Department of Biomedical Engineering, Northwestern University, 2145 Sheridan Road, Evanston, Illinois 60208, United States.
Department of Mechanical Engineering, Northwestern University, 2145 Sheridan Road, Evanston, Illinois 60208, United States.
Langmuir. 2020 Mar 10;36(9):2291-2299. doi: 10.1021/acs.langmuir.9b03149. Epub 2020 Feb 25.
Self-assembled nanocarriers have inspired a range of applications for bioimaging, diagnostics, and drug delivery. The noninvasive visualization and characterization of these nanocarriers are important to understand their structure to function relationship. However, the quantitative visualization of nanocarriers in the sample's native environment remains challenging with the use of existing technologies. Single-molecule localization microscopy (SMLM) has the potential to provide both high-resolution visualization and quantitative analysis of nanocarriers in their native environment. However, nonspecific binding of fluorescent probes used in SMLM can introduce artifacts, which imposes challenges in the quantitative analysis of SMLM images. We showed the feasibility of using spectroscopic point accumulation for imaging in nanoscale topography (sPAINT) to visualize self-assembled polymersomes (PS) with molecular specificity. Furthermore, we analyzed the unique spectral signatures of Nile Red (NR) molecules bound to the PS to reject artifacts from nonspecific NR bindings. We further developed quantitative spectroscopic analysis for cluster extraction (qSPACE) to increase the localization density by 4-fold compared to sPAINT; thus, reducing variations in PS size measurements to less than 5%. Finally, using qSPACE, we quantitatively imaged PS at various concentrations in aqueous solutions with ∼20 nm localization precision and 97% reduction in sample misidentification relative to conventional SMLM.
自组装纳米载体在生物成像、诊断和药物输送方面激发了一系列应用。为了了解其结构与功能关系，对这些纳米载体进行非侵入性可视化和特征描述非常重要。然而，利用现有技术，在样品的天然环境中对纳米载体进行定量可视化仍然具有挑战性。单分子定位显微镜（SMLM）具有在天然环境中对纳米载体进行高分辨率可视化和定量分析的潜力。然而，SMLM 中使用的荧光探针的非特异性结合会引入伪影，这给 SMLM 图像的定量分析带来了挑战。我们展示了使用光谱点积累成像纳米形貌（sPAINT）以分子特异性可视化自组装聚合物囊泡（PS）的可行性。此外，我们分析了与 PS 结合的尼罗红（NR）分子的独特光谱特征，以排除非特异性 NR 结合产生的伪影。我们进一步开发了用于簇提取的定量光谱分析（qSPACE），与 sPAINT 相比，将定位密度提高了 4 倍；因此，将 PS 尺寸测量的变化减少到小于 5%。最后，使用 qSPACE，我们在水溶液中以约 20nm 的定位精度对各种浓度的 PS 进行了定量成像，与传统 SMLM 相比，样品误识别率降低了 97%。
