Real-time nanomicroscopy via three-dimensional single-particle tracking.

We developed a new method for real-time, three-dimensional tracking of fluorescent particles. The instrument is based on a laser-scanning confocal microscope where the focus of the laser beam is scanned or orbited around the particle. Two confocal pinholes are used to simultaneously monitor regions immediately above and below the particle and a feedback loop is used to keep the orbit centered on the particle. For moderate count rates, this system can track particles with 15 nm spatial resolution in the lateral dimensions and 50 nm in the axial dimension at a temporal resolution of 32 ms. To investigate the interaction of the tracked particles with cellular components, we have combined our orbital tracking microscope with a dual-color, wide-field setup. Dual-color fluorescence wide-field images are recorded simultaneously in the same image plane as the particle being tracked. The functionality of the system was demonstrated by tracking fluorescent-labeled artificial viruses in tubulin-eGFP expressing HUH7 cells. The resulting trajectories can be used to investigate the microtubule network with super resolution.

[1]  H. Berg.  How to track bacteria. , 1971, The Review of scientific instruments.

[2]  N O Petersen,et al.  Scanning fluorescence correlation spectroscopy. I. Theory and simulation of aggregation measurements. , 1986, Biophysical journal.

[3]  M. Schlesinger,et al.  Scanning fluorescence correlation spectroscopy. II. Application to virus glycoprotein aggregation. , 1986, Biophysical journal.

[4]  M. Saxton,et al.  Lateral diffusion in an archipelago. Single-particle diffusion. , 1993, Biophysical journal.

[5]  M. Saxton.  Anomalous diffusion due to obstacles: a Monte Carlo study. , 1994, Biophysical journal.

[6]  M. Saxton,et al.  Single-particle tracking: models of directed transport. , 1994, Biophysical journal.

[7]  Werner Baumgartner,et al.  Characterization of Photophysics and Mobility of Single Molecules in a Fluid Lipid Membrane , 1995 .

[8]  M. Saxton,et al.  Single-particle tracking: effects of corrals. , 1995, Biophysical journal.

[9]  E Gratton,et al.  Scanning two-photon fluctuation correlation spectroscopy: particle counting measurements for detection of molecular aggregation. , 1996, Biophysical journal.

[10]  Gerald Kada,et al.  Properties of lipid microdomains in a muscle cell membrane visualized by single molecule microscopy , 2000, The EMBO journal.

[11]  T. Kues,et al.  Imaging and tracking of single GFP molecules in solution. , 2000, Biophysical journal.

[12]  J. Enderlein.  Tracking of fluorescent molecules diffusing within membranes , 2000 .

[13]  M. Hallek,et al.  Real-Time Single-Molecule Imaging of the Infection Pathway of an Adeno-Associated Virus , 2001, Science.

[14]  Akihiro Kusumi,et al.  Phospholipids undergo hop diffusion in compartmentalized cell membrane , 2002, The Journal of cell biology.

[15]  Alexandr Jonás,et al.  Three-dimensional tracking of fluorescent nanoparticles with subnanometer precision by use of off-focus imaging. , 2003, Optics letters.

[16]  E Gratton,et al.  Scanning FCS, a novel method for three-dimensional particle tracking. , 2003, Biochemical Society transactions.

[17]  Enrico Gratton,et al.  Distance measurement by circular scanning of the excitation beam in the two‐photon microscope , 2004, Microscopy research and technique.

[18]  S. Ram,et al.  Simultaneous imaging of different focal planes in fluorescence microscopy for the study of cellular dynamics in three dimensions , 2004, IEEE Transactions on NanoBioscience.

[19]  Feng Zhang,et al.  Assembly of endocytic machinery around individual influenza viruses during viral entry , 2004, Nature Structural &Molecular Biology.

[20]  Enrico Gratton,et al.  3-D particle tracking in a two-photon microscope: application to the study of molecular dynamics in cells. , 2005, Biophysical journal.

[21]  M. Ogris,et al.  Cryoconserved shielded and EGF receptor targeted DNA polyplexes: cellular mechanisms. , 2005, European journal of pharmaceutics and biopharmaceutics : official journal of Arbeitsgemeinschaft fur Pharmazeutische Verfahrenstechnik e.V.

[22]  Mark Marsh,et al.  Virus Entry: Open Sesame , 2006, Cell.

[23]  Sung Wan Kim,et al.  Current status of polymeric gene delivery systems. , 2006, Advanced drug delivery reviews.

[24]  Kevin Braeckmans,et al.  The transport of nanosized gene carriers unraveled by live-cell imaging. , 2006, Angewandte Chemie.

[25]  W. Moerner,et al.  Suppressing Brownian motion of individual biomolecules in solution. , 2006, Proceedings of the National Academy of Sciences of the United States of America.

[26]  Zhuo Gan,et al.  Elucidation of intracellular recycling pathways leading to exocytosis of the Fc receptor, FcRn, by using multifocal plane microscopy , 2007, Proceedings of the National Academy of Sciences.

[27]  H. Balci,et al.  Three-dimensional particle tracking via bifocal imaging. , 2007, Nano letters.

[28]  C. Bräuchle,et al.  Cellular dynamics of EGF receptor-targeted synthetic viruses. , 2007, Molecular therapy : the journal of the American Society of Gene Therapy.

[29]  James H. Werner,et al.  Three-dimensional tracking of individual quantum dots , 2007 .

[30]  X. Zhuang,et al.  Virus trafficking – learning from single-virus tracking , 2007, Nature Reviews Microbiology.

[31]  Y. Katayama,et al.  Simultaneous Transport of Different Localized mRNA Species Revealed by Live‐Cell Imaging , 2008, Traffic.