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Real‐Time Nanomicroscopy via Three‐Dimensional Single‐Particle Tracking
Author(s) -
Katayama Yoshihiko,
Burkacky Ondrej,
Meyer Martin,
Bräuchle Christoph,
Gratton Enrico,
Lamb Don C.
Publication year - 2009
Publication title -
chemphyschem
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.016
H-Index - 140
eISSN - 1439-7641
pISSN - 1439-4235
DOI - 10.1002/cphc.200900436
Subject(s) - tracking (education) , confocal , microscope , optics , particle (ecology) , temporal resolution , image resolution , resolution (logic) , image plane , fluorescence , field of view , materials science , laser , physics , computer vision , computer science , artificial intelligence , psychology , pedagogy , oceanography , image (mathematics) , geology
Abstract 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.