Abstract

We present a fast-updating Lissajous image reconstruction methodology that uses an increased image frame rate beyond the pattern repeat rate generally used in conventional Lissajous image reconstruction methods. The fast display rate provides increased dynamic information and reduced motion blur, as compared to conventional Lissajous reconstruction, at the cost of single-frame pixel density. Importantly, this method does not discard any information from the conventional Lissajous image reconstruction, and frames from the complete Lissajous pattern can be displayed simultaneously. We present the theoretical background for this image reconstruction methodology along with images and video taken using the algorithm in a custom-built miniaturized multiphoton microscopy system.

© 2011 Optical Society of America

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References

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  1. D. L. Dickensheets and G. S. Kino, “Micromachined scanning confocal optical microscope,” Opt. Lett. 21, 764–766(1996).
    [CrossRef] [PubMed]
  2. F. Helmchen, M. S. Fee, D. W. Tank, and W. Denk, “A miniature head-mounted two-photon microscope: high-resolution brain imaging in freely moving animals,” Neuron 31, 903–912(2001).
    [CrossRef] [PubMed]
  3. B. A. Flusberg, J. C. Jung, E. D. Cocker, E. P. Anderson, and M. J. Schnitzer, “In vivo brain imaging using a portable 3.9 gram two-photon fluorescence microendoscope,” Opt. Lett. 30, 2272–2274 (2005).
    [CrossRef] [PubMed]
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    [CrossRef] [PubMed]
  5. T.-M. Liu, M.-C. Chan, I. H. Chen, S.-H. Chia, and C.-K. Sun, “Miniaturized multiphoton microscope with a 24 Hz frame-rate,” Opt. Express 16, 10501–10506 (2008).
    [CrossRef] [PubMed]
  6. J. A. Lissajous, “Note sur un nouveau moyen de mettre en évidence le mouvement vibratoire des corps,” C. R. Hebd. Seances Acad. Sci. 41, 93–95 (1855).
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    [CrossRef] [PubMed]
  8. C. L. Hoy, W. Piyawattabanetha, H. Ra, O. Solgaard, and A. Ben-Yakar, “Optical design and imaging performance testing of a 9.6 mm diameter femtosecond laser microsurgery probe,” Opt. Express 19, 10536–10552 (2011).
    [PubMed]
  9. C. Landis, “Determinants of the critical flicker-fusion threshold,” Physiol. Rev. 34, 259–286 (1954).
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2010 (1)

J. T. C. Liu, M. J. Mandella, N. O. Loewke, H. Haeberle, H. Ra, W. Piyawattanametha, O. Solgaard, G. S. Kino, and C. H. Contag, “Micromirror-scanned dual-axis confocal microscope utilizing a gradient-index relay lens for image guidance during brain surgery,” J. Biomed. Opt. 15, 026029 (2010).
[CrossRef] [PubMed]

2008 (2)

2005 (1)

2001 (1)

F. Helmchen, M. S. Fee, D. W. Tank, and W. Denk, “A miniature head-mounted two-photon microscope: high-resolution brain imaging in freely moving animals,” Neuron 31, 903–912(2001).
[CrossRef] [PubMed]

1996 (1)

1954 (1)

C. Landis, “Determinants of the critical flicker-fusion threshold,” Physiol. Rev. 34, 259–286 (1954).
[PubMed]

1855 (1)

J. A. Lissajous, “Note sur un nouveau moyen de mettre en évidence le mouvement vibratoire des corps,” C. R. Hebd. Seances Acad. Sci. 41, 93–95 (1855).

Anderson, E. P.

Ben-Yakar, A.

Chan, M.-C.

Chen, I. H.

Chen, P.

Chia, S.-H.

Cocker, E. D.

Contag, C. H.

J. T. C. Liu, M. J. Mandella, N. O. Loewke, H. Haeberle, H. Ra, W. Piyawattanametha, O. Solgaard, G. S. Kino, and C. H. Contag, “Micromirror-scanned dual-axis confocal microscope utilizing a gradient-index relay lens for image guidance during brain surgery,” J. Biomed. Opt. 15, 026029 (2010).
[CrossRef] [PubMed]

Denk, W.

F. Helmchen, M. S. Fee, D. W. Tank, and W. Denk, “A miniature head-mounted two-photon microscope: high-resolution brain imaging in freely moving animals,” Neuron 31, 903–912(2001).
[CrossRef] [PubMed]

Dickensheets, D. L.

Durr, N. J.

Fee, M. S.

