Abstract

We present a real-time target-locking confocal microscope that follows an object moving along an arbitrary path, even as it simultaneously changes its shape, size and orientation. This Target-locking Acquisition with Realtime Confocal (TARC) microscopy system integrates fast image processing and rapid image acquisition using a Nipkow spinning-disk confocal microscope. The system acquires a 3D stack of images, performs a full structural analysis to locate a feature of interest, moves the sample in response, and then collects the next 3D image stack. In this way, data collection is dynamically adjusted to keep a moving object centered in the field of view. We demonstrate the system’s capabilities by target-locking freely-diffusing clusters of attractive colloidal particles, and actively-transported quantum dots (QDs) endocytosed into live cells free to move in three dimensions, for several hours. During this time, both the colloidal clusters and live cells move distances several times the length of the imaging volume.

© 2007 Optical Society of America

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  1. P. J. Lu, "Confocal Scanning Optical Microscopy and Nanotechnology," in Handbook of Microscopy for Nanotechnology, N. Yao, and Z. L.Wang, eds. (Kluwer, 2005), pp. 3-24.
  2. P. J. Lu, J. C. Conrad, H. M. Wyss, A. B. Schofield, and D. A. Weitz, "Fluids of Clusters in Attractive Colloids," Phys. Rev. Lett. 96, 028306 (2006).
    [CrossRef] [PubMed]
  3. X. S. Xie, J. Yu, and W. Y. Yang, "Living Cells as Test Tubes," Science 312, 228-230 (2006).
    [CrossRef] [PubMed]
  4. M. E. Wickham, M. Rug, S. A. Ralph, N. Klonis, G. I. McFadden, L. Tilley, and A. F. Cowman, "Trafficking and assembly of the cytoadherence complex in Plasmodium falciparum-infected human erythrocytes," EMBO J. 20, 5636-5649 (2001).
    [CrossRef] [PubMed]
  5. B. Gligorijevic, R. McAllister, J. S. Urbach, and P. D. Roepe, "Spinning Disk Confocal Microscopy of Live, Intraerythrocytic Malarial Parasites. 1. Quantification of Hemozoin Development for Drug Sensitive versus Resistant Malaria," Biochemistry 45, 12400-12410 (2006).
    [CrossRef] [PubMed]
  6. B. Gligorijevic, R. McAllister, J. S. Urbach, and P. D. Roepe, "Spinning Disk Confocal Microscopy of Live, Intraerythrocytic Malarial Parasites. 2. Altered Vacuolar Volume Regulation in Drug Resistant Malaria," Biochemistry 45, 12411-12423 (2006).
    [CrossRef] [PubMed]
  7. T. A. Camesano, M. J. Natan, B. E. Logan, "Observation of Changes in Bacterial Cell Morphology Using Tapping Mode Atomic Force Microscopy," Langmuir 16, 4563-4572 (2000).
    [CrossRef]
  8. N. Arhel, A. Genovesio, K.-A. Kim, S. Miko, E. Perret, J.-C. Olivo-Marin, S. Shorte, and P. Charneau, "Quantitative four-dimensional tracking of cytoplasmic and nuclear HIV-1 complexes," Nat. Meth. 3, 817-823 (2006).
    [CrossRef]
  9. H. Berg, "How to track bacteria," Rev. Sci. Instrum. 42, 868-71 (1971).
    [CrossRef] [PubMed]
  10. I. M. Peters, B. G. de Grooth, J. M. Schins, C. G. Figdor, and J. Greve, "Three dimensional single-particle tracking with nanometer resolution," Rev. Sci. Instrum. 69, 2762-2766 (1998).
    [CrossRef]
  11. G. Rabut, J. Ellenberg, "Automatic real-time three-dimensional cell tracking by fluorescence microscopy," J. Microsc. 216, 131-137 (2005).
  12. V. Levi, Q. Q. Ruan, and E. Gratton, "3-D Particle Tracking in a Two-Photon Microscope: Application to the Study of Molecular Dynamics in Cells," Biophys. J. 88, 2919-2928 (2005).
    [CrossRef] [PubMed]
  13. H. Cang, C. M. Wong, C. S. Xu, A. H. Rizvi, and H. Yang, "Confocal three dimensional tracking of a single nanoparticle with concurrent spectroscopic readouts," Appl. Phys. Lett. 88, 223901 (2006).
    [CrossRef]
  14. T. Ragan, H. Huang, P. So, and E. Gratton, "3D Particle Tracking on a Two-Photon Microscope," J. Fluorescence 16, 325-336 (2006).
    [CrossRef]
  15. A. Egner, V. Andresen and S. W. Hell, "Comparison of the axial resolution of practical Nipkow-disk confocal fluorescence microscopy with that of multifocal multiphoton microscopy: theory and experiment," J. Microsc. 206, 24-32 (2002).
    [CrossRef] [PubMed]
  16. E. Wang, C. M. Babbey and K.W. Dunn, "Performance comparison between the high-speed Yokogawa spinning disc confocal system and single-point scanning confocal systems," J. Microsc. 218, 148-159 (2005).
    [CrossRef] [PubMed]
  17. J. C. Crocker, and D. G. Grier, "Methods of Digital Video Microscopy for Colloidal Studies," J. Colloid Interface Sci. 179, 298-310 (1996).
    [CrossRef]
  18. X. L. Nan, P. A. Sims, P. Chen, X. S. Xie, "Observation of Individual Microtubule Motor Steps in Living Cells with Endocytosed Quantum Dots," J. Phys. Chem. B. 109, 24220-24224 (2005).
    [CrossRef] [PubMed]

