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The common realization of the necessity to reduce the use of mineral sources is promoting the use of alternative fuels. Big efforts are being made to replace petroleum derivatives in the internal combustion engines (ICEs). For this purpose it is mandatory to evaluate the behavior of non-conventional fuels in the ICEs. The optical diagnostics have proven to be a powerful tool to analyze the processes that take place inside the engine. In particular, 2d imaging in the infrared range can reveal new details about the effect of the fuel properties since this technique is still not very common. In this work, a comparison between commercial diesel fuel and two non-conventional fuels has been made in an optically accessible diesel engine. The non-conventional fuels are: the first generation biofuel Rapeseed Methyl Ester (RME) and an experimental blend of diesel and a fuel with high glycerol content (HG).