumentation and medical applications,” Ann. Biomed. Eng. 40(2), 378–397 (2012).
[Crossref] [PubMed]

2011 (3)

2010 (1)

K.-S. Lee, P. Vanderwall, and J. P. Rolland, “Two-photon microscopy with dynamic focusing objective using a liquid lens,” Proc. SPIE 7569, 756923 (2010).
[Crossref]

2009 (1)

A. A. Tanbakuchi, A. R. Rouse, J. A. Udovich, K. D. Hatch, and A. F. Gmitro, “Clinical confocal microlaparoscope for real-time in vivo optical biopsies,” J. Biomed. Opt. 14(4), 044030 (2009).
[Crossref] [PubMed]

2007 (1)

2005 (1)

2003 (1)

P. M. Lane, R. P. Elliott, and C. E. MacAulay, “Confocal microendoscopy with chromatic sectioning,” Proc. SPIE 4959, 23–26 (2003).
[Crossref]

2002 (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(10), 1168–1172 (2002).
[Crossref] [PubMed]

2000 (1)

R. A. Drezek, T. Collier, C. K. Brookner, A. Malpica, R. Lotan, R. R. Richards-Kortum, and M. Follen, “Laser scanning confocal microscopy of cervical tissue before and after application of acetic acid,” Am. J. Obstet. Gynecol. 182(5), 1135–1139 (2000).
[Crossref] [PubMed]

Applegate, B. E.

Bentley, J.

Bixler, J. N.

J. M. Jabbour, M. A. Saldua, J. N. Bixler, and K. C. Maitland, “Confocal endomicroscopy: instrumentation and medical applications,” Ann. Biomed. Eng. 40(2), 378–397 (2012).
[Crossref] [PubMed]

Booth, M. J.

Botcherby, E. J.

Brookner, C. K.

R. A. Drezek, T. Collier, C. K. Brookner, A. Malpica, R. Lotan, R. R. Richards-Kortum, and M. Follen, “Laser scanning confocal microscopy of cervical tissue before and after application of acetic acid,” Am. J. Obstet. Gynecol. 182(5), 1135–1139 (2000).
[Crossref] [PubMed]

Carrasco-Zevallos, O.

Chen, H. S.

Cheng, S.

J. M. Jabbour, S. Cheng, B. H. Malik, R. Cuenca, J. A. Jo, J. Wright, Y. S. Cheng, and K. C. Maitland, “Fluorescence lifetime imaging and reflectance confocal microscopy for multiscale imaging of oral precancer,” J. Biomed. Opt. 18(4), 046012 (2013).
[Crossref] [PubMed]

Cheng, Y. S.

J. M. Jabbour, S. Cheng, B. H. Malik, R. Cuenca, J. A. Jo, J. Wright, Y. S. Cheng, and K. C. Maitland, “Fluorescence lifetime imaging and reflectance confocal microscopy for multiscale imaging of oral precancer,” J. Biomed. Opt. 18(4), 046012 (2013).
[Crossref] [PubMed]

Collier, T.

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(10), 1168–1172 (2002).
[Crossref] [PubMed]

R. A. Drezek, T. Collier, C. K. Brookner, A. Malpica, R. Lotan, R. R. Richards-Kortum, and M. Follen, “Laser scanning confocal microscopy of cervical tissue before and after application of acetic acid,” Am. J. Obstet. Gynecol. 182(5), 1135–1139 (2000).
[Crossref] [PubMed]

Cuenca, R.

J. M. Jabbour, S. Cheng, B. H. Malik, R. Cuenca, J. A. Jo, J. Wright, Y. S. Cheng, and K. C. Maitland, “Fluorescence lifetime imaging and reflectance confocal microscopy for multiscale imaging of oral precancer,” J. Biomed. Opt. 18(4), 046012 (2013).
[Crossref] [PubMed]

Descour, M.

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(10), 1168–1172 (2002).
[Crossref] [PubMed]

Drezek, R. A.

R. A. Drezek, T. Collier, C. K. Brookner, A. Malpica, R. Lotan, R. R. Richards-Kortum, and M. Follen, “Laser scanning confocal microscopy of cervical tissue before and after application of acetic acid,” Am. J. Obstet. Gynecol. 182(5), 1135–1139 (2000).
[Crossref] [PubMed]

Dunbar, L. A.

Dunsby, C.

Elliott, R. P.

P. M. Lane, R. P. Elliott, and C. E. MacAulay, “Confocal microendoscopy with chromatic sectioning,” Proc. SPIE 4959, 23–26 (2003).
[Crossref]

Fahrbach, F. O.

