Abstract

There has been an increasing interest for observing fast biological phenomena such as cell movements in circulations and action potentials. The laser scanning confocal microscopy offers a good spatial resolution and optical sectioning ability to observe various in vivo animal models. We developed a high speed laser scanning confocal microscope capable of acquiring 512 by 512 pixel images at 200 fps (frames per second). We have incorporated a fast rotating polygonal scanning mirror with 128 facets for the X-axis scanner. In order to increase the throughput of the Y-axis scanner, we applied a bi-directional scanning method for vertical scanning. This made it possible to scan along the Y-axis two times during each scanner motion cycle. For the image acquisition, we used a custom photomultiplier tube amplifier with a broad frequency band. In addition, custom imaging software was written for the new microscope. In order to verify the acquisition speed of the developed confocal microscope, a resolution target moving at a series of constant speeds and a sedated mouse with slight movements due to heartbeats were observed. By comparing successive frames, the frame acquisition speeds were calculated.

© 2013 OSA

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References

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  1. B. James, “Foundations of confocal scanned imaging in light microscopy,” in Handbook of Biological Confocal Microscopy, Shinya Inoue, ed. (Plenum Press, 1990).
  2. M. Minsky, “Microscopy apparatus,” U.S. Patent 3013467 (1961).
  3. C. M.  Brown, R. B.  Dalal, B.  Hebert, M. A.  Digman, A. R.  Horwitz, E.  Gratton, “Raster image correlation spectroscopy (RICS) for measuring fast protein dynamics and concentrations with a commercial laser scanning confocal microscope,” J. Microsc. 229(1), 78–91 (2008).
    [CrossRef] [PubMed]
  4. P. D. J.  Moens, E.  Gratton, I. L.  Salvemini, “Fluorescence Correlation Spectroscopy, Raster Image Correlation Spectroscopy, and Number and Brightness on a Commercial Confocal Laser Scanning Microscope with Analog Detectors (Nikon C1),” Microsc Res Techniq. 74, 377–388 (2011).
  5. S. H.  Choi, W. H.  Kim, Y. J.  Lee, W. J.  Lee, J. D.  Yang, J. W.  Shim, J. W.  Kim, H.  Lee, “Visualization of Epidermis and Dermal Cells in ex vivo Human Skin Using the Confocal and Two-photon Microscopy,” j. Opt. Soc. Korea (South) 15, 61–67 (2011).
  6. J. A.  Spencer, D. P.  Biss, D.  Côté, C. P.  Lin, “In vivo Cell Tracking With Video Rate Multimodality Laser Scanning Microscopy,” IEEE J. Sel. Top. Quant 14, 10–18 (2008).
  7. S. M.  Choi, W. H.  Kim, D.  Côté, C. W.  Park, H.  Lee, “Blood cell assisted in vivo Particle Image Velocimetry using the confocal laser scanning microscope,” Opt. Express 19(5), 4357–4368 (2011).
    [CrossRef] [PubMed]
  8. T.  Tanaami, S.  Otsuki, N.  Tomosada, Y.  Kosugi, M.  Shimizu, H.  Ishida, “High-speed 1-frame/ms scanning confocal microscope with a microlens and Nipkow disks,” Appl. Opt. 41, 4704–4708 (2002).
  9. E.  Wang, C. M.  Babbey, K. W.  Dunn, “Performance comparison between the high-speed Yokogawa spinning disc confocal system and single-point scanning confocal systems,” J. Microsc. 218(Pt 2), 148–159 (2005).
    [CrossRef] [PubMed]
  10. M. C.  Adams, W. C.  Salmon, S. L.  Gupton, C. S.  Cohan, T.  Wittmann, N.  Prigozhina, C. M.  Waterman-Storer, “A high-speed multispectral spinning-disk confocal microscope system for fluorescent speckle microscopy of living cells,” Methods 29(1), 29–41 (2003).
    [CrossRef] [PubMed]

2011 (3)

P. D. J.  Moens, E.  Gratton, I. L.  Salvemini, “Fluorescence Correlation Spectroscopy, Raster Image Correlation Spectroscopy, and Number and Brightness on a Commercial Confocal Laser Scanning Microscope with Analog Detectors (Nikon C1),” Microsc Res Techniq. 74, 377–388 (2011).

