title: 3D orbital tracking in a modified two-photon microscope: an application to the tracking of intracellular vesicles.
abstract: The objective of this video protocol is to discuss how to perform and analyze a three-dimensional fluorescent orbital particle tracking experiment using a modified two-photon microscope(1). As opposed to conventional approaches (raster scan or wide field based on a stack of frames), the 3D orbital tracking allows to localize and follow with a high spatial (10 nm accuracy) and temporal resolution (50 Hz frequency response) the 3D displacement of a moving fluorescent particle on length-scales of hundreds of microns(2). The method is based on a feedback algorithm that controls the hardware of a two-photon laser scanning microscope in order to perform a circular orbit around the object to be tracked: the feedback mechanism will maintain the fluorescent object in the center by controlling the displacement of the scanning beam(3-5). To demonstrate the advantages of this technique, we followed a fast moving organelle, the lysosome, within a living cell(6,7). Cells were plated according to standard protocols, and stained using a commercially lysosome dye. We discuss briefly the hardware configuration and in more detail the control software, to perform a 3D orbital tracking experiment inside living cells. We discuss in detail the parameters required in order to control the scanning microscope and enable the motion of the beam in a closed orbit around the particle. We conclude by demonstrating how this method can be effectively used to track the fast motion of a labeled lysosome along microtubules in 3D within a live cell. Lysosomes can move with speeds in the range of 0.4-0.5 µm/sec, typically displaying a directed motion along the microtubule network(8).
authors.affiliation: Biomedical Engineering, Laboratory for Fluorescence Dynamics, University of California, Irvine.
authors.forename: Andrea
authors.initials: A
authors.lastname: Anzalone
authors.affiliation: Biomedical Engineering, Laboratory for Fluorescence Dynamics, University of California, Irvine.
authors.forename: Paolo
authors.initials: P
authors.lastname: Annibale
authors.affiliation: Biomedical Engineering, Laboratory for Fluorescence Dynamics, University of California, Irvine; egratton@uci.edu.
authors.forename: Enrico
authors.initials: E
authors.lastname: Gratton
mesh_terms: D000818:Animals; D016466:CHO Cells; D058948:Cell Tracking / Q000295:instrumentation / Q000379:methods; D003412:Cricetulus; D005456:Fluorescent Dyes / Q000032:analysis / Q000737:chemistry; D021621:Imaging, Three-Dimensional / Q000295:instrumentation / Q000379:methods; D008247:Lysosomes / Q000737:chemistry* / Q000502:physiology; D036641:Microscopy, Fluorescence, Multiphoton / Q000295:instrumentation* / Q000379:methods*; D008870:Microtubules / Q000737:chemistry / Q000648:ultrastructure; D009038:Motion
chemical_list: D005456:Fluorescent Dyes