Spatial light modulation using cost efficient digital micromirror devices (DMD) is finding broad applications in fluorescence microscopy due to the reduction of phototoxicity and bleaching and the ability to manipulate proteins in optogenetic experiments. However, precise illumination by DMDs and their application to single-molecule localization microscopy (SMLM) remained a challenge because of non-linear distortions between the DMD and camera coordinate systems caused by optical components in the excitation and emission path. Here we develop a fast and easy to implement calibration procedure that determines these distortions and matches the DMD and camera coordinate system with a precision below the optical diffraction limit. As a result, a region from a fluorescence image can be selected with a higher precision for illumination compared to a rigid transformation allowed by manual alignment of the DMD. We first demonstrate the application of our precisely calibrated light modulation by performing a proof of concept fluorescence recovery after photobleaching experiment with the endoplasmic reticulum-localized protein IRE1 fused to GFP in budding yeast (S. cerevisiae). Next, we develop a spatially informed photoactivation approach for SMLM in which only regions of the cell that contain photoactivatable fluorescent proteins are selected for photoactivation. The reduced exposure of the cells to 405 nm light increased the possible imaging time by 44% until phototoxic effects cause a dominant fluorescence background and a change in cell morphology. As a result, the mean number of reliable single-molecule localizations was also significantly increased by 28%. Since the localization precision and the ability for single-molecule tracking is not altered compared to traditional photoactivation of the entire field of view, spatially informed photoactivation significantly improves the quality of SMLM images and single-molecule tracking data. Our precise calibration method therefore lays the foundation for improved SMLM with active feedback photoactivation far beyond the applications in this work.

由于光毒性和漂白的减少以及在光遗传学实验中操纵蛋白质的能力，使用具有成本效益的数字微镜器件 (DMD) 的空间光调制在荧光显微镜中得到了广泛的应用。然而，DMD 的精确照明及其在单分子定位显微镜 (SMLM) 中的应用仍然是一个挑战，因为 DMD 和相机坐标系之间的非线性失真是由激发和发射路径中的光学元件引起的。在这里，我们开发了一种快速且易于实施的校准程序，可以确定这些失真，并以低于光学衍射极限的精度匹配 DMD 和相机坐标系。因此，与手动对齐 DMD 所允许的刚性变换相比，可以以更高的精度从荧光图像中选择区域进行照明。我们首先通过在出芽酵母中与 GFP 融合的内质网定位蛋白 IRE1 进行光漂白实验后进行概念荧光恢复来证明我们精确校准的光调制的应用。酿酒酵母）。接下来，我们为 SMLM 开发了一种空间信息光激活方法，其中仅选择包含光激活荧光蛋白的细胞区域进行光激活。细胞暴露于 405 nm 光的减少使可能的成像时间增加了 44%，直到光毒性效应导致显性荧光背景和细胞形态发生变化。结果，可靠的单分子定位的平均数量也显着增加了 28%。由于与整个视场的传统光激活相比，定位精度和单分子跟踪能力没有改变，空间信息光激活显着提高了 SMLM 图像和单分子跟踪数据的质量。