Publications 2015

In Vivo Multiphoton Microscopy of Basal Cell Carcinoma

Balu M, Zachary CB, Harris RM, krasieva TB, König K, Tromberg BJ and Kelly KM.

JAMA Dermatol. 151(10):1068-74, 2015 October. DOI: 10.1001/jamadermatol.2015.0453.


Basal cell carcinomas (BCCs) are diagnosed by clinical evaluation, which can include dermoscopic evaluation, biopsy, and histopathologic examination. Recent translation of multiphoton microscopy (MPM) to clinical practice raises the possibility of noninvasive, label-free in vivo imaging of BCCs that could reduce the time from consultation to treatment.


Two-photon imaging of intact living plants during freezing with a flexible multiphoton tomograph

Weinigel M and König K.

Laser Physics Letter 12(5):025601, 2015 January. DOI: 10.1088/1612-2011/12/2/025601


We describe the combination of a flexible multiphoton tomograph (MPTflex) with a heating and cooling stage. The stage allows temperature control in the range of (−196 °C) (77 K) to +600 °C (873 K) with selectable heating/freezing rates between 0.01 K min−1 and 150 K min−1. To illustrate the imaging capabilities of the combined system, fluorescence intensity and lifetime of intrinsic molecules from a plant leaf were imaged with submicron resolution during freezing in vivo without detaching the leaf from the plant. An increase of fluorescence intensity and decay times with decreasing temperature was observed. The measurements illustrate the usefulness of multiphoton imaging as a non-invasive online tool to investigate temperature-induced effects. The flexible multiphoton tomograph with its adjustable mechano-optical arm and scan head allows imaging at otherwise hardly accessible sample regions.


Impact of refractive index mismatches on coherent anti-Stokes Raman scattering and multiphoton autofluorescence tomography of human skin in vivo

Weinigel M, Breunig HG, Darvin ME, Klemp M, Röwert-Huber J, Lademann J and König K.

Phys Med Biol. 60(17):6881-99, 2015 September. DOI: 10.1088/0031-9155/60/17/6881. Epub 2015 Aug 25


Optical non-linear multimodal tomography is a powerful diagnostic imaging tool to analyse human skin based on its autofluorescence and second-harmonic generation signals. Recently, the field of clinical non-linear imaging has been extended by adding coherent anti-Stokes Raman scattering (CARS)-a further optical sectioning method for the detection of non-fluorescent molecules. However, the heterogeneity of refractive indices of different substances in complex tissues like human skin can have a strong influence on CARS image formation and requires careful clinical interpretation of the detected signals. Interestingly, very regular patterns are present in the CARS images, which have no correspondence to the morphology revealed by autofluorescence at the same depth. The purpose of this paper is to clarify this phenomenon and to sensitize users for possible artefacts. A further part of this paper is the detailed comparison of CARS and autofluorescence images of healthy human skin in vivo covering the complete epidermis and part of the upper dermis by employing the flexible medical non-linear tomograph MPTflex CARS.


Sperm metabolism is altered during storage by female insects: evidence from two-photon autofluorescence lifetime measurements in bedbugs

Reinhardt K, Breunig HG, Uchugonova A and König K.

J R Soc Interface. 12(110):0609, 2015 September. DOI: 10.1098/rsif.2015.0609.


We explore the possibility of characterizing sperm cells without the need to stain them using spectral and fluorescence lifetime analyses after multi-photon excitation in an insect model. The autofluorescence emission spectrum of sperm of the common bedbug, Cimex lectularius, was consistent with the presence of flavins and NAD(P)H. The mean fluorescence lifetimes showed smaller variation in sperm extracted from the male (tau m, τm = 1.54-1.84 ns) than in that extracted from the female sperm storage organ (tau m, τm = 1.26-2.00 ns). The fluorescence lifetime histograms revealed four peaks. These peaks (0.18, 0.92, 2.50 and 3.80 ns) suggest the presence of NAD(P)H and flavins and show that sperm metabolism can be characterized using fluorescence lifetime imaging. The difference in fluorescence lifetime variation between the sexes is consistent with the notion that female animals alter the metabolism of sperm cells during storage. It is not consistent, however, with the idea that sperm metabolism represents a sexually selected character that provides females with information about the male genotype.


