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.

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.

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.

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.

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.

Translucency and Color Stability of a Simplified Shade Nanohybrid Composite after Ultrasonic Scaling and Air-Powder Polishing.

We aimed to assess the influence of professional dental prophylaxis on the translucency and color stability of a novel simplified shade nanohybrid composite material. Sixty composite disks (5 mm in diameter and 2 mm thick) of light (n = 30) and dark (n = 30) shades were prepared. The specimens were randomly divided into the following three groups (n = 10) according to the prophylaxis procedure used: ultrasonic scaling, air-powder polishing with sodium bicarbonate, and controls. The specimens were submitted to translucency and color analysis based on the CIELab system. Two measurements were performed before and after 48-h storage in coffee. Translucency values of untreated light and dark specimens were 9.15 ± 0.38 and 5.28 ± 1.10, respectively. Air-powder polishing decreased the translucency of the light composite specimens. Storage in coffee resulted in color changes (∆E) ranging between 2.69 and 12.05 and a mean translucency decrease ranging between -0.88 and -6.91. The samples in the light group tended to exhibit greater staining; the treatment method had no effect on ∆E. It can be concluded that light-shade composite restorations are more prone to translucency and color changes resulting from air-powder polishing and contact with staining media. However, further research using other composites and powders is required.

Effect of Collagen Matrix on Doxorubicin Distribution and Cancer Cells' Response to Treatment in 3D Tumor Model.

The extracellular matrix (ECM) plays an important role in regulation of many aspects of tumor growth and response to therapies. However, the specifics of the interaction of chemotherapeutic agents with cancer cells in the presence of collagen, the major component of ECM, is still poorly investigated. In this study, we explored distribution of doxorubicin (DOX) and its effects on cancer cells' metabolism in the presence of collagen with different structures in 3D models. For this, a combination of second harmonic generation imaging of collagen and multiphoton fluorescence microscopy of DOX, and metabolic cofactor NAD(P)H was used. It was found that collagen slowed down the diffusion of DOX and thus decreased the cellular drug uptake. Besides nuclei, DOX also targeted mitochondria leading to inhibition of oxidative phosphorylation, which was more pronounced in the cells growing in the absence of collagen. As a result, the cells in collagen displayed better viability upon treatment with DOX. Taken together, our data illustrate that tumor collagen contributes to heterogeneous and sub-optimal response to DOX and highlight the challenges in improving drug delivery and efficacy.

Hydrophobic Rose Bengal Derivatives Exhibit Submicromolar-to-Subnanomolar Activity against Enveloped Viruses.

Rose Bengal (RB) is an anionic xanthene dye with multiple useful biological features, including photosensitization properties. RB was studied extensively as a photosensitizer, mostly for antibacterial and antitumor photodynamic therapy (PDT). The application of RB to virus inactivation is rather understudied, and no RB derivatives have been developed as antivirals. In this work, we used a synthetic approach based on a successful design of photosensitizing antivirals to produce RB derivatives for virus photoinactivation. A series of n-alkyl-substituted RB derivatives was synthesized and evaluated as antiviral photosensitizers. The compounds exhibited similar 1O2 generation rate and efficiency, but drastically different activities against SARS-CoV-2, CHIKV, and HIV; with comparable cytotoxicity for different cell lines. Submicromolar-to-subnanomolar activities and high selectivity indices were detected for compounds with C4-6 alkyl (SARS-CoV-2) and C6-8 alkyl (CHIKV) chains. Spectrophotometric assessment demonstrates low aqueous solubility for C8-10 congeners and a significant aggregation tendency for the C12 derivative, possibly influencing its antiviral efficacy. Initial evaluation of the synthesized compounds makes them promising for further study as viral inactivators for vaccine preparations.

Label-free imaging of M1 and M2 macrophage phenotypes in the human dermis in vivo using two-photon excited FLIM.

