The development of new imaging tools, molecules and modalities is crucial to understanding biological processes and the localised cellular impact of bioactive compounds. A small molecule photosensitiser, DC473, has been designed to be both highly fluorescent and to exhibit a strong Raman signal in the cell-silent region of the Raman spectrum due to a diphenylacetylene structure. DC473 has been utilised to perform a range of novel tandem fluorescence and Raman (fluoRaman) imaging probe experiments, enabling a thorough examination of the compound’s cellular localisation, exemplified in colorectal cancer cells (SW480). This multifunctional fluoRaman imaging modality has revealed the presence of the compound in lipid droplets and only weak signal in the cytosol, where both Raman and fluorescence results show the presence of the fluoRaman imaging probe. In addition, Raman microscopy detected the compound in a cell compartment we labelled as the nucleolus, where fluorescence microscopy did not detect the fluoRaman probe due to solvatochromatic effects in a local polar environment. This last finding was only possible with the use of tandem confocal Raman and fluorescence methods. By following the approach detailed herein, incorporation of strong Raman functional groups into fluorophores can enable a plethora of fluoRaman experiments, shedding further light on an imaging probe or potential drug compound’s cellular behaviour and biological activity
