Infrared Microscopy: "We don’t know why certain pharmaceuticals bind especially well while others bind barely at all"

Interview with Professor Joachim Heberle, Free University of Berlin

Professor Joachim Heberle from the Free University of Berlin wants to make the smallest protein structures visible under the microscope. He wants to accomplish this with an infrared microscope, originating in the field of physics. He told which technology is behind all this and what he also wants to examine with it in the future.


Joachim Heberle; © private

Professor Joachim Heberle, Freie Universität Berlin, Department of Physics; © private Professor Heberle, you study protein complexes with an infrared microscope that’s still quite new in the field of biomedicine. Why is it not possible to examine the structures you are analyzing with conventional transmitted or reflected light microscopes?

Joachim Heberle: We are looking at protein complexes that are smaller than the diffraction limit. This means that they are smaller than the wavelength of visible light, which can no longer be resolved according to the Abbe criterion. At this point, there are fluorescence microscopes that use visible light and that are able to go below the diffraction limit, but these types of microscopes always require dyed samples. This is not needed with the infrared microscope. We look at the molecular vibrational spectrum and are thus able to differentiate proteins from lipids for instance. How does an infrared microscope work?

Heberle: Instead of visible light, the infrared microscope uses infrared radiation, which has very low energy and does not heat up the protein at all or only very little. What makes this microscope so special is that you are able to disperse infrared light via a fine needlepoint from a laser source and for the diffused infrared light to interact with the specimen. The light is being absorbed by the sample so that you are able to determine the lateral resolution via the fineness of the tip. In extreme cases, this means that the finer the tip, the better the lateral – that is the laterally expanded - resolution. We can show structures of up to 30 nanometers with this technology. Which structures have you researched so far?

Heberle: So far, we were able to study isolated protein complexes like amyloid structures for example. We can distinguish them from viral structures for instance. From the outside, these structures appear typologically similar, but their molecular structure is very different. My research focus is particularly on integral membrane proteins. They are the primary target for approximately 70 percent of all drugs on the market. We would like to be able to make these membrane proteins visible with the microscope and to be able to differentiate them from the surrounding lipid bilayer. For the moment, this is still research for the future, because you need an even higher lateral resolution for this. However, then we might be able to microscopically study an entire membrane without having to tint it beforehand. For which type of research would you like to use this device in the future?

Heberle: We hope that we will someday be able to enter cellular research, but for the moment, we are still in the early stage. I am interested in obtaining a fundamental biophysical understanding of biological membranes, their structure and their characteristics. As physicists, we want to completely understand the biological membrane in its complexity. To do this, structural and imaging procedures such as infrared nano-spectroscopy are essential. You can also combine them with mechanical measurement methods by extracting proteins from the membrane for instance and measuring their forces and the infrared spectrum to get an idea on how the structure changes when you pull at the protein.
© Elmar Hubrich (FU Berlin, Exp. Molekulare Biophysik)/Iban Amenebar (CIC nanoGUNE, San Sebastian)
(left) Topography of a native membrane patch (here the purple membrane of Halobacterium salinarum) adsorbed onto a silicon substrate as recorded by atomic force microscopy (AFM). Black corresponds to the substrate and grey colors are the membrane patches. The white structures are smaller membrane fragments atop the larger fragment. (right) Infrared near-field image recorded at a frequency of 1,660 cm-1 which corresponds to the C=O stretching mode of the protein polypeptide (here the transmembrane protein bacteriorhodopsin). Yellow color corresponds to the absorbance by a single patch and red to two layers of a membrane. Blue is the non-absorbing background of the silicon substrate; © Elmar Hubrich (FU Berlin, Exp. Molekulare Biophysik)/Iban Amenebar (CIC nanoGUNE, San Sebastian)
: So the microscope is suited for pharmacological research?

Heberle: That’s correct. Many of these membrane proteins are not yet well understood in their functions and dynamics. This is why we also don’t know why certain pharmaceuticals bond especially well while others bind barely at all – there are still no answers to these questions. The infrared microscope could definitely make a significant contribution in finding these answers. How important is interdisciplinary collaboration to you?

Heberle: The method that my colleague Rainer Hillenbrand from San Sebastian developed, introduced a procedure from solid-state physics into biophysics or biomedicine, respectively. This shows already how important interdisciplinarity is. The samples that we are testing come from the field of molecular biology or biomedicine. There is interdisciplinary collaboration with cell biology labs that provide cancer cells or slices from brains of rats or mice affected by Alzheimer’s disease for example. So the questions stem from biomedicine, the technology comes from the field of physics and as far as the functioning mechanisms are concerned, chemical or biochemical knowledge is often required. In which direction would you like to further develop this device?

Heberle: On the one hand, we would still like to achieve a higher lateral resolution for this device, meaning beyond 30 nanometers. After all, many proteins are barely five to ten nanometers in size. On the other hand, we would of course like to be able to study proteins in a water environment. We are currently still in the development stage for this. This is not easy, because water strongly absorbs the infrared radiation of the microscope. However, water is precisely what determines the structure of a biological cell. This means, we have to adapt the measurement method accordingly. However, I am confident that this will work in the near future.

Photo: Simone Ernst; Copyright: B. Frommann

© B. Frommann

The interview was conducted by Simone Ernst and translated by Elena O'Meara.