An ultracompact high-resolution multiphoton cryomicroscope with a femtosecond near infrared fiber laser has been utilized to study the cellular autofluorescence during freezing and thawing of cells. Cooling resulted in an increase of the intracellular fluorescence intensity followed by morphological modifications at temperatures below -10 °C, depending on the application of the cryoprotectant DMSO and the cooling rate. Furthermore, fluorescence lifetime imaging revealed an increase of the mean lifetime with a decrease in temperature. Non-destructive, label-free optical biopsies of biomaterial in ice can be obtained with sub-20 mW mean powers.
In vivo histology: Optical biopsies with chemical contrast using clinical multiphoton/coherent anti-Stokes Raman scattering tomography
Weinigel M, Breunig HG, Kellner-Höfer M, Bückle R, Darvin ME, Klemp M, Lademann J and König K.
Laser Physics Letter 11(5):055601, 2014 March. DOI: 10.1088/1612-2011/11/5/055601
The majority of existing coherent anti-Stokes Raman scattering (CARS) imaging systems are still huge and complicated laboratory systems and neither compact nor user-friendly nor mobile medically certified CARS systems. We have developed a new flexible multiphoton/CARS tomograph for imaging in a clinical environment. The system offers exceptional 360° flexibility with a very stable setup and enables label free 'in vivo histology' with chemical contrast within seconds. It can be completely operated by briefly trained non-laser experts. The imaging capability and flexibility of the novel in vivo tomograph are shown on optical biopsies with subcellular resolution and chemical contrast of patients suffering from psoriasis and squamous cell carcinoma.
Distinguishing between benign and malignant melanocytic nevi by in vivo multiphoton microscopy
Balu M, Kelly KM, Zachary CB, Harris RM, Krasieva TB, König K, Durkin AJ and Tromberg BJ.
Cancer Res. 74(10):2688-97, 2014 May 15. DOI: 10.1158/0008-5472.CAN-13-2582. Epub 2014 Mar 31.
Monitoring of atypical nevi is an important step in early detection of melanoma, a clinical imperative in preventing the disease progression. Current standard diagnosis is based on biopsy and histopathologic examination, a method that is invasive and highly dependent upon physician experience. In this work, we used a clinical multiphoton microscope to image in vivo and noninvasively melanocytic nevi at three different stages: common nevi without dysplastic changes, dysplastic nevi with structural and architectural atypia, and melanoma. We analyzed multiphoton microscopy (MPM) images corresponding to 15 lesions (five in each group) both qualitatively and quantitatively. For the qualitative analysis, we identified the morphologic features characteristic of each group. MPM images corresponding to dysplastic nevi and melanoma were compared with standard histopathology to determine correlations between tissue constituents and morphology and to evaluate whether standard histopathology criteria can be identified in the MPM images. Prominent qualitative correlations included the morphology of epidermal keratinocytes, the appearance of nests of nevus cells surrounded by collagen fibers, and the structure of the epidermal-dermal junction. For the quantitative analysis, we defined a numerical multiphoton melanoma index (MMI) based on three-dimensional in vivo image analysis that scores signals derived from two-photon excited fluorescence, second harmonic generation, and melanocyte morphology features on a continuous 9-point scale. Indices corresponding to common nevi (0-1), dysplastic nevi (1-4), and melanoma (5-8) were significantly different (P < 0.05), suggesting the potential of the method to distinguish between melanocytic nevi in vivo.
Comparison of in vivo and ex vivo laser scanning microscopy and multiphoton tomography application for human and porcine skin imaging
Darvin ME, Richter H, Zhu YJ, Meinke MC, Knorr F, Gonchukov SA, König K and Lademann J.
Two state-of-the-art microscopic optical methods, namely, confocal laser scanning microscopy in the fluorescence and reflectance regimes and multiphoton tomography in the autofluorescence and second harmonic generation regimes, are compared for porcine skin ex vivo and healthy human skin in vivo. All skin layers such as stratum corneum (SC), stratum spinosum (SS), stratum basale (SB), papillary dermis (PD) and reticular dermis (RD) as well as transition zones between these skin layers are measured noninvasively at a high resolution, using the above mentioned microscopic methods. In the case of confocal laser scanning microscopy (CLSM), measurements in the fluorescence regime were performed by using a fluorescent dye whose topical application on the surface is well suited for the investigation of superficial SC and characterisation of the skin barrier function. For investigations of deeply located skin layers, such as SS, SB and PD, the fluorescent dye must be injected into the skin, which markedly limits fluorescence measurements using CLSM. In the case of reflection CLSM measurements, the obtained results can be compared to the results of multiphoton tomography (MPT) for all skin layers excluding RD. CLSM cannot distinguish between dermal collagen and elastin measuring their superposition in the RD. By using MPT, it is possible to analyse the collagen and elastin structures separately, which is important for the investigation of anti-aging processes. The resolution of MPT is superior to CLSM. The advantages and limitations of both methods are discussed and the differences and similarities between human and porcine skin are highlighted.
In Vivo Imaging of ZnO Nanoparticles from Sunscreen on Human Skin with a Mobile Multiphoton Tomograph
Reports on the toxicity of inorganic nanoparticles used in sunscreen, in particular of zinc oxide and titanium dioxide, have raised concerns about a possible particle penetration through the skin barrier. We used two-photon imaging to visualize the distribution of zinc-oxide nanoparticles after topical application of a commercially available sunscreen on human skin in vivo and to investigate a possible penetration of nanoparticles beyond the stratum corneum. Two-photon imaging is in particular suitable for these investigations since the excitation and the nonlinear signal light from zinc-oxide nanoparticles as well as the endogenous skin autofluorescence are all spectrally well-separated which allows a clear identification of the signal origin by detection in two-spectral channels. Furthermore, microscopic modifications in the cutaneous structure like skin wrinkles which exhibit different thicknesses of the dermal layers and at the same time are regions where nanoparticle accumulation can be specifically investigated. The results indicate no penetration of nanoparticle through the barrier of the stratum corneum of healthy skin even in microscopic wrinkles.