Duocastella Martí, Arnold Craig B, Puchalla Jason
Department of Nanophysics, Istituto Italiano di Tecnologia, Genoa, Via Morego 30, 16163, Italy.
Department of Mechanical and Aerospace Engineering, Princeton University, Olden St, Princeton, NJ, 08544, USA.
Microsc Res Tech. 2017 Feb;80(2):250-259. doi: 10.1002/jemt.22795. Epub 2016 Nov 6.
Light-sheet fluorescence microscopy (LSFM) is an optical sectioning technique capable of rapid three-dimensional (3D) imaging of a wide range of specimens with reduced phototoxicity and superior background rejection. However, traditional light-sheet generation approaches based on elliptical or circular Gaussian beams suffer an inherent trade-off between light-sheet thickness and area over which this thickness is preserved. Recently, an increase in light-sheet uniformity was demonstrated using rapid biaxial Gaussian beam scanning along the lateral and beam propagation directions. Here we apply a similar scanning concept to an elliptical beam generated by a cylindrical lens. In this case, only z-scanning of the elliptical beam is required and hence experimental implementation of the setup can be simplified. We introduce a simple dimensionless uniformity statistic to better characterize scanned light-sheets and experimentally demonstrate custom tailored uniformities up to a factor of 5 higher than those of unscanned elliptical beams. This technique offers a straightforward way to generate and characterize a custom illumination profile that provides enhanced utilization of the detector dynamic range and field of view, opening the door to faster and more efficient 2D and 3D imaging.
光片荧光显微镜（LSFM）是一种光学切片技术，能够对各种标本进行快速三维（3D）成像，同时降低光毒性并具有出色的背景抑制能力。然而，基于椭圆或圆形高斯光束的传统光片生成方法在光片厚度与保持该厚度的区域之间存在固有的权衡。最近，通过沿横向和光束传播方向进行快速双轴高斯光束扫描，证明了光片均匀性有所提高。在这里，我们将类似的扫描概念应用于由柱面透镜产生的椭圆光束。在这种情况下，仅需要对椭圆光束进行z扫描，因此可以简化设置的实验实施。我们引入了一个简单的无量纲均匀性统计量，以更好地表征扫描光片，并通过实验证明定制的均匀性比未扫描的椭圆光束高出5倍。该技术提供了一种直接的方法来生成和表征定制照明轮廓，从而提高探测器动态范围和视野的利用率，为更快、更高效的二维和三维成像打开了大门。
