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基于镜的TIRF与共聚焦显微镜的整合:smFRET实验的经济方法

Pratibha Agarwala¹, Dibyendu K Sasmal¹

¹Department of Chemistry, Indian Institute of Technology Jodhpur, Jodhpur, RJ, India.

Current protocols

|August 20, 2025

概括

本研究详细介绍了一种成本效益高的基于镜的全内反射光显微镜 (p-TIRF). 这种设置增强了现有的同焦显微镜,用于先进的单分子研究.

科学领域:

生物物理
显微镜
分子生物学

背景情况:

全内部反射光显微镜 (TIRF) 提供了高分辨率的实时分子动态观测.
基于镜的TIRF (p-TIRF) 可用于研究宏分子复合体,囊泡和蛋白质-DNA相互作用.
通过分析单个分子层面的分子相互作用,TIRF显微镜提供了对细胞机制的洞察.

研究的目的:

为构建基于镜的TIRF设置提供详细且具有成本效益的协议.
使用共享光源调整现有的共聚焦显微镜进行TIRF成像.
提供构建,组装,调整p-TIRF设置,并为单分子光共振能量转移 (smFRET) 实验准备样本的教程.

主要方法:

基于镜的TIRF模块与共焦显微镜的集成.
使用共享光源进行对焦和TIRF模式.
对p-TIRF设置构建,对齐和smFRET样本准备的逐步协议.

主要成果:

一个功能和成本效益的p-TIRF设置成功构建并与共聚焦显微镜集成.
详细的协议使实验室能够扩大其成像能力.
该装置适用于先进的单分子实验,包括smFRET.

结论:

这项协议使现有共聚焦显微镜的实验室能够采用先进的TIRF成像技术.
具有成本效益的p-TIRF设置可方便对生物过程进行高分辨率单分子研究.
整合为研究细胞内的分子机制提供了一个多功能平台.

关键词:

基于镜的全内反射显微镜单个分子检测一个分子FRET

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