Footwear impression evidence in dust is often difficult to locate in ambient light and is a fragile medium that both collection and enhancement techniques can destroy or distort. The collection of footwear impression evidence always begins with non-destructive photographic techniques; however, current methods are limited to oblique lighting of the impression followed by an attempt to photograph in situ12,15,17. For the vast majority of footwear impressions, an interactive collection method, and thus a potentially destructive procedure, is subsequently carried out to gather the evidence18. Therefore, alternative non-destructive means for the preservation and enhancement of footwear impressions in dust merits further attention. Previous research performed with reflected ultraviolet (UV) photography and reflected ultraviolet imaging systems (RUVIS) has shown that there are additional non-destructive methodologies that can be applied to the search for and documentation of footwear impressions in dust34,36,37. Unfortunately, these prior studies did not include robust comparisons to traditional oblique white light, instead choosing to focus on different UV wavelengths. This study, however, seeks to evaluate the use of a RUVIS device paired with a 254 nanometer (nm) UV light source to locate 2-D footwear impressions in dust on multiple substrates against standard oblique white light techniques and assess the visibility of the impression and amount of background interference present. The optimal angle of incident UV light for each substrate was also investigated. Finally, this study applied an image enhancement technique in order to evaluate its usefulness when looking at the visibility of a footwear impression and the amount of background interference present for enhanced white light and RUVIS pictures of footwear impressions in dust. A collection of eight different substrate types was gathered for investigation, including vinyl composition tile (VCT), ceramic tile, marble tile, magazine paper, steel sheet metal, vinyl flooring, wood flooring, and carpet. Heel impressions were applied to the various substrates utilizing vacuum collected dust and normal walking pressure. Each substrate was then explored and photographed in ambient fluorescent light, oblique white light at 0, 15, 30, and 0 with the light source below the surface plane of the substrate, and 254 nm UV light at 0, 15, 30, 45, 60, 75, 90 and 0 with the light source below the surface plane of the substrate. All pictures were evaluated for clarity and visible detail of the footwear impression and the amount of background interference present, selecting for the best images within a lighting condition group. Additional intra- and intergroup comparisons were carried out to explore differences created by the various lighting conditions. Enhanced images were then created with the best scored pictures and evaluated for additional modifications in impression visibility and background interference. Photographs of footwear impressions in dust illuminated with ambient fluorescent light proved to be the most difficult conditions under which a footwear impression could be visualized. However, both oblique white light and 254 nm UV light lighting conditions showed improvements in either visualization or background dropout, or both, over ambient light conditions. An assessment of the white light and 254 nm UV light RUVIS images also demonstrated that the best angles for the light source for all substrates were oblique 0 and oblique 0 below the surface plane of the substrate lighting. It was found that white light photographs generally provided higher visibility ratings, while RUVIS 254 nm UV light photographs provided better grades for reducing background interference. Enhanced images of white light conditions provided generally poorer quality and quantity of details, while enhanced RUVIS images seemed to improve upon these areas. The use of a RUVIS to capture photographs of footwear impression evidence in dust was found to be a successful secondary non-destructive technique that can be paired with traditional oblique white light procedures. Additionally, the use of below the surface plane of the substrate lighting techniques were found to improve either visibility or background dropout, or both, over standard 0 oblique lighting, depending on the light source, and should be employed, when applicable. Finally, further investigation into digital photo-editing enhancement techniques for footwear impression evidence in dust is needed.