Optimazation of the measurement procedure during multiphoton tomography of human skin in vivo

Springer S, Zieger M, Koenig K, Kaatz M, Lademann J und Darvin ME.

Skin Res Technol.: 1-7, 2015 October 12. DOI: 10.1111/srt.12273


BACKGROUND: The in vivo multiphoton tomography has evolved into a useful tool for the non-invasive investigation of morphological and biophysical characteristics of human skin. Until now, changes of skin have been evaluated mainly by clinical and histological techniques. The current study addresses the effects of a changed acquisition time for single scans in a Z-stack on the directly related qualitative and quantitative interpretability of the data. METHODS: A test area of the skin was used for scanning 12 Z-stacks of 10 volunteers aged between 25 and 34 years. The stacks were taken up to a depth of 220 μm at increments of 10 μm at four different times, 1, 3, 7, 13 s, per scan. Subsequently, the second harmonic generation (SHG)-to-autofluorescence aging index of dermis (SAAID) was evaluated at three different measuring depths, i.e. at the maximum of SHG as well as at depths of 60 and 150 μm.
RESULTS: The evaluation did not reveal any significant differences in the SAAID behavior between the Z-stacks of each test area scanned at different acquisition times. However, the acquisition time of 1 s/frame increases the measurement stability without influencing the SAAID behavior. The resolution of subcellular structures decreases significantly at scan times ≤3 s, whereas the acquisition time from 7 to 13 s warrants a high image quality.
CONCLUSION: The study has shown that there are no significant differences between the scan speeds per scan in a Z-stack and the resulting SAAID. Acquisition times of 7 s are suitable for the morphological evaluation whereas a further extension to 13 s does not result in any benefits. A scan time per image of 1 s is sufficient for the quantitative evaluation of SAAID thus substantially reducing the possible influence of movement artifacts.



Comparison of morphologic criteria for actinic keratosis and squamous cell carcinoma using in vivo multiphoton tomography

 Klemp M, Meinke MC, Weinigel M, Röwert-Huber HJ,König K, Ulrich M, Lademann J and Darvin ME.

Exp Dermatol. 25(3):218-22, 2016 March. DOI: 10.1111/exd.12912. Epub 2016 Feb 10.

The routine diagnostic procedure of actinic keratosis (AK) and invasive squamous cell carcinoma (SCC) is a histological examination after taking a biopsy. In the past decades, non-invasive optical methods for skin examination have been developed. Patients with clinical diagnosis of AK or SCC were examined. The morphological criteria were determined for healthy, AK and SCC skin and compared for statistically significant differences. In this study, the applicability of multiphoton tomography (MPT) as an in vivo diagnostic tool for AK and SCC was evaluated. Changes in the morphology of the keratinocytes such as broadened epidermis, large intercellular spaces, enlarged nucleus and a large variance in cell shape could easily be recognized. The cell nuclei of AK and SCC were significantly larger compared to healthy skin cells in all cell layers. The nucleus-cytoplasm ratio was also significantly higher for AK and SCC than for the healthy skin cells. It was even higher in SCC compared to spinous and basal cell layer of AK. The cell density in AK and SCC was significantly lower than in the basal and spinous cell layers of healthy skin. In SCC, the cell density was significantly lower than in AK. Concerning the intercellular spaces, significant differences were found for AK and healthy skin in spinous and basal cell layer and for SCC compared to AK and healthy skin. In this study, MPT proved to be a valuable non-invasive imaging method for in vivo detection and discrimination of AK and SCC from healthy skin.