Macrophages (ΜΦs) are important immune effector cells that promote (M1 ΜΦs) or inhibit (M2 ΜΦs) inflammation and are involved in numerous physiological and pathogenic immune responses. Their precise role and relevance, however, are not fully understood for lack of noninvasive quantification methods. Here, we show that two-photon excited fluorescence lifetime imaging (TPE-FLIM), a label-free noninvasive method, can visualize ΜΦs in the human dermis in vivo. We demonstrate in vitro that human dermal ΜΦs exhibit specific TPE-FLIM properties that distinguish them from the main components of the extracellular matrix and other dermal cells. We visualized ΜΦs, their phenotypes and phagocytosis in the skin of healthy individuals in vivo using TPE-FLIM. Additionally, machine learning identified M1 and M2 MФs with a sensitivity of 0.88±0.04 and 0.82±0.03 and a specificity of 0.89±0.03 and 0.90±0.03, respectively. In clinical research, TPE-FLIM can advance the understanding of the role of MФs in health and disease.

Modulation of Membrane Microviscosity by Protein-Mediated Carotenoid Delivery as Revealed by Time-Resolved Fluorescence Anisotropy.

Carotenoids are potent antioxidants with a wide range of biomedical applications. However, their delivery into human cells is challenging and relatively inefficient. While the use of natural water-soluble carotenoproteins capable to reversibly bind carotenoids and transfer them into membranes is promising, the quantitative estimation of the delivery remains unclear. In the present work, we studied echinenone (ECN) delivery by cyanobacterial carotenoprotein AnaCTDH (C-terminal domain homolog of the Orange Carotenoid Protein from Anabaena), into liposome membranes labelled with BODIPY fluorescent probe. We observed that addition of AnaCTDH-ECN to liposomes led to the significant changes in the fast-kinetic component of the fluorescence decay curve, pointing on the dipole-dipole interactions between the probe and ECN within the membrane. It may serve as an indirect evidence of ECN delivery into membrane. To study the delivery in detail, we carried out molecular dynamics modeling of the localization of ECN within the lipid bilayer and calculate its orientation factor. Next, we exploited FRET to assess concentration of ECN delivered by AnaCTDH. Finally, we used time-resolved fluorescence anisotropy to assess changes in microviscosity of liposomal membranes. Incorporation of liposomes with ß-carotene increased membrane microviscosity while the effect of astaxanthin and its mono- and diester forms was less pronounced. At temperatures below 30 °C addition of AnaCTDH-ECN increased membrane microviscosity in a concentration-dependent manner, supporting the protein-mediated carotenoid delivery mechanism. Combining all data, we propose FRET-based analysis and assessment of membrane microviscosity as potent approaches to characterize the efficiency of carotenoids delivery into membranes.

Albumin microbubbles conjugated with zinc and aluminum phthalocyanine dyes for enhanced photodynamic activity.

Gas-liquid interfaces are reaching a particular interest in biomedicine. Microbubbles, ultrasound contrast agents of clinical routine, gained increasing attention as theranostic platforms due to the preserved acoustic response, drug conjugation capabilities, and applicability in biological barrier opening. A combination of microbubbles and photodynamic therapy agents can enhance the photodynamic effect, yet the evaluation of agent conjugation on microbubble stabilization and photodynamic effect is needed. Hence, two commercially available phthalocyanine photosensitizers - Holosens® (ZnPc) and Photosens® (AlPc) - were coupled with bovine serum albumin before microbubble synthesis. We demonstrated an albumin: phthalocyanine ratio of 1:1 and covalent attachment for ZnPc, a ratio of 1:3 with electrostatic binding for AlPc. Submicron-sized microbubbles (air- and SF6- filled) had a diameter of 0.8 µm. Albumin-phthalocyanine conjugates increased the microbubble concentration and shelf-life stability compared to plain ones. We hypothesized that phthalocyanine fluorescence lifetime values decreased after conjugation with microbubbles due to narrow distance between conjugates in the shell. Agents based on AlPc demonstrated higher photodynamic activity than agents based on ZnPc, and microbubbles preserved acoustic stability in human blood plasma. The biodistribution of AlPc-conjugated microbubbles was evaluated. We conclude that our microbubble platforms demonstrate greater photodynamic activity and prolonged stability for further applications in photodynamic therapy.