Follen, M.

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(10), 1168–1172 (2002).
[Crossref] [PubMed]

R. A. Drezek, T. Collier, C. K. Brookner, A. Malpica, R. Lotan, R. R. Richards-Kortum, and M. Follen, “Laser scanning confocal microscopy of cervical tissue before and after application of acetic acid,” Am. J. Obstet. Gynecol. 182(5), 1135–1139 (2000).
[Crossref] [PubMed]

French, P. M.

Gao, F.

Gmitro, A. F.

A. A. Tanbakuchi, A. R. Rouse, J. A. Udovich, K. D. Hatch, and A. F. Gmitro, “Clinical confocal microlaparoscope for real-time in vivo optical biopsies,” J. Biomed. Opt. 14(4), 044030 (2009).
[Crossref] [PubMed]

Grewe, B. F.

Guo, S.

Hatch, K. D.

A. A. Tanbakuchi, A. R. Rouse, J. A. Udovich, K. D. Hatch, and A. F. Gmitro, “Clinical confocal microlaparoscope for real-time in vivo optical biopsies,” J. Biomed. Opt. 14(4), 044030 (2009).
[Crossref] [PubMed]

Helmchen, F.

Huisken, J.

Jabbour, J. M.

J. M. Jabbour, S. Cheng, B. H. Malik, R. Cuenca, J. A. Jo, J. Wright, Y. S. Cheng, and K. C. Maitland, “Fluorescence lifetime imaging and reflectance confocal microscopy for multiscale imaging of oral precancer,” J. Biomed. Opt. 18(4), 046012 (2013).
[Crossref] [PubMed]

J. M. Jabbour, M. A. Saldua, J. N. Bixler, and K. C. Maitland, “Confocal endomicroscopy: instrumentation and medical applications,” Ann. Biomed. Eng. 40(2), 378–397 (2012).
[Crossref] [PubMed]

Jo, J. A.

J. M. Jabbour, S. Cheng, B. H. Malik, R. Cuenca, J. A. Jo, J. Wright, Y. S. Cheng, and K. C. Maitland, “Fluorescence lifetime imaging and reflectance confocal microscopy for multiscale imaging of oral precancer,” J. Biomed. Opt. 18(4), 046012 (2013).
[Crossref] [PubMed]

Juskaitis, R.

Kumar, S.

Lane, P. M.

P. M. Lane, R. P. Elliott, and C. E. MacAulay, “Confocal microendoscopy with chromatic sectioning,” Proc. SPIE 4959, 23–26 (2003).
[Crossref]

Lee, K.-S.

K.-S. Lee, P. Vanderwall, and J. P. Rolland, “Two-photon microscopy with dynamic focusing objective using a liquid lens,” Proc. SPIE 7569, 756923 (2010).
[Crossref]

Liang, C.

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(10), 1168–1172 (2002).
[Crossref] [PubMed]

Lin, Y. H.

Lotan, R.

R. A. Drezek, T. Collier, C. K. Brookner, A. Malpica, R. Lotan, R. R. Richards-Kortum, and M. Follen, “Laser scanning confocal microscopy of cervical tissue before and after application of acetic acid,” Am. J. Obstet. Gynecol. 182(5), 1135–1139 (2000).
[Crossref] [PubMed]

Lyon, A. R.

Ma, Y.

Mac Raighne, A.

MacAulay, C. E.

P. M. Lane, R. P. Elliott, and C. E. MacAulay, “Confocal microendoscopy with chromatic sectioning,” Proc. SPIE 4959, 23–26 (2003).
[Crossref]

MacLeod, K. T.

Maitland, K. C.

C. Olsovsky, R. Shelton, O. Carrasco-Zevallos, B. E. Applegate, and K. C. Maitland, “Chromatic confocal microscopy for multi-depth imaging of epithelial tissue,” Biomed. Opt. Express 4(5), 732–740 (2013).
[Crossref] [PubMed]

J. M. Jabbour, S. Cheng, B. H. Malik, R. Cuenca, J. A. Jo, J. Wright, Y. S. Cheng, and K. C. Maitland, “Fluorescence lifetime imaging and reflectance confocal microscopy for multiscale imaging of oral precancer,” J. Biomed. Opt. 18(4), 046012 (2013).
[Crossref] [PubMed]

J. M. Jabbour, M. A. Saldua, J. N. Bixler, and K. C. Maitland, “Confocal endomicroscopy: instrumentation and medical applications,” Ann. Biomed. Eng. 40(2), 378–397 (2012).
[Crossref] [PubMed]

Malik, B. H.

