Error-Correction Method for High-Throughput Sizing of Nanoscale Vesicles with Single-Molecule Localization Microscopy.
Error-Correction Method for High-Throughput Sizing of Nanoscale Vesicles with Single-Molecule Localization Microscopy.
复制标题
使用单分子定位显微镜高通量测定纳米级囊泡的误差校正方法。
DOI：
10.1021/acs.jpcb.2c09053
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发表时间：
2023
期刊：
影响因子：
--
通讯作者：
Chiu,DanielT
中科院分区：
文献类型：
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作者：
Jung,Seung-Ryoung;Kim,James;Vojtech,Lucia;Vaughan,JoshuaC;Chiu,DanielT
单分子定位显微镜（SMLM）允许生物纳米结构（如细胞器和细胞外囊泡（EV））的超分辨率成像，绘图，计数和尺寸确定，但由于点扩散函数的失真和有限的光子数引起的单分子定位误差，尺寸小于100 nm的结构可能不准确。在这里，我们展示了一种方法来纠正定位误差时，大小囊泡和其他球形纳米粒子与SMLM和比较大小的结果，使用两个囊泡标记计划。我们使用平均近似理论推导出一个简单的方程，使用半高宽（FWHM）校正颗粒尺寸测量的二维SMLM，验证的方法，通过大小链霉亲和素包被的聚苯乙烯纳米珠与SMLM techniquedSTORM有和没有误差校正，使用透射电子显微镜（TEM）进行比较，然后应用该方法大小的小精液EV。对于小于50 nm的珠粒，通过dSTORM测量的纳米珠粒尺寸变得越来越不准确（大于TEM值）。误差校正方法相对于TEM的尺寸差异从没有误差校正的15%降低到40 nm珠的误差校正的7%，从44%降低到30 nm珠的9%，以及从66%降低到20 nm珠的15%。用亲脂性膜染料（MemBright 700）和Alexa Fluor 488-抗-CD 63抗体缀合物标记精液EV，并使用两种染料bydSTORM分别测定大小。误差校正的外泌体直径小于未校正的值：膜染料的平均直径为72 nm对79 nm;抗体缀合染料的平均直径为84 nm对97 nm。当使用膜染料时，平均误差校正直径比当使用抗体缀合染料时小12 nm，这可能是由于抗体的大尺寸。因此，误差校正方法和紧凑的膜标记方案减少了SMLM对囊泡大小的高估。这种误差校正方法具有低计算成本，因为它不需要校正各个眨眼事件，并且它与所有SMLM技术（例如，PALM、STORM和DNA-PAINT）。
Single-molecule localization microscopy (SMLM) allows super-resolution imaging, mapping, counting, and sizing of biological nanostructures such as cell organelles and extracellular vesicles (EVs), but sizing structures smaller than ∼100 nm can be inaccurate due to single-molecule localization error caused by distortion of the point spread function and limited photon number. Here we demonstrate a method to correct localization error when sizing vesicles and other spherical nanoparticles with SMLM and compare sizing results using two vesicle labeling schemes. We use mean approximation theory to derive a simple equation using full width at half-maximum (FWHM) for correcting particle sizes measured by two-dimensional SMLM, validate the method by sizing streptavidin-coated polystyrene nanobeads with the SMLM techniquedSTORM with and without error correction, using transmission electron microscopy (TEM) for comparison, and then apply the method to sizing small seminal EVs. Nanobead sizes measured bydSTORM became increasingly less accurate (larger than TEM values) for beads smaller than 50 nm. The error-correction method reduced the size difference versus TEM from 15% without error correction to 7% with error correction for 40 nm beads, from 44% to 9% for 30 nm beads, and from 66% to 15% for 20 nm beads. Seminal EVs were labeled with a lipophilic membrane dye (MemBright 700) and with an Alexa Fluor 488-anti-CD63 antibody conjugate, and were sized separately using both dyes bydSTORM. Error-corrected exosome diameters were smaller than uncorrected values: 72 nm vs 79 nm mean diameter with membrane dyes; 84 nm vs 97 nm with the antibody-conjugated dyes. The mean error-corrected diameter was 12 nm smaller when using the membrane dye than when using the antibody-conjugated dye likely due to the large size of the antibody. Thus, both the error-correction method and the compact membrane labeling scheme reduce overestimation of vesicle size by SMLM. This error-correction method has a low computational cost as it does not require correction of individual blinking events, and it is compatible with all SMLM techniques (e.g., PALM, STORM, and DNA-PAINT).