Use of ex vivo confocal laser scanning microscopy to differentiate actinic keratoses and cutaneous squamous cell carcinomas
Skin cancers are becoming increasingly common as people live longer and have higher levels of long-term sun exposure. CLI chatted to Dr Viktor Schnabel (Leipzig University, Leipzig, Germany) to find out more about how modern imaging technologies, particularly ex vivo confocal laser scanning microscopy, are being used to improve accurate diagnosis and treatment of skin lesions such as cutaneous squamous cell carcinomas and their precursor lesions, actinic keratoses.
What are cutaneous squamous cell carcinoma and actinic keratosis, and how do they relate to other common forms of skin cancer?
Cutaneous squamous cell carcinomas (cSCCs) and basal cell carcinomas (BCCs) are the most common skin cancers worldwide. The increasing incidence over recent years is largely attributed to population ageing, cumulative lifetime ultraviolet exposure and improved detection. Both cSCCs and BCCs are part of the non-melanoma skin cancers.
Despite their generally favourable prognosis, these tumours can cause significant morbidity if diagnosis and treatment are delayed. We were particularly interested in cSCC and its precursor lesions, namely actinic keratoses. Actinic keratoses arise from dysplastic keratinocytes within the epidermis and are generally considered part of a continuum of keratinocyte carcinogenesis. If atypical cells occupy the full thickness of the epidermis without breaching the basement membrane, the lesion is classified as in situ cSCC. Once tumour cells invade through the basement membrane into the underlying dermis, the lesion is considered invasive cSCC. Although the risk of progression of an individual lesion is relatively low, patients with multiple actinic keratoses have a substantially increased risk of developing invasive cSCC. In Germany, approximately 70 000 to 100 000 new cases of cSCC are diagnosed each year, making cSCC the second most common skin cancer after BCC. This growing incidence also places an increasing burden on healthcare systems due to the need for repeated consultations, surveillance and treatment. In their early stages, these tumours are usually easy to treat and rarely cause significant problems. However, if left untreated, they may continue to grow, invade deeper tissues, and cause substantial local destruction. In advanced cases, the tumour can extend into underlying structures such as muscle, cartilage, or even bone. Another important aspect of cSCC is its potential to metastasize. While the overall risk is relatively low, it increases with advanced disease, highlighting the importance of early diagnosis and treatment.
Layers of skin
Why is accurate diagnosis necessary?
Accurate diagnosis is essential to distinguish between benign lesions, precursor lesions such as actinic keratoses, and invasive cSCCs. This distinction directly influences treatment decisions, surgical planning, including margin assessment, and long-term follow-up strategies.
Furthermore, patients who develop one keratinocyte carcinoma are at increased risk of developing additional skin cancers over time. A particularly challenging situation is field cancerization, where chronically UV-damaged skin contains numerous clinically visible and subclinical lesions. In these patients, it is important to identify which lesions represent low-risk actinic keratoses and which may already have progressed to invasive disease. Therefore, accurate diagnosis is closely linked to risk stratification and helps clinicians prioritize treatment for the lesions most likely to require intervention.
By improving lesion selection, unnecessary biopsies or excisions can potentially be avoided, which benefits both patients and healthcare systems.
The key clinical message is that these lesions are generally highly treatable when detected early. Early diagnosis often allows for less extensive surgery, lower morbidity, and improved long-term outcomes.
How is diagnosis normally achieved?
The current gold standard for diagnosing cSCC and actinic keratosis is histopathological examination. While clinicians can often develop a strong clinical suspicion based on the appearance of a lesion, it is not always possible to reliably distinguish between benign lesions, precursor lesions and invasive tumours by clinical examination alone.
Therefore, a biopsy or excision specimen is typically obtained and processed for microscopic evaluation. After fixation and tissue processing, sections are stained, most commonly with hematoxylin and eosin (H&E), and examined by a pathologist or dermatopathologist.
Histopathology allows detailed assessment of tissue architecture and cellular morphology, enabling reliable differentiation between actinic keratosis, in situ cSCC, invasive cSCC and other skin tumours. In selected cases, additional immunohistochemical stains may be required to further characterize poorly differentiated lesions.
Overall, histopathology remains a highly reliable and well-established diagnostic method. However, the process requires tissue sampling and laboratory processing, meaning that a definitive diagnosis is often only available hours to days after the biopsy has been performed.
Red crusty lesions of actinic keratosis caused by sun (UV) exposure
What are the limitations of the current diagnosis pathway?
