Conducting Multiple Imaging Modes with One Fluorescence Microscope

J Vis Exp. 2018 Oct 28:(140):58320. doi: 10.3791/58320.

Abstract

Fluorescence microscopy is a powerful tool to detect biological molecules in situ and monitor their dynamics and interactions in real-time. In addition to conventional epi-fluorescence microscopy, various imaging techniques have been developed to achieve specific experimental goals. Some of the widely used techniques include single-molecule fluorescence resonance energy transfer (smFRET), which can report conformational changes and molecular interactions with angstrom resolution, and single-molecule detection-based super-resolution (SR) imaging, which can enhance the spatial resolution approximately ten to twentyfold compared to diffraction-limited microscopy. Here we present a customer-designed integrated system, which merges multiple imaging methods in one microscope, including conventional epi-fluorescent imaging, single-molecule detection-based SR imaging, and multi-color single-molecule detection, including smFRET imaging. Different imaging methods can be achieved easily and reproducibly by switching optical elements. This set-up is easy to adopt by any research laboratory in biological sciences with a need for routine and diverse imaging experiments at a reduced cost and space relative to building separate microscopes for individual purposes.

Publication types

  • Research Support, N.I.H., Extramural
  • Video-Audio Media

MeSH terms

  • Color
  • Fluorescence Resonance Energy Transfer
  • Microscopy, Fluorescence / methods*
  • Multimodal Imaging / methods*
  • Single Molecule Imaging