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

Department of Chemistry, Virginia Commonwealth University, 1001 West Main Street, Richmond, VA 23284, USA
Author to whom correspondence should be addressed.
Methods Protoc. 2018, 1(4), 40;
Received: 29 September 2018 / Revised: 31 October 2018 / Accepted: 31 October 2018 / Published: 3 November 2018
(This article belongs to the Special Issue Single-Molecule Techniques)
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. View Full-Text
Keywords: fluorescence microscope; pTIRF; single-molecule FRET; single-molecule detection fluorescence microscope; pTIRF; single-molecule FRET; single-molecule detection
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Gibbs, D.R.; Kaur, A.; Megalathan, A.; Sapkota, K.; Dhakal, S. Build Your Own Microscope: Step-By-Step Guide for Building a Prism-Based TIRF Microscope. Methods Protoc. 2018, 1, 40.

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