Computational protocol: Multimodal label-free imaging of living dermal equivalents including dermal papilla cells

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Protocol publication

[…] For more detailed information, the changes in the collagen structure and the quality of the cell components were assessed with the use of MPT and FLIM. The two-photon excited fluorescence intensity and FLIM images were obtained using a multiphoton tomography MPTflex (JenLab GmbH, Germany) equipped with a tunable 80 MHz, 200 fs Ti:Sa laser MaiTai, and a TCSPC-based FLIM module (Becker & Hickl GmbH, Germany). The images were acquired through a 40×, 1.3 NA, oil immersion objective.NAD(P)H fluorescence was excited at a wavelength of 740 nm and detected in the range 410–650 nm. The average power applied to the sample was ~ 12 mW.Second harmonic generation (SHG) was excited at 740 nm and detected in the range 373–387 nm to identify oriented collagen fibers in the dermal equivalent. Quantitative analysis of the SHG signal intensity distribution for the collagen was performed using first and second order statistical methods (Integration Density, Energy and Coherence) in ImageJ (National Institutes of Health, USA). For quantitative analysis of the SHG signal the intensity distribution for the collagen at each time point in the dermal equivalents under study (from 5 to 18 regions of interest (ROIs)) was inspected. Higher integration density, coherence, and energy values correspond to structures that are less isotropic and more oriented. [...] Statistical analysis was performed using STATISTICA 64 software, version 10 (StatSoft Inc., Tulsa, OK, USA). Mean and standard deviation (SD) values were used to express the data. Differences in the mean values were tested for significance using the Student’s t test or one-way analysis of variance (ANOVA) with Fisher’s post hoc test (p ≤ 0.05). […]

Pipeline specifications

Software tools ImageJ, Statistica
Applications Miscellaneous, Microscopic phenotype analysis
Chemicals NADP