Single particle tracking through highly scattering media with multiplexed two-photon excitation

Evan Perillo; Yen-Liang Liu; Cong Liu; Hsin-Chih Yeh; Andrew K. Dunn

doi:10.1117/12.2079897

9 March 2015 Single particle tracking through highly scattering media with multiplexed two-photon excitation

Evan Perillo, Yen-Liang Liu, Cong Liu, Hsin-Chih Yeh, Andrew K. Dunn

Proceedings Volume 9331, Single Molecule Spectroscopy and Superresolution Imaging VIII; 933107 (2015) https://doi.org/10.1117/12.2079897
Event: SPIE BiOS, 2015, San Francisco, California, United States

Abstract

3D single-particle tracking (SPT) has been a pivotal tool to furthering our understanding of dynamic cellular processes in complex biological systems, with a molecular localization accuracy (10-100 nm) often better than the diffraction limit of light. However, current SPT techniques utilize either CCDs or a confocal detection scheme which not only suffer from poor temporal resolution but also limit tracking to a depth less than one scattering mean free path in the sample (typically <15μm). In this report we highlight our novel design for a spatiotemporally multiplexed two-photon microscope which is able to reach sub-diffraction-limit tracking accuracy and sub-millisecond temporal resolution, but with a dramatically extended SPT range of up to 200 μm through dense cell samples. We have validated our microscope by tracking (1) fluorescent nanoparticles in a prescribed motion inside gelatin gel (with 1% intralipid) and (2) labeled single EGFR complexes inside skin cancer spheroids (at least 8 layers of cells thick) for ~10 minutes. Furthermore we discuss future capabilities of our multiplexed two-photon microscope design, specifically to the extension of (1) simultaneous multicolor tracking (i.e. spatiotemporal co-localization analysis) and (2) FRET studies (i.e. lifetime analysis). The high resolution, high depth penetration, and multicolor features of this microscope make it well poised to study a variety of molecular scale dynamics in the cell, especially related to cellular trafficking studies with in vitro tumor models and in vivo.

Citation Download Citation

Evan Perillo, Yen-Liang Liu, Cong Liu, Hsin-Chih Yeh, and Andrew K. Dunn "Single particle tracking through highly scattering media with multiplexed two-photon excitation", Proc. SPIE 9331, Single Molecule Spectroscopy and Superresolution Imaging VIII, 933107 (9 March 2015); https://doi.org/10.1117/12.2079897

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