2006 (7)

P. J. Lu, J. C. Conrad, H. M. Wyss, A. B. Schofield, and D. A. Weitz, "Fluids of Clusters in Attractive Colloids," Phys. Rev. Lett. 96, 028306 (2006).
[CrossRef] [PubMed]

X. S. Xie, J. Yu, and W. Y. Yang, "Living Cells as Test Tubes," Science 312, 228-230 (2006).
[CrossRef] [PubMed]

B. Gligorijevic, R. McAllister, J. S. Urbach, and P. D. Roepe, "Spinning Disk Confocal Microscopy of Live, Intraerythrocytic Malarial Parasites. 1. Quantification of Hemozoin Development for Drug Sensitive versus Resistant Malaria," Biochemistry 45, 12400-12410 (2006).
[CrossRef] [PubMed]

B. Gligorijevic, R. McAllister, J. S. Urbach, and P. D. Roepe, "Spinning Disk Confocal Microscopy of Live, Intraerythrocytic Malarial Parasites. 2. Altered Vacuolar Volume Regulation in Drug Resistant Malaria," Biochemistry 45, 12411-12423 (2006).
[CrossRef] [PubMed]

N. Arhel, A. Genovesio, K.-A. Kim, S. Miko, E. Perret, J.-C. Olivo-Marin, S. Shorte, and P. Charneau, "Quantitative four-dimensional tracking of cytoplasmic and nuclear HIV-1 complexes," Nat. Meth. 3, 817-823 (2006).
[CrossRef]

H. Cang, C. M. Wong, C. S. Xu, A. H. Rizvi, and H. Yang, "Confocal three dimensional tracking of a single nanoparticle with concurrent spectroscopic readouts," Appl. Phys. Lett. 88, 223901 (2006).
[CrossRef]

T. Ragan, H. Huang, P. So, and E. Gratton, "3D Particle Tracking on a Two-Photon Microscope," J. Fluorescence 16, 325-336 (2006).
[CrossRef]

2005 (4)

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

X. L. Nan, P. A. Sims, P. Chen, X. S. Xie, "Observation of Individual Microtubule Motor Steps in Living Cells with Endocytosed Quantum Dots," J. Phys. Chem. B. 109, 24220-24224 (2005).
[CrossRef] [PubMed]

G. Rabut, J. Ellenberg, "Automatic real-time three-dimensional cell tracking by fluorescence microscopy," J. Microsc. 216, 131-137 (2005).

V. Levi, Q. Q. Ruan, and E. Gratton, "3-D Particle Tracking in a Two-Photon Microscope: Application to the Study of Molecular Dynamics in Cells," Biophys. J. 88, 2919-2928 (2005).
[CrossRef] [PubMed]

2002 (1)

A. Egner, V. Andresen and S. W. Hell, "Comparison of the axial resolution of practical Nipkow-disk confocal fluorescence microscopy with that of multifocal multiphoton microscopy: theory and experiment," J. Microsc. 206, 24-32 (2002).
[CrossRef] [PubMed]

2001 (1)

M. E. Wickham, M. Rug, S. A. Ralph, N. Klonis, G. I. McFadden, L. Tilley, and A. F. Cowman, "Trafficking and assembly of the cytoadherence complex in Plasmodium falciparum-infected human erythrocytes," EMBO J. 20, 5636-5649 (2001).
[CrossRef] [PubMed]

2000 (1)

T. A. Camesano, M. J. Natan, B. E. Logan, "Observation of Changes in Bacterial Cell Morphology Using Tapping Mode Atomic Force Microscopy," Langmuir 16, 4563-4572 (2000).
[CrossRef]

1998 (1)

I. M. Peters, B. G. de Grooth, J. M. Schins, C. G. Figdor, and J. Greve, "Three dimensional single-particle tracking with nanometer resolution," Rev. Sci. Instrum. 69, 2762-2766 (1998).
[CrossRef]

1996 (1)

J. C. Crocker, and D. G. Grier, "Methods of Digital Video Microscopy for Colloidal Studies," J. Colloid Interface Sci. 179, 298-310 (1996).
[CrossRef]

1971 (1)

H. Berg, "How to track bacteria," Rev. Sci. Instrum. 42, 868-71 (1971).
[CrossRef] [PubMed]

Andresen, V.

