Build Your Own Microscope: Step-By-Step Guide for Building a Prism-Based TIRF Microscope

Methods and Protocols

Kaur, Anisa¹  Gibbs, DaltonR.² 

[1] Author to whom correspondence should be addressed;Department of Chemistry, Virginia Commonwealth University, 1001 West Main Street, Richmond, VA 23284, USA

关键词: fluorescence microscope;    pTIRF;    single-molecule FRET;    single-molecule detection;   

DOI  :  10.3390/mps1040040

学科分类：生物技术

来源: mdpi

【 摘 要 】

Prism-based total internal reflection fluorescence (pTIRF) microscopy is one of the most widely used techniques for the single molecule analysis of a vast range of samples including biomolecules, nanostructures, and cells, to name a few. It allows for excitation of surface bound molecules/particles/quantum dots via evanescent field of a confined region of space, which is beneficial not only for single molecule detection but also for analysis of single molecule dynamics and for acquiring kinetics data. However, there is neither a commercial microscope available for purchase nor a detailed guide dedicated for building this microscope. Thus far, pTIRF microscopes are custom-built with the use of a commercially available inverted microscope, which requires high level of expertise in selecting and handling sophisticated instrument-parts. To directly address this technology gap, here we describe a step-by-step guide on how to build and characterize a pTIRF microscope for in vitro single-molecule imaging, nanostructure analysis and other life sciences research.

【 授权许可】

CC BY