Large area fractional laser treatment (LAFLT) is a new fat removal and weight loss concept, which applies microscopic thermal injuries to large parts of the skin. These injuries induce a controlled metabolic response which temporarily increases the energy expenditure (EE), thereby reducing overall weight. In a mouse model, we investigated the dosimetry settings to induce this metabolic reaction. Therefore, mice were housed in metabolic cages and their body composition was analyzed. Results showed a significant EE increase (31% - 91%, p < 0.05) over a 6-day period which seems to suggest that an enhanced metabolic state can be achieved.
KEYWORDS: Optical coherence tomography, Skin, In vitro testing, Temperature metrology, Mode conditioning cables, Signal processing, Scattering, Acquisition tracking and pointing, 3D modeling, Motion models
Microscopic optical coherence tomography (OCT) provides three-dimensional, high-resolution imaging but lacks (sub-) cellular contrast. Dynamic-microscopic OCT (dmOCT) is an approach exploiting dynamic changes of the scattering behavior in metabolically active cells. However, the underlying cellular processes responsible for those intensity fluctuations and hence the dynamic signals are not finally identified yet. Here, we present the effects of different temperatures and metabolic reagents on dmOCT images of an in-vitro human skin model. Our data indicates a dependency of the dmOCT signals on metabolic activity rather than Brownian motion and suggests dependency on the metabolic state.
The evaluation of complex metabolic changes of individual live cells and heterogeneous cell cultures is not feasible using traditional methods due to their destructive behavior and lack of spatial information. Two-photon excited fluorescence of intrinsic fluorophores such as nicotinamide adenine dinucleotide (NADH) facilitate a label-free and non-destructive evaluation of metabolic activity. This study explores the phasor approach in combination with two-photon fluorescence lifetime imaging microscopy (FLIM) as a potential method to evaluate pharmacologically induced metabolic changes that occur during the browning of adipocytes. The possibility of browning of white adipose cells is a desirable prospect for the treatment of obesity and related disorders. Here, we compared the results obtained by Fourier-based phasor analysis with the traditional exponential FLIM analysis as well as results of an extracellular flux analyzer. The alteration of glycolytic function and oxidative phosphorylation after treatment with pharmacological reagents significantly shift the contribution of each of the fluorescence lifetime components to the total fluorescence intensity. Further, we showed that the ratios of the lifetime components obtained by the phasor approach reflect the shift in mitochondrial and cytosolic NADH concentration. The phasor analysis agrees with traditional assessments, such as the optical free-to-bound NADH ratio as well as the oxygen consumption rate and extracellular acidification rate as determined by the extracellular flux analyzer. Our results support the concept that non-invasive sensing of fat metabolism and browning of fat may be possible by analyzing the fluorescence lifetime of NADH using the phasor approach.
Cryolipolysis is a well-established cosmetic procedure for non-invasive local fat reduction. This technique selectively destroys subcutaneous fat cells using controlled cooling. Thickness measurements of subcutaneous fat were conducted using a mouse model. For detailed examination of mouse skin optical coherence tomography (OCT) was performed, which is a non-invasive imaging modality. Due to a high number of image slices manual delineation is not feasible. Therefore, automatic segmentation algorithms are required. In this work an algorithm for the automatic 3D segmentation of the subcutaneous fat layer is presented, which is based on a random forest classification followed by a graph-based refinement step. Our approach is able to accurately segment the subcutaneous fat layer with an overall average symmetric surface distance of 11.80±6.05 μm and Dice coefficient of 0.921 ± 0.045. Furthermore, it was shown that the graph-based refining step leads to increased accuracy and robustness of the segmentation results of the random forest classifier.
Traditional assessments of cellular metabolism are often destructive, time consuming and without visual information. Fluorescence lifetime imaging microscopy (FLIM) provides a highly sensitive, non-invasive, and label-free alternative.
This study uses FLIM in combination with two-photon microscopy to investigate pharmacological induced metabolic changes of adipocytes via changes in the fluorescence of the metabolic co-factors NADH and FAD. In agreement with recent publications NADH fluorescence suggests the presence of four distinct lifetimes in cell culture and tissue with two unbound and two protein bound states which show different responses to treatment with metabolic modifiers. We evaluated the effects on NADH fluorescence lifetime after systematic manipulations to change the balance between oxidative and glycolytic metabolism using five pharmacological reagents - Oligomycin, 2-DG, FCCP, Rotenone, and Glucose - which interact with different parts of the metabolic pathway. We established several ratios between the four distinct lifetimes of NADH after treatment and compared the results to oxygen consumption rate and extracellular acidification rate.
We demonstrated, for the first time, a correlation between the two unbound fluorescence lifetimes components and glycolytic and oxidative metabolic activity with a significant higher sensitivity compared to the commonly used free-to-bound ratio of NADH. Analyzing all four lifetime components of NADH has the potential to become a powerful tool to evaluate metabolic activity of adipocytes with subcellular resolution.
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