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30 December 2019 Achieving 3D FRAP using multiphoton polygon scanning microscopy
Yean J. Lim, Yongxiao Li, Woei M. Lee
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Proceedings Volume 11202, Biophotonics Australasia 2019; 1120217 (2019) https://doi.org/10.1117/12.2539683
Event: ANZCOP, 2019, Melbourne, Australia
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.
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Yean J. Lim, Yongxiao Li, and Woei M. Lee "Achieving 3D FRAP using multiphoton polygon scanning microscopy", Proc. SPIE 11202, Biophotonics Australasia 2019, 1120217 (30 December 2019); https://doi.org/10.1117/12.2539683
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KEYWORDS
Microscopy
Diffusion
Multiphoton microscopy
Microscopes
Molecules
Image resolution
Multiphoton fluorescence microscopy
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