Muscles are the actuators that drive human movement. However, despite many decades of work, we still cannot readily assess the forces that muscles transmit during human movement. Direct measurements of muscle–tendon loads are invasive and modeling approaches require many assumptions. Here, we introduce a non-invasive approach to assess tendon loads by tracking vibrational behavior. We first show that the speed of shear wave propagation in tendon increases with the square root of axial stress. We then introduce a remarkably simple shear wave tensiometer that uses micron-scale taps and skin-mounted accelerometers to track tendon wave speeds in vivo. Tendon wave speeds are shown to modulate in phase with active joint torques during isometric exertions, walking, and running. The capacity to non-invasively assess muscle–tendon loading can provide new insights into the motor control and biomechanics underlying movement, and could lead to enhanced clinical treatment of musculoskeletal injuries and diseases.

Multiphoton excited tissue fluorescence summarises the emission of all naturally occurring endogenous fluorescent bio-molecules with their often overlapping fluorescence spectra. Common fluorescence intensity measurements could not be utilised to distinguish between different fluorophores or metabolic states. To overcome this limitation, we investigated new procedures of selective melanin imaging and spectral fluorescence lifetime imaging in combination with high resolution multiphoton laser tomography. Overall 46 melanocytic lesions of human skin were analysed. We suggested that fluorescence light, detected in such a way, may yield additional information for melanoma diagnostics. Remarkable differences in lifetime behaviour of keratinocytes in contrast to melanocytes were observed. Fluorescence lifetime distribution was found in correlation with the intracellular amount of melanin. Spectral analysis of melanoma revealed a main fluorescence peak around 470 nm in combination with an additional peak close to 550 nm throughout all epidermal layers. Excitation at 800 nm shows a selectively observable fluorescence of melanin containing cells and offers the possibility of cell classification. Procedures of selective imaging as well as spectral fluorescence lifetime imaging by means of multiphoton laser tomography support diagnostic decisions and may improve the process of non-invasive early detection of melanoma.Key words: melanoma -melanocytic skin lesions -multiphoton laser tomography -selective melanin imaging -spectral fluorescence lifetime imaging Please cite this paper as: Spectral fluorescence lifetime detection and selective melanin imaging by multiphoton laser tomography for melanoma diagnosis.

Two-photon medical imaging has found its way into dermatology as an excellent method for noninvasive skin cancer detection without need of contrast agents as well as for in situ drug screening of topically-applied cosmetical and pharmaceutical components. There is an increasing demand to apply the multiphoton technology also for deep-tissue skin imaging as well as for intracorporal imaging. We report on the first clinical use of multiphoton endoscopes, in particular of a miniaturized rigid two-photon GRIN lens endoscope. The microendoscope was attached to the multiphoton tomograph DermaInspect and employed to detect the extracellular matrix proteins collagen and elastin in the human dermis of volunteers and patients with ulcera by in vivo second harmonic generation and in vivo two-photon autofluorescence.

The effect of the inclusion of flufenamic acid in poly(lactide-co-glycolide) nanoparticles on the transport of flufenamic acid into excised human skin was investigated. Penetration and permeation data were acquired using two different in vitro test systems: the Saarbrücken penetration model, where the skin acts as its own receptor medium, and the Franz diffusion cell, where the receptor medium is a buffer solution. For the stratum corneum, no differences were found between nanoencapsulated and free drug. Drug accumulation in the deeper skin layers and drug transport across human epidermis were slightly delayed for the nanoencapsulated drug compared to the free drug after shorter incubation times (<12 h). In contrast, after longer incubation times (>12 h), the nanoencapsulated drug showed a statistically significantly enhanced transport and accumulation (p < 0.05). Additionally, nanoencapsulated flufenamic acid was visualized by multiphoton fluorescence microscopy. Particles were found homogeneously distributed on the skin surface and within the dermatoglyphs, but no nanoparticles were detected within or between the corneocytes.

The novel femtosecond laser multiphoton imaging system DermaInspect forin vivotomography of human skin was used to study the diffusion and intradermal accumulation of topically applied cosmetic and pharmaceutical components. Near-infrared 80 MHz picojoule femtosecond laser pulses were employed to excite endogenous fluorophores and fluorescent components of a variety of ointments via a two-photon excitation process. In addition, collagen was imaged by second harmonic generation. A high submicron spatial resolution and 50 ps temporal resolution was achieved using galvoscan mirrors and piezodriven focusing optics together with a time-correlated single-photon counting module with a fast microchannel plate detector. Individual intratissue cells, intracellular mitochondria, melanosomes, and the morphology of the nuclei as well as extracellular matrix elements were clearly visualized due to NAD(P)H, melanin, elastin, and collagen imaging and the calculation of fluorescence lifetime images. Nanoparticles and intratissue drugs were detected by two-photon-excited fluorescence. In addition, hydration effects and UV effects were studied by monitoring modifications of cellular morphology and autofluorescence. The system was used to observe the diffusion through the stratum corneum and the accumulation and release of functionalized nanoparticles along hair shafts and epidermal ridges. The novel noninvasive 4-D multiphoton tomography tool provides high-resolution optical biopsies with subcellular resolution, and offers for the first time the possibility to study in situthe diffusion through the skin barrier, long-term pharmacokinetics, and cellular response to cosmetic and pharmaceutical products.

In vivo and in vitro multiphoton imaging was used to perform high resolution optical sectioning of human hair by nonlinear excitation of endogenous as well as exogenous fluorophores. Multiphoton fluorescence lifetime imaging (FLIM) based on time-resolved single photon counting and near-infrared femtosecond laser pulse excitation was employed to analyze the various fluorescent hair components. Time-resolved multiphoton imaging of intratissue pigments has the potential (i) to identify endogenous keratin and melanin, (ii) to obtain information on intrahair dye accumulation, (iii) to study bleaching effects, and (iv) to monitor the intratissue diffusion of pharmaceutical and cosmetical components along hair shafts.

It has recently been shown that shear wave speed in tendons is directly dependent on axial stress. Hence, wave speed could be used to infer tendon load provided that the wave speed-stress relationship can be calibrated and remains robust across loading conditions. The purpose of this study was to investigate the effects of loading rate and fluid immersion on the wave speed-stress relationship in ex vivo tendons, and to assess potential calibration techniques. Tendon wave speed and axial stress were measured in 20 porcine digital flexor tendons during cyclic (0.5, 1.0 and 2.0 Hz) or static axial loading. Squared wave speed was highly correlated to stress (r 2 avg = 0.98) and was insensitive to loading rate (p = 0.57). The constant of proportionality is the effective density, which reflects the density of the tendon tissue and additional effective mass added by the adjacent fluid. Effective densities of tendons vibrating in a saline bath averaged 1680 kg/m 3 and added mass effects caused wave speeds to be 22% lower on average in a saline bath than in air. The rootmean-square error between predicted and measured stress was 0.67 MPa (6.7% of maximum stress) when using tendon-specific calibration parameters. These errors increased to 1.31 MPa

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