A. Egner, V. Andresen and S. W. Hell, "Comparison of the axial resolution of practical Nipkow-disk confocal fluorescence microscopy with that of multifocal multiphoton microscopy: theory and experiment," J. Microsc. 206, 24-32 (2002).
[CrossRef] [PubMed]

Arhel, N.

N. Arhel, A. Genovesio, K.-A. Kim, S. Miko, E. Perret, J.-C. Olivo-Marin, S. Shorte, and P. Charneau, "Quantitative four-dimensional tracking of cytoplasmic and nuclear HIV-1 complexes," Nat. Meth. 3, 817-823 (2006).
[CrossRef]

Babbey, C. M.

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

Berg, H.

H. Berg, "How to track bacteria," Rev. Sci. Instrum. 42, 868-71 (1971).
[CrossRef] [PubMed]

Camesano, T. A.

T. A. Camesano, M. J. Natan, B. E. Logan, "Observation of Changes in Bacterial Cell Morphology Using Tapping Mode Atomic Force Microscopy," Langmuir 16, 4563-4572 (2000).
[CrossRef]

Cang, H.

H. Cang, C. M. Wong, C. S. Xu, A. H. Rizvi, and H. Yang, "Confocal three dimensional tracking of a single nanoparticle with concurrent spectroscopic readouts," Appl. Phys. Lett. 88, 223901 (2006).
[CrossRef]

Charneau, P.

N. Arhel, A. Genovesio, K.-A. Kim, S. Miko, E. Perret, J.-C. Olivo-Marin, S. Shorte, and P. Charneau, "Quantitative four-dimensional tracking of cytoplasmic and nuclear HIV-1 complexes," Nat. Meth. 3, 817-823 (2006).
[CrossRef]

Chen, P.

X. L. Nan, P. A. Sims, P. Chen, X. S. Xie, "Observation of Individual Microtubule Motor Steps in Living Cells with Endocytosed Quantum Dots," J. Phys. Chem. B. 109, 24220-24224 (2005).
[CrossRef] [PubMed]

Conrad, J. C.

P. J. Lu, J. C. Conrad, H. M. Wyss, A. B. Schofield, and D. A. Weitz, "Fluids of Clusters in Attractive Colloids," Phys. Rev. Lett. 96, 028306 (2006).
[CrossRef] [PubMed]

Cowman, A. F.

M. E. Wickham, M. Rug, S. A. Ralph, N. Klonis, G. I. McFadden, L. Tilley, and A. F. Cowman, "Trafficking and assembly of the cytoadherence complex in Plasmodium falciparum-infected human erythrocytes," EMBO J. 20, 5636-5649 (2001).
[CrossRef] [PubMed]

Crocker, J. C.

J. C. Crocker, and D. G. Grier, "Methods of Digital Video Microscopy for Colloidal Studies," J. Colloid Interface Sci. 179, 298-310 (1996).
[CrossRef]

de Grooth, B. G.

I. M. Peters, B. G. de Grooth, J. M. Schins, C. G. Figdor, and J. Greve, "Three dimensional single-particle tracking with nanometer resolution," Rev. Sci. Instrum. 69, 2762-2766 (1998).
[CrossRef]

Dunn, K.W.

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

Egner, A.

A. Egner, V. Andresen and S. W. Hell, "Comparison of the axial resolution of practical Nipkow-disk confocal fluorescence microscopy with that of multifocal multiphoton microscopy: theory and experiment," J. Microsc. 206, 24-32 (2002).
[CrossRef] [PubMed]

Ellenberg, J.

G. Rabut, J. Ellenberg, "Automatic real-time three-dimensional cell tracking by fluorescence microscopy," J. Microsc. 216, 131-137 (2005).

Figdor, C. G.

I. M. Peters, B. G. de Grooth, J. M. Schins, C. G. Figdor, and J. Greve, "Three dimensional single-particle tracking with nanometer resolution," Rev. Sci. Instrum. 69, 2762-2766 (1998).
[CrossRef]

Genovesio, A.