The main limitation of the current diagnostic pathway is the time required to obtain a definitive diagnosis. Although histopathology is highly accurate, tissue processing and microscopic evaluation typically require several hours to days before results become available, particularly in outpatient settings.
Another limitation is that the procedure is invasive, as it requires a biopsy or excision of tissue. While these procedures are generally well tolerated, they may cause discomfort, require local anaesthesia, and can result in scarring, particularly in cosmetically sensitive areas such as the face.
Furthermore, the conventional workflow often requires multiple patient visits: one for the biopsy, another to discuss the results, and, if necessary, additional appointments for treatment. This can delay clinical decision-making and increase the burden on both patients and healthcare systems.
Therefore, while histopathology remains the diagnostic gold standard, there is considerable interest in technologies that can provide reliable diagnostic information more rapidly and, ideally, with fewer procedures.
How can diagnosis be improved?
New imaging technologies
Over the past decade, imaging technologies have advanced considerably and are increasingly being integrated into dermatologic diagnostics. Because the skin is directly accessible, dermatology is particularly well suited for the application of non-invasive and minimally invasive imaging techniques. Several complementary technologies are now available.
In vivo confocal laser scanning microscopy
In vivo confocal laser scanning microscopy (CLSM) is a non-invasive imaging technique that provides near cellular-level resolution of skin structures. A laser scans the skin and generates optical sections that allow detailed morphological assessment of epidermal and superficial dermal structures.
The technique is well established in dermatology and is particularly valuable for the evaluation of melanocytic lesions and other diagnostically challenging skin tumours. However, one important limitation is its relatively limited penetration depth, which is generally restricted to the epidermis and superficial dermis. As a result, assessment of tumour invasion can be challenging in lesions with marked hyperkeratosis or in anatomical sites with a thick epidermis, such as the palms and soles. In addition, image acquisition and interpretation can be relatively time-consuming.
Ex vivo CLSM
Ex vivo CLSM, which was investigated in our study, uses freshly excised tissue rather than imaging the lesion directly on the patient. After a biopsy or excision, the specimen is rapidly stained, typically using acridine orange, and scanned using both reflectance and fluorescence lasers. The resulting images are digitally processed to resemble conventional H&E histology.
A major advantage of this approach is speed. High-resolution, histology-like images can be generated within minutes, allowing near real-time assessment of freshly excised tissue. The rapid availability of diagnostic information may help streamline clinical workflows, facilitate treatment planning and reduce delays between biopsy, diagnosis and definitive treatment. In selected situations, results can be discussed with patients during the same visit, potentially reducing the number of follow-up appointments. Importantly, the same specimen can subsequently undergo routine histopathological processing, enabling direct comparison of the ex vivo CLSM results with the diagnostic gold standard.
In our study, ex vivo CLSM demonstrated high diagnostic accuracy for the detection of actinic keratoses, cSCCs and BCCs. Beyond primary diagnosis, ex vivo CLSM has also shown promise for intraoperative margin assessment, where rapid evaluation of excised tissue may support surgical decision-making. While conventional histopathology remains the reference standard, ex vivo CLSM has the potential to substantially accelerate diagnostic workflows and support clinical decision-making.
Optical coherence tomography
Other imaging modalities are also becoming increasingly important in dermatology. Optical coherence tomography (OCT) enables non-invasive cross-sectional imaging of the skin and provides greater imaging depth than CLSM, making it particularly useful for assessing tumour architecture and invasion depth.
More recently, line-field confocal optical coherence tomography (LC-OCT) has emerged as one of the most promising developments in dermatologic imaging. LC-OCT combines the near-cellular resolution of confocal microscopy with the greater imaging depth of OCT, allowing visualization of skin structures in both vertical and horizontal planes. Recent studies have demonstrated excellent diagnostic performance for both neoplastic and inflammatory skin diseases, making LC-OCT a valuable tool for non-invasive skin cancer diagnostics and disease monitoring.
Beyond structural imaging techniques, functional imaging approaches such as hyperspectral imaging are also being investigated. These technologies provide information on tissue composition, oxygenation and vascular characteristics and may further support non-invasive lesion classification and risk stratification in the future.
Taken together, technologies such as CLSM, ex vivo CLSM, OCT, LC-OCT and hyperspectral imaging are expanding the possibilities of dermatologic diagnostics and may help to provide faster, more precise and less invasive patient care. While our study focused on cSCC and actinic keratoses, many of these technologies are increasingly being applied to other skin tumours and inflammatory skin diseases, highlighting their broad potential within dermatology.
What are the future perspectives in this field?
Streamlining the care pathway
One of the major challenges in dermatology is the increasing number of patients with skin cancer and precancerous lesions. Modern imaging technologies may help improve risk stratification, allowing clinicians to identify high-risk lesions more efficiently and prioritize treatment accordingly. This has the potential to streamline patient pathways, reduce unnecessary procedures and improve healthcare resource utilization.
Digital image analysis and artificial intelligence
Another exciting development is the integration of artificial intelligence into dermatologic imaging. Because technologies such as CLSM, LC-OCT and hyperspectral imaging generate digital datasets, they are ideally suited for computer-assisted image analysis. AI-based tools may support lesion classification, improve diagnostic consistency and assist clinicians in identifying lesions that require further investigation or treatment.
In parallel, professional societies are increasingly recognizing the role of imaging technologies in dermatology. For example, Germany recently published the S1 Guideline for Imaging Diagnostics in Skin Diseases (see Bibliography), reflecting the growing clinical importance of these techniques.
Teledermatology and telepathology
Digital imaging also opens new opportunities for teledermatology and telepathology. High-quality images can be acquired locally and assessed remotely by experienced specialists, potentially improving access to expert diagnostics in underserved regions. Combined with AI-assisted image analysis, these approaches may further support rapid lesion triage and clinical decision-making.
Expanding applications in dermatology
Although our study focused on the differentiation between actinic keratosis and invasive cSCC, these technologies are increasingly being applied to other areas of dermatology. Promising applications include basal cell carcinoma, melanocytic lesions, inflammatory skin diseases and certain infectious dermatoses. Continued technological development and improved image analysis may further expand their diagnostic utility in the coming years.
Overall, modern imaging technologies are already transforming dermatologic diagnostics and are likely to play an increasingly important role in delivering faster, more precise and more individualized patient care.
Ex vivo laser scanning microscopy workflow
- ✓ Tumour excision
- ✓ Transport specimen to the microscope (in 0.9% NaCl)
- ✓ Cut specimen
- ✓ Immerse in acridine orange (30 s)
- ✓ Wash in 0.9% NaCl (30 s)
- ✓ Place on the microscope stage
- ✓ Enter patient data and perform scan
- ✓ Results
Duration: just a few minutes
Bibliography
1. Schnabel V, Hempel C, Ziemer M, Simon JC, Grunewald S. Diagnostic accuracy of ex vivo confocal laser scanning microscopy for routine detection of cutaneous squamous cell carcinoma and actinic keratoses. Cancers (Basel). 2026;18(9):1458 (https://pubmed.ncbi.nlm.nih.gov/42122253/).
2. Deußing M, Schuh S, Thamm J, Winkler D, Schneider S et al. S1 guideline for imaging diagnostics for skin diseases. J Dtsch Dermatol Ges. 2025;23:1616–1630 (https://doi.org/10.1111/ddg.15883).
3. Vladimirova G, Ruini C, Kapp F, Kendziora B, Ergün EZ et al. Ex vivo confocal laser scanning microscopy: a diagnostic technique for easy real-time evaluation of benign and malignant skin tumours. J Biophotonics. 2022;15:e202100372 (https://doi.org/10.1002/jbio.202100372).
4. Pérez-Anker J, Toll A, Puig S, Malvehy J. Six steps to reach optimal scanning in ex vivo confocal microscopy. J Am Acad Dermatol. 2022;86:188–189 (https://doi.org/10.1016/j.jaad.2021.01.044).
5. Schüürmann M, Stecher MM, Paasch U, Simon JC, Grunewald S. Evaluation of digital staining for ex vivo confocal laser scanning microscopy. J Eur Acad Dermatol Venereol. 2020;34:1496–1499 (https://doi.org/10.1111/jdv.16085).
6. Grupp M, Illes M, Mentzel J, Simon JC, Paasch U, Grunewald S. Routine application of ex vivo confocal laser scanning microscopy with digital staining for examination of surgical margins in basal cell carcinomas. J Dtsch Dermatol Ges. 2021;19:685–692 (https://doi.org/10.1111/ddg.14374).
The interviewee
Dr Viktor Schabel, MD
Senior Physician
Department of Dermatology, Venereology and Allergology, University Medical Center Leipzig (AöR), 04103 Leipzig, Germany
Email address: viktor.schnabel@medizin.uni-leipzig.de
