1166 Guillaume Wille et al.


Figure 6. Monte-Carlo simulation of the beam dispersion on a carbon samples (thickness 80 nm, 2000 electrons, beam radius 10nm) at (a)5,(b) 15, and (c) 25 kV, and bright field (BF)/dark field (DF) images of a biofilm in the same con- ditions – detail (white rectangle) is a magnified view of the dotted white rectangle.


the same area on one biofilm floc sample under the same imaging conditions (accelerating voltage, beam width) (Fig. 6). These simulations show the influence of the accel- erating voltage on the STEM-in-SEM resolution and enabled comparison of the STEM-DF images obtained from the same area on one biofilm sample at these accelerating voltage conditions. Due to the low mean Z of the biological sample (composed mainly of carbon, hydrogen, nitrogen, oxygen), most of the electrons are transmitted with little or no energy loss at high accelerating voltage, but the dispersion of the electron trajectories increased strongly when lowering the high voltage. This effect provides an increase of the image contrast, as does the addition of osmium tetroxide as a contrasting agent for cell membranes during sample pre- paration. An accelerating voltage of 30 kV was chosen because it provided the best resolution and good contrast in STEM-in-SEM on biological samples.


Imaging with STEM-in-SEM STEM in the SEM offers the ability to simultaneously collect images using the STEM, SE, and BSE detectors. This com- bination of detectors is a great advantage for the


discrimination of contrasts in the STEM-DF/BF images. An example of STEM-in-SEM observation (DF, BF) of the same area of a biofilm (an 80nm microtomic section of a resin embedded biofilm floc sample) is presented in Figure 7, and compared with BSE and SE images (all images were simul- taneously collected). Some bright particles are clearly visible in BF or DF images (with reverse contrast in the other). Due to the origin of contrast in STEM images, these bright par- ticles can only be associated to a difference in composition or to diffraction contrast. BSE images of these particles can be used to discriminate between a strong diffraction contrast and high Z. SE image contrast was weak, no contrast was observed using this detector. This can be explained by an absence of topography and a very low difference in mean atomic Z which contributes a little to the signal detected by the SE detector. On this image, bright particles observed on the BSE image, together with DF images, were analyzed by EDS point analysis (on STEM-in-SEM—data not shown) and mapping (on STEM in TEM) (Fig. 8). The particles were composed of calcium and phosphorus. A comparison of TEM (Philips CM20 – 120kV) and STEM-in-SEM (Tescan Mira – 30 kV) was performed.


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