Fluorescence saturation imaging microscopy: molecular fingerprinting with a standard confocal microscope.

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.

In vivo assessment of bladder cancer with diffuse reflectance and fluorescence spectroscopy: A comparative study.

OBJECTIVES: The aim of this work is to assess the performance of multimodal spectroscopic approach combined with single core optical fiber for detection of bladder cancer during surgery in vivo. METHODS: Multimodal approach combines diffuse reflectance spectroscopy (DRS), fluorescence spectroscopy in the visible (405 nm excitation) and near-infrared (NIR) (690 nm excitation) ranges, and high-wavenumber Raman spectroscopy. All four spectroscopic methods were combined in a single setup. For 21 patients with suspected bladder cancer or during control cystoscopy optical spectra of bladder cancer, healthy bladder wall tissue and/or scars were measured. Classification of cancerous and healthy bladder tissue was performed using machine learning methods. RESULTS: Statistically significant differences in relative total haemoglobin content, oxygenation, scattering, and visible fluorescence intensity were found between tumor and normal tissues. The combination of DRS and visible fluorescence spectroscopy allowed detecting cancerous tissue with sensitivity and specificity of 78% and 91%, respectively. The addition of features extracted from NIR fluorescence and Raman spectra did not improve the quality of classification. CONCLUSIONS: This study demonstrates that multimodal spectroscopic approach allows increasing sensitivity and specificity of bladder cancer detection in vivo. The developed approach does not require special probes and can be used with single-core optical fibers applied for laser surgery.

Correction: Body composition analysis via spatially resolved NIR spectroscopy with multifrequency bioimpedance precision.

Correction for 'Body composition analysis via spatially resolved NIR spectroscopy with multifrequency bioimpedance precision' by Evgeny Shirshin et al., Anal. Methods, 2024, 16, 175-178, https://doi.org/10.1039/D3AY01901B.

Skin dehydration monitoring with optical spectroscopy allows assessment of water content in the organism: Thermal and physical loads, diuretic therapy.

This study investigates the relationship between body hydration levels and skin hydration using spatially resolved diffuse reflectance spectroscopy. The research involved monitoring skin dehydration and rehydration under various conditions, including thermal and physical loads on healthy volunteers, and diuretic therapy in patients with edema syndrome. Findings indicate a correlation between body mass reduction and skin hydration: a 1% loss in body mass corresponds to a 10% decrease in skin hydration. During thermal stress, water absorption at 970 nm decreased monotonically without recovery. Physical activity resulted in approximately 10% changes in skin water content within 20 min, followed by rehydration. Patients with edema syndrome exhibited the most substantial decrease in water absorption amplitude, at nearly 30%, during diuretic treatment. These results support optical spectroscopy as a non-invasive tool for assessing body hydration, with implications for developing portable hydration monitoring devices for clinical and sports applications.

Body composition analysis via spatially resolved NIR spectroscopy with multifrequency bioimpedance precision.

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.

Photoluminescence of Two-Dimensional MoS2 Nanosheets Produced by Liquid Exfoliation.

Extraordinary properties of two-dimensional materials make them attractive for applications in different fields. One of the prospective niches is optical applications, where such types of materials demonstrate extremely sensitive performance and can be used for labeling. However, the optical properties of liquid-exfoliated 2D materials need to be analyzed. The purpose of this work is to study the absorption and luminescent properties of MoS2 exfoliated in the presence of sodium cholate, which is the most often used surfactant. Ultrasound bath and mixer-assisted exfoliation in water and dimethyl sulfoxide were used. The best quality of MoS2 nanosheets was achieved using shear-assisted liquid-phase exfoliation as a production method and sodium cholate (SC) as a surfactant. The photoluminescent properties of MoS2 nanosheets varied slightly when changing the surfactant concentrations in the range C(SC) = 0.5-2.5 mg/mL. This work is of high practical importance for further enhancement of MoS2 photoluminescent properties via chemical functionalization.

First 24-Membered Macrocyclic 1,10-Phenanthroline-2,9-Diamides-An Efficient Switch from Acidic to Alkaline Extraction of f-Elements.

