Visualising the distribution of chemical substances on samples with non-flat surfaces
The chemical analysis of biological tissues with three-dimensional shapes has previously been a major problem. Researchers from three different institutes of the Beutenberg Campus in Jena, Germany, together have now improved mass spectrometry imaging in a way that the distribution of molecules can be visualized on warped, hairy, or coarse surfaces. Prof. Hans Peter Saluz of the Leibniz Institute for Natural Product Research and Infection Biology - Hans Knöll Institute (HKI) joined as scientific expert with long lasting experience in imaging and technology development at the interface between biology, physics and chemistry.
In addition, he is the supervisor of Benjamin Bartels, the first author of this study. The source of the laser-based technique was custom-built in the group of Dr. Ales Svatos in the Max Planck Institute for Chemical Ecology (MPI-CE) to accommodate the topography of non-flat samples. By employing a distance sensor, a height profile of the surface is recorded before the actual chemical imaging. Dr. Norbert Danz from the Fraunhofer Institute for Applied Optics and Precision Engineering (IOF) designed a specific lens system for infrared lasers.
Laser ablation electrospray ionization (LAESI) is a method that can be applied in mass spectrometry imaging to investigate the distribution of many different chemical compounds within a biological sample. The laser is used to remove a small fraction of the sample by local heating. The illuminated part of the sample bursts and the escaping vapor is ionized by an electrically charged mist to make the vapor contents detectable in a mass spectrometer. "The spatially confined laser probing enables us to assemble chemical information very much like pixels form an image," Ales Svatos, the leader of the new study, explains the technical principles of the technique.
The distribution of chemical compounds, for example in flowers, leaves, stalks and other parts of a plant are of major importance in ecological research. Many of these chemical compounds, used to communicate between individual organisms, are so called secondary metabolites which are produced by plants and other organisms to attract pollinators or to fend off herbivores or pathogens, for example. It is important to know if or not a plant produces these substances, but the location, where within the plant tissue the molecules accumulate, can also be crucial. Is a defensive substance distributed evenly in a plant leaf or are there special glands that provide protection by secreting this chemical? In which parts of an insect exoskeleton are toxins or pheromones for communicating with their conspecifics, symbionts or competitors specifically enriched? Scientists in Jena seek to unravel how microbes and higher organisms interact with each other on a molecular level, with the aim to rebalance ecological systems. Thus, the three involved institutes MPI-CE, HKI and IOF provide a sound basis with their excellent cooperation.
"The biggest challenge in analytics is preserving the constitution of a sample throughout the analytical process. More often than not, sample preparation influences the result by altering the sample's chemical constitution. Typical preparation steps include sectioning a sample into thin, flat slices because flatness is required to guarantee optimal laser focus, a key parameter in reliable analysis.", Benjamin Bartels, PhD student in the Mass Spectrometry Research Group at the MPI-CE, points out the limitations of previous setups.
But most biological samples have surfaces which are far from being flat: For example, plant leaves often have hairy structures or they are warped. Caterpillars can also be hairy, and they are generally rather bulgy than flat. The Jena scientists have therefore adapted the LAESI technique to non-flat surfaces to open up the possibility of performing chemical imaging of samples with pronounced three-dimensional shapes while maintaining the reliability of classical measurements.
The improved laboratory setup measures the height profile of the surface in question prior to the actual mass spectrometry imaging. The recorded height profiles can be used to correct the distance between the focusing lens of the laser and the sample's surface. In this way one of the essential parameters for reliable laser probing is kept constant throughout the experiment on samples with three-dimensional structure which were previously not subjectable to such analysis. "This means, that we can now investigate molecular distributions on a much bigger range of accessible surfaces. I am thinking of insect exoskeletons or microbial colonies within their natural environment. We can now also compare the contents of different trichomes of a leaf," Benjamin Bartels emphasizes the advantages of the LAESI enhancements.
In the near future the researchers plan to implement further improvements and refinements. It is their goal to use LAESI also in routine measurements of non-flat surfaces.