## 4 Discussion  
The accurate diagnosis and timely management of actinic keratoses (AKs) are essential to prevent their progression into invasive squamous cell carcinoma [34]. Since NMSC is frequently surrounded by UV-induced skin alterations like AKs, it is therefore important to evaluate the diagnostic workflow of these precancerous lesions for a simple and fast risk stratification. When the dermatologist/practitioner has no laboratory or pathologist  
Figure 4: EVCM imaging of collagen fiber patterns in digital HE (**a**–**c**) and corresponding FCF fluorescence mode (**d**–**f**). White circles depict exemplarily regular collagen fibers (**a**,**d**) and altered collagen fibers with blurred lines (**b**,**e**), as well as damaged collagen fibers represented as degeneration patterns (**c**,**f**). Figure 5: Scatter plot showing the quantitative results of the fiber brightness index (=FBI) in actinic keratoses (AK) and healthy skin imaged with the FCF and regular staining protocol. *** = \(p\) < 0.001.  
for cryosections nearby, EVCM may be a valid adjunction to fresh frozen sectioning or traditional histopathology. This imaging approach aligns with the growing interest in minimizing costly and time-consuming traditional histopathological procedures. Therefore, our study investigated the feasibility of PRO score assessment in EVCM images. EVCM therefore may provide dermatologists with a valuable tool to confidently diagnose and differentiate the stages of AKs in only a few minutes, providing a possibility for bedside assessment and prompt treatment initiation. The use of a new staining protocol using FCF Fast Green furthermore proved to be a valid method for the visualization of UV-induced collagen alterations, offering the potential to assess the degree of collagen damage and its association with AK pathogenesis. The observed disarray and fragmentation of collagen fibers within AK lesions are consistent with previous histopathological studies that have highlighted collagen remodeling as a hallmark of sun-induced skin damage [35, 36, 37]. These changes disrupt the skin's structural integrity, leading to functional changes, accompanied by an underlying inflammatory and oxidative response that affects the components of the extracellular matrix and triggers the activation of proteolytic enzymes such as metalloproteinases, potentially creating an environment conducive to the progression of precancerous lesions [38]. The quantitative analyses of collagen parameters confirmed our first visual impressions and revealed significant differences between AK lesions and healthy control skin. The decreased collagen density and altered orientation observed in AKs are consistent with the tissue remodeling characteristic of precancerous lesions. The increased collagen fragmentation further highlights the severity of collagen damage in these lesions. The early identification of precancerous lesions may therefore allow for proactive interventions and prevention of the progression to invasive malignancies. The ability of FCF Fast Green-enhanced EVCM to unveil collagen modifications that may, at an early stage, precede visible cellular atypia offers a unique advantage in enhancing the temporal resolution of diagnostics. These quantitative insights may contribute to more objective diagnostic criteria for AK assessment and potentially aid in risk stratification, reinforcing the diagnostic value of collagen imaging. While in anhydrous conditions FCF Fast Green primarily targets collagen fibers, the capability of FGs binding affinity to collagen is significantly decreased in watery conditions. In this case, not only collagen but also elastic fibers are dyed. To maximize collagen affinity, our adjusted staining protocol included an additional step with rinsing in ethanol for 30 s for adequate tissue sample dehydration. The choice of imaging dye is not a one-size-fits-all decision and depends on the specific formulation of the question.