S. H.  Choi, W. H.  Kim, Y. J.  Lee, W. J.  Lee, J. D.  Yang, J. W.  Shim, J. W.  Kim, H.  Lee, “Visualization of Epidermis and Dermal Cells in ex vivo Human Skin Using the Confocal and Two-photon Microscopy,” j. Opt. Soc. Korea (South) 15, 61–67 (2011).

S. M.  Choi, W. H.  Kim, D.  Côté, C. W.  Park, H.  Lee, “Blood cell assisted in vivo Particle Image Velocimetry using the confocal laser scanning microscope,” Opt. Express 19(5), 4357–4368 (2011).
[CrossRef] [PubMed]

2008 (2)

J. A.  Spencer, D. P.  Biss, D.  Côté, C. P.  Lin, “In vivo Cell Tracking With Video Rate Multimodality Laser Scanning Microscopy,” IEEE J. Sel. Top. Quant 14, 10–18 (2008).

C. M.  Brown, R. B.  Dalal, B.  Hebert, M. A.  Digman, A. R.  Horwitz, E.  Gratton, “Raster image correlation spectroscopy (RICS) for measuring fast protein dynamics and concentrations with a commercial laser scanning confocal microscope,” J. Microsc. 229(1), 78–91 (2008).
[CrossRef] [PubMed]

2005 (1)

E.  Wang, C. M.  Babbey, K. W.  Dunn, “Performance comparison between the high-speed Yokogawa spinning disc confocal system and single-point scanning confocal systems,” J. Microsc. 218(Pt 2), 148–159 (2005).
[CrossRef] [PubMed]

2003 (1)

M. C.  Adams, W. C.  Salmon, S. L.  Gupton, C. S.  Cohan, T.  Wittmann, N.  Prigozhina, C. M.  Waterman-Storer, “A high-speed multispectral spinning-disk confocal microscope system for fluorescent speckle microscopy of living cells,” Methods 29(1), 29–41 (2003).
[CrossRef] [PubMed]

2002 (1)

Adams, M. C.

M. C.  Adams, W. C.  Salmon, S. L.  Gupton, C. S.  Cohan, T.  Wittmann, N.  Prigozhina, C. M.  Waterman-Storer, “A high-speed multispectral spinning-disk confocal microscope system for fluorescent speckle microscopy of living cells,” Methods 29(1), 29–41 (2003).
[CrossRef] [PubMed]

Babbey, C. M.

E.  Wang, C. M.  Babbey, K. W.  Dunn, “Performance comparison between the high-speed Yokogawa spinning disc confocal system and single-point scanning confocal systems,” J. Microsc. 218(Pt 2), 148–159 (2005).
[CrossRef] [PubMed]

Biss, D. P.

J. A.  Spencer, D. P.  Biss, D.  Côté, C. P.  Lin, “In vivo Cell Tracking With Video Rate Multimodality Laser Scanning Microscopy,” IEEE J. Sel. Top. Quant 14, 10–18 (2008).

Brown, C. M.

C. M.  Brown, R. B.  Dalal, B.  Hebert, M. A.  Digman, A. R.  Horwitz, E.  Gratton, “Raster image correlation spectroscopy (RICS) for measuring fast protein dynamics and concentrations with a commercial laser scanning confocal microscope,” J. Microsc. 229(1), 78–91 (2008).
[CrossRef] [PubMed]

Choi, S. H.

S. H.  Choi, W. H.  Kim, Y. J.  Lee, W. J.  Lee, J. D.  Yang, J. W.  Shim, J. W.  Kim, H.  Lee, “Visualization of Epidermis and Dermal Cells in ex vivo Human Skin Using the Confocal and Two-photon Microscopy,” j. Opt. Soc. Korea (South) 15, 61–67 (2011).

Choi, S. M.

Cohan, C. S.

M. C.  Adams, W. C.  Salmon, S. L.  Gupton, C. S.  Cohan, T.  Wittmann, N.  Prigozhina, C. M.  Waterman-Storer, “A high-speed multispectral spinning-disk confocal microscope system for fluorescent speckle microscopy of living cells,” Methods 29(1), 29–41 (2003).
[CrossRef] [PubMed]

Côté, D.

S. M.  Choi, W. H.  Kim, D.  Côté, C. W.  Park, H.  Lee, “Blood cell assisted in vivo Particle Image Velocimetry using the confocal laser scanning microscope,” Opt. Express 19(5), 4357–4368 (2011).
[CrossRef] [PubMed]

J. A.  Spencer, D. P.  Biss, D.  Côté, C. P.  Lin, “In vivo Cell Tracking With Video Rate Multimodality Laser Scanning Microscopy,” IEEE J. Sel. Top. Quant 14, 10–18 (2008).

