Open AccessDual-color dynamic tracking of GM-CSF receptors/JAK2 kinases signaling activation using temporal focusing multiphoton fluorescence excitation and astigmatic imaging
Author(s) -
FanChing Chien,
Chunshan Liu,
Chi-Hsiang Lien,
Yang-Hong Dai,
J. J. Yen
Publication year - 2015
Publication title -
optics express
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.394
H-Index - 271
ISSN - 1094-4087
DOI - 10.1364/oe.23.030943
Subject(s) - optics , photobleaching , excitation , microscopy , fluorescence , materials science , microscope , fluorescence lifetime imaging microscopy , optical tweezers , fluorescence microscope , optical sectioning , tracking (education) , physics , quantum mechanics , psychology , pedagogy
The dual-color dynamic particle tracking approach that uses temporal focusing multiphoton fluorescence excitation and two-channel astigmatic imaging is utilized to track molecular trajectories in three dimensions to explore molecular interactions. Images of two fluorophores were obtained to extract their positions by optical sectioning excitation using a fast temporal focusing multiphoton excitation microscope (TFMPEM) and by the simultaneous collection of data in two channels. The presented pair of cylindrical lenses, which was used to adjust the astigmatism effect with the minimum shifting of the imaging plane, was more feasible and flexible than single cylindrical lens for aligning two separate detection channels in astigmatic imaging. The lateral and axial positioning resolutions were observed to be approximately 9-13 nm and 23-30 nm respectively, for the two fluorescence channels. The dynamic movement and binding behavior of clusters of GM-CSF receptors and JAK2 kinases in HeLa cells in the presence of GM-CSF ligands were observed. Therefore, the proposed dual-color tracking strategy is useful for the dynamic study of molecular interactions in living specimens with a fast frame rate, less photobleaching, better penetration depth, and minimum optical trapping force.