J. M. Jabbour, S. Cheng, B. H. Malik, R. Cuenca, J. A. Jo, J. Wright, Y. S. Cheng, and K. C. Maitland, “Fluorescence lifetime imaging and reflectance confocal microscopy for multiscale imaging of oral precancer,” J. Biomed. Opt. 18(4), 046012 (2013).
[Crossref] [PubMed]

Malpica, A.

R. A. Drezek, T. Collier, C. K. Brookner, A. Malpica, R. Lotan, R. R. Richards-Kortum, and M. Follen, “Laser scanning confocal microscopy of cervical tissue before and after application of acetic acid,” Am. J. Obstet. Gynecol. 182(5), 1135–1139 (2000).
[Crossref] [PubMed]

McCabe, E. M.

Neil, M. A.

Olsovsky, C.

Ouzounov, D. G.

Paterson, C.

Peyghambarian, N.

Peyman, G.

Richards-Kortum, R.

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(10), 1168–1172 (2002).
[Crossref] [PubMed]

Richards-Kortum, R. R.

R. A. Drezek, T. Collier, C. K. Brookner, A. Malpica, R. Lotan, R. R. Richards-Kortum, and M. Follen, “Laser scanning confocal microscopy of cervical tissue before and after application of acetic acid,” Am. J. Obstet. Gynecol. 182(5), 1135–1139 (2000).
[Crossref] [PubMed]

Rivera, D. R.

Rolland, J. P.

K.-S. Lee, P. Vanderwall, and J. P. Rolland, “Two-photon microscopy with dynamic focusing objective using a liquid lens,” Proc. SPIE 7569, 756923 (2010).
[Crossref]

Rouse, A. R.

A. A. Tanbakuchi, A. R. Rouse, J. A. Udovich, K. D. Hatch, and A. F. Gmitro, “Clinical confocal microlaparoscope for real-time in vivo optical biopsies,” J. Biomed. Opt. 14(4), 044030 (2009).
[Crossref] [PubMed]

Rupp, R. A.

Saldua, M. A.

J. M. Jabbour, M. A. Saldua, J. N. Bixler, and K. C. Maitland, “Confocal endomicroscopy: instrumentation and medical applications,” Ann. Biomed. Eng. 40(2), 378–397 (2012).
[Crossref] [PubMed]

Savidis, N.

Scharf, T.

Schmid, B.

Schwiegerling, J.

Shelton, R.

Sikkel, M. B.

Sung, K. B.

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(10), 1168–1172 (2002).
[Crossref] [PubMed]

Tanbakuchi, A. A.

A. A. Tanbakuchi, A. R. Rouse, J. A. Udovich, K. D. Hatch, and A. F. Gmitro, “Clinical confocal microlaparoscope for real-time in vivo optical biopsies,” J. Biomed. Opt. 14(4), 044030 (2009).
[Crossref] [PubMed]

Thompson, A. J.

Udovich, J. A.

A. A. Tanbakuchi, A. R. Rouse, J. A. Udovich, K. D. Hatch, and A. F. Gmitro, “Clinical confocal microlaparoscope for real-time in vivo optical biopsies,” J. Biomed. Opt. 14(4), 044030 (2009).
[Crossref] [PubMed]

van ’t Hoff, M.

Vanderwall, P.

K.-S. Lee, P. Vanderwall, and J. P. Rolland, “Two-photon microscopy with dynamic focusing objective using a liquid lens,” Proc. SPIE 7569, 756923 (2010).
[Crossref]

Voigt, F. F.

Webb, W. W.

Wilding, D.

Wilson, T.

Wright, J.

J. M. Jabbour, S. Cheng, B. H. Malik, R. Cuenca, J. A. Jo, J. Wright, Y. S. Cheng, and K. C. Maitland, “Fluorescence lifetime imaging and reflectance confocal microscopy for multiscale imaging of oral precancer,” J. Biomed. Opt. 18(4), 046012 (2013).
[Crossref] [PubMed]

Wu, Q.

Xu, C.

Xu, J.

Yang, C.

Yang, L.

Yang, M.

Yu, X.

Zhang, X.

