Taylor Michael A, Vanwalleghem Gilles C, Favre-Bulle Itia A, Scott Ethan K
School of Biomedical Sciences, The University of Queensland, St. Lucia, QLD, Australia.
J Biophotonics. 2018 Dec;11(12):e201800088. doi: 10.1002/jbio.201800088. Epub 2018 Aug 7.
Light-sheet microscopy is used extensively in developmental biology and neuroscience. One limitation of this approach is that absorption and scattering produces shadows in the illuminating light sheet, resulting in stripe artifacts. Here, we introduce diffuse light-sheet microscopes that use a line diffuser to randomize the light propagation within the image plane, allowing the light sheets to reform after obstacles. We incorporate diffuse light sheets in two existing configurations: selective plane illumination microscopy in which the sample is illuminated with a static sheet of light, and digitally scanned light sheet (DSLS) in which a thin Gaussian beam is scanned across the image plane during each acquisition. We compare diffuse light-sheet microscopes to their conventional counterparts for calcium imaging of neural activity in larval zebrafish. We show that stripe artifacts can cast deep shadows that conceal some neurons, and that the stripes can flicker, producing spurious signals that could be interpreted as biological activity. Diffuse light-sheets mitigate these problems, illuminating the blind spots produced by stripes and removing artifacts produced by the stripes' movements. The upgrade to diffuse light sheets is simple and inexpensive, especially in the case of DSLS, where it requires the addition of one optical element.
光片显微镜在发育生物学和神经科学中得到广泛应用。这种方法的一个局限性在于，吸收和散射会在照明光片中产生阴影，从而导致条纹伪影。在此，我们介绍了漫射光片显微镜，它使用线扩散器使图像平面内的光传播随机化，使光片在遇到障碍物后重新形成。我们将漫射光片应用于两种现有配置中：一种是选择性平面照明显微镜，即用静态光片照射样品；另一种是数字扫描光片（DSLS），即在每次采集过程中，一束细高斯光束在图像平面上扫描。我们将漫射光片显微镜与其传统对应物进行比较，用于斑马鱼幼体神经活动的钙成像。我们发现条纹伪影会投射出深阴影，从而掩盖一些神经元，并且条纹会闪烁，产生可能被解释为生物活动的虚假信号。漫射光片可缓解这些问题，照亮条纹产生的盲点并消除条纹移动产生的伪影。升级到漫射光片简单且成本低廉，特别是在DSLS的情况下，只需添加一个光学元件即可。
