# Novel Microscopic Approach to Particle Size Evaluation in Colloidal Systems
## 3 Results and Discussion
Therefore,since the measurements are independent of the thickness, all the analyses reported in this study will be carried out at 21 \(\upmu\)m.  
The dimensions of the solid particles (asphaltenes) are significantly smaller than the minimum thickness reached between the two slides, therefore problems of the possibility of deformation (crushing) of any asphaltene aggregates interacting together [39; 40], due to compression during sample preparation, should not exist. However, elongated clusters can be found in bitumen, presenting an interior with rippled patterns and sub-nanometer edges which are dispersed in a quite consistent matrix. These clusters are made up of asphaltene molecules showing different levels of aggregation as they are patterned into a hierarchical aggregation system [41]. This self-assembly phenomenon of asphaltenes at high levels of aggregation presents particles having a prolate ellipsoidal shape extending up to a few thousand nanometers [39; 42]. For each sample, at least 10 images were captured at different points in order to obtain and thus elaborate over 10,000 data points that guarantee a significant value for each asphaltene dimension. In fact, Figure 2 shows the excellent reproducibility of the data obtained, even though they were carried out on the same sample at different thicknesses. To validate the effectiveness of the OM technique, measurements of the dimensions of the asphaltenes present in the virgin bitumen sample were carried out using two other previously mentioned techniques already commonly used in scientific literature on this subject. Figure 3 shows the measurements using the three different techniques, OM, CM, and AFM, while the Figure 4 reports optical microscopy (OM) and confocal microscopy (CM) data of aged bitumen. It was observed that the results of the three techniques gave similar results, validating the use of OM to outline the trend of the size of the asphaltenes present in bitumen. In the case of the CM technique, it is possible to observe many more particles, as this technology allows us to acquire the image and then process the image itself digitally, therefore zooming in digitally and not using the lens. On the other hand, with OM and AFM techniques, it was not possible to have the same number of data points because these techniques operate at a higher magnification which only covers a smaller area. As a result of this, the quantity of observable particles is decidedly more limited. In fact, for some types of bitumen whose dimensions are visible to the instrument (>0.5 nm), the OM technique is advantageous and is accredited as a less expensive and excellent complex asphaltene size measuring method when compared to the other techniques. However, for smaller dimensions, the other two techniques turn out to be the only ones that can be used and work best for this case scenario. Figure 2: Different thicknesses of the virgin bitumen sample via optical microscope. From the results, it is demonstrated that the asphaltene size measurements were independent of the sample thickness. This allows the analysis of any sample without concerning its thickness. We know that the asphaltenic fraction of bitumen is the most susceptible to oxidation, in fact by incorporating oxygen, they tend to form increasingly larger clusters. We expect that after the aging process, the distribution should shift significantly towards larger particles. The dimensions of the oxidized bitumen significantly increase compared to those of the asphaltenes of the virgin bitumen; in particular, the asphaltene size in the range 1.4-2.5 \(\upmu\)m\({}^{2}\). This is due to the increase of polar functional groups of the molecules in bitumen [43] with their ultimate aggregation and constrained dynamics typically caused by the aging process.