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.
Katayama
Yoshihiko
Y
Department for Chemistry and Biochemistry and Center for Nanoscience (CeNS), Ludwig-Maximilians-Universität München, Butenandtstrasse 11, 81377 Munich, Germany.
Burkacky
Ondrej
O
Meyer
Martin
M
Bräuchle
Christoph
C
Gratton
Enrico
E
Lamb
Don C
DC
D045744:Cell Line, Tumor; D004247:DNA / Q000737:chemistry; D049452:Green Fluorescent Proteins / Q000737:chemistry; D006801:Humans; D008856:Microscopy, Fluorescence / Q000379:methods*; D036103:Nanotechnology; D014404:Tubulin / Q000737:chemistry
D014404:Tubulin; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D004247:DNA