Single-Molecule Fluorescence Imaging on the Cell Membrane

Single-Molecule Fluorescence Imaging on the Cell Membrane Using a Super High Numerical Aperture (NA) Objective Lens

Introduction

Recent advances in cell preparation and microscope optical systems have enabled imaging of single biomolecules in a live cell. Molecular dynamics, such as the binding of a physiologically active ligand to a cell, dimerization of signal molecules, and the formation of a molecular complex, can be visualized at the single molecule level in live a cell using objective lenses with a super high numerical aperture. In this study, researchers used an Olympus super high NA objective lens for fluorescence imaging of intermolecular interactions in ion channels on the cell membrane at the single molecule level.

Super high NA objective lens TIRF application

Fluorescent-protein (FP) tagged ion channel subunits are expressed in Xenopus oocytes and observed at the single molecule level by TIRF microscopy (Figure 1, left). Stochastic bleaching events of individual FPs can be observed as ‘bleaching steps’ (Figure 1, right). The number of subunits in a single ion channel complex can be determined by counting the bleaching steps from individual fluorescent spots.

Images of the fluorescent proteins Kv4.2-mCherry Figure 2, left) and mEGFP-DPP10 (Figure 2, middle) expressed in a Xenopus oocyte were observed at the single molecule level using TIRF microscopy and super high NA objective lenses.  Each red spot represents a single Kv4.2 channel (tetramer).  Some of the green spots overlap with the red spots (white arrowheads in Figure 2, right) indicating that Kv4.2 and DPP10 form a complex.  By counting bleaching events of mEGFP from a single fluorescent spot, the number of subunits in the complex can be counted. ¹  In the Figure 2 graph, four bleaching events (green arrows) were observed from a Kv4.2-mCherry/mEGFP-DPP10 spot, suggesting four DPP10 subunits were included in the complex.
1 Ulbrich, Maximilian H., and Ehud Y. Isacoff. “Subunit counting in membrane-bound proteins.” Nature methods 4, no. 4 (2007): 319–321

Conclusion

A high numerical aperture objective lens designed only for evanescent illumination can produce remarkably high contrast images even with weak fluorescent light because of the efficient formation of an evanescent wave field with a shallow penetration depth. While this type of observation requires the quantitative measurement of minute changes in fluorescence intensity due to fluorescence loss at the single molecule level, observation using a super high NA objective lens with special immersion oil and coverslips facilitates images of intermolecular interactions in ion channels in Xenopus oocyte membranes. Since these images have a high signal-to-noise ratio, changes in fluorescence intensity can be quantitatively measured.