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Determination of the molecular composition of the skin is crucial for numerous tasks in medicine, pharmacology, dermatology and cosmetology. Confocal Raman microspectroscopy is a sensitive method for the evaluation of molecular depth profiles in the skin in vivo. Since the Raman spectra of most of the skin constituents significantly superimpose, a spectral decomposition by a set of predefined library components is usually performed to disentangle their contributions. However, the incorrect choice of the number and type of components or differences between the spectra of the basic components measured in vitro and in vivo can lead to incorrect results of the decomposition procedure. Here, we investigate an alternative data-driven approach based on a non-negative matrix factorization (NNMF) algorithm of depth-resolved Raman spectra of skin that does not require a priori information of spectral data for the analysis. Using the model and experimentally measured depth-resolved Raman spectra of the upper epidermis in vivo, we show that NNMF provides depth profiles of endogenous molecular components and exogenous agents penetrating through the upper epidermis for the spectra and concentration. Moreover, we demonstrate that this approach is capable of providing new information on the molecular profiles of the skin. [ABSTRACT FROM AUTHOR]

Fluorescence quenching (FQ) is extensively used for quantitative assessment of partition coefficients (KOC) of polycyclic aromatic hydrocarbons (PAHs) to natural organic materials – humic substances (HS). The presence of bound PAHs with incompletely quenched fluorescence would lead to underestimation of the KOC values measured by this technique. The goal of this work was to prove the validity of this assumption using an original experimental setup, which implied FQ measurements upon excitation into two distinct vibronically coupled electronic states. Pyrene was used as a fluorescent probe, and aquatic fulvic acid (SRFA) and leonardite humic acid (CHP) were used as the humic materials with low and high binding affinity for pyrene, respectively. Excitation of pyrene into the forbidden (S0–S1) and allowed (S0–S2) electronic states yielded two pairs of nonidentical FQ curves. This was indicative of incomplete quenching of the bound pyrene, and the divergence of the two FQ curves was much more pronounced for CHP as compared to SRFA. The two component model of fluorescence response formation was proposed to estimate the KOC values from the data obtained. The resulting pyrene KOC value for CHP (220 ± 20) g L−1 was a factor 3 higher compared to the KOC value determined with the use of the Stern–Volmer formalism (68 ± 2) g L−1. At the same time for aquatic FA the difference in FQ curves was almost negligible, which enables the use of the Stern–Volmer formalism for weakly interacting HS and PAHs. [ABSTRACT FROM AUTHOR]

As high-intensity solar radiation can lead to extensive damage of the photosynthetic apparatus, cyanobacteria have developed various protection mechanisms to reduce the effective excitation energy transfer (EET) from the antenna complexes to the reaction center. One of them is non-photochemical quenching (NPQ) of the phycobilisome (PB) fluorescence. In Synechocystis sp. PCC6803 this role is carried by the orange carotenoid protein (OCP), which reacts to high-intensity light by a series of conformational changes, enabling the binding of OCP to the PBs reducing the flow of energy into the photosystems. In this paper the mechanisms of energy migration in two mutant PB complexes of Synechocystis sp. were investigated and compared. The mutant CK is lacking phycocyanin in the PBs while the mutant ΔPSI/PSII does not contain both photosystems. Fluorescence decay spectra with picosecond time resolution were registered using a single photon counting technique. The studies were performed in a wide range of temperatures - from 4 to 300 K. The time course of NPQ and fluorescence recovery in darkness was studied at room temperature using both steady-state and time-resolved fluorescence measurements. The OCP induced NPQ has been shown to be due to EET from PB cores to the red form of OCP under photon flux densities up to 1000 μmol photons m- 2 s- 1. The gradual changes of the energy transfer rate from allophycocyanin to OCP were observed during the irradiation of the sample with blue light and consequent adaptation to darkness. This fact was interpreted as the revelation of intermolecular interaction between OCP and PB binding site. At low temperatures a significantly enhanced EET from allophycocyanin to terminal emitters has been shown, due to the decreased back transfer from terminal emitter to APC. The activation of OCP not only leads to fluorescence quenching, but also affects the rate constants of energy transfer as shown by model based analysis of the decay associated spectra. The results indicate that the ability of OCP to quench the fluorescence is strongly temperature dependent. This article is part of a Special Issue entitled: Photosynthesis Research for Sustainability: Keys to Produce Clean Energy. [ABSTRACT FROM AUTHOR]

