Département de Physique, de Génie Physique et d'Optique, Centre d'Optique, Photonique et Laser, Université Laval Québec, QC, Canada ; Centre de Recherche de l'Institut Universitaire en Santé Mentale de Québec Québec, QC, Canada.
Centre de Recherche de l'Institut Universitaire en Santé Mentale de Québec Québec, QC, Canada.
Front Cell Neurosci. 2014 May 20;8:139. doi: 10.3389/fncel.2014.00139. eCollection 2014.
Two-photon microscopy has revolutionized functional cellular imaging in tissue, but although the highly confined depth of field (DOF) of standard set-ups yields great optical sectioning, it also limits imaging speed in volume samples and ease of use. For this reason, we recently presented a simple and retrofittable modification to the two-photon laser-scanning microscope which extends the DOF through the use of an axicon (conical lens). Here we demonstrate three significant benefits of this technique using biological samples commonly employed in the field of neuroscience. First, we use a sample of neurons grown in culture and move it along the z-axis, showing that a more stable focus is achieved without compromise on transverse resolution. Second, we monitor 3D population dynamics in an acute slice of live mouse cortex, demonstrating that faster volumetric scans can be conducted. Third, we acquire a stereoscopic image of neurons and their dendrites in a fixed sample of mouse cortex, using only two scans instead of the complete stack and calculations required by standard systems. Taken together, these advantages, combined with the ease of integration into pre-existing systems, make the extended depth-of-field imaging based on Bessel beams a strong asset for the field of microscopy and life sciences in general.
双光子显微镜技术已经彻底改变了组织中的功能细胞成像，但尽管标准设置具有高度受限的景深（DOF），能够实现出色的光学切片，但它也限制了体积样本的成像速度和易用性。出于这个原因，我们最近对双光子激光扫描显微镜进行了简单的改装和改造，通过使用轴棱锥（锥形透镜）来扩展景深。在这里，我们使用神经科学领域常用的生物样本展示了这项技术的三个重要优势。首先，我们使用培养的神经元样本在 z 轴上移动，结果表明，在不影响横向分辨率的情况下，可以实现更稳定的焦点。其次，我们在活体小鼠皮层的急性切片中监测 3D 群体动力学，证明可以进行更快的体积扫描。第三，我们使用仅两次扫描而不是标准系统所需的完整堆栈和计算，从固定的小鼠皮层样本中获取神经元及其树突的立体图像。总之，这些优势，加上易于集成到现有系统中，使得基于贝塞尔光束的扩展景深成像成为显微镜和生命科学领域的有力工具。
