摘要
Localisation microscopy overcomes the diffraction limit by measuring the position of individual molecules to obtain optical images with a lateral resolution better than 30 nm. Single molecule localisation microscopy was originally demonstrated only in two dimensions but has recently been extended to three dimensions. Here we develop a new approach to three-dimensional （3D） localisation microscopy by engineering of the point-spread function （PSF） of a fluorescence microscope. By introducing a linear phase gradient between the two halves of the objective pupil plane the PSF is split into two lateral lobes whose relative position depends on defocus. Calculations suggested that the phase gradient resulting from the very small tolerances in parallelism of conventional slides made from float glass would be sufficient to generate a two-lobed PSF. We demonstrate that insertion of a suitably chosen microscope slide that occupies half the objective aperture combined with a novel fast fitting algorithm for 3D localisation estimation allows nanoscopic imaging with detail resolution well below 100 nm in all three dimensions （standard deviations of 20, 16, and 42 nm in x, y, and z directions, respectively）. The utility of the approach is shown by imaging the complex 3D distribution of microtubules in cardiac muscle cells that were stained with conventional near infrared fluorochromes. The straightforward optical setup, minimal hardware requirements and large axial localisation range make this approach suitable for many nanoscopic imaging applications.
本地化显微镜学由测量单个分子的位置比 30 nm 更好与侧面的分辨率获得光图象克服衍射限制。单个分子本地化显微镜学原来仅仅在二种尺寸被表明，但是最近被扩大了到三种尺寸。这里，我们开发一条新途径到三维(3D ) 由一台荧光显微镜的点传播功能(PSF ) 的工程的本地化显微镜学。由介绍在客观学生飞机的二一半之间的一个线性阶段坡度， PSF 被分成位置取决于其亲戚的二片侧面的脑叶散焦。计算建议从小忍耐导致用漂流玻璃做的常规幻灯片的并行的阶段坡度将是足够的产生二脑叶的 PSF。我们表明占据与快为 3D 本地化评价适合算法的一篇小说相结合的半客观的孔的一个合适选择的显微镜幻灯片的那插入在所有三种尺寸在 100 nm 下面与详细分辨率允许 nanoscopic 成像很好(在 x 的 20 ， 16 ，和 42 nm 的标准差， y ，和 z 方向，分别地)。途径的用途被成像在与常规近红外线的 fluorochromes 被染色的心肌房间看微导管的复杂 3D 分发。直接光安装，最小的硬件要求和大轴的本地化范围为许多 nanoscopic 成像应用程序使这条途径合适。