F. Helmchen, M. S. Fee, D. W. Tank, and W. Denk, “A miniature head-mounted two-photon microscope: high-resolution brain imaging in freely moving animals,” Neuron 31, 903–912(2001).
[CrossRef] [PubMed]

Flusberg, B. A.

Haeberle, H.

J. T. C. Liu, M. J. Mandella, N. O. Loewke, H. Haeberle, H. Ra, W. Piyawattanametha, O. Solgaard, G. S. Kino, and C. H. Contag, “Micromirror-scanned dual-axis confocal microscope utilizing a gradient-index relay lens for image guidance during brain surgery,” J. Biomed. Opt. 15, 026029 (2010).
[CrossRef] [PubMed]

Helmchen, F.

F. Helmchen, M. S. Fee, D. W. Tank, and W. Denk, “A miniature head-mounted two-photon microscope: high-resolution brain imaging in freely moving animals,” Neuron 31, 903–912(2001).
[CrossRef] [PubMed]

Hoy, C. L.

Jung, J. C.

Kino, G. S.

J. T. C. Liu, M. J. Mandella, N. O. Loewke, H. Haeberle, H. Ra, W. Piyawattanametha, O. Solgaard, G. S. Kino, and C. H. Contag, “Micromirror-scanned dual-axis confocal microscope utilizing a gradient-index relay lens for image guidance during brain surgery,” J. Biomed. Opt. 15, 026029 (2010).
[CrossRef] [PubMed]

D. L. Dickensheets and G. S. Kino, “Micromachined scanning confocal optical microscope,” Opt. Lett. 21, 764–766(1996).
[CrossRef] [PubMed]

Landis, C.

C. Landis, “Determinants of the critical flicker-fusion threshold,” Physiol. Rev. 34, 259–286 (1954).
[PubMed]

Lissajous, J. A.

J. A. Lissajous, “Note sur un nouveau moyen de mettre en évidence le mouvement vibratoire des corps,” C. R. Hebd. Seances Acad. Sci. 41, 93–95 (1855).

Liu, J. T. C.

J. T. C. Liu, M. J. Mandella, N. O. Loewke, H. Haeberle, H. Ra, W. Piyawattanametha, O. Solgaard, G. S. Kino, and C. H. Contag, “Micromirror-scanned dual-axis confocal microscope utilizing a gradient-index relay lens for image guidance during brain surgery,” J. Biomed. Opt. 15, 026029 (2010).
[CrossRef] [PubMed]

Liu, T.-M.

Loewke, N. O.

J. T. C. Liu, M. J. Mandella, N. O. Loewke, H. Haeberle, H. Ra, W. Piyawattanametha, O. Solgaard, G. S. Kino, and C. H. Contag, “Micromirror-scanned dual-axis confocal microscope utilizing a gradient-index relay lens for image guidance during brain surgery,” J. Biomed. Opt. 15, 026029 (2010).
[CrossRef] [PubMed]

Mandella, M. J.

J. T. C. Liu, M. J. Mandella, N. O. Loewke, H. Haeberle, H. Ra, W. Piyawattanametha, O. Solgaard, G. S. Kino, and C. H. Contag, “Micromirror-scanned dual-axis confocal microscope utilizing a gradient-index relay lens for image guidance during brain surgery,” J. Biomed. Opt. 15, 026029 (2010).
[CrossRef] [PubMed]

Piyawattabanetha, W.

Piyawattanametha, W.

J. T. C. Liu, M. J. Mandella, N. O. Loewke, H. Haeberle, H. Ra, W. Piyawattanametha, O. Solgaard, G. S. Kino, and C. H. Contag, “Micromirror-scanned dual-axis confocal microscope utilizing a gradient-index relay lens for image guidance during brain surgery,” J. Biomed. Opt. 15, 026029 (2010).
[CrossRef] [PubMed]

C. L. Hoy, N. J. Durr, P. Chen, W. Piyawattanametha, H. Ra, O. Solgaard, and A. Ben-Yakar, “Miniaturized probe for femtosecond laser microsurgery and two-photon imaging,” Opt. Express 16, 9996–10005 (2008).
[CrossRef] [PubMed]

Ra, H.

Schnitzer, M. J.

Solgaard, O.

Sun, C.-K.

Tank, D. W.

F. Helmchen, M. S. Fee, D. W. Tank, and W. Denk, “A miniature head-mounted two-photon microscope: high-resolution brain imaging in freely moving animals,” Neuron 31, 903–912(2001).
[CrossRef] [PubMed]

C. R. Hebd. Seances Acad. Sci. (1)

J. A. Lissajous, “Note sur un nouveau moyen de mettre en évidence le mouvement vibratoire des corps,” C. R. Hebd. Seances Acad. Sci. 41, 93–95 (1855).

