Although prostate cancer is the most diagnosed cancer in men worldwide, there is geographical variance in both incidence and morbidity, with higher trends in Westernised developed countries. In particular, the high levels of the omega-6 polyunsaturated fatty acid arachidonic acid (AA) in Western diets has been shown to promote aggressive prostate cancer in vitro. However, the exact mechanism through which AA induces the aggressive phenotype has not been fully characterised. Here Fourier transform infrared microspectroscopy (FTâIRM) coupled with fluorescence microscopy (FM) was used to follow AA metabolism in prostate cancer cell lines. Using partially deuterated AA, (d8-AA), with a distinctive C-D stretch seen at 2251 cmâ1 providing molecular specificity, coupled with Nile Red Fluorescence imaging, it has been shown that, unlike the non-invasive prostate cancer cell lines PNT2 and LNCaP, invasive prostate cancer lines PC-3M, PC-3. LNCaP C4-2B and DU145 readily uptake and metabolise AA, producing prostaglandin E2 (PGE2) via the cyclooxygenase-2 (COX-2) pathway. Inhibition of the COX-2 pathway with NS938 reduces the invasive stimulus of AA and blocks the uptake of AA. Similarly, the addition of the omega-3 poly unsaturated fatty acid Docosahexaenoic acid (DHA), previously shown to inhibit AA induced invasion, inhibited cellular AA uptake in invasive cell line PC-3. In conclusion, it has been demonstrated that FTâIRM can be utilised to follow metabolomics processes within a prostate model and provide an insight to the molecular pathways underlying the metabolome. This could play a pivotal role in understanding the chemistry and behaviour of the initiation of metastatic prostate cancer.