A three-dimensional (3D) single fluorescent particle tracking strategy based on temporal focusing multiphoton excitation microscopy (TFMPEM) combined with astigmatism imaging is proposed for delivering nanoscale-level axial information that reveals 3D trajectories of single fluorospheres in the axially-resolved multiphoton excitation volume without z-axis scanning. It provides the dynamical ability by measuring the diffusion coefficient of fluorospheres in glycerol solutions with a position standard deviation of 14 nm and 21 nm in the lateral and axial direction and a frame rate of 100 Hz. Moreover, the optical trapping force based on the TFMPEM is minimized to avoid the interference in the tracing measurements compared to that in the spatial focusing MPE approaches. Therefore, we presented a three dimensional single particle tracking strategy to overcome the limitation of the time resolution of the multiphoton imaging using fast frame rate of TFMPEM, and provide three dimensional locations of multiple particles using an astigmatism method.
Chi-Hsiang Lien, Chun-Yu Lin, Shean-Jen Chen, and Fan-Ching Chien, "Dynamical measurements of motion behavior of free fluorescent sphere using the wide field temporal focusing microscopy with astigmatism method (Conference Presentation)," Proc. SPIE 10069, Multiphoton Microscopy in the Biomedical Sciences XVII, 100691V (Presented at SPIE BiOS: February 01, 2017; Published: 24 April 2017); https://doi.org/10.1117/12.2252323.5380060933001.
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