Dataset Information

Construction of a Three-Color Prism-Based TIRF Microscope to Study the Interactions and Dynamics of Macromolecules.

ABSTRACT: Single-molecule total internal reflection fluorescence (TIRF) microscopy allows for the real-time visualization of macromolecular dynamics and complex assembly. Prism-based TIRF microscopes (prismTIRF) are relatively simple to operate and can be easily modulated to fit the needs of a wide variety of experimental applications. While building a prismTIRF microscope without expert assistance can pose a significant challenge, the components needed to build a prismTIRF microscope are relatively affordable and, with some guidance, the assembly can be completed by a determined novice. Here, we provide an easy-to-follow guide for the design, assembly, and operation of a three-color prismTIRF microscope which can be utilized for the study of macromolecular complexes, including the multi-component protein-DNA complexes responsible for DNA repair, replication, and transcription. Our hope is that this article can assist laboratories that aspire to implement single-molecule TIRF techniques, and consequently expand the application of this technology.

SUBMITTER: Fairlamb MS

PROVIDER: S-EPMC8301196 | biostudies-literature | 2021 Jun

REPOSITORIES: biostudies-literature

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Construction of a Three-Color Prism-Based TIRF Microscope to Study the Interactions and Dynamics of Macromolecules.

Fairlamb Max S MS Whitaker Amy M AM Bain Fletcher E FE Spies Maria M Freudenthal Bret D BD

Biology 20210623 7

Single-molecule total internal reflection fluorescence (TIRF) microscopy allows for the real-time visualization of macromolecular dynamics and complex assembly. Prism-based TIRF microscopes (prismTIRF) are relatively simple to operate and can be easily modulated to fit the needs of a wide variety of experimental applications. While building a prismTIRF microscope without expert assistance can pose a significant challenge, the components needed to build a prismTIRF microscope are relatively affor ...[more]

PMID: 34201434

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Project description:Gene function discovery in plants, as other plant science quests, is aided by tools that image, document, and measure plant phenotypes. Tools that acquire images of plant organs and tissues at the microscopic level have evolved from qualitative documentation tools, to advanced tools where software-assisted analysis of images extracts quantitative information that allows statistical analyses. They are useful to perform morphometric studies that describe plant physical characteristics and quantify phenotypes, aiding gene function discovery. In parallel, non-destructive, versatile, robust, and user friendly technologies have also been developed for surface topography analysis and quality control in the industrial manufacture sector, such as optoelectronic three-dimensional (3D) color microscopes. These microscopes combine optical lenses, electronic image sensors, motorized stages, graphics engines, and user friendly software to allow the visualization and inspection of objects of diverse sizes and shapes from different angles. This allow the integration of different automatically obtained images along the Z axis of an object, into a single image with a large depth-of-field, or a 3D model in color. In this work, we explored the performance of an optoelectronic microscope to study plant morphological phenotypes and plant surfaces in different model species. Furthermore, as a "proof-of-concept," we included the phenotypic characterization (morphometric analyses at the organ level, color, and cell size measurements) of Arabidopsis mutant leaves. We found that the microscope tested is a suitable, practical, and fast tool to routinely and precisely analyze different plant organs and tissues, producing both high-quality, sharp color images and morphometric and color data in real time. It is fully compatible with live plant tissues (no sample preparation is required) and does not require special conditions, high maintenance, nor complex training. Therefore, though barely reported in plant scientific studies, optoelectronic microscopes should emerge as convenient and useful tools for phenotypic characterization in plant sciences.

Dataset Information

Construction of a Three-Color Prism-Based TIRF Microscope to Study the Interactions and Dynamics of Macromolecules.

Publications

Construction of a Three-Color Prism-Based TIRF Microscope to Study the Interactions and Dynamics of Macromolecules.

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