###### Abstract  
Actinic keratoses (AKs) represent a common skin cancer in situ associated with chronic sun exposure. Early diagnosis and management of AKs are crucial to prevent their progression to invasive squamous cell carcinoma. Therefore, we investigated AK PRO score assessment using ex vivo confocal laser microscopy (EVCM) coupled with a novel fluorescent dye, FCF Fast Green, to explore its potential for the precise imaging and discrimination of collagen fibers. AK PRO assessment using EVCM demonstrated excellent conformity (95.8%) with histopathologic examination. The additional utilization of FCF Fast Green dye had no impact on AK visualization but showed a high affinity for collagen fibers enabling clear differentiation of collagen alterations between healthy and sun-damaged skin. The enhanced visualization of collagen fiber changes may aid clinicians in accurately identifying AKs and differentiating them from benign skin lesions. dramatology; non-invasive imaging; reflectance confocal microscopy; skin cancer; bedside histology; actinic keratoses; fluorescent dye; collagen imaging +
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To develop this area of research, and considering the fact that the interaction between the epidermis and dermis, including tumor invasiveness, arises at the dermoepidermal junction zone [11], a histomorphological classification of atypical keratinocytes based on the \"basal growth pattern\", called (PRO I-III), has been proposed [12]. The PRO score is a frequently used clinical scoring system for AK-based histology findings at the DEJ [13]. While an early-stage PRO I can be recognized by \"clustered and atypical keratinocytes in the basal epidermal layers\", PRO II reveals \"small hemispherical buds from the basal epidermis emerge into the upper papillary dermis\". In PRO III, \"spiky or filiform papillary elongations of atypical keratinocytes extend into the upper dermis\" [13; 14]. While visual grading systems often show a high interobserver variability, a study conducted by [PERSON] et al., including 21 dermatologists and pathologists, demonstrated a robust inter-rater reliability of AK PRO assessment [12]. Nevertheless, all these classifications are based on histological examinations, which can be costly and time-consuming and may delay a proper treatment initiation. In response, new imaging techniques have gained momentum in recent years, aiming to provide faster and enhanced diagnostic accuracy [15]. Ex vivo confocal laser microscopy (EVCM) has emerged as a powerful diagnostic method offering near-real-time and rapid imaging of freshly excised tissue at cellular and subcellular level. By employing near-infrared lasers and detecting reflected light along fluorescence signals, EVCM provides high-resolution vertical images nearly similar to conventional histopathology [16]. While this technique has already demonstrated its utility in various dermatological applications, including the intraoperative assessment of excision margins as an adjunctive method to Mohs microscopic surgery [17; 18], the assessment of melanocytic lesions [19; 20], inflammatory skin disorders [21], and superficial skin cancers [17; 18; 19; 20; 22; 23; 24; 25], the evaluation of PRO score assessment in actinic keratoses has not yet been investigated. To further enhance the specificity and sensitivity of reflectance microscopy, numerous fluorescent dyes with specific binding properties have been developed [17; 18; 19; 20; 24; 26; 27]. Among these, FCF Fast Green (FG)--a synthetic substance originally used for coloring food items--has shown its capability in the precise visualization of collagen and elastic fibers. FCF Fast Green fluorescent dye emits a fluorescent signal in the far-red range at 638 nm, while also being able to be combined with other antibody-coupled labels and/or nuclear stains that also emit signals at shorter wavelengths. When applied and kept in anhydrous conditions, FG is highly selective to collagen.