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{"title":"Light Sheet Fluorescence Microscopy (LSFM)","authors":"Michael W. Adams, Andrew F. Loftus, Sarah E. Dunn, Matthew S. Joens, James A.J. Fitzpatrick","doi":"10.1002/0471142956.cy1237s71","DOIUrl":null,"url":null,"abstract":"<p>The development of confocal microscopy techniques introduced the ability to optically section fluorescent samples in the axial dimension, perpendicular to the image plane. These approaches, via the placement of a pinhole in the conjugate image plane, provided superior resolution in the axial (z) dimension resulting in nearly isotropic optical sections. However, increased axial resolution, via pinhole optics, comes at the cost of both speed and excitation efficiency. Light sheet fluorescent microscopy (LSFM), a century-old idea made possible with modern developments in both excitation and detection optics, provides sub-cellular resolution and optical sectioning capabilities without compromising speed or excitation efficiency. Over the past decade, several variations of LSFM have been implemented each with its own benefits and deficiencies. Here we discuss LSFM fundamentals and outline the basic principles of several major light-sheet-based imaging modalities (SPIM, inverted SPIM, multi-view SPIM, Bessel beam SPIM, and stimulated emission depletion SPIM) while considering their biological relevance in terms of intrusiveness, temporal resolution, and sample requirements. © 2015 by John Wiley & Sons, Inc.</p>","PeriodicalId":11020,"journal":{"name":"Current Protocols in Cytometry","volume":"71 1","pages":""},"PeriodicalIF":0.0000,"publicationDate":"2015-01-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/0471142956.cy1237s71","citationCount":"21","resultStr":null,"platform":"Semanticscholar","paperid":null,"PeriodicalName":"Current Protocols in Cytometry","FirstCategoryId":"1085","ListUrlMain":"https://onlinelibrary.wiley.com/doi/10.1002/0471142956.cy1237s71","RegionNum":0,"RegionCategory":null,"ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q1","JCRName":"Health Professions","Score":null,"Total":0}
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Abstract
The development of confocal microscopy techniques introduced the ability to optically section fluorescent samples in the axial dimension, perpendicular to the image plane. These approaches, via the placement of a pinhole in the conjugate image plane, provided superior resolution in the axial (z) dimension resulting in nearly isotropic optical sections. However, increased axial resolution, via pinhole optics, comes at the cost of both speed and excitation efficiency. Light sheet fluorescent microscopy (LSFM), a century-old idea made possible with modern developments in both excitation and detection optics, provides sub-cellular resolution and optical sectioning capabilities without compromising speed or excitation efficiency. Over the past decade, several variations of LSFM have been implemented each with its own benefits and deficiencies. Here we discuss LSFM fundamentals and outline the basic principles of several major light-sheet-based imaging modalities (SPIM, inverted SPIM, multi-view SPIM, Bessel beam SPIM, and stimulated emission depletion SPIM) while considering their biological relevance in terms of intrusiveness, temporal resolution, and sample requirements. © 2015 by John Wiley & Sons, Inc.
薄片荧光显微镜(LSFM)
共聚焦显微镜技术的发展引入了在垂直于成像平面的轴向尺寸上对荧光样品进行光学切片的能力。这些方法通过在共轭成像平面上放置针孔,在轴向(z)尺寸上提供了优越的分辨率,从而产生了几乎各向同性的光学切片。然而,通过针孔光学增加轴向分辨率,是以速度和激发效率为代价的。光片荧光显微镜(LSFM)是一个有着百年历史的想法,随着激发和检测光学的现代发展,它提供了亚细胞分辨率和光学切片能力,而不会影响速度或激发效率。在过去的十年中,已经实现了LSFM的几种变体,每种变体都有自己的优点和不足。在这里,我们讨论了LSFM的基本原理,并概述了几种主要的基于光片的成像模式(SPIM,倒置SPIM,多视图SPIM,贝塞尔光束SPIM和受激发射耗尽SPIM)的基本原理,同时考虑了它们在侵入性,时间分辨率和样品要求方面的生物学相关性。©2015 by John Wiley &儿子,Inc。
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