Correlation of AFM/SEM/EDS Images
performed with the same P900H60 calibration grating. Te use of the same grating, calibrated using a metrological AFM [12], on both instruments ensures the measurements from each are comparable and traceable to the SI meter. Te voltage was set at 3 kV and the working distance
(WD) fixed at 3 mm. Te pixel size was set to 4.6 nm with a total cycle time to record an image of 28.4 seconds. Tis short recording time ensured contamination did not occur during scanning [13,14]. Moreover, to prevent such contamination, which can be critical for both SEM and EDS measurements, further precautions were taken. Te sample was placed in the SEM vacuum chamber the night preceding both SEM and EDS measurements, and plasma cleaning of the SEM cham- ber was performed. Energy dispersive X-ray spectrometry. Te SEM was
also equipped with an Oxford windowless UltimMax Extreme EDS detector adapted for chemical nano-analysis. Tis is a 100 mm2
detector that allows identification of the elementary
chemical composition nanoparticle by nanoparticle (up to 15 nm or 20 nm particles) at very low voltage (of the order of 2–3 kV). Indeed, as it is possible to work at very low voltage the interaction volume is consequently reduced allowing very high spatial resolution. Te EDS chemical maps presented in the following para-
graphs were acquired at 5 kV and 7 mm WD. Depending on the SEM’s stability, spatial driſts can occur during long acqui- sition times (typically several minutes) making chemical mapping at very high magnification difficult. Tus, correct- ing this driſt is crucial in obtaining proper results. To over- come this issue, the Oxford AZtecLive soſtware provides a driſt correction routine. Tis routine is applied for the acqui- sition presented here. Image processing: colocalization and particle analysis. Premium 8.2 soſtware (developed by Digital
MountainsLab®
Surf) was used to perform colocalization of the images acquired by the different instruments. Te following steps were carried out to process the images:
• First, AFM and SEM images, as well as the EDS maps associated with each element, were loaded and processed independently. Tus, the AFM image was leveled. Te SEM image and the EDS maps were scaled. Both were also converted into intensity (grayscale) maps. A color was attributed for each EDS map.
• Ten, using the MountainsLab® soſtware colocalization
tool, the different images were superimposed, two by two. Te aim was to create a multi-channel image composed of different layers (AFM, SEM, EDS maps). To take into account the image scale issues and driſting associated with each imaging technique, the MountainsLab®
“Points
positioning” tool was used. Tis tool allows refining of the colocalization by manually indicating the position of several remarkable points on the different images. As a result, the soſtware compensates the distortion of the different layers (images) to ensure that the manually placed points are superimposed.
• Te combined images were superimposed to create the final multi-layered image dataset. • Finally, the MountainsLab®
“Particle analysis” tool was
used to obtain the dimensional properties (area, perimeter, equivalent diameter) of the particles in the image, separately for each population.
Results Images from SEM, AFM, and EDS maps are presented
in Figures 3 and 4. Te object studied for the colocalization process was an agglomerate. As mentioned previously, in the SEM image two populations are clearly identifiable by their shapes in the SEM image (Figure 3, leſt): nanorods and particles of isotropic form. Resulting from the two-stage deposition procedure, the agglomerate appears to be a nanorod population covered by other nanoparticles. Te lateral dimensions of the agglomerate are nearly 6.5 μm×8 µm. Tese dimensions enable easy identification of the object thanks to the optical microscope mounted on the AFM. Te AFM image (Figure 3,
Figure 3: SEM (left) and AFM images (right) giving dimensional information on the mixed agglomerate. 48
www.microscopy-today.com • 2021 May
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