Researchers from Katholieke Universiteit Leuven in Belgium and from Pasteur Institute in Paris recently combined atomic force microscopy (AFM), x-ray photoelectron spectroscopy and secondary ion mass spectrometry to study the human fungal pathogen Aspergillus fumigatus. They found surface structure and surface chemical composition differences between mutants and wild-type versions of the microbe, results that agree with earlier biochemical data and that also provide insight into the cell wall architecture.The threefold characterization approach could produce information hard to get by other means, said team member Yves F. Dufrêne, a professor at the university. “It could provide info on the cell surface function and how these surfaces may be altered by drugs or chemicals.” He added that the three techniques themselves are not new and often have been used separately in cellular investigations. The novelty is the application of the three to the same set of samples. This demonstration showed that the techniques are complementary and that they correlate to one another. Of the three techniques, only AFM allows nanoscale-resolution imaging of live cells. In this process, a sharp probe tip attached to a cantilever is drawn across a cell, causing a measurable deflection in the cantilever. An appropriate tip enables chemical and molecular recognition imaging. However, the technique is limited to the surface of a cell and can image only that to which the tip is sensitive. In contrast, secondary ion mass spectrometry and x-ray photoelectron spectroscopy provide complete chemical composition information at both the surface and at some depth. Secondary ion mass spectrometry bombards the surface with an ion beam, ejecting secondary ions that can be analyzed. It offers submicron lateral resolution, which is crucial when studying cells. However, it is not easy to extract quantitative data from the measured signal. X-ray photoelectron spectroscopy uses a focused beam to produce photoemitted electrons from a sample’s surface. Extracting such data is something x-ray photoelectron spectroscopy readily allows, although, unfortunately, it does so with poor lateral resolution. Both spectrometry methods must be done under a vacuum, which prevents them from being used on live cells. The three techniques present different parts of the total picture. The researchers, therefore, set out to combine them despite radically dissimilar principles behind each method. The surface analysis, however, did not involve combining equipment or approaches, noted Dufrêne. “There are still three separate instruments in which we put the samples.” The investigators collected AFM images using an instrument from Veeco of Santa Barbara, Calif. They imaged immobilized cells using as little force as possible — about 250 pN — to minimize surface damage. For x-ray photoelectron spectroscopy, they used a system from Kratos Analytical of Manchester, UK, collecting data over an area roughly 700 × 300 μm. Finally, they used a time-of-flight spectrometry system for the last method. In this, a pulsed gallium-ion beam swept over an area 120 μm2. They applied the three surface-characterization techniques to wild-type strain and three mutant types of A. fumigatus. Of the three mutants, one lacked the RodA gene, another lacked the RodB gene, and the third lacked both. Based on scanning electron microscopy studies, the wild strain and the mutant lacking the RodB gene were known to produce morphologically similar rodlets. It also was known that the other two mutants completely lacked these surface features. It is thought that these and other surface characteristics are key to adhesion to host cells during infection and other cellular functions. The researchers first investigated the surface structure of the various cell types using high-resolution atomic force microscopy. As expected, the images revealed the presence of rodlets several hundred nanometers long and 10 nm in diameter in the wild strain and in the mutant lacking the RodB gene. In contrast, the surface of the other mutants was granular. The structure of cell surfaces are shown via in situ atomic force microscopy imaging. These high-resolution deflection images over a 500 × 500-nm area were recorded in deionized water for the surface of the human fungal pathogen Aspergillus fumigatus. Panel (A) shows the wild-type strain, while (B) is a mutant lacking the RodB gene. Figure (C) is a mutant lacking the RodA gene, while (D) is a double mutant lacking both RodA and RodB. Reprinted with permission from Langmuir.They investigated the surface chemical composition using x-ray photoelectron spectroscopy, extracting information on the biomolecular composition of the surface. They found the major constituents to be polysaccharides and proteins, along with some lipidlike compounds. The wild strain and the mutant lacking the RodB gene were much richer in proteins than the other mutants, a result consistent with the presence of rodlets. Lastly, they probed the surface using secondary ion mass spectrometry. With this method, they compared normalized intensities of amino acid peaks and found among the various strains differences that correlated with the data obtained with the other techniques. The work was published online Feb. 1 in Langmuir. Based on these results, the researchers concluded that RodA and RodB genes play distinct roles in modulating the nature and amount of proteins at the cell’s surface. Such information is an example of one of the ultimate goals of the research, Dufrêne noted . “We would like to analyze large sets of genetic mutants or microbial samples for screening purposes and phenotypic analysis.” Those efforts could be aided by technological improvements in the three probe techniques. Atomic force microscopy, for example, could benefit from modifications to the tip’s chemistry and biology. A version of secondary ion mass spectrometry, called nanoSIMS, could allow 100-nm resolution. Finally, cryogenic techniques would allow x-ray photoelectron measurements of cells closer to their native hydrated state.