## 2 Materials and Methods  
Study Participants: Participants were recruited at the Department of Dermatology and Allergy of the University Hospital at Ludwig Maximilian University (LMU) in Munich between March and September 2023. Patients included in our study had skin types varying from Fitzpatrick type I to III. Each patient gave written informed consent before inclusion in the study, which was approved by the local ethics committee of the LMU university hospital (Ref.-Nr. 19-150). Patients with clinically suspected actinic keratoses (AKs) underwent standard biopsy procedures, resulting in 61 samples. Within this number, 13 cases eventually proved to be Bowens disease, invasive squamous cell carcinoma, or basal cell carcinoma in histology and EVCM. To maintain the focus on actinic keratoses, these lesions were excluded from our study, ensuring that only cases with histologically confirmed AK (=48 lesions) were included. The locations of the lesions were mainly on the capillitium, face, and forearms. A total of 32 healthy control skin samples with no history of chronic sun exposure were collected from wound closures and Burow triangles. Sample sizes ranged from 2 to 10 mm. Ex vivo confocal laser scanning microscopy (EVCM): Prior to imaging, one cohort of the skin samples (25 AK lesions and 17 healthy skin samples) underwent a new staining protocol consisting of incubation in ethanol (0.7 mmol/L), acridine orange (AO) (0.04 mmol/L, Sigma-Aldrich, St. Louis, MO, USA), FCF Fast Green (0.067 mmol/L, Sigma-Aldrich, St. Louis, MO, USA), and NaCl (0.09 mmol/L) for 30 s each. Due to the incubation in ethanol, a sample dehydration with optimal binding of FCF Fast Green to collagen fibers was achieved. The other cohort (23 AK lesions and 15 healthy skin samples) underwent a previously established staining protocol using acridine orange (0.1 mmol/L, Sigma-Aldrich, St. Louis, MO, USA) followed by phosphate-buffered saline (0.1 mmol/L, Dulbecco's Phosphate Buffered Saline; PBS; pH 7.4) and citric acid (0.1 mmol/L) for so-called aceto-whitening for 30 s each [20]. All samples were fully immersed in the staining solutions, which covered the entire cut surface. Ex vivo confocal laser scanning microscopy (EVCM) imaging was performed using the Vivascope 2500 G-4 device (Vivascope, Munich, Germany) with two laser wavelengths at 488 nm (blue) and 638 nm (red). Therefore, the tissue probes were placed on object slides ([PERSON], Emmendingen, Germany), mounted with sponges as well as magnets, and sectioned in vertical mode to reveal all skin layers according to standard histological procedures [31; 32]. While the blue 488 nm laser excites acridine orange-stained structures, generating a fluorescence image, the reflection of the red 638 laser usually produces a reflectance image. With the use of the new FCF Fast Green dye, which fluoresces at 638 nm, the red laser can be used either in reflectance mode (with the pinhole filter open) or in fluorescence mode with FCF Fast Green staining (pinhole filter activated) (Figure 1a). Analogously to previous studies, both of these fluorescence images can be merged together to produce a digital HE (dHE) image similar to a traditional histopathological examination (Figure 1b-d) [24]. Histological analysis and correlation:  
After imaging, the skin samples from both cohorts were immediately fixed in 4% buffered formalin and subsequently processed for routine paraffin embedding. A histological analysis was conducted on paraffin-embedded skin sections stained with hematoxylin and eosin (H&E). A histological assessment of PRO score was performed by an experienced dermatopathologist blinded to the clinical and imaging data. EVCM image analysis:  
All images were analyzed by two experienced examiners (L.B., M.D. ), who were blinded to the clinical diagnosis and staining protocol. To mitigate biases, the examiners reviewed both AO and FCF images for all cases in a randomized order. Nevertheless, good interobserver agreement was found for all parameters.