de Zwaan Klarinda, Huo Ran, Hensgens Myron N F, Wienecke Hannah Lena, Tekpınar Miyase, Geertsema Hylkje, Grußmayer Kristin
Department of Bionanoscience, Kavli Institute of Nanoscience Delft, Delft University of Technology, 2629 HZ Delft, The Netherlands.
Department of Imaging Physics, Delft University of Technology, 2628 CJ Delft, The Netherlands.
ACS Nano. 2025 Apr 8;19(13):13149-13159. doi: 10.1021/acsnano.4c18502. Epub 2025 Mar 27.
Super-resolution microscopy facilitates the visualization of cellular structures at a resolution approaching the molecular level. Especially, super-resolution techniques based on the localization of single molecules have relatively modest instrument requirements and are thus good candidates for adoption in bioimaging. However, their low-throughput nature hampers their applicability in biomolecular research and screening. Here, we propose a workflow for more efficient data collection, starting with the scanning of large areas using fast fluctuation-based imaging, followed by single-molecule localization microscopy of selected cells. To achieve this workflow, we exploit the versatility of DNA oligo hybridization kinetics with DNA-PAINT probes to tailor the fluorescent blinking toward high-throughput and high-resolution imaging. Additionally, we employ super-resolution optical fluctuation imaging (SOFI) to analyze statistical fluctuations in the DNA-PAINT binding kinetics, thereby tolerating much denser blinking and facilitating accelerated imaging speeds. Thus, we demonstrate 30-300-fold faster imaging of different cellular structures compared to conventional DNA-PAINT imaging, albeit at a lower resolution. Notably, by tuning the image medium and data processing though, we can flexibly switch between high-throughput SOFI (scanning an FOV of 0.65 mm × 0.52 mm within 4 min of total acquisition time) and super-resolution DNA-PAINT microscopy and thereby demonstrate that combining DNA-PAINT and SOFI enables one to adapt image resolution and acquisition time based on the imaging needs. We envision this approach to be especially powerful when combined with multiplexing and 3D imaging.
超分辨率显微镜能够以接近分子水平的分辨率实现细胞结构的可视化。特别是，基于单分子定位的超分辨率技术对仪器的要求相对较低，因此是生物成像中很有潜力被采用的技术。然而，其低通量的特性限制了它们在生物分子研究和筛选中的应用。在此，我们提出一种更高效的数据采集工作流程，首先使用基于快速涨落的成像技术对大面积区域进行扫描，然后对选定的细胞进行单分子定位显微镜成像。为实现这一工作流程，我们利用DNA寡核苷酸杂交动力学与DNA-PAINT探针的多功能性，使荧光闪烁适用于高通量和高分辨率成像。此外，我们采用超分辨率光学涨落成像（SOFI）来分析DNA-PAINT结合动力学中的统计涨落，从而能够容忍更密集的闪烁并加快成像速度。因此，与传统的DNA-PAINT成像相比，我们展示了对不同细胞结构快30-300倍的成像速度，尽管分辨率较低。值得注意的是，通过调整成像介质和数据处理方式，我们可以在高通量SOFI（在4分钟的总采集时间内扫描0.65毫米×0.52毫米的视野）和超分辨率DNA-PAINT显微镜之间灵活切换，从而证明将DNA-PAINT和SOFI相结合能够根据成像需求调整图像分辨率和采集时间。我们设想，当与多路复用和三维成像相结合时，这种方法将特别强大。
