Abstract

A significant challenge for in vivo imaging is to remove movement artifacts. These movements (typically due to either respiration and cardiac-related movement or surface chemical response) are normally limited to the axial direction, and hence features move in and out of the focal plane. This presents a real problem for high-resolution optically sectioned imaging techniques such as confocal and multiphoton microscopy. To overcome this we have developed an actively locked focus-tracking system based around a deformable membrane mirror. This has a significant advantage over more conventional focus-tracking techniques where the microscope objective is dithered, since the active element is not in direct, or indirect, contact with the sample. To examine the operational limits and to demonstrate possible applications for this form of focus locking, sample oscillation and movement are simulated for two different biological applications. We were able to track focus over a 400μm range (limited by the range of the piezomounted objective) with a rms precision on the focal depth of 0.31μm±0.05μm.

© 2008 Optical Society of America

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References

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  1. A. R. Carter, G. M. King, T. A. Ulrich, W. Halsey, D. Alchenberger, and T. T. Perkins, Appl. Opt. 46, 421 (2007).
    [CrossRef] [PubMed]
  2. L. Sherman, J. Y. Ye, O. Albert, and T. B. Norris, J. Microsc. 206, 65 (2002).
    [CrossRef] [PubMed]
  3. M. J. Booth, M. A. A. Neil, R. Juskaitis, T. Wilson, Proc. Natl. Acad. Sci. USA 99, 5788 (2002).
    [CrossRef] [PubMed]
  4. P. N. Marsh, D. Burns, and G. Girkin, Opt. Express 11, 1123 (2003).
    [CrossRef] [PubMed]
  5. A. J. Wright, D. Burns, B. A. Patterson, S. P. Poland, G. Valentine, and J. M. Girkin, Microsc. Res. Tech. 67, 36 (2005).
    [CrossRef] [PubMed]
  6. B. Potsaid, Y. Bellouard, and J. T. Wen, Opt. Express 13, 6504 (2005).
    [CrossRef] [PubMed]
  7. D. P. Biss, D. Sumorok, S. A. Burns, R. H. Webb, Y. Zhou, T. G. Bifano, D. Cǒté, I. Veilleux, P. Zamiri, and C. P. Lin, Opt. Lett. 32, 659 (2007).
    [CrossRef] [PubMed]
  8. A. J. Wright, B. A. Patterson, S. P. Poland, J. M. Girkin, G. M. Gibson, and M. J. Padgett, Opt. Express 14, 222 (2006).
    [CrossRef] [PubMed]
  9. G. Smith, Institute of Biomedical and Life Sciences, Glasgow University, Scotland (personal communica-tion, 2007).
  10. S. P. Poland, D. Hughes, C. Longbottom, and J. M. Girkin are preparing a paper to be called "A protocol to monitor acid etching in dental tissue using a confocal microscope."

2007 (2)

2006 (1)

2005 (2)

B. Potsaid, Y. Bellouard, and J. T. Wen, Opt. Express 13, 6504 (2005).
[CrossRef] [PubMed]

A. J. Wright, D. Burns, B. A. Patterson, S. P. Poland, G. Valentine, and J. M. Girkin, Microsc. Res. Tech. 67, 36 (2005).
[CrossRef] [PubMed]

2003 (1)

2002 (2)

L. Sherman, J. Y. Ye, O. Albert, and T. B. Norris, J. Microsc. 206, 65 (2002).
[CrossRef] [PubMed]

M. J. Booth, M. A. A. Neil, R. Juskaitis, T. Wilson, Proc. Natl. Acad. Sci. USA 99, 5788 (2002).
[CrossRef] [PubMed]

Appl. Opt. (1)

J. Microsc. (1)

L. Sherman, J. Y. Ye, O. Albert, and T. B. Norris, J. Microsc. 206, 65 (2002).
[CrossRef] [PubMed]

Microsc. Res. Tech. (1)

A. J. Wright, D. Burns, B. A. Patterson, S. P. Poland, G. Valentine, and J. M. Girkin, Microsc. Res. Tech. 67, 36 (2005).
[CrossRef] [PubMed]

Opt. Express (3)

Opt. Lett. (1)

Proc. Natl. Acad. Sci. USA (1)

M. J. Booth, M. A. A. Neil, R. Juskaitis, T. Wilson, Proc. Natl. Acad. Sci. USA 99, 5788 (2002).
[CrossRef] [PubMed]

Other (2)

G. Smith, Institute of Biomedical and Life Sciences, Glasgow University, Scotland (personal communica-tion, 2007).

S. P. Poland, D. Hughes, C. Longbottom, and J. M. Girkin are preparing a paper to be called "A protocol to monitor acid etching in dental tissue using a confocal microscope."

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Figures (4)

Fig. 1
Fig. 1

Optical configuration incorporating the DMM. Light exiting the scan head system is directed onto the DMM before subsequently being focused onto the sample by the objective via a polarizing beam splitter and 1 4 waveplate. Light reflected from the sample follows its original path back to the scan head where it passes through a pinhole and is detected by a PMT.

Fig. 2
Fig. 2

(a) Simulated axial oscillation of a small rat arteriole without focus lock. The graph depicts the axial movement to the sample along with the signal response measured on the PMT when no feedback loop, or focus lock, is applied. (b) Simulated axial oscillation of a small rat arteriole with focus lock ( rms = 0.48 μ m ) . The graph depicts the axial movement of the sample, as well as the axial correction and signal response when a focus lock is applied.

Fig. 3
Fig. 3

(a) Reflection confocal image showing an etched enamel surface ( 141 μm × 94 μ m ) . Dental enamel is mainly composed of hydroxyapatite crystals that form prismatic interlinking structures. (b) Enamel surface layer depth against etching time using citric acid (pH 2.0). This represents an extremely vigorous acid-etching treatment that is representative of drinks with the highest acidicity.

Fig. 4
Fig. 4

Focus-locking ability of the system when simulating dental etching at a rate of (a) 0.6 μ m s and (b) 21.3 μ m s .

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