Efficient visualization of the vascular system is of key importance in biomedical research into tumor angiogenesis, cerebrovascular alterations, and other angiopathies. Optoacoustic (OA) angiography offers a promising solution combining molecular optical contrast with high resolution and deep penetration of ultrasound. However, its hybrid nature implies complex data collection and processing workflows, with significant variability in methodologies across developers and users. To streamline interoperability, we introduce SKYQUANT 3D, a Python-based set of instructions for the Thermo Fisher Scientific Amira/Avizo 3D Visualization & Analysis Software. Our workflow simplifies the batch processing of volumetric optoacoustic angiography images, extracting meaningful quantitative information while also providing statistical analysis and graphical representation of the results. Quantification performance of SKYQUANT 3D is demonstrated using functional preclinical and clinical in vivo 3D OA angiographic tests involving ambient temperature variations and repositioning of the imaged limb., (© 2024 The Author(s). Journal of Biophotonics published by Wiley‐VCH GmbH.)

Non-invasive assessment of haemoglobin (Hb) level in blood is a hot spot in the point-of-care biomedical diagnostics. Several optical methods are suggested as a solution, some of them being approved for clinical use. Still, there is no consensus on the accuracy of optical techniques, the quality of Hb assessment on different tissue sites, and on the ability of combined use of several optical techniques to improve the quality of Hb level prediction. In this work we examined the capabilities of two optical techniques-diffuse reflectance spectroscopy and RGB-imaging of the skin and fingernails areas-in detecting low blood Hb level. The test sample consisted of 240 adult volunteers with 70 volunteers exhibiting Hb level lower than 120 g/L. We show that using simple descriptors of the diffuse reflectance spectrum of the forearm skin and fingernails is applicable for predicting low blood Hb concentration (ROC-AUC = 0.84 ± 0.08), while RGB-imaging shows similar performance when applied to the fingernail areas (ROC-AUC = 0.83 ± 0.07), which can be considered perspective for clinical use and screening properties. We also report that while the joint use of predictions from two optical methods slightly improves the accuracy of non-invasive Hb level assessment (ROC-AUC = 0.86 ± 0.07), the effect is not as high as one might expect from combining predictions of truly independent modalities, indicating the limit of the accuracy one can expect with multimodal optical approach. We review this case and propose possible solutions towards more sensitive non-invasive optical determination of hemoglobin., (© 2024. The Author(s).)

Anaemia, a decrease in total concentration of haemoglobin (Hb) in blood, affects substantial percentage of the population worldwide. Currently, the gold standard for determining the Hb level is the invasive analysis of venous blood. Yet, more and more research groups demonstrate the possibility of non-invasive Hb assessment using white light imaging of tissue sites where Hb is the main chromophore, in particular, fingernails. Despite the promising declarations, non-invasive Hb assessment via RGB-imaging is still poorly used in practice. The main reason is the difficulty in establishing the true accuracy of the methods presented in different works since they are tested on private datasets collected under different experimental conditions. Here we present an open dataset containing RGB images of skin and fingernails for patients with a known level of Hb, thus providing a single benchmark for researchers and engineers in the field, aimed at fostering translation of non-invasive imaging methods to the bedside., (© 2024. The Author(s).)

Significance: Autofluorescence characteristics of the reduced nicotinamide adenine dinucleotide and oxidized flavin cofactors are important for the evaluation of the metabolic status of the cells. The approaches that involve a detailed analysis of both spectral and time characteristics of the autofluorescence signals may provide additional insights into the biochemical processes in the cells and biological tissues and facilitate the transition of spectral fluorescence lifetime imaging into clinical applications., Aim: We present the experiments on multispectral fluorescence lifetime imaging with a detailed analysis of the fluorescence decays and spectral profiles of the reduced nicotinamide adenine dinucleotide and oxidized flavin under a single excitation wavelength aimed at understanding whether the use of multispectral detection is helpful for metabolic imaging of cancer cells., Approach: We use two-photon spectral fluorescence lifetime imaging microscopy. Starting from model solutions, we switched to cell cultures treated by metabolic inhibitors and then studied the metabolism of cells within tumor spheroids., Results: The use of a multispectral detector in combination with an excitation at a single wavelength of 750 nm allows the identification of fluorescence signals from three components: free and bound NAD(P)H, and flavins based on the global fitting procedure. Multispectral data make it possible to assess not only the lifetime but also the spectral shifts of emission of flavins caused by chemical perturbations. Altogether, the informative parameters of the developed approach are the ratio of free and bound NAD(P)H amplitudes, the decay time of bound NAD(P)H, the amplitude of flavin fluorescence signal, the fluorescence decay time of flavins, and the spectral shift of the emission signal of flavins. Hence, with multispectral fluorescence lifetime imaging, we get five independent parameters, of which three are related to flavins., Conclusions: The approach to probe the metabolic state of cells in culture and spheroids using excitation at a single wavelength of 750 nm and a fluorescence time-resolved spectral detection with the consequent global analysis of the data not only simplifies image acquisition protocol but also allows to disentangle the impacts of free and bound NAD(P)H, and flavin components evaluate changes in their fluorescence parameters (emission spectra and fluorescence lifetime) upon treating cells with metabolic inhibitors and sense metabolic heterogeneity within 3D tumor spheroids., (© 2024 The Authors.)