J. Biomed. Opt. (1)

J. T. C. Liu, M. J. Mandella, N. O. Loewke, H. Haeberle, H. Ra, W. Piyawattanametha, O. Solgaard, G. S. Kino, and C. H. Contag, “Micromirror-scanned dual-axis confocal microscope utilizing a gradient-index relay lens for image guidance during brain surgery,” J. Biomed. Opt. 15, 026029 (2010).
[CrossRef] [PubMed]

Neuron (1)

F. Helmchen, M. S. Fee, D. W. Tank, and W. Denk, “A miniature head-mounted two-photon microscope: high-resolution brain imaging in freely moving animals,” Neuron 31, 903–912(2001).
[CrossRef] [PubMed]

Opt. Express (3)

Opt. Lett. (2)

Physiol. Rev. (1)

C. Landis, “Determinants of the critical flicker-fusion threshold,” Physiol. Rev. 34, 259–286 (1954).
[PubMed]

Supplementary Material (3)

» Media 1: MOV (1830 KB)     
» Media 2: MOV (1829 KB)     
» Media 3: MOV (216 KB)     

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Figures (6)

Fig. 1
Fig. 1

Conventional Lissajous reconstructions for various pattern repeat rates and pixel sizes. The color map represents the number of times a given pixel is sampled before one cycle of the Lissajous pattern completes. For the conventional case where f F = f P , this represents the number of times a pixel is sampled in each frame. White pixels denote unsampled regions of the image. Data generated by MATLAB simulation.

Fig. 2
Fig. 2

Sampling statistics for conventionally reconstructed Lissajous images with resonant frequencies at f x = 2260 Hz and f y = 980 Hz . (a) Percentage of pixels in each frame sampled for a given frame rate. (b) Percentage of pixels sampled ten times or more in each frame at a given frame rate. The solid, dotted, and dashed curves represent 512 × 512 , 256 × 256 , and 128 × 128 pixel images, respectively. Data generated by MATLAB simulation.

Fig. 3
Fig. 3

Sampling statistics for Lissajous images reconstructed using our fast-updating method with scanning frequencies of f x = 2260 Hz and f y = 979 Hz and a 512 × 512 image size. (a) Percentage of pixels in each frame sampled for a given frame rate. (b) Percentage of pixels sampled ten times or more in each frame at a given frame rate. The gray circles denote the values for each frame at a given frame rate, while the solid line displays the average for each frame rate. Data generated by MATLAB simulation.

Fig. 4
Fig. 4

Flow chart illustrating our fast-updating Lissajous image reconstruction algorithm. User inputs are shaded gray and located on the left. User-selectable options are denoted by diamond process blocks. The MEMS driving signal and the data collection are synchronized by triggering from the same master clock in the software and by linking the data acquisition cards physically with a RTSI cable.

Fig. 5
Fig. 5

Two-photon fluorescence images of 1 μm fluorescent beads deposited on glass. (a) Raw frame using the fast-updating image reconstruction algorithm updating at 7 Hz . (b) Frame taken using the moving average algorithm, averaging seven raw frames and updating at 7 Hz . (c) Frame taken using the conventional Lissajous image reconstruction methodology, updating at the pattern repeat rate of 1 Hz . Contrast has been increased on the moving average image to match that of the unaveraged images. Scale bars are 10 μm .

Fig. 6
Fig. 6

Images from real-time videos (Media 1, Media 2, Media 3) of 1 μm fluorescent beads dried on a glass microscope slide and translated manually at varying speeds. The videos demonstrate the effect of each image reconstruction algorithm. (a) Our fast- updating algorithm updating at 7 Hz . (b) Moving average algorithm updating at 7 Hz displaying a seven frame moving average. (c) Conventional 1 Hz image reconstruction. Note that the conventional algorithm video contains motion artifacts not present in the fast-updating algorithm video. The image reconstruction size was reduced here to 128 pixels × 128 pixels to demonstrate the denser sampling that can be achieved with small FOV. Contrast has been adjusted on the moving average image to match that of the unaveraged images. Scale bars are 5 μm .

Equations (6)

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x ( t ) = 1 2 X [ sin ( 2 π f x t + ϕ x ) + 1 ] ,
y ( t ) = 1 2 Y [ sin ( 2 π f y t + ϕ y ) + 1 ] .
f P = f x n x = f y n y ,
x ˙ ( t ) = π f x X [ cos ( 2 π f x t + ϕ x ) ] ,
y ˙ ( t ) = π f y Y [ cos ( 2 π f y t + ϕ y ) ] .
v max = π P f x 2 + f y 2 ,

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