Quantifying distortions in two-photon remote focussing microscope images using a volumetric calibration specimen.
Quantifying distortions in two-photon remote focussing microscope images using a volumetric calibration specimen.
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DOI：
10.3389/fphys.2014.00384
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发表时间：
2014
影响因子：
4
通讯作者：
Wilson T
中科院分区：
文献类型：
--
作者：
Corbett AD;Burton RA;Bub G;Salter PS;Tuohy S;Booth MJ;Wilson T
远程聚焦显微镜允许锐利，在焦点图像，以高速从外部的物镜透镜的焦平面采集，而没有任何搅拌的标本。然而，如果没有仔细的光学对准，远程聚焦显微镜的优点可能会受到影响的深度相关的缩放文物的介绍。为了在点扫描远程聚焦显微镜中实现理想的对准，横向（XY）扫描镜对必须在远心布置中成像到参考物镜和成像物镜的后焦平面上。然而，对于许多商用物镜，可能难以精确地定位后焦平面的位置。本文研究了这种限制对活体心脏组织三维数据集保真度的影响，特别是失真的引入。这些失真限制了直接从原始体积数据获得的肌节测量的准确性。变形的起源首先确定通过模拟的远程聚焦显微镜。使用一种新的三维校准标本，然后可以量化实验的失真的大小作为一个功能的目标错位。最后，通过首先近似，然后补偿整个心脏啮齿动物研究的成像数据中的失真，肌节长度（SL）测量的方差减少了近50%。
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%.
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影响因子：
48
作者：
Abrahamsson, Sara;Chen, Jiji;Hajj, Bassam;Stallinga, Sjoerd;Katsov, Alexander Y.;Wisniewski, Jan;Mizuguchi, Gaku;Soule, Pierre;Mueller, Florian;Darzacq, Claire Dugast;Darzacq, Xavier;Wu, Carl;Bargmann, Cornelia I.;Agard, David A.;Dahan, Maxime;Gustafsson, Mats G. L.
通讯作者：
Gustafsson, Mats G. L.
影响因子：
3.8
作者：
Salter, P. S.;Baum, M.;Booth, M. J.
通讯作者：
Booth, M. J.
影响因子：
3.8
作者：
Jesacher, Alexander;Booth, Martin J.
通讯作者：
Booth, Martin J.
影响因子：
3.8
作者：
Botcherby, Edward J.;Booth, Martin J.;Wilson, Tony
通讯作者：
Wilson, Tony
影响因子：
8.6
作者：
Salter, P. S.;Carbone, G.;Raynes, E. P.
通讯作者：
Raynes, E. P.