N. Arhel, A. Genovesio, K.-A. Kim, S. Miko, E. Perret, J.-C. Olivo-Marin, S. Shorte, and P. Charneau, "Quantitative four-dimensional tracking of cytoplasmic and nuclear HIV-1 complexes," Nat. Meth. 3, 817-823 (2006).
[CrossRef]

Gligorijevic, B.

B. Gligorijevic, R. McAllister, J. S. Urbach, and P. D. Roepe, "Spinning Disk Confocal Microscopy of Live, Intraerythrocytic Malarial Parasites. 2. Altered Vacuolar Volume Regulation in Drug Resistant Malaria," Biochemistry 45, 12411-12423 (2006).
[CrossRef] [PubMed]

B. Gligorijevic, R. McAllister, J. S. Urbach, and P. D. Roepe, "Spinning Disk Confocal Microscopy of Live, Intraerythrocytic Malarial Parasites. 1. Quantification of Hemozoin Development for Drug Sensitive versus Resistant Malaria," Biochemistry 45, 12400-12410 (2006).
[CrossRef] [PubMed]

Gratton, E.

T. Ragan, H. Huang, P. So, and E. Gratton, "3D Particle Tracking on a Two-Photon Microscope," J. Fluorescence 16, 325-336 (2006).
[CrossRef]

V. Levi, Q. Q. Ruan, and E. Gratton, "3-D Particle Tracking in a Two-Photon Microscope: Application to the Study of Molecular Dynamics in Cells," Biophys. J. 88, 2919-2928 (2005).
[CrossRef] [PubMed]

Greve, J.

I. M. Peters, B. G. de Grooth, J. M. Schins, C. G. Figdor, and J. Greve, "Three dimensional single-particle tracking with nanometer resolution," Rev. Sci. Instrum. 69, 2762-2766 (1998).
[CrossRef]

Grier, D. G.

J. C. Crocker, and D. G. Grier, "Methods of Digital Video Microscopy for Colloidal Studies," J. Colloid Interface Sci. 179, 298-310 (1996).
[CrossRef]

Hell, S. W.

A. Egner, V. Andresen and S. W. Hell, "Comparison of the axial resolution of practical Nipkow-disk confocal fluorescence microscopy with that of multifocal multiphoton microscopy: theory and experiment," J. Microsc. 206, 24-32 (2002).
[CrossRef] [PubMed]

Huang, H.

T. Ragan, H. Huang, P. So, and E. Gratton, "3D Particle Tracking on a Two-Photon Microscope," J. Fluorescence 16, 325-336 (2006).
[CrossRef]

Kim, K.-A.

N. Arhel, A. Genovesio, K.-A. Kim, S. Miko, E. Perret, J.-C. Olivo-Marin, S. Shorte, and P. Charneau, "Quantitative four-dimensional tracking of cytoplasmic and nuclear HIV-1 complexes," Nat. Meth. 3, 817-823 (2006).
[CrossRef]

Klonis, N.

M. E. Wickham, M. Rug, S. A. Ralph, N. Klonis, G. I. McFadden, L. Tilley, and A. F. Cowman, "Trafficking and assembly of the cytoadherence complex in Plasmodium falciparum-infected human erythrocytes," EMBO J. 20, 5636-5649 (2001).
[CrossRef] [PubMed]

Levi, V.

V. Levi, Q. Q. Ruan, and E. Gratton, "3-D Particle Tracking in a Two-Photon Microscope: Application to the Study of Molecular Dynamics in Cells," Biophys. J. 88, 2919-2928 (2005).
[CrossRef] [PubMed]

Logan, B. E.

T. A. Camesano, M. J. Natan, B. E. Logan, "Observation of Changes in Bacterial Cell Morphology Using Tapping Mode Atomic Force Microscopy," Langmuir 16, 4563-4572 (2000).
[CrossRef]

Lu, P. J.

P. J. Lu, J. C. Conrad, H. M. Wyss, A. B. Schofield, and D. A. Weitz, "Fluids of Clusters in Attractive Colloids," Phys. Rev. Lett. 96, 028306 (2006).
[CrossRef] [PubMed]

McAllister, R.

B. Gligorijevic, R. McAllister, J. S. Urbach, and P. D. Roepe, "Spinning Disk Confocal Microscopy of Live, Intraerythrocytic Malarial Parasites. 2. Altered Vacuolar Volume Regulation in Drug Resistant Malaria," Biochemistry 45, 12411-12423 (2006).
[CrossRef] [PubMed]

B. Gligorijevic, R. McAllister, J. S. Urbach, and P. D. Roepe, "Spinning Disk Confocal Microscopy of Live, Intraerythrocytic Malarial Parasites. 1. Quantification of Hemozoin Development for Drug Sensitive versus Resistant Malaria," Biochemistry 45, 12400-12410 (2006).
[CrossRef] [PubMed]

McFadden, G. I.

