High-Resolution Imaging of Dried and Living Single Bacterial Cell Surfaces: Artifact or Not?
Dominik Greif,* Daniel Wesner, Dario Anselmetti, and Jan Regtmeier Experimental Biophysics and Applied Nanoscience, Bielefeld University, Universitätsstraße 25, 33615 Bielefeld, Germany
*
dgreif@physik.uni-bielefeld.de
Introduction When studying highly resolved scanning electron
microscope images of cell surfaces, the question arises, whether the observed patterns are real or just artifacts of the cell preparation process. Te following steps are usually necessary for preparation: fixation, drying, and metal coating. Each step might introduce different artifacts. Clever techniques have been developed to dry cells as gently as possible, for example critical point drying with different organic solvents and CO2. Instrument manufacturers also have taken account of this issue, for example, through the realization of the environmental scanning electron microscope (ESEM), operating with a low-vacuum environment saturated with water so that samples might stay hydrated. Another approach is the extreme high-resolution scanning electron microscope (XHR SEM), where the electron beam is decelerated shortly before reaching the sample. Tis technique requires no metal coating of the sample. Cryo-SEM also may be used, where no sample preparation is required beyond freezing in a high-pressure freezer or other cryo-fixation device. Ten the cell can be examined in the frozen, hydrated state using a cryostage. However, at least some kind of preparation is necessary for SEM imaging, and we wanted to find out what changes the preparation makes on the cell surface.
Methods and Materials To elucidate the different possible artifacts of each sample
preparation step, complementary techniques were used to image the surface of the model bacterium Sinorhizobium meliloti [1]. S. meliloti is a gram negative soil bacterium of 0.5 to 3 µm in length, which lives in symbiosis with certain host plants, helping them to reduce and fix nitrogen. In our study, the following scanning electron microscopy
techniques were used: SEM and XHR SEM. Tey were complemented by atomic force microscopy (AFM) to image dried cells under ambient conditions, as well as viable cells in liquid. Tis means that conditions ranged from living bacteria in liquids to fixed bacteria in high vacuum. Each step of cell preparation was investigated (fixation, drying, metal coating) by imaging the resulting cell surface patterns. We found that the observed wrinkled protrusions in SEM images were not generated de novo but evolved from similar and naturally present structures on the surface of living bacteria [1]. Detailed analysis of AFM images of living bacteria exhibited surface structures of the size of single proteins, emphasizing the usefulness of AFM for high-resolution cell imaging.
Results Conventional SEM images of S. meliloti showed prominent
wave-like surface patterns when the cells were fixed, dried, and metal-coated with a 12 nm gold layer in vacuum (see Figure 1).
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Figure 2: XHR SEM image of S. meliloti fixed, dried, and imaged in vacuum without metal coating. Elongated wrinkles cover most of the bacterium. In addition, several single dots (some examples marked by arrows) are distributed over the surface.
doi:10.1017/S1551929511000836
www.microscopy-today.com • 2011 September
Figure 1: SEM image of fixed, dried, and metal-coated S. meliloti. The bacterial surface is decorated with wrinkled protrusions.
Te observed wavy structures had a width of 29 ± 12 nm and a length of approximately 92 ± 31 nm (mean ± SD). To figure out if these patterns were caused by metal coating, uncoated bacteria were examined with XHR SEM (fixed, dried, in vacuum). In Figure 2 elongated surface protrusions can be seen with a width of 18 ± 5 nm and a length of 80 ± 27 nm. Additionally, the XHR SEM images allowed the detection of distinct round protrusions with a diameter of 21 ± 4 nm. Te interpretation of these images is not trivial because structures several nanometers below the surface might also be visible, producing fainter contrast on the surface.
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