Dalal, R. B.

C. M.  Brown, R. B.  Dalal, B.  Hebert, M. A.  Digman, A. R.  Horwitz, E.  Gratton, “Raster image correlation spectroscopy (RICS) for measuring fast protein dynamics and concentrations with a commercial laser scanning confocal microscope,” J. Microsc. 229(1), 78–91 (2008).
[CrossRef] [PubMed]

Digman, M. A.

C. M.  Brown, R. B.  Dalal, B.  Hebert, M. A.  Digman, A. R.  Horwitz, E.  Gratton, “Raster image correlation spectroscopy (RICS) for measuring fast protein dynamics and concentrations with a commercial laser scanning confocal microscope,” J. Microsc. 229(1), 78–91 (2008).
[CrossRef] [PubMed]

Dunn, K. W.

E.  Wang, C. M.  Babbey, K. W.  Dunn, “Performance comparison between the high-speed Yokogawa spinning disc confocal system and single-point scanning confocal systems,” J. Microsc. 218(Pt 2), 148–159 (2005).
[CrossRef] [PubMed]

Gratton, E.

P. D. J.  Moens, E.  Gratton, I. L.  Salvemini, “Fluorescence Correlation Spectroscopy, Raster Image Correlation Spectroscopy, and Number and Brightness on a Commercial Confocal Laser Scanning Microscope with Analog Detectors (Nikon C1),” Microsc Res Techniq. 74, 377–388 (2011).

C. M.  Brown, R. B.  Dalal, B.  Hebert, M. A.  Digman, A. R.  Horwitz, E.  Gratton, “Raster image correlation spectroscopy (RICS) for measuring fast protein dynamics and concentrations with a commercial laser scanning confocal microscope,” J. Microsc. 229(1), 78–91 (2008).
[CrossRef] [PubMed]

Gupton, S. L.

M. C.  Adams, W. C.  Salmon, S. L.  Gupton, C. S.  Cohan, T.  Wittmann, N.  Prigozhina, C. M.  Waterman-Storer, “A high-speed multispectral spinning-disk confocal microscope system for fluorescent speckle microscopy of living cells,” Methods 29(1), 29–41 (2003).
[CrossRef] [PubMed]

Hebert, B.

C. M.  Brown, R. B.  Dalal, B.  Hebert, M. A.  Digman, A. R.  Horwitz, E.  Gratton, “Raster image correlation spectroscopy (RICS) for measuring fast protein dynamics and concentrations with a commercial laser scanning confocal microscope,” J. Microsc. 229(1), 78–91 (2008).
[CrossRef] [PubMed]

Horwitz, A. R.

C. M.  Brown, R. B.  Dalal, B.  Hebert, M. A.  Digman, A. R.  Horwitz, E.  Gratton, “Raster image correlation spectroscopy (RICS) for measuring fast protein dynamics and concentrations with a commercial laser scanning confocal microscope,” J. Microsc. 229(1), 78–91 (2008).
[CrossRef] [PubMed]

Ishida, H.

Kim, J. W.

S. H.  Choi, W. H.  Kim, Y. J.  Lee, W. J.  Lee, J. D.  Yang, J. W.  Shim, J. W.  Kim, H.  Lee, “Visualization of Epidermis and Dermal Cells in ex vivo Human Skin Using the Confocal and Two-photon Microscopy,” j. Opt. Soc. Korea (South) 15, 61–67 (2011).

Kim, W. H.

S. H.  Choi, W. H.  Kim, Y. J.  Lee, W. J.  Lee, J. D.  Yang, J. W.  Shim, J. W.  Kim, H.  Lee, “Visualization of Epidermis and Dermal Cells in ex vivo Human Skin Using the Confocal and Two-photon Microscopy,” j. Opt. Soc. Korea (South) 15, 61–67 (2011).

S. M.  Choi, W. H.  Kim, D.  Côté, C. W.  Park, H.  Lee, “Blood cell assisted in vivo Particle Image Velocimetry using the confocal laser scanning microscope,” Opt. Express 19(5), 4357–4368 (2011).
[CrossRef] [PubMed]

Kosugi, Y.

Lee, H.