M. E. Wickham, M. Rug, S. A. Ralph, N. Klonis, G. I. McFadden, L. Tilley, and A. F. Cowman, "Trafficking and assembly of the cytoadherence complex in Plasmodium falciparum-infected human erythrocytes," EMBO J. 20, 5636-5649 (2001).
[CrossRef] [PubMed]

Miko, S.

N. Arhel, A. Genovesio, K.-A. Kim, S. Miko, E. Perret, J.-C. Olivo-Marin, S. Shorte, and P. Charneau, "Quantitative four-dimensional tracking of cytoplasmic and nuclear HIV-1 complexes," Nat. Meth. 3, 817-823 (2006).
[CrossRef]

Nan, X. L.

X. L. Nan, P. A. Sims, P. Chen, X. S. Xie, "Observation of Individual Microtubule Motor Steps in Living Cells with Endocytosed Quantum Dots," J. Phys. Chem. B. 109, 24220-24224 (2005).
[CrossRef] [PubMed]

Natan, M. J.

T. A. Camesano, M. J. Natan, B. E. Logan, "Observation of Changes in Bacterial Cell Morphology Using Tapping Mode Atomic Force Microscopy," Langmuir 16, 4563-4572 (2000).
[CrossRef]

Olivo-Marin, J.-C.

N. Arhel, A. Genovesio, K.-A. Kim, S. Miko, E. Perret, J.-C. Olivo-Marin, S. Shorte, and P. Charneau, "Quantitative four-dimensional tracking of cytoplasmic and nuclear HIV-1 complexes," Nat. Meth. 3, 817-823 (2006).
[CrossRef]

Perret, E.

N. Arhel, A. Genovesio, K.-A. Kim, S. Miko, E. Perret, J.-C. Olivo-Marin, S. Shorte, and P. Charneau, "Quantitative four-dimensional tracking of cytoplasmic and nuclear HIV-1 complexes," Nat. Meth. 3, 817-823 (2006).
[CrossRef]

Peters, I. M.

I. M. Peters, B. G. de Grooth, J. M. Schins, C. G. Figdor, and J. Greve, "Three dimensional single-particle tracking with nanometer resolution," Rev. Sci. Instrum. 69, 2762-2766 (1998).
[CrossRef]

Rabut, G.

G. Rabut, J. Ellenberg, "Automatic real-time three-dimensional cell tracking by fluorescence microscopy," J. Microsc. 216, 131-137 (2005).

Ragan, T.

T. Ragan, H. Huang, P. So, and E. Gratton, "3D Particle Tracking on a Two-Photon Microscope," J. Fluorescence 16, 325-336 (2006).
[CrossRef]

Ralph, S. A.

M. E. Wickham, M. Rug, S. A. Ralph, N. Klonis, G. I. McFadden, L. Tilley, and A. F. Cowman, "Trafficking and assembly of the cytoadherence complex in Plasmodium falciparum-infected human erythrocytes," EMBO J. 20, 5636-5649 (2001).
[CrossRef] [PubMed]

Rizvi, A. H.

H. Cang, C. M. Wong, C. S. Xu, A. H. Rizvi, and H. Yang, "Confocal three dimensional tracking of a single nanoparticle with concurrent spectroscopic readouts," Appl. Phys. Lett. 88, 223901 (2006).
[CrossRef]

Roepe, P. D.

B. Gligorijevic, R. McAllister, J. S. Urbach, and P. D. Roepe, "Spinning Disk Confocal Microscopy of Live, Intraerythrocytic Malarial Parasites. 2. Altered Vacuolar Volume Regulation in Drug Resistant Malaria," Biochemistry 45, 12411-12423 (2006).
[CrossRef] [PubMed]

B. Gligorijevic, R. McAllister, J. S. Urbach, and P. D. Roepe, "Spinning Disk Confocal Microscopy of Live, Intraerythrocytic Malarial Parasites. 1. Quantification of Hemozoin Development for Drug Sensitive versus Resistant Malaria," Biochemistry 45, 12400-12410 (2006).
[CrossRef] [PubMed]

Ruan, Q. Q.

V. Levi, Q. Q. Ruan, and E. Gratton, "3-D Particle Tracking in a Two-Photon Microscope: Application to the Study of Molecular Dynamics in Cells," Biophys. J. 88, 2919-2928 (2005).
[CrossRef] [PubMed]

Rug, M.

