Quantifying distortions in two-photon remote focussing microscope images using a volumetric calibration specimen
Corbett, Alexander D.
Burton, Rebecca A.B.
Salter, Patrick S.
Booth, Martin J.
Frontiers in Physiology
Copyright © 2014 Corbett, Burton, Bub, Salter, Tuohy, Booth and Wilson. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY): http://creativecommons.org/licenses/by/4.0/. The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
Remote focussing microscopy allows sharp, in-focus images to be acquired at high speed from outside of the focal plane of an objective lens without any agitation of the specimen. However, without careful optical alignment, the advantages of remote focussing microscopy could be compromised by the introduction of depth-dependent scaling artifacts. To achieve an ideal alignment in a point-scanning remote focussing microscope, the lateral (XY) scan mirror pair must be imaged onto the back focal plane of both the reference and imaging objectives, in a telecentric arrangement. However, for many commercial objective lenses, it can be difficult to accurately locate the position of the back focal plane. This paper investigates the impact of this limitation on the fidelity of three-dimensional data sets of living cardiac tissue, specifically the introduction of distortions. These distortions limit the accuracy of sarcomere measurements taken directly from raw volumetric data. The origin of the distortion is first identified through simulation of a remote focussing microscope. Using a novel three-dimensional calibration specimen it was then possible to quantify experimentally the size of the distortion as a function of objective misalignment. Finally, by first approximating and then compensating the distortion in imaging data from whole heart rodent studies, the variance of sarcomere length (SL) measurements was reduced by almost 50%.
Medical Research Council (MRC)
Engineering and Physical Sciences Research Council (EPSRC)
Biotechnology and Biological Sciences Research Council (BBSRC)
British Heart Foundation Centre of Research Excellence, Oxford
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Vol. 5, article 384