In vivo two-photon-excited cellular fluorescence of melanin, NAD(P)H, and keratin enables an accurate differential diagnosis of seborrheic keratosis and pigmented cutaneous melanoma.

SIGNIFICANCE: Seborrheic keratoses (SKs) are harmless pigmented skin lesions (PSLs) that may be confused clinically not only with other benign conditions but also with cutaneous melanoma (CM). As SKs are one of the most common neoplasms in adults, the importance of their correct diagnosis is high. Misclassifying SK as malignant is not rare and leads to a high number of unnecessary biopsies. On the other hand, misdiagnosing CM as SK may have a large impact on prognosis or therapy. AIM: In the non-invasive technique of dermatofluoroscopy, the fluorophores in melanocytes and keratinocytes are excited in vivo with nanosecond laser pulses and the resulting spectrally resolved, melanin-dominated fluorescence signals are used to differentiate between pigmented benign lesions and CM. APPROACH: In this single-center, non-interventional study, 33 PSLs of 20 patients were scanned with dermatofluoroscopy in vivo. For all included cases, dermatofluoroscopic signals were compared to pathology classification. RESULTS: The characteristic spectral features of SK were identified, where the signals are dominated by keratin, NAD(P)H, and melanin. The fluorescence spectra of SKs differed substantially from those of CM: a characteristic spectrum of SK has been identified in 27 of 28 SKs. CONCLUSIONS: The high-accuracy differential diagnosis between CM and SK is possible with dermatofluoroscopy.

Diagnostic Performance of a Support Vector Machine for Dermatofluoroscopic Melanoma Recognition: The Results of the Retrospective Clinical Study on 214 Pigmented Skin Lesions.

The need for diagnosing malignant melanoma in its earliest stages results in an increasing number of unnecessary excisions. Objective criteria beyond the visual inspection are needed to distinguish between benign and malignant melanocytic tumors in vivo. Fluorescence spectra collected during the prospective, multicenter observational study ("FLIMMA") were retrospectively analyzed by the newly developed machine learning algorithm. The formalin-fixed paraffin-embedded (FFPE) tissue samples of 214 pigmented skin lesions (PSLs) from 144 patients were examined by two independent pathologists in addition to the first diagnosis from the FLIMMA study, resulting in three histopathological results per sample. The support vector machine classifier was trained on 17,918 fluorescence spectra from 49 lesions labeled as malignant (1) and benign (0) by three histopathologists. A scoring system that scales linearly with the number of the "malignant spectra" was designed to classify the lesion as malignant melanoma (score > 28) or non-melanoma (score ≤ 28). Finally, the scoring algorithm was validated on 165 lesions to ensure model prediction power and to estimate the diagnostic accuracy of dermatofluoroscopy in melanoma detection. The scoring algorithm revealed a sensitivity of 91.7% and a specificity of 83.0% in diagnosing malignant melanoma. Using additionally the image segmentation for normalization of lesions' region of interest, a further improvement of sensitivity of 95.8% was achieved, with a corresponding specificity of 80.9%.

LA-ICP-MS Allows Quantitative Microscopy of Europium-Doped Iron Oxide Nanoparticles and is a Possible Alternative to Ambiguous Prussian Blue Iron Staining.

The development of iron oxide nanoparticles for biomedical applications requires accurate histological evaluation. Prussian blue iron staining is widely used but may be unspecific when tissues contain substantial endogenous iron. Here we tested whether microscopy by laser ablation coupled to inductively coupled plasma mass spectrometry (LA-ICP-MS) is sensitive enough to analyze accumulation of very small iron oxide particles (VSOP) doped with europium in tissue sections. For synthesis of VSOP, a fraction of Fe3+ (5 wt%) was replaced by Eu3+, resulting in particles with 0.66 mol% europium relative to iron (Eu-VSOP) but with otherwise similar properties as VSOP. Eu-VSOP or VSOP was intravenously injected into ApoE-/- mice on Western cholesterol diet and accumulated in atherosclerotic plaques of these animals. Prussian blue staining was positive for ApoE-/- mice with particle injection but also for controls. LA-ICP-MS microscopy resulted in sensitive and specific detection of the europium of Eu-VSOP in liver and atherosclerotic plaques. Furthermore, calibration with Eu-VSOP allowed calculation of iron and particle concentrations in tissue sections. The combination of europium-doped iron oxide particles and LA-ICP-MS microscopy provides a new tool for specific and quantitative analysis of particle distribution at the tissue level and allows correlation with other elements such as endogenous iron.

