The persistent presence of pathogenic bacteria is one of the main obstacles to wound healing. Detection of wound bacteria relies on sampling methods, which delay confirmation by several days. However, a novel handheld fluorescence imaging device has recently enabled real-time detection of bacteria in wounds based on their intrinsic fluorescence characteristics, which differ from those of background tissues. This device illuminates the wound with violet (405 nm) light, causing tissues and bacteria to produce endogenous, characteristic fluorescence signals that are filtered and displayed on the device screen in real-time. The resulting images allow for rapid assessment and documentation of the presence, location, and extent of fluorescent bacteria at moderate-to-heavy loads. This information has been shown to assist in wound assessment and guide patient-specific treatment plans. However, proper image interpretation is essential to assessing this information. To properly identify regions of bacterial fluorescence, users must understand: (1) Fluorescence signals from tissues (e.g., wound tissues, tendon, bone) and fluids (e.g., blood, pus); (2) fluorescence signals from bacteria (red or cyan); (3) the rationale for varying hues of both tissue and bacterial fluorescence; (4) image artifacts that can occur; and (5) some potentially confounding signals from non-biological materials (e.g., fluorescent cleansing solutions). Therefore, this tutorial provides clinicians with a rationale for identifying common wound fluorescence characteristics. Clinical examples are intended to help clinicians with image interpretation—with a focus on image artifacts and potential confounders of image interpretation—and suggestions of how to overcome such challenges when imaging wounds in clinical practice.

Background/Objectives: Accurate and reproducible wound measurement is essential for monitoring healing and guiding treatment decisions. Conventional ruler-based techniques are prone to geometric overestimation and operator variability. This study evaluated the accuracy and reproducibility of the MolecuLightDX wound imaging device for measuring wound surface area and depth compared with ruler-based measurements and ground truth digital photography methods. Methods: This investigation comprised two companion studies: a prospective, paired, multicenter clinical study comparing MolecuLightDX measurement with the ruler method against an image-based ground truth, and a bench and clinical validation of the AutoDepth feature against a calibrated three-dimensional optical scanner. The area of study included 17 benchtop wound models and enrolled 27 patients (33 wounds; area range: 0.56–23.04 cm²) across two wound care centers, and the AutoDepth study included 17 benchtop wound models and 34 clinical wounds (depth range: 0.06–4.13 cm). Accuracy, intra- and inter-user variability, and agreement were assessed using the mean percentage error (MPE), coefficient of variation (CV), intraclass correlation coefficients (ICC), and Bland–Altman analysis. Results: The device demonstrated high accuracy and reproducibility for both wound surface area and depth measurements compared with ruler-based and ground truth digital photography methods. The MPE for surface area was <10%, representing a tenfold improvement over ruler estimation (77.9%). For wound area, intra- and inter-user CVs were <10%, and for depth, ICCs were ≈0.99. Conclusions: The MolecuLightDX device provides accurate and consistent wound area and depth measurements across diverse wound types, demonstrating superior accuracy and reproducibility compared with conventional ruler-based methods and supporting its integration into wound assessment workflows.