Am. J. Obstet. Gynecol. (1)

R. A. Drezek, T. Collier, C. K. Brookner, A. Malpica, R. Lotan, R. R. Richards-Kortum, and M. Follen, “Laser scanning confocal microscopy of cervical tissue before and after application of acetic acid,” Am. J. Obstet. Gynecol. 182(5), 1135–1139 (2000).
[Crossref] [PubMed]

Ann. Biomed. Eng. (1)

J. M. Jabbour, M. A. Saldua, J. N. Bixler, and K. C. Maitland, “Confocal endomicroscopy: instrumentation and medical applications,” Ann. Biomed. Eng. 40(2), 378–397 (2012).
[Crossref] [PubMed]

Appl. Opt. (2)

Biomed. Opt. Express (2)

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(10), 1168–1172 (2002).
[Crossref] [PubMed]

J. Biomed. Opt. (2)

J. M. Jabbour, S. Cheng, B. H. Malik, R. Cuenca, J. A. Jo, J. Wright, Y. S. Cheng, and K. C. Maitland, “Fluorescence lifetime imaging and reflectance confocal microscopy for multiscale imaging of oral precancer,” J. Biomed. Opt. 18(4), 046012 (2013).
[Crossref] [PubMed]

A. A. Tanbakuchi, A. R. Rouse, J. A. Udovich, K. D. Hatch, and A. F. Gmitro, “Clinical confocal microlaparoscope for real-time in vivo optical biopsies,” J. Biomed. Opt. 14(4), 044030 (2009).
[Crossref] [PubMed]

Opt. Express (4)

Opt. Lett. (3)

Proc. SPIE (2)

P. M. Lane, R. P. Elliott, and C. E. MacAulay, “Confocal microendoscopy with chromatic sectioning,” Proc. SPIE 4959, 23–26 (2003).
[Crossref]

K.-S. Lee, P. Vanderwall, and J. P. Rolland, “Two-photon microscopy with dynamic focusing objective using a liquid lens,” Proc. SPIE 7569, 756923 (2010).
[Crossref]

Other (1)

Optotune Application Note, “Optical focusing in microscopy with Optotune’s focus tunable lens EL-10-30”, (Optotune, 2013), http://www.optotune.com/images/products/Optotune application note EL-10-30 for microscopy.pdf .

Supplementary Material (3)

» Media 1: AVI (459 KB)     
» Media 2: AVI (1450 KB)     
» Media 3: AVI (5572 KB)     

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Figures (5)

Fig. 1
Fig. 1 Zemax design showing the illumination arm of the confocal system (Media 1) including the ETL positioned adjacent to the scanning mirrors (SM). With negative ETL focal lengths, the imaging planes lie further from the lens or deeper in the sample (a), and with positive ETL focal lengths, the imaging planes lie closer to the lens and sample surface (b). The insets show the change in the numerical aperture and focal position at the sample space as ETL changes focal length from (a) f = −127 mm to (b) f = + 44.3 mm. L1: Simplified objective lens, L2/L3: beam expander.

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Fig. 2
Fig. 2 Measured axial position of the focal plane of the confocal microscope relative to the nominal focal position of the objective lens as a function of ETL control current. 50 mA of ETL current corresponds to no focusing power of the ETL (f = ∞) and the focus in sample space positioned at the working distance of the objective lens. Increasing axial position indicates increased depth in the sample. 150 mA ETL control current corresponds to an axial focal position above the nominal focal position by 215 µm. 0 mA ETL control current corresponds to an axial focal position below the nominal focal position by 40 µm.

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Fig. 3
Fig. 3 Theoretical and measured FWHM lateral and axial resolutions as a function of focal position.

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Fig. 4
Fig. 4 Confocal images and video (Media 2) from stage scan (a,b,e,f,i,j) and ETL scan (c,d,g,h,k,l) through oral mucosa ex vivo taken from 72 µm (a-d), 106 µm (e-h), and 154 µm (i-l) below the surface of the tissue, corresponding to axial focal positions of z = −143, −109, and −61 µm, respectively. Zoom in images (b,c,f,g,j,k) clearly show nuclei as bright spots surrounded by dark cytoplasmic media. Corresponding histology image is shown (m) cut vertically through the epithelium. Arrows in confocal and histology images point to papillary regions of connective tissue between rete ridges. Scale bars: 100 µm in (a) for (d,e,h,i,l), 25 µm in (b) for (c,f,g,j,k), and 100 µm in (m).

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Fig. 5
Fig. 5 In vivo confocal video (Media 3) acquired at 0.2 Hz ETL axial scan through 177 µm of normal skin, and images from approximately 92 µm (a) and 161 µm (b) below the surface of the tissue. Scale bar: 100 µm.

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Equations (1)

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z=−0.0091 i 2 −0.297i+38

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