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

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]

Appl. Phys. Lett.

• 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

• 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.

• 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.

• 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.

• 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

• 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.

• 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.

• 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

• 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.

• 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.

• 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.

• 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

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

Other

• 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.

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