Abstract：
Confining light illumination in the three dimensions of space is a challenge for various applications. Among these, optogenetic methods developed for live experiments in cell biology would benefit from such a localized illumination as it would improve the spatial resolution of diffusive photosensitive proteins leading to spatially constrained biological responses in specific subcellular organelles. Here, we describe a method to create and move a focused evanescent spot, at the interface between a glass substrate and an aqueous sample, across the field of view of a high numerical aperture microscope objective, using a digital micro-mirror device (DMD). We show that, after correcting the optical aberrations, light is confined within a spot of sub-micron lateral size and ∼100 nm axial depth above the coverslip, resulting in a volume of illumination drastically smaller than the one generated by a standard propagative focus. This evanescent focus is sufficient to induce a more intense and localized recruitment compared to a propagative focus on the optogenetic system CRY2-CIBN, improving the resolution of its pattern of activation.
摘要：
在三维空间中限制光照明是各种应用的挑战。其中,为细胞生物学中的活实验开发的光遗传学方法将受益于这种局部照明，因为它将提高扩散光敏蛋白的空间分辨率，从而导致特定亚细胞细胞器中空间受限的生物反应。这里,我们描述了一种创建和移动聚焦消逝点的方法，在玻璃基板和水性样品之间的界面处，在高数值孔径显微镜物镜的视野中，使用数字微镜器件（DMD）。我们证明,校正光学像差后，光线被限制在盖玻片上方亚微米横向尺寸和100纳米轴向深度的斑点内，导致照明量大大小于由标准传播焦点产生的照明量。与光遗传系统CRY2-CIBN上的繁殖聚焦相比，这种渐逝聚焦足以诱导更强烈和局部的募集，提高其激活模式的分辨率。
