Schodt David J, Farzam Farzin, Liu Sheng, Lidke Keith A
Department of Physics and Astronomy, University of New Mexico, Albuquerque, New Mexico 87106, USA.
Contributed equally.
Biomed Opt Express. 2022 Dec 20;14(1):429-440. doi: 10.1364/BOE.477501. eCollection 2023 Jan 1.
We describe a dedicated microscope for automated sequential localization microscopy which we term Sequential Super-resolution Microscope (SeqSRM). This microscope automates precise stage stabilization on the order of 5-10 nanometers and data acquisition of all user-selected cells on a coverslip, limiting user interaction to only cell selection and buffer exchanges during sequential relabeling. We additionally demonstrate that nanometer-scale changes to cell morphology affect the fidelity of the resulting multi-target super-resolution overlay reconstructions generated by sequential super-resolution microscopy, and that regions affected by these shifts can be reliably detected and masked out using brightfield images collected periodically throughout the experiment. The SeqSRM enables automated multi-target imaging on multiple user-selected cells without the need for multiple distinct fluorophores and emission channels, while ensuring that the resulting multi-target localization data accurately reflect the relative organization of the underlying targets.
我们描述了一种用于自动序列定位显微镜的专用显微镜，我们将其称为序列超分辨率显微镜（SeqSRM）。该显微镜能自动将精确的载物台稳定在5-10纳米的量级，并对盖玻片上所有用户选择的细胞进行数据采集，在序列重新标记过程中，将用户交互限制为仅细胞选择和缓冲液交换。我们还证明，细胞形态的纳米级变化会影响序列超分辨率显微镜生成的多目标超分辨率叠加重建的保真度，并且在整个实验过程中定期收集的明场图像能够可靠地检测和掩盖受这些变化影响的区域。SeqSRM能够在多个用户选择的细胞上进行自动多目标成像，无需多个不同的荧光团和发射通道，同时确保生成的多目标定位数据准确反映基础目标的相对组织。
