In the last several years, scientists have developed and harnessed new technologies that have enabled them to peer into cells, and now into molecules, with clarity undreamed of just a decade before. Phrases like "super-resolution" and "single-molecule" now describe microscopy.

In last week's issue of Cell Reports, Matthias Wulf and Stephan Pless of the University of Copenhagen describe a new way to "see" the movements of individual proteins in cells. We talked to them about their technique and about the artwork they commissioned to visualize it.

Stephen Matheson: Your paper describes a new experimental approach and method. What's the problem the technique aims to solve? How do you think it will be used?

Matthias Wulf and Stephan Pless: A surprisingly high number of proteins encoded in the human genome code for membrane proteins, such as ion channels. They're involved in many processes, such as heartbeat generation, neuronal firing, and insulin secretion. Using electrophysiology, their activity can be measured with single-molecule resolution, but our understanding of the protein movements—called conformational changes—that precede, accompany, or follow transitions from closed ion channels to open ion-conducting channels is limited.

To detect these conformational changes, fluorescent and environmentally sensitive indicators called fluorophores can be used to translate conformational movements into changes in fluorescence signal intensity. Comparison of the time course of fluorescence changes (i.e., conformational changes) with ionic currents indicates whether conformational changes are linked to specific channel states.

However, conventional methods have failed to generate fluorescence signals large enough to enable sufficiently high time resolution. Furthermore, the interpretation of fluorescence signals from ligand-activated ion channels is limited by a slow solution exchange to apply and remove the ligand.

Our high-sensitivity patch-clamp fluorometry (hsPCF) setup aims to address these shortcomings. It enables fast solution exchange, large fluorescence signals, and a high time resolution to resolve fast conformational changes in ion channels. We assume that the advantages of hsPCF will also be beneficial to studies of other membrane proteins, such as transporters, pumps, and G protein-coupled receptors.

SM: Any interesting stories about how the approach was developed?

MW and SP: Initially, we were mostly focused on a fast solution exchange system in addition to a sensitive and fast optical detector. It was just by chance that we came across a digital mirror device (DMD), which allows you to focus the excitation light beam on only a small fraction of the full field of view. We speculated that this should improve the fluorescence signal, although no one had tested the DMD for this purpose and the idea required a considerable investment. But after assembling the prototype, we were extremely happy (and relieved) to see that the DMD substantially increases fluorescence signals, reduces background fluorescence and optical artifacts caused by the movement of the perfusion tool.

SM: What are the next steps?

MW and SP: We will continue to use hsPCF to elucidate conformational changes in a variety of ligand-gated ion channels. In parallel, we will expand the utility of our setup to also track ligand binding in small channel populations in real time.

SM: Can you show us the rig?

MW and SP:

SM: You created a great image for the cover of Cell Reports. It wasn't chosen, which is a shame, so we featured it at the top of this blog. Please tell us how the image was developed. How did the scientists and artist work together?



MW and SP: We wanted the cover suggestion to be a considerably simplified illustration of hsPCF. We provided a rough sketch to professional graphic designer Henning Dahlhoff to help us with the realization. We went through a couple of iterations in terms of size and position of objects, as well as colors, and then asked Henning to add a glossy touch to the whole arrangement.

A pharmacist by training, Matthias Wulf did his undergraduate studies in Germany and the United States before joining the Pless laboratory as a PhD student in 2015. Stephan Pless obtained his undergraduate degree in Germany before moving to Australia for his PhD, followed by a postdoc in Canada. Since 2014, he's been an Associate Professor at the University of Copenhagen with a focus on ion channel function and pharmacology.