Induced pluripotent stem cells (iPSC) are a promising tool for personalized cell therapy, in particular, in the field of dermatology. Metabolic plasticity of iPSC are not completely understood due to the fact that iPSC have a mixed mitochondrial phenotype, which still resembles that of somatic cells. In this study we investigated the metabolic changes in iPSC undergoing differentiation in two directions, dermal and epidermal, using two-photon fluorescence microscopy combined with FLIM. Directed differentiation of iPSC into dermal fibroblasts and keratinocyte progenitor cells was induced. Cellular metabolism was examined on the basis of the fluorescence of the metabolic cofactors NAD(P)H and FAD. The optical redox ratio (FAD/NAD(P)H) and the fluorescence lifetimes of NAD(P)H and FAD were traced using two-photon fluorescence microscopy combined with FLIM. Evaluation of the intracellular pH was carried out with the fluorescent pH sensor SypHer-2 and fluorescence microscopy. In this study, evaluation of the metabolic status of iPSC during dermal and epidermal differentiation was accomplished for the first time with the use of optical metabolic imaging. Based on the data on the FAD/NAD(P)H redox ratio and on the fluorescence lifetimes of protein-bound form of NAD(P)H and closed form of FAD, we registered a metabolic shift toward a more oxidative status in the process of iPSC differentiation into dermal fibroblasts and keratinocyte progenitor cells. Biosynthetic processes occurring in dermal fibroblasts associated with the synthesis of fibronectin and versican, that stimulate increased energy metabolism and lower the intracellular pH. No intracellular pH shift is observed in the culture of keratinocyte progenitor cells, which reflects the incomplete process of differentiation in this type of cells. Presented results provide the basis for further understanding the metabolic features of iPSC during differentiation process, which is essential for developing new treatment strategies in cell therapy and tissue engineering.

Conventional techniques are insufficient precisely to describe the internal structure, the heterogeneous cell populations, and the dynamics of biological processes occurring in diseased liver during surgery. There is a need for a rapid and safe method for the successful diagnosis of liver disease in order to plan surgery and to help avoid postoperative liver failure. We analyze the progression of both acute (cholestasis) and chronic (fibrosis) liver pathology using multiphoton microscopy with fluorescence lifetime imaging and second-harmonic generation modes combined with time-of-flight secondary ion mass spectrometry chemical analysis to obtain new data about pathological changes to hepatocytes at the cellular and molecular levels. All of these techniques allow the study of cellular metabolism, lipid composition, and collagen structure without staining the biological materials or the incorporation of fluorescent or other markers, enabling the use of these methods in a clinical situation. The combination of multiphoton microscopy and mass spectrometry provides more complete information about the liver structure and function than could be assessed using either method individually. The data can be used both to obtain new criteria for the identification of hepatic pathology and to develop a rapid technique for liver quality analysis in order to plan surgery and to help avoid postoperative liver failure in clinic. © The Authors. Published by SPIE under a Creative Commons Attribution 4.0 Unported License. Distribution or reproduction of this work in whole or in part requires full attribution of the original publication, including its DOI.

The article is devoted to the development of an EUV microscope using a wavelength of 13.84 nm. Due to the use of a mirror lens with a large numerical aperture, NA = 0.27, and a short depth of focus, it has been possible to carry out z-tomography of bio-samples for the first time with this type of microscope. A 3D image was reconstructed, and a pixel resolution of 140 nm was obtained. A new simple algorithm for the 3D reconstruction of absorption images from z-tomography data has been proposed that takes into account lens aberrations and a point spread function. The algorithm reduces the inverse absorption task to the corresponding well-studied task of fluorescence microscopy, with an error of 10% for cells up to 10 µm thick.

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The emission spectra of krypton plasma in the range of 8 – 14 nm upon excitation of a pulsed gas jet by 1.06-mm Nd : YAG laser radiation with a pulse energy of 0.85 J, pulse duration of 5.2 ns, and repetition rate of 10 Hz are investigated. The krypton emission spectrum is a wide (8 – 14 nm) band, peaking at 10.3 nm, which is formed by a series of much narrower lines. The observed lines are identified, and the fraction of laser pulse energy converted into the (8 – 14)-nm emission band and emitted into half-space (2π sr) is determined. The maximum conversion efficiency is found to be 21 %. The expected throughputs of lithographic systems with sources based on Sn, Xe, and Kr ions for different wavelengths, corresponding to the emission peaks of ions of these materials, are compared.