M. E. Wickham, M. Rug, S. A. Ralph, N. Klonis, G. I. McFadden, L. Tilley, and A. F. Cowman, "Trafficking and assembly of the cytoadherence complex in Plasmodium falciparum-infected human erythrocytes," EMBO J. 20, 5636-5649 (2001).
[CrossRef] [PubMed]

Schins, J. M.

I. M. Peters, B. G. de Grooth, J. M. Schins, C. G. Figdor, and J. Greve, "Three dimensional single-particle tracking with nanometer resolution," Rev. Sci. Instrum. 69, 2762-2766 (1998).
[CrossRef]

Schofield, A. B.

P. J. Lu, J. C. Conrad, H. M. Wyss, A. B. Schofield, and D. A. Weitz, "Fluids of Clusters in Attractive Colloids," Phys. Rev. Lett. 96, 028306 (2006).
[CrossRef] [PubMed]

Shorte, S.

N. Arhel, A. Genovesio, K.-A. Kim, S. Miko, E. Perret, J.-C. Olivo-Marin, S. Shorte, and P. Charneau, "Quantitative four-dimensional tracking of cytoplasmic and nuclear HIV-1 complexes," Nat. Meth. 3, 817-823 (2006).
[CrossRef]

Sims, P. A.

X. L. Nan, P. A. Sims, P. Chen, X. S. Xie, "Observation of Individual Microtubule Motor Steps in Living Cells with Endocytosed Quantum Dots," J. Phys. Chem. B. 109, 24220-24224 (2005).
[CrossRef] [PubMed]

So, P.

T. Ragan, H. Huang, P. So, and E. Gratton, "3D Particle Tracking on a Two-Photon Microscope," J. Fluorescence 16, 325-336 (2006).
[CrossRef]

Tilley, L.

M. E. Wickham, M. Rug, S. A. Ralph, N. Klonis, G. I. McFadden, L. Tilley, and A. F. Cowman, "Trafficking and assembly of the cytoadherence complex in Plasmodium falciparum-infected human erythrocytes," EMBO J. 20, 5636-5649 (2001).
[CrossRef] [PubMed]

Urbach, J. S.

B. Gligorijevic, R. McAllister, J. S. Urbach, and P. D. Roepe, "Spinning Disk Confocal Microscopy of Live, Intraerythrocytic Malarial Parasites. 2. Altered Vacuolar Volume Regulation in Drug Resistant Malaria," Biochemistry 45, 12411-12423 (2006).
[CrossRef] [PubMed]

B. Gligorijevic, R. McAllister, J. S. Urbach, and P. D. Roepe, "Spinning Disk Confocal Microscopy of Live, Intraerythrocytic Malarial Parasites. 1. Quantification of Hemozoin Development for Drug Sensitive versus Resistant Malaria," Biochemistry 45, 12400-12410 (2006).
[CrossRef] [PubMed]

Wang, E.

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

Weitz, D. A.

P. J. Lu, J. C. Conrad, H. M. Wyss, A. B. Schofield, and D. A. Weitz, "Fluids of Clusters in Attractive Colloids," Phys. Rev. Lett. 96, 028306 (2006).
[CrossRef] [PubMed]

Wickham, M. E.

M. E. Wickham, M. Rug, S. A. Ralph, N. Klonis, G. I. McFadden, L. Tilley, and A. F. Cowman, "Trafficking and assembly of the cytoadherence complex in Plasmodium falciparum-infected human erythrocytes," EMBO J. 20, 5636-5649 (2001).
[CrossRef] [PubMed]

Wong, C. M.

H. Cang, C. M. Wong, C. S. Xu, A. H. Rizvi, and H. Yang, "Confocal three dimensional tracking of a single nanoparticle with concurrent spectroscopic readouts," Appl. Phys. Lett. 88, 223901 (2006).
[CrossRef]

Wyss, H. M.

P. J. Lu, J. C. Conrad, H. M. Wyss, A. B. Schofield, and D. A. Weitz, "Fluids of Clusters in Attractive Colloids," Phys. Rev. Lett. 96, 028306 (2006).
[CrossRef] [PubMed]

Xie, X. S.

X. S. Xie, J. Yu, and W. Y. Yang, "Living Cells as Test Tubes," Science 312, 228-230 (2006).
[CrossRef] [PubMed]

X. L. Nan, P. A. Sims, P. Chen, X. S. Xie, "Observation of Individual Microtubule Motor Steps in Living Cells with Endocytosed Quantum Dots," J. Phys. Chem. B. 109, 24220-24224 (2005).
[CrossRef] [PubMed]

Xu, C. S.

