The investigation of the asphalt binder microstructure can provide an alternative approach to characterize the material. However, various factors prior to its analysis need to be considered. This involves the identification of the influence by the sample preparation process in regards to how factors like heating time and temperature as well as resting time can affect the resulting microstructure. Furthermore, most microscopic images are used without any post process analysis, which makes it difficult to judge the observed surface morphologies. Nevertheless, there is an increasing trend in utilizing different analysis techniques to quantify the respective microstructures. The goal of this study is to address these parameters and quantify their respective influence via a combination of optical inverse dark- and brightfield microscopy and post process particle analysis. The results show that small binder quantities, typically used for microscopy, can oxidize within one or two minutes of heating or when selecting higher heating temperatures above 150°C. Therefore, staying below these parameters will ensure that the sample surface has not experienced any ageing. Furthermore, the resting time study revealed that the microstructure becomes stable after 30 minutes of resting, making binder characterization via microscopy much more convenient. For imaging analysis an overall area covered by bee structure enables universal characterization and comparison, while providing good differentiation between binders of different origin. These findings should set the basis for future research, where microscopy can be used to identify and tackle important questions related to changes caused by ageing, modification or rejuvenation.