S. H.  Choi, W. H.  Kim, Y. J.  Lee, W. J.  Lee, J. D.  Yang, J. W.  Shim, J. W.  Kim, H.  Lee, “Visualization of Epidermis and Dermal Cells in ex vivo Human Skin Using the Confocal and Two-photon Microscopy,” j. Opt. Soc. Korea (South) 15, 61–67 (2011).

S. M.  Choi, W. H.  Kim, D.  Côté, C. W.  Park, H.  Lee, “Blood cell assisted in vivo Particle Image Velocimetry using the confocal laser scanning microscope,” Opt. Express 19(5), 4357–4368 (2011).
[CrossRef] [PubMed]

Lee, W. J.

S. H.  Choi, W. H.  Kim, Y. J.  Lee, W. J.  Lee, J. D.  Yang, J. W.  Shim, J. W.  Kim, H.  Lee, “Visualization of Epidermis and Dermal Cells in ex vivo Human Skin Using the Confocal and Two-photon Microscopy,” j. Opt. Soc. Korea (South) 15, 61–67 (2011).

Lee, Y. J.

S. H.  Choi, W. H.  Kim, Y. J.  Lee, W. J.  Lee, J. D.  Yang, J. W.  Shim, J. W.  Kim, H.  Lee, “Visualization of Epidermis and Dermal Cells in ex vivo Human Skin Using the Confocal and Two-photon Microscopy,” j. Opt. Soc. Korea (South) 15, 61–67 (2011).

Lin, C. P.

J. A.  Spencer, D. P.  Biss, D.  Côté, C. P.  Lin, “In vivo Cell Tracking With Video Rate Multimodality Laser Scanning Microscopy,” IEEE J. Sel. Top. Quant 14, 10–18 (2008).

Moens, P. D. J.

P. D. J.  Moens, E.  Gratton, I. L.  Salvemini, “Fluorescence Correlation Spectroscopy, Raster Image Correlation Spectroscopy, and Number and Brightness on a Commercial Confocal Laser Scanning Microscope with Analog Detectors (Nikon C1),” Microsc Res Techniq. 74, 377–388 (2011).

Otsuki, S.

Park, C. W.

Prigozhina, N.

M. C.  Adams, W. C.  Salmon, S. L.  Gupton, C. S.  Cohan, T.  Wittmann, N.  Prigozhina, C. M.  Waterman-Storer, “A high-speed multispectral spinning-disk confocal microscope system for fluorescent speckle microscopy of living cells,” Methods 29(1), 29–41 (2003).
[CrossRef] [PubMed]

Salmon, W. C.

M. C.  Adams, W. C.  Salmon, S. L.  Gupton, C. S.  Cohan, T.  Wittmann, N.  Prigozhina, C. M.  Waterman-Storer, “A high-speed multispectral spinning-disk confocal microscope system for fluorescent speckle microscopy of living cells,” Methods 29(1), 29–41 (2003).
[CrossRef] [PubMed]

Salvemini, I. L.

P. D. J.  Moens, E.  Gratton, I. L.  Salvemini, “Fluorescence Correlation Spectroscopy, Raster Image Correlation Spectroscopy, and Number and Brightness on a Commercial Confocal Laser Scanning Microscope with Analog Detectors (Nikon C1),” Microsc Res Techniq. 74, 377–388 (2011).

Shim, J. W.

S. H.  Choi, W. H.  Kim, Y. J.  Lee, W. J.  Lee, J. D.  Yang, J. W.  Shim, J. W.  Kim, H.  Lee, “Visualization of Epidermis and Dermal Cells in ex vivo Human Skin Using the Confocal and Two-photon Microscopy,” j. Opt. Soc. Korea (South) 15, 61–67 (2011).

Shimizu, M.

Spencer, J. A.

J. A.  Spencer, D. P.  Biss, D.  Côté, C. P.  Lin, “In vivo Cell Tracking With Video Rate Multimodality Laser Scanning Microscopy,” IEEE J. Sel. Top. Quant 14, 10–18 (2008).

Tanaami, T.

Tomosada, N.

Wang, E.

E.  Wang, C. M.  Babbey, K. W.  Dunn, “Performance comparison between the high-speed Yokogawa spinning disc confocal system and single-point scanning confocal systems,” J. Microsc. 218(Pt 2), 148–159 (2005).
[CrossRef] [PubMed]

Waterman-Storer, C. M.

