Spectral fluorescence lifetime detection and selective melanin imaging by multiphoton laser tomography for melanoma diagnosis.

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.

Calibration of the shear wave speed-stress relationship in ex vivo tendons.

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 (r2avg = 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/m3 and added mass effects caused wave speeds to be 22% lower on average in a saline bath than in air. The root-mean-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 (13.1% of maximum stress) when calibrating based on group-compiled data from ten tendons. These results support the feasibility of calculating absolute tendon stresses from wave speed squared based on linear calibration relationships.

Gauging force by tapping tendons.

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 fluorescence lifetime imaging of human hair.

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.

Multiparametric high-resolution imaging of barley embryos by multiphoton microscopy and magnetic resonance micro-imaging.

Nonlinear optical microscopy and magnetic resonance imaging (MRI) address different properties of the sample and operate on different geometrical scales. MRI maps density and mobility of molecules tracking specific molecular signatures. Multiphoton imaging profits from the nonlinear absorption of light in the focus of a femtosecond laser source stimulating the autofluorescence of biomolecules. As this effect relies on a high light intensity, the accessible field of view is limited, but the resolution is very high (a few hundred nanometers). Here, we aim to link the different accessible scales and properties addressed in the different techniques to obtain a synoptic view. As model specimen we studied embryos of barley. Multiphoton stimulated autofluorescence images and images of second harmonic generation are achieved even down to low magnification (10x), low numerical aperture (N.A. 0.25) conditions. The overview images allowed morphological assignments and fluorescence lifetime imaging provides further information to identify accumulation of endogenous fluorophores. The second, complementary contribution from high-resolution MR images provides a 3D model and shows the embedding of the embryo in the grain. Images of the proton density were acquired using a standard 3D spin-echo imaging pulse sequence. Details directly comparable to the low magnification optical data are visible. Eventually, passing from the MR images of the whole grain via low magnification to high resolution autofluorescence data bridges the scale barrier, and might provide the possibility to trace transport and accumulation of, e.g., nutrients from large structure of the plant to the (sub-) cellular level.