A reaction of acyl chlorides derived from 1,10-phenanthroline-2,9-dicarboxylic acids with piperazine allows the preparation of the corresponding 24-membered macrocycles in good yield. The structural and spectral properties of these new macrocyclic ligands were thoroughly investigated, revealing promising coordination properties towards f-elements (Am, Eu). It was shown that the prepared ligands can be used for selective extraction of Am(III) from alkaline-carbonate media in presence of Eu(III) with an SFAm/Eu up to 40. Their extraction efficiency is higher than calixarene-type extraction of the Am(III) and Eu(III) pair. Composition of macrocycle-metal complex with Eu(III) was investigated by luminescence and UV-vis spectroscopy. The possibility of such ligands to form complexes of L:Eu = 1:2 stoichiometry is revealed.

Monitoring the skin structure during edema in vivo with spatially resolved diffuse reflectance spectroscopy.

Significance: Edema occurs in the course of various skin diseases. It manifests itself in changes in water concentrations in skin layers: dermis and hypodermis and their thicknesses. In medicine and cosmetology, objective tools are required to assess the skin's physiological parameters. The dynamics of edema and the skin of healthy volunteers were studied using spatially resolved diffuse reflectance spectroscopy (DRS) in conjunction with ultrasound (US). Aim: In this work, we have developed a method based on DRS with a spatial resolution (SR DRS), allowing us to simultaneously assess water content in the dermis, dermal thickness, and hypodermal thickness. Approach: An experimental investigation of histamine included edema using SR DRS under the control of US was conducted. An approach for skin parameter determination was studied and confirmed using Monte-Carlo simulation of diffuse reflectance spectra for a three-layered system with the varying dermis and hypodermis parameters. Results: It was shown that an interfiber distance of 1 mm yields a minimal relative error of water content determination in the dermis equal to 9.3%. The lowest error of hypodermal thickness estimation was achieved with the interfiber distance of 10 mm. Dermal thickness for a group of volunteers (7 participants, 21 measurement sites) was determined using SR DRS technique with an 8.3% error using machine learning approaches, taking measurements at multiple interfiber distances into account. Hypodermis thickness was determined with root mean squared error of 0.56 mm for the same group. Conclusions: This study demonstrates that measurement of the skin diffuse reflectance response at multiple distances makes it possible to determine the main parameters of the skin and will serve as the basis for the development and verification of an approach that works in a wide range of skin structure parameters.

Diffuse reflectance spectroscopy of the cartilage tissue in the fourth optical window.

Studies of the optical properties of biological tissues in the infrared range have demonstrated significant potential for diagnostic tasks. One of the insufficiently explored ranges for diagnostic problems at the moment is the fourth transparency window, or short wavelength infrared region II (SWIR II). A Cr2+:ZnSe laser with tuning capability in the range from 2.1 to 2.4 µm was developed to explore the possibilities in this region. The capability of diffuse reflectance spectroscopy to analyze water and collagen content in biosamples was investigated using the optical gelatin phantoms and the cartilage tissue samples during their drying process. It was demonstrated that decomposition components of the optical density spectra correlated with the partial content of the collagen and water in the samples. The present study indicates the possibility of using this spectral range for the development of diagnostic methods, in particular, for observation of the changes in the content of cartilage tissue components in degenerative diseases such as osteoarthritis.

Protein-Mediated Carotenoid Delivery Suppresses the Photoinducible Oxidation of Lipofuscin in Retinal Pigment Epithelial Cells.

Lipofuscin of retinal pigment epithelium (RPE) cells is a complex heterogeneous system of chromophores which accumulates as granules during the cell's lifespan. Lipofuscin serves as a source of various cytotoxic effects linked with oxidative stress. Several age-related eye diseases such as macular degeneration of the retina, as well as some severe inherited eye pathologies, are accompanied by a significant increase in lipofuscin granule concentration. The accumulation of carotenoids in the RPE could provide an effective antioxidant protection against lipofuscin cytotoxic manifestations. Given the highly lipophilic nature of carotenoids, their targeted delivery to the vulnerable tissues can potentially be assisted by special proteins. In this study, we demonstrate how protein-mediated delivery of zeaxanthin using water-soluble Bombyx mori carotenoid-binding protein (BmCBP-ZEA) suppresses the photoinducible oxidative stress in RPE cells caused by irradiation of lipofuscin with intense white light. We implemented fluorescence lifetime imaging of the RPE cell culture ARPE-19 fed with lipofuscin granules and then irradiated by white light with and without the addition of BmCBP-ZEA. We demonstrate that after irradiation the mean fluorescence lifetime of lipofuscin significantly increases, while the presence of BmCBP-ZEA at 200 nM concentration suppresses the increase in the average lifetime of lipofuscin fluorescence, indicating an approx. 35% inhibition of the oxidative stress. This phenomenon serves as indirect yet important evidence of the efficiency of the protein-mediated carotenoid delivery into pigment epithelium cells.