M. C.  Adams, W. C.  Salmon, S. L.  Gupton, C. S.  Cohan, T.  Wittmann, N.  Prigozhina, C. M.  Waterman-Storer, “A high-speed multispectral spinning-disk confocal microscope system for fluorescent speckle microscopy of living cells,” Methods 29(1), 29–41 (2003).
[CrossRef] [PubMed]

Wittmann, T.

M. C.  Adams, W. C.  Salmon, S. L.  Gupton, C. S.  Cohan, T.  Wittmann, N.  Prigozhina, C. M.  Waterman-Storer, “A high-speed multispectral spinning-disk confocal microscope system for fluorescent speckle microscopy of living cells,” Methods 29(1), 29–41 (2003).
[CrossRef] [PubMed]

Yang, J. D.

S. H.  Choi, W. H.  Kim, Y. J.  Lee, W. J.  Lee, J. D.  Yang, J. W.  Shim, J. W.  Kim, H.  Lee, “Visualization of Epidermis and Dermal Cells in ex vivo Human Skin Using the Confocal and Two-photon Microscopy,” j. Opt. Soc. Korea (South) 15, 61–67 (2011).

Appl. Opt. (1)

IEEE J. Sel. Top. Quant (1)

J. A.  Spencer, D. P.  Biss, D.  Côté, C. P.  Lin, “In vivo Cell Tracking With Video Rate Multimodality Laser Scanning Microscopy,” IEEE J. Sel. Top. Quant 14, 10–18 (2008).

J. Microsc. (2)

E.  Wang, C. M.  Babbey, K. W.  Dunn, “Performance comparison between the high-speed Yokogawa spinning disc confocal system and single-point scanning confocal systems,” J. Microsc. 218(Pt 2), 148–159 (2005).
[CrossRef] [PubMed]

C. M.  Brown, R. B.  Dalal, B.  Hebert, M. A.  Digman, A. R.  Horwitz, E.  Gratton, “Raster image correlation spectroscopy (RICS) for measuring fast protein dynamics and concentrations with a commercial laser scanning confocal microscope,” J. Microsc. 229(1), 78–91 (2008).
[CrossRef] [PubMed]

j. Opt. Soc. Korea (South) (1)

S. H.  Choi, W. H.  Kim, Y. J.  Lee, W. J.  Lee, J. D.  Yang, J. W.  Shim, J. W.  Kim, H.  Lee, “Visualization of Epidermis and Dermal Cells in ex vivo Human Skin Using the Confocal and Two-photon Microscopy,” j. Opt. Soc. Korea (South) 15, 61–67 (2011).

Methods (1)

M. C.  Adams, W. C.  Salmon, S. L.  Gupton, C. S.  Cohan, T.  Wittmann, N.  Prigozhina, C. M.  Waterman-Storer, “A high-speed multispectral spinning-disk confocal microscope system for fluorescent speckle microscopy of living cells,” Methods 29(1), 29–41 (2003).
[CrossRef] [PubMed]

Microsc Res Techniq. (1)

P. D. J.  Moens, E.  Gratton, I. L.  Salvemini, “Fluorescence Correlation Spectroscopy, Raster Image Correlation Spectroscopy, and Number and Brightness on a Commercial Confocal Laser Scanning Microscope with Analog Detectors (Nikon C1),” Microsc Res Techniq. 74, 377–388 (2011).

Opt. Express (1)

Other (2)

B. James, “Foundations of confocal scanned imaging in light microscopy,” in Handbook of Biological Confocal Microscopy, Shinya Inoue, ed. (Plenum Press, 1990).

M. Minsky, “Microscopy apparatus,” U.S. Patent 3013467 (1961).

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Figures (6)

Fig. 1
Fig. 1

Schematic diagram of high speed laser scanning confocal microscopy.

Fig. 2
Fig. 2

The analog galvanometer mirror control voltage, HSYNC and VSYNC generation electro-circuit for bi-directional slow axis scanning.

Fig. 3
Fig. 3

The analog galvanometer mirror control voltage signal and SYNC signal timing.

Fig. 4
Fig. 4

High speed laser scanning confocal microscopy images of a resolution target moving at the following constant speeds: (a) 30 fps (b) 100 fps (c) 200 fps

Fig. 5
Fig. 5

Apparent elongation and shortening of a pattern on the resolution target along the Y-axis for three different frame rates. The red box indicates the non-distorted size of the pattern under investigation. (a) 30 fps, (b) 200 fps

Fig. 6
Fig. 6

in-vivo mouse skin showing structures and movement. (a) 30 fps, (b) 100 fps, (c) 200 fps

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