Molecular specificity in fluorescence imaging of cells and tissues can be increased by measuring parameters other than intensity. For instance, fluorescence lifetime imaging became a widespread modality for biomedical optics. Previously, we suggested using the fluorescence saturation effect at pulsed laser excitation to map the absorption cross-section as an additional molecular contrast in two-photon microscopy [Opt. Lett.47(17), 4455 (2022).10.1364/OL.465605]. Here, it is shown that, somewhat counterintuitive, fluorescence saturation can be observed under cw excitation in a standard confocal microscopy setup. Mapping the fluorescence saturation parameter allows obtaining additional information about the fluorophores in the system, as demonstrated by the example of peptide hydrogel, stained cells and unstained thyroid gland. The suggested technique does not require additional equipment and can be implemented on confocal systems as is., Competing Interests: The authors declare no conflicts of interest., (© 2024 Optica Publishing Group.)

Near-infrared spectroscopy (NIRS) is often criticized due to its insufficient accuracy in determining body composition compared to the gold standard methods. In this work, we show that the use of multiple source-detector distances, as well as the simultaneous use of physiological and optical features, can significantly improve the accuracy of determination of fat and lean mass percentage in the human body using NIR spectroscopy. The study performed on the n = 292 cohort revealed the mean absolute errors of 3.5% for fat content and 3.3% for soft lean mass percentage prediction ( r = 0.93) using the multifrequency bioimpedance analysis (BIA) as a reference. Hence, NIRS can serve as an independent reliable method for body composition analysis with precision close to that of advanced multifrequency BIA.

The extracellular matrix (ECM), in which collagen is the most abundant protein, impacts many aspects of tumor physiology, including cellular metabolism and intracellular pH (pHi), as well as the efficacy of chemotherapy. Meanwhile, the role of collagen in differential cell responses to treatment within heterogeneous tumor environments remains poorly investigated. In the present study, we simultaneously monitored the changes in pHi and metabolism in living colorectal cancer cells in vitro upon treatment with a chemotherapeutic combination, FOLFOX (5-fluorouracil, oxaliplatin and leucovorin). The pHi was followed using the new pH-sensitive probe BC-Ga-Ir, working in the mode of phosphorescence lifetime imaging (PLIM), and metabolism was assessed from the autofluorescence of the metabolic cofactor NAD(P)H using fluorescence lifetime imaging (FLIM) with a two-photon laser scanning microscope. To model the ECM, 3D collagen-based hydrogels were used, and comparisons with conventional monolayer cells were made. It was found that FOLFOX treatment caused an early temporal intracellular acidification (reduction in pHi), followed by a shift to more alkaline values, and changed cellular metabolism to a more oxidative state. The presence of unstructured collagen markedly reduced the cytotoxic effects of FOLFOX, and delayed and diminished the pHi and metabolic responses. These results support the observation that collagen is a factor in the heterogeneous response of cancer cells to chemotherapy and a powerful regulator of their metabolic behavior.

Mammalian spermatozoa are highly energized cells in which most of the proteins and activated signaling cascades are involved in the metabolic pathways. Flavin adenine dinucleotide (FAD) has one of the most important roles in the correct functional activity of spermatozoa since it acts as a cofactor for flavoenzymes, critical for proper metabolism and predominantly located in mitochondria. Non-invasive, vital and non-traumatic examination of sperm FAD level and microenvironment could be performed by fluorescence lifetime imaging microscopy (FLIM). In this study, we assessed the metabolic status of spermatozoa from healthy donors and found that FLIM could be used to segregate and separate the male germ cells according to the type of metabolic activity which corresponds with spermatozoa motility measured in standard spermogram tests., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Nucleobase crystals demonstrate unique intrinsic fluorescence properties in the visible spectral range. This is in contrast to their monomeric counterparts. Moreover, some nucleobases were found to exhibit red edge excitation shift. This behavior is uncommon in the field of organic supramolecular materials and could have implications in fields such as therapeutics of metabolic disorders and materials science., Competing Interests: There are no conflicts to declare., (This journal is © The Royal Society of Chemistry.)