Stages of OCP-FRP Interactions in the Regulation of Photoprotection in Cyanobacteria, Part 1: Time-Resolved Spectroscopy.

Most cyanobacteria utilize a water-soluble Orange Carotenoid Protein (OCP) to protect their light-harvesting complexes from photodamage. The Fluorescence Recovery Protein (FRP) is used to restore photosynthetic activity by inactivating OCP via dynamic OCP-FRP interactions, a multistage process that remains underexplored. In this work, applying time-resolved spectroscopy, we demonstrate that the interaction of FRP with the photoactivated OCP begins early in the photocycle. Interacting with the compact OCP state, FRP completely prevents the possibility of OCP domain separation and formation of the signaling state capable of interacting with the antenna. The structural element that prevents FRP binding and formation of the complex is the short α-helix at the beginning of the N-terminal domain of OCP, which masks the primary site in the C-terminal domain of OCP. We determined the rate of opening of this site and show that it remains exposed long after the relaxation of the red OCP states. Observations of the OCP transitions on the ms time scale revealed that the relaxation of the orange photocycle intermediates is accompanied by an increase in the interaction of the carotenoid keto group with the hydrogen bond donor tyrosine-201. Our data refine the current model of photoinduced OCP transitions and the interaction of its intermediates with FRP.

Intrinsic fluorescence of nucleobase crystals.

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.

Fluorescence lifetime imaging microscopy as an instrument for human sperm assessment.

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.

Site-specific modification of N-terminal α-amino groups of succinylated collagen.

Polymer based protein engineering provides an attractive strategy to endow novel properties to protein and overcome the inherent limitations of both counterparts. The exquisite control of site and density of attached polymers on the proteins is crucial for the bioactivities and properties of the protein-polymer bioconjugates, but is still a challenge. Collagen is the major structural protein in extracellular matrix of animals. Based on the advancements of polymer-based protein engineering, collagen bioconjugates has been widely fabricated and applied as biomaterials. However, the site-specific synthesis of well-defined collagen-polymer bioconjugates is still not achieved. Herein, a versatile strategy for the specific modification of N-terminal α-amino groups in collagen was developed. Firstly, all reactive amino groups of tropocollagen (collagen with telopeptides) were protected by succinic anhydride. Then, the telopeptides were digested to give the active N-terminal α-amino groups, which were subsequently attached with poly(N-isopropylacrylamide) (PNIPAAm) via "grafting from" method based on the atom transfer radical polymerization (ATRP). The site-specific N-terminal PNIPAAm modified succinylated collagen was prepared and its structure, thermal responsive behaviour, and properties was explored.

5-(Perylen-3-ylethynyl)uracil as an antiviral scaffold: Potent suppression of enveloped virus reproduction by 3-methyl derivatives in vitro.

Amphipathic nucleoside and non-nucleoside derivatives of pentacyclic aromatic hydrocarbon perylene are known as potent non-cytotoxic broad-spectrum antivirals. Here we report 3-methyl-5-(perylen-3-ylethynyl)-uracil-1-acetic acid and its amides, a new series of compounds based on a 5-(perylen-3-ylethynyl)-uracil scaffold. The compounds demonstrate pronounced in vitro activity against arthropod-borne viruses, namely tick-borne encephalitis virus (TBEV) and yellow fever virus (YFV), in plaque reduction assays with EC50 values below 1.9 and 1.3 nM, respectively, and Chikungunya virus (CHIKV) in cytopathic effect inhibition test with EC50 values below 3.2 µM. The compounds are active against respiratory viruses as well: severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2) in cytopathic effect inhibition test and influenza A virus (IAV) in virus titer reduction experiments are inhibited - EC50 values below 51 nM and 2.2 µM, respectively. The activity stems from the presence of a hydrophobic perylene core, and all of the synthesized compounds exhibit comparable 1O2 generation rates. Nonetheless, activity can vary by orders of magnitude depending on the hydrophilic part of the molecule, suggesting a complex mode of action. A time-of-addition experiment and fluorescent imaging indicate that the compounds inhibit viral fusion in a dose-dependent manner. The localization of the compound in the lipid bilayers and visible damage to the viral envelope suggest the membrane as the primary target. Dramatic reduction of antiviral activity with limited irradiation or under treatment with antioxidants further cements the idea of photoinduced ROS-mediated viral envelope damage being the mode of antiviral action.

