Scanning electron microscopy (SEM) and cryo-scanning electron microscopy (Cryo-SEM)

Cell shapes of E. coli BL21 and E. coli BL21(DE3) were observed using a SEM microscope. In the case of SEM imaging, a special protocol for bacterial preparation was established to avoid several centrifugation steps that are required during the exchange of solvents. In samples where the bacteria were suspended with the nanorods, the centrifugal force could intensify the piercing of the bacteria by the nanorods that would falsify the real pictures of the cells. The bacterial cells E. coli BL21 (control and after exposure to NR every 24 hours) were centrifuged (4 minutes, 4000 rpm). The bacterial pellet was suspended in a sterile, 50-fold diluted solution of physiological saline in MQ water. A suspension of the bacteria (5 µl) was placed on (previously washed in acetone, ethanol, MQ water) silicon plates. After 60 seconds half the liquid was discarded to obtain a thin film on the surface of the plate. Just as the edges of the spot were starting to dry out, the plates were gently flushed by a rowing movement in filtered MQ water to remove the excess salt from the surface. Silicon plates were attached to the metal holder by silver paste. Images were taken using the FEI Nova NanoSEM 450 (USA) scanning electron microscope. In the case of cryo-SEM, E. coli BL21(DE3) cells after two exposures to NR (each exposure for 24 hours) were analyzed. Images were taken with the use of the JEOL JSM-7001F TTLS (JEOL Ltd., Japan) scanning electron microscope equipped with the PP3000T cryo-SEM preparation system which enables the preparation, loading, processing, and transfer of cryo specimens into the SEM chamber. The sample was prepared by dropping 50 µl of bacterial suspension on the metal holder. The sample was rapidly frozen by plunging the holder into slush nitrogen (the temperature around −210 °C) and transferred under a vacuum to the preparation chamber mounted onto the SEM. Inside the preparation chamber (−185 °C), the specimen was fractured to expose a fresh surface. Then it was sublimated and coated with a thin platinum layer. Finally, the sample was transferred under a vacuum into the SEM cryo stage (−190 °C) where the surface was imaged applying an accelerating voltage of 10 kV and secondary electron (SEI) detector.