H. Cang, C. M. Wong, C. S. Xu, A. H. Rizvi, and H. Yang, "Confocal three dimensional tracking of a single nanoparticle with concurrent spectroscopic readouts," Appl. Phys. Lett. 88, 223901 (2006).
[CrossRef]

Yang, H.

H. Cang, C. M. Wong, C. S. Xu, A. H. Rizvi, and H. Yang, "Confocal three dimensional tracking of a single nanoparticle with concurrent spectroscopic readouts," Appl. Phys. Lett. 88, 223901 (2006).
[CrossRef]

Yang, W. Y.

X. S. Xie, J. Yu, and W. Y. Yang, "Living Cells as Test Tubes," Science 312, 228-230 (2006).
[CrossRef] [PubMed]

Yu, J.

X. S. Xie, J. Yu, and W. Y. Yang, "Living Cells as Test Tubes," Science 312, 228-230 (2006).
[CrossRef] [PubMed]

Appl. Phys. Lett. (1)

H. Cang, C. M. Wong, C. S. Xu, A. H. Rizvi, and H. Yang, "Confocal three dimensional tracking of a single nanoparticle with concurrent spectroscopic readouts," Appl. Phys. Lett. 88, 223901 (2006).
[CrossRef]

Biochemistry (2)

B. Gligorijevic, R. McAllister, J. S. Urbach, and P. D. Roepe, "Spinning Disk Confocal Microscopy of Live, Intraerythrocytic Malarial Parasites. 1. Quantification of Hemozoin Development for Drug Sensitive versus Resistant Malaria," Biochemistry 45, 12400-12410 (2006).
[CrossRef] [PubMed]

B. Gligorijevic, R. McAllister, J. S. Urbach, and P. D. Roepe, "Spinning Disk Confocal Microscopy of Live, Intraerythrocytic Malarial Parasites. 2. Altered Vacuolar Volume Regulation in Drug Resistant Malaria," Biochemistry 45, 12411-12423 (2006).
[CrossRef] [PubMed]

Biophys. J. (1)

V. Levi, Q. Q. Ruan, and E. Gratton, "3-D Particle Tracking in a Two-Photon Microscope: Application to the Study of Molecular Dynamics in Cells," Biophys. J. 88, 2919-2928 (2005).
[CrossRef] [PubMed]

EMBO J. (1)

M. E. Wickham, M. Rug, S. A. Ralph, N. Klonis, G. I. McFadden, L. Tilley, and A. F. Cowman, "Trafficking and assembly of the cytoadherence complex in Plasmodium falciparum-infected human erythrocytes," EMBO J. 20, 5636-5649 (2001).
[CrossRef] [PubMed]

J. Colloid Interface Sci. (1)

J. C. Crocker, and D. G. Grier, "Methods of Digital Video Microscopy for Colloidal Studies," J. Colloid Interface Sci. 179, 298-310 (1996).
[CrossRef]

J. Fluorescence (1)

T. Ragan, H. Huang, P. So, and E. Gratton, "3D Particle Tracking on a Two-Photon Microscope," J. Fluorescence 16, 325-336 (2006).
[CrossRef]

J. Microsc. (3)

A. Egner, V. Andresen and S. W. Hell, "Comparison of the axial resolution of practical Nipkow-disk confocal fluorescence microscopy with that of multifocal multiphoton microscopy: theory and experiment," J. Microsc. 206, 24-32 (2002).
[CrossRef] [PubMed]

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

G. Rabut, J. Ellenberg, "Automatic real-time three-dimensional cell tracking by fluorescence microscopy," J. Microsc. 216, 131-137 (2005).

J. Phys. Chem. B. (1)

X. L. Nan, P. A. Sims, P. Chen, X. S. Xie, "Observation of Individual Microtubule Motor Steps in Living Cells with Endocytosed Quantum Dots," J. Phys. Chem. B. 109, 24220-24224 (2005).
[CrossRef] [PubMed]

Langmuir (1)

T. A. Camesano, M. J. Natan, B. E. Logan, "Observation of Changes in Bacterial Cell Morphology Using Tapping Mode Atomic Force Microscopy," Langmuir 16, 4563-4572 (2000).
[CrossRef]

Nat. Meth. (1)

N. Arhel, A. Genovesio, K.-A. Kim, S. Miko, E. Perret, J.-C. Olivo-Marin, S. Shorte, and P. Charneau, "Quantitative four-dimensional tracking of cytoplasmic and nuclear HIV-1 complexes," Nat. Meth. 3, 817-823 (2006).
[CrossRef]

Phys. Rev. Lett. (1)