Grading cartilage damage with diffuse reflectance spectroscopy: Optical markers and mechanical properties.

Osteoarthritis (OA) is one of the most common joint diseases worldwide. Unfortunately, clinical methods lack the ability to detect OA in the early stages. Timely detection of the knee joint degradation at the level of tissue changes can prevent its progressive damage. Here, diffuse reflectance spectroscopy (DRS) in the NIR range was used to obtain optical markers of the cartilage damage grades and to assess its mechanical properties. It was observed that the water content obtained by DRS strongly correlates with the cartilage thickness (R = .82) and viscoelastic relaxation time (R = .7). Moreover, the spectral parameters, including water content (OH-band), protein content (CH-band), and scattering parameters allowed for discrimination between the cartilage damage grades (10-4 < P ≤ 10-3 ). The developed approach may become a valuable addition to arthroscopy, helping to identify lesions at the microscopic level in the early stages of OA and complement the surgical analysis.

Multifarious analytical capabilities of the UV/Vis protein fluorescence in blood plasma.

Autofluorescence of blood plasma has been broadly considered as a prospective disease screening method. However, the assessment of such intrinsic fluorescence is mostly phenomenological, and its origin is still not fully understood, complicating its use in the clinical practice. Here we present the detailed evaluation of analytical capabilities, variability, and formation of blood plasma protein fluorescence based on the open dataset of excitation-emission matrices measured for ∼300 patients with suspected colorectal cancer, and our supporting model experiments. Using high-resolution size-exclusion chromatography coupled with comprehensive spectral analysis, we demonstrate, for the first time, the dominant role of HSA in the formation of blood plasma fluorescence in the visible spectral range (excitation wavelength >350 nm), presumably caused by its oxidative modifications. Furthermore, the diagnostic value of the tryptophan emission, as well as of the tyrosine fluorescence and visible fluorescence of proteins is shown by building a tree-based classification model that uses a small subset of physically interpretable fluorescence features for distinguishing between the control group and cancer patients with >80% accuracy. The obtained results extend current understanding and approaches used for the analysis of blood plasma fluorescence and pave the way for novel autofluorescence-based disease screening methods.

Monitoring the Intracellular pH and Metabolic State of Cancer Cells in Response to Chemotherapy Using a Combination of Phosphorescence Lifetime Imaging Microscopy and Fluorescence Lifetime Imaging Microscopy.

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.

Tunable Self-Assembled Peptide Hydrogel Sensor for Pharma Cold Supply Chain.

Defrost sensors are a crucial element for proper functioning of the pharmaceutical cold chain. In this paper, the self-assembled peptide-based hydrogels were used to construct a sensitive defrost sensor for the transportation and storage of medications and biomaterials. The turbidity of the peptide hydrogel was employed as a marker of the temperature regime. The gelation kinetics under different conditions was studied to detect various stages of hydrogel structural transitions aimed at tuning the system properties. The developed sensor can be stored at room temperature for a long period, irreversibly indicates whether the product has been thawed, and can be adjusted to a specific temperature range and detection time.

Sensing Cells-Peptide Hydrogel Interaction In Situ via Scanning Ion Conductance Microscopy.

Peptide-based hydrogels were shown to serve as good matrices for 3D cell culture and to be applied in the field of regenerative medicine. The study of the cell-matrix interaction is important for the understanding of cell attachment, proliferation, and migration, as well as for the improvement of the matrix. Here, we used scanning ion conductance microscopy (SICM) to study the growth of cells on self-assembled peptide-based hydrogels. The hydrogel surface topography, which changes during its formation in an aqueous solution, were studied at nanoscale resolution and compared with fluorescence lifetime imaging microscopy (FLIM). Moreover, SICM demonstrated the ability to map living cells inside the hydrogel. A zwitterionic label-free pH nanoprobe with a sensitivity > 0.01 units was applied for the investigation of pH mapping in the hydrogel to estimate the hydrogel applicability for cell growth. The SICM technique that was applied here to evaluate the cell growth on the peptide-based hydrogel can be used as a tool to study functional living cells.