Determination of blood circulation times of superparamagnetic iron oxide nanoparticles by T2* relaxometry using ultrashort echo time (UTE) MRI.

Blood circulation is an important determinant of the biodistribution of superparamagnetic iron oxide nanoparticles. Here we present a magnetic resonance imaging (MRI) technique based on the use of ultrafast echo times (UTE) for the noninvasive determination of blood half-lives at high particle concentrations, when conventional pulse sequences fail to produce a useful MR signal. Four differently coated iron oxide nanoparticles were administered intravenously at a dose of 500 µmol Fe/kg bodyweight and UTE images of C57BL/6 mice were acquired on a 1-T ICON scanner (Bruker). T2* relaxometry was done by acquiring UTE images with echo times of 0.1, 0.8 and 1.6 ms. Blood circulation time was then determined by fitting an exponential curve to the time course of the measured relaxation rates. Circulation time was shortest for particles coated with malic acid (t1/2=23 min) and longest for particles coated with tartaric acid (t1/2=63 min). UTE-based T2* relaxometry allows noninvasive determination of blood circulation time and is especially useful when high particle concentrations are present.

Synthesis of acid-stabilized iron oxide nanoparticles and comparison for targeting atherosclerotic plaques: evaluation by MRI, quantitative MPS, and TEM alternative to ambiguous Prussian blue iron staining.

To further optimize citrate-stabilized VSOPs (very small iron oxide particles, developed for MR angiography) for identification of atherosclerotic plaques, we modified their surface during synthesis using eight other acids for electrostatic stabilization. This approach preserves effective production for clinical application. Five particles were suitable to be investigated in targeting plaques of apoE(-/-) mice. Accumulation was evaluated by ex vivo MRI, TEM, and quantitatively by magnetic particle spectroscopy (MPS). Citric- (VSOP), etidronic-, tartaric-, and malic-acid-coated particles accumulated in atherosclerotic plaques with highest accumulation for VSOP (0.2‰ of injected dose). Targets were phagolysosomes of macrophages and of altered endothelial cells. In vivo MRI with VSOP allowed for definite plaque identification. Prussian blue staining revealed abundant endogenous iron in plaques, indistinguishable from particle iron. In apoE(-/-) mice, VSOPs are still the best anionic iron oxide particles for imaging atherosclerotic plaques. MPS allows for quantification of superparamagnetic nanoparticles in such small specimens. FROM THE CLINICAL EDITOR: The presence of vulnerable plaques in arteries is important for the prediction of acute coronary events. VSOP (very small iron oxide particles, developed for MR angiography) have been shown to be very sensitive in identifying atherosclerotic plaques. The authors studied here further modification to the surface of VSOP during synthesis and compared their efficacy.

Imaging of magnetic microfield distortions allows sensitive single-cell detection.

Cell tracking with magnetic resonance imaging (MRI) is mostly performed using superparamagnetic iron oxide (SPIO) nanoparticle-labeled cells. However, negative contrast in T2*-weighted imaging is inherently problematic as a homogeneous background signal is required to visualize the negative signal. In a magnetic field, SPIO-labeled cells develop their own magnetization, distorting the main field. We show here a method to visualize these distortions and use them to identify single cells with increased sensitivity and certainty compared to T2* images. We labeled HeLa cells with SPIOs, suspended labeled cells in agarose to make phantoms, and performed high-resolution gradient-echo MRI. Phase images were processed to enhance the visibility of single cells. To quantify SPIO content, we generated a map of frequency differences. MRI of cell phantoms showed that single cells could be detected at concentrations ranging from 200 to 10,000 cells mL(-1). Postprocessing of the magnetic resonance phase images reveals characteristic microfield distortions, increasing dramatically the sensitivity of cell recognition, compared to unprocessed T2* images. Calculating frequency shifts and comparing microfield distortions to simulations permit estimation of the nanoparticle load of single cells. We expect the ability to detect and quantify the iron load of single cells to prove useful in studies of cell trafficking, especially in rare cell populations.