Search Results (40)

The search for substances that increase the immunity of bees is becoming a necessity in the era of various environmental threats and the declining immunocompetence of these insects. Therefore, we tested the biological and physicochemical properties of 7-diethylamino-4-hydroxycoumarin (7DOC). In a cage test, two groups of bees were created: a control group fed with sugar syrup and an experimental group fed with sugar syrup with the addition of 7DOC. In each group, the longevity of the bees was determined and the protein concentrations and antioxidant activities in the bees’ hemolymph were determined. The bees fed with 7DOC lived 2.7 times longer than those in the control group. The protein concentrations and activities of SOD, CAT, GPx and GST, as well as the TAC levels, were significantly higher in the hemolymph of the supplemented workers. To confirm these potent biological properties of 7DOC, the UV-Vis spectra, emission and excitation of fluorescence, synchronous spectra and finally the fluorescence lifetimes of this compound were measured using the time-correlated single photon counting method, in various environments differing in polarity and in the environment applied in bee research. This compound was shown to be sensitive to changes in solvent polarity. The spectroscopic assays were complemented with crystallographic tests of the obtained monocrystals of the aforementioned compounds, which attested to the aggregation effects observed in the spectra measurements for the selected coumarin. The research results confirm that this compound has the potential to be implemented in apiary management, which will be our application goal, but further research into apiary conditions is required. Full article

Single-molecule fluorescence spectroscopy offers unique capabilities for the low-concentration sensing and probing of molecular dynmics. However, employing such a methodology for versatile sensing and diagnostics under point-of-care demands device miniaturization to lab-on-a-chip size. In this study, we numerically design metalenses with high numerical aperture (NA = 1.1), which are composed of silicon nitride nanostructures deposited on a waveguide and can selectively focus guided light into an aqueous solution at two wavelengths of interest in the spectral range of 500–780 nm. Despite the severe chromatic focal shift in the lateral directions owing to the wavelength-dependent propagation constant in a waveguide, segmented on-chip metalenses provide perfectly overlapping focal volumes that meet the requirements for epi-fluorescence light collection. We demonstrate that the molecule detection efficiencies of metalenses designed for the excitation and emission wavelengths of ATTO 490LS, Alexa 555, and APC-Cy7 tandem fluorophores are sufficient to collect several thousand photons per second per molecule at modest excitation rate constants. Such sensitivity provides reliable diffusion fluorescence correlation spectroscopy analysis of single molecules on a chip to extract their concentration and diffusion properties in the nanomolar range. Achromatic on-chip metalenses open new avenues for developing ultra-compact and sensitive devices for precision medicine and environmental monitoring. Full article

ATTO 565, a Rhodamine-type dye, has garnered significant attention due to its remarkable optical properties, such as a high fluorescence quantum yield, and the fact that it is a relatively stable structure and has low biotoxicity. ATTO 565 has found extensive applications in combination with microscopy technology. In this review, the chemical and optical properties of ATTO 565 are introduced, along with the principles behind them. The functionality of ATTO 565 in confocal microscopy, stimulated emission depletion (STED) microscopy, single-molecule tracking (SMT) techniques, two-photon excitation–stimulated emission depletion microscopy (TPