Keratoacanthoma (KA) is a common epidermal tumor that originates from the hair follicle of the skin. It is generally considered as a benign neoplasm, but in rare cases, it can also transform into squamous cell carcinoma. Although surgical excision with a safety margin is considered to be the gold standard treatment for most subtypes of KA, several other treatment options are also available. Intralesional therapy is one of these options, which could be cosmetically and functionally a better alternative to surgical removal, while it provides similar outcomes. It is more effective than topical treatments, yet fewer side effects may be seen than in systemic treatments. Based on the literature, the most commonly used intralesional agent is methotrexate, followed by 5‐fluorouracil and interferon alpha. Regardless of the advantages, which make intralesional therapy a desirable treatment alternative, guidelines for the intralesional treatment of KA are not yet established. A histopathological confirmation before the start of treatment is still recommended to prevent any possible misdiagnosis of KA for SCC. In our present study, we set out to review the current state of the art of the intralesional treatment of KA.

Basal cell carcinoma (BCC) is the most frequent malignant neoplasm in the Caucasian population. There are several therapeutic options for BCC, but surgical excision is considered gold standard treatment. As BCCs often have poorly defined borders, the clinical assessment of the tumor margins can be challenging. Therefore, there is an increasing demand for efficient in vivo imaging techniques for the evaluation of tumor borders prior to and during surgeries. In the near future, nonlinear microscopy techniques might meet this demand. We measured the two-photon excitation fluorescence (TPEF) signal of nicotinamide adenine dinucleotide hydride (NADH) and elastin and second harmonic generation (SHG) signal of collagen on 10 ex vivo healthy control and BCC skin samples and compared the images by different quantitative image analysis methods. These included integrated optical density (IOD) measurements on TPEF and SHG images and application of fast Fourier transform (FFT), CT-FIRE and CurveAlign algorithms on SHG images to evaluate the collagen structure. In the BCC samples, we found significantly lower IOD of both the TPEF and SHG signals and higher collagen orientation index utilizing FFT. CT-FIRE algorithm revealed increased collagen fiber length and decreased fiber angle while CurveAlign detected higher fiber alignment of collagen fibers in BCC. These results are in line with previous findings which describe pronounced changes in the collagen structure of BCC. In the future, these novel image analysis methods could be integrated in handheld nonlinear microscope systems, for sensitive and specific identification of BCC.

Obesity related metabolic syndrome and type 2 diabetes have severe consequences on our skin. Latest developments in nonlinear microscopy allow the use of noninvasive, label free imaging methods, such as second harmonic generation (SHG) and coherent anti-Stokes Raman scattering (CARS), for early diagnosis of metabolic syndrome-related skin complications by 3D imaging of the skin and the connective tissue. Our aim was to study effects of various types of diet-induced obesity in mice using these methods. We examined mice on different diets for 32 weeks. The collagen morphology was evaluated four times by SHG microscopy, and adipocytes were examined once at the end of experiment by CARS method. A strong correlation was found between the body weight and the adipocyte size, while we found that the SHG intensity of dermal collagen reduces considerably with increasing body weight. Obese mice on high-fat diet showed worse results than those on high-fat - high-fructose diet. Animals on high-fructose diet did not gain more weight than those on ordinary diet despite of the increased calorie intake, but their collagen damage was nonetheless significant. Obesity and high sugar intake damages the skin, mainly the dermal connective tissue and subcutaneous adipose tissue, which efficiently can be monitored by SHG and CARS microscopy.

Basal cell carcinoma (BCC) is the most common malignancy in Caucasians. Nonlinear microscopy has been previously utilized for the imaging of BCC, but the captured images do not correlate with H&E staining. Recently, Freudiger et al. introduced a novel method to visualize tissue morphology analogous to H&E staining, using coherent anti-Stokes Raman scattering (CARS) technique. In our present work, we introduce a novel algorithm to post-process images obtained from dual vibration resonance frequency (DVRF) CARS measurements to acquire high-quality pseudo H&E images of BCC samples. We adapted our CARS setup to utilize the distinct vibrational properties of CH (mainly in proteins) and CH bonds (primarily in lipids). In a narrowband setup, the central wavelength of the pump laser is set to 791 nm and 796 nm to obtain optimal excitation. Due to the partial overlap of the excitation spectra and the 5-10 nm FWHM spectral bandwidth of our lasers, we set the wavelengths to 790 nm (proteins) and 800 nm (lipids). Nonresonant background from water molecules also reduces the chemical selectivity which can be significantly improved if we subtract the DVRF images from each other. As a result, we acquired two images: one for "lipids" and one for" proteins" when we properly set a multiplication factor to minimize the non-specific background. By merging these images, we obtained high contrast H&E "stained" images of BBC's. Nonlinear microscope systems upgraded for real time DVRF CARS measurements, providing pseudo H&E images can be suitable for in vivo assessment of BCC in the future.

Ehlers-Danlos syndrome (EDS) is the name for a heterogenous group of rare genetic connective tissue disorders with an overall incidence of 1 in 5000. The histological characteristics of EDS have been previously described in detail in the late 1970s and early 1980s. Since that time, the classification of EDS has undergone significant changes, yet the description of the histological features of collagen morphology in different EDS subtypes has endured the test of time. Nonlinear microscopy techniques can be utilized for non-invasive in vivo label-free imaging of the skin. Among these techniques, two-photon absorption fluorescence (TPF) microscopy can visualize endogenous fluorophores, such as elastin, while the morphology of collagen fibers can be assessed by second-harmonic generation (SHG) microscopy. In our present work, we performed TPF and SHG microscopy imaging on ex vivo skin samples of one patient with classical EDS and two patients with vascular EDS and two healthy controls. We detected irregular, loosely dispersed collagen fibers in a non-parallel arrangement in the dermis of the EDS patients, while as expected, there was no noticeable impairment in the elastin content. Based on further studies on a larger number of patients, in vivo nonlinear microscopic imaging could be utilized for the assessment of the skin status of EDS patients in the future.