P. J. Lu, J. C. Conrad, H. M. Wyss, A. B. Schofield, and D. A. Weitz, "Fluids of Clusters in Attractive Colloids," Phys. Rev. Lett. 96, 028306 (2006).
[CrossRef] [PubMed]

Rev. Sci. Instrum. (2)

H. Berg, "How to track bacteria," Rev. Sci. Instrum. 42, 868-71 (1971).
[CrossRef] [PubMed]

I. M. Peters, B. G. de Grooth, J. M. Schins, C. G. Figdor, and J. Greve, "Three dimensional single-particle tracking with nanometer resolution," Rev. Sci. Instrum. 69, 2762-2766 (1998).
[CrossRef]

Science (1)

X. S. Xie, J. Yu, and W. Y. Yang, "Living Cells as Test Tubes," Science 312, 228-230 (2006).
[CrossRef] [PubMed]

Other (1)

P. J. Lu, "Confocal Scanning Optical Microscopy and Nanotechnology," in Handbook of Microscopy for Nanotechnology, N. Yao, and Z. L.Wang, eds. (Kluwer, 2005), pp. 3-24.

Supplementary Material (2)

» Media 1: MOV (2406 KB)     
» Media 2: MOV (2149 KB)     

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Figures (4)

Fig. 1.
Fig. 1.

Block diagram of the TARC system (not to scale). Major components indicated by black boxes. The beam path is labeled in green (excitation) and orange (emission), with lenses in light blue. TTL signal connections are indicated with blue lines; RS-232, with red; and IEEE1394 firewire, with purple.

Fig. 2.
Fig. 2.

Pulse sequence for the acquisition of two 3D image stacks, each with three images. Data acquisition begins at T 1, when the pulse generator opens the laser shutter by raising Shutter Signal to a TTL-high value, which it maintains during the course of acquiring the first stack. At T 2, after delaying for Laser On Delay (= T 2 -T 1), the pulse generator sends a Confocal Trigger/Camera Trigger pulse to synchronize the confocal spinning disk and begin exposure of the CCD camera. At T 3, after delaying for Piezo Delay (= T 3 -T 2), the pulse generator then sends a Piezo Trigger pulse to move the piezo to the next position. At T 4, after delaying for Inter-frame Spacing (= T 4 - T 2) relative to T 2, the pulse generator sends another Confocal Trigger/Camera Trigger pulse to start acquisition for the next frame. And again, the piezo is then moved with a Piezo Trigger pulse following the end of acquisition of the second frame, after a delay of PiezoDelay relative to T 4. This process repeats for each frame in the 3D image stack. After the final frame in each stack is collected (i.e. the third frame here), the pulse generator sends several more Piezo Trigger pulses to move the objective back to the starting position in small steps: with immersion objectives, mechanical coupling via the viscous index-matching liquid will cause the sample to slip if the objective is moved too quickly. After the final piezo pulse, when the objective has returned to the starting position, the pulse generator waits for Laser Off Delay (= T 6 -T 5) before dropping Shutter Signal back to the TTL-low value, cutting off the laser and preventing sample bleaching during the waiting time between stacks (= T 7 -T 6). At T 7, after a delay of Interstack Spacing (= T 7 -T 1) relative to the acquisition start of the previous stack at T 1, the shutter is again opened and the acquisition of the second 3D image stack commences.

Fig. 3.
Fig. 3.

Target-locking freely-diffusing clusters of colloidal spheres. (a)-(e) 3D reconstructions and (inset) 2D confocal images (24×24 μm2) of a growing cluster. In 3D reconstructions, monomers and dimers are transparent grey, and the color of larger clusters indicates their number of spheres, following the color bar at the left of the graph in (g). In (a), a small cluster enters the volume in addition to the largest central cluster, and the TARC system properly follows the larger central cluster after (b) the smaller cluster has departed the imaging volume. (c) Later, another small cluster enters the volume and (d) merges to form a much larger cluster, which then (e) rotates and contracts. (f) 3D plot of the trajectory of the cluster’s center of mass. In all cases, the TARC system successfully follows the largest cluster in the imaging volume and (g) determines the mass (number of particles; black line) and displacement of its center of mass relative to its initial position (green line) through time. Yellow arrows indicate times of structures depicted in (a)-(e). [Media 1]

Fig. 4.
Fig. 4.

Target-locking actively-transported QDs in a freely-moving cell. Confocal images of a human lung cancer cell, with cell membrane highlighted in green, and quantum dots undergoing active transport in red, at (a) 1020 seconds and (b) 2950 seconds elapsed time. (c) Displacement from original position, with yellow arrows indicating times depicted in (a)-(b). (d) 3D trajectory plot of the center of the cell. [Media 2]

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