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Achieving 3D FRAP using multiphoton polygon scanning microscopy
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Paper Abstract

Fluorescence recovery after photobleaching (FRAP) has been developed to measure molecular diffusion in living cells. However, conventional FRAP using a single stationary beam guided by a pair of galvanometer mirrors is not tailored for raster scanning microscopy. Furthermore, it has been shown that a single point of 2D FRAP only acquires molecular diffusion within a given imaging plane and does not fully capture the full molecular dynamics. Here, we address these limitations with a custom-built 2-photon polygon scanning microscope that features volumetric scanning with a frame rate of 20 fps and 170 nm pixel size. Importantly, our system allows photomanipulation to selectively measure FRAP from the diffusion dynamics of fluorescent molecules in a 3D sample. To demonstrate these capabilities, we performed rapid axial scans of fluorescent beads in suspension, achieving a volumetric scan rate of less than a second. FRAP functionality was verified in vitro on sulforhodamine-labelled giant unilamellar vesicles and diffusion kinetics determined from the rate of fluorescence recovery. The resolution and speed introduced from polygon scanning microscopy coupled with photomanipulation capabilities sets a precedent for 2-photon 3D FRAP imaging.

Paper Details

Date Published: 30 December 2019
PDF: 2 pages
Proc. SPIE 11202, Biophotonics Australasia 2019, 1120217 (30 December 2019); doi: 10.1117/12.2539683
Show Author Affiliations
Yean J. Lim, The Australian National Univ. (Australia)
Yongxiao Li, The Australian National Univ. (Australia)
Woei M. Lee, The Australian National Univ. (Australia)


Published in SPIE Proceedings Vol. 11202:
Biophotonics Australasia 2019
Ewa M. Goldys; Brant C. Gibson, Editor(s)

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