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Updated: Sep 12, 2025

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光学偏差的系统性表征揭示了冷FLM定位忠实性

Hongjia Li, Lauren Ann Metskas, Fang Huang

bioRxiv : the preprint server for biology

|August 6, 2025

概括

低温相对光和电子显微镜 (cryo-CLEM) 面临的光学偏差限制了分子定位的准确性. 通过点差函数建模来缓解这些扭曲,可以提高精度到10纳米以下.

科学领域:

细胞生物学细胞生物学
结构生物学结构生物学
显微镜的使用方法

背景情况:

低温相对照光和电子显微镜 (cryo-CLEM) 集成了光和电子显微镜,用于在位细胞分析.
将单分子局部化与冷CLEM相结合,可以在细胞超结构内提供高分辨率的分子定位.
低温光光显微镜 (cryo-FLM) 被光学偏差所阻碍,影响了定位精度和下游的低温电子显微镜.

研究的目的:

系统地分析商用冷FLM系统中的光学偏差.
识别影响点差函数 (PSF) 精度的光学扭曲源.
评估缓解冷FLM局部化错误的策略.

主要方法:

在商用冷FLM系统中对光学偏差进行定量分析.
调查系统的不完美,折射率不匹配和样本异质性作为偏差源.
应用空间匹配和自适应的PSF模型来纠正光学扭曲.

主要成果:

发现了显著的光学误差,导致侧向误差高达90nm和轴向误差超过300nm.
这些异常源于系统的不完美,折射率不匹配和样本异质.
PSF建模将本地化误差降低到十纳米或更少,显著提高了准确性.

结论:

准确的现场PSF建模对于在冷FLM中实现纳米尺度分子定位至关重要.
开发的实验管道作为一种工具来评估冷-CLEM和相关工作流程中的光学性能.
偏差感应的冷FLM和冷CLEM策略对于玻璃化标本中精确的分子定位至关重要.

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