Integrating Prism‐based TIRF with Confocal Microscopy: An Economical Approach for smFRET Experiments

Abstract Total internal reflection fluorescence (TIRF) microscopy enables the observation of complex bioassemblies and macromolecular dynamics in high spatial‐temporal resolution at the single‐molecule level in real time. Through TIRF illumination, fluorophores near a sample substrate are selectively excited within an evanescent field, thereby overcoming the axial diffraction limit of light. Prism‐based TIRF (p‐TIRF) microscopes are relatively straightforward to construct and can be readily adapted to accommodate a wide range of experimental applications, including the examination of macromolecular complexes, the study of the behavior of vesicles and small organelles, and the investigation of protein‐DNA complexes at the single‐molecule level. These experiments can give unique insights into the mechanisms driving the molecular interactions that underline many fundamental activities within the cell by providing information on fluctuation distributions and unusual events. In this paper, we present a detailed and cost‐effective protocol for constructing a p‐TIRF setup using an existing confocal microscope, utilizing the same light source for both modalities. Additionally, we provide a step‐by‐step tutorial on building, assembling, and aligning the p‐TIRF setup and preparing the sample for single‐molecule fluorescence resonance energy transfer (smFRET) experiments. This article will be particularly helpful for laboratories equipped with a confocal microscope seeking to expand their experimental capabilities by integrating TIRF‐based approaches. © 2025 Wiley Periodicals LLC. Basic Protocol : Microscopy setup for TIRF Support Protocol 1 : Construction of prism holder and prism holder carrier arm Support Protocol 2 : Preparation of sample chamber with sample Support Protocol 3 : Slide preparation and KOH etching Support Protocol 4 : Preparation of biotinylated DNA Holliday junctions immobilized on slides Support Protocol 5 : Preparation of DNA Holliday junctions