# Novel Microscopic Approach to Particle Size Evaluation in Colloidal Systems
Optical and confocal laser scanning microscopy techniques were used to investigate the size distribution of the asphaltene particles in a paving-grade bitumen subjected to an aging process. The changes in the dimensions of the asphaltenes during the process of aging were evaluated to estimate the effect of oxidation on the asphaltene fraction of the bitumen system. To do so, the standard atomic force microscopy technique, generally recognized as the pivotal technique to investigate asphaltene size distribution in bitumen, has been taken into account for comparative purposes. The novelty of this work lies in the development of a new, relatively easy sample preparation method making it easier and more straightforward to directly observe colloidal particles (asphaltenes) using optical microscopy (OM). This sample preparation method coupled with software image analysis and elaboration using data-based software brings about determination of the area values of the elliptical colloidal particles, thereby providing particle dimensions and size distributions. Generally, the bitumen samples analyzed by AFM are investigated in intermittent-contact (IC) mode (tapping) [14; 15]. Compared to many other microscopic techniques, this device can resolve surfaces below the Abbe diffraction limit by simply scanning or tapping the surface with a nanometer-sized tip. It has thus become one of the most popular techniques to investigate the microstructure of bitumen and tackle the question mentioned above on the origin or reason for the microstructure [16]. In the present study, the AFM analysis was used as an evaluation template to determine the effectiveness of the confocal and optical microscopy methods in measuring the asphaltene size of the bitumen system [17; 18; 19]. Confocal laser scanning microscopy (CM) particularly exploits the principle of measurements which are dynamically focused based on a focus-detection technique. This technique is also a scanning optical microscopy technique due to the fact that the light beam illuminates only one spot (with a diameter of (\(\approx\)0.5 \(\mu\)m) at a time and the sample surface must be scanned or rotated to generate the image pixel by pixel. Three-dimensional measurements using CM can also be carried out by performing a raster scan of the laser spot over the measured surface [20]. Optical microscopy (OM) imaging requires no scanning and thus can be acquired rapidly, even in real-time, limited only by the camera frame rate. At low magnification, even though the internal structure is no longer resolved, such microstructures appear as near-point-like optical scattering centers. All microstructures within the optical absorption depth contribute to the observed optical scattering, thus optical probes provide access to subsurface structures. Finally, optical microscopy investigations require no mechanical contact with the sample surface so they can characterize bitumen microstructures equally well at any temperature, whether the sample is frozen or heated beyond its melting point [21]. In addition, these instruments are very popular and relatively cheap. In fact, optical microscopes are commonly found in science laboratories regardless of the research theme. Bitumen's viscoelastic performance and especially the aging phenomenon have been investigated by several researchers and scholars using an array of physicochemical and mechanical methods due to the direct correlation of these analyses with asphalt behavior and performance. Microscopic approaches to investigating the microstructural patterns and changes associated with oxidative aging are becoming increasingly popular, making it very instrumental to achieving an in-depth understanding of the intricacies of bitumen's structure pre- and post-aging. In the scientific literature, several studies have been published involving the use of atomic force microscopy (AFM) or confocal laser scanning microscopy (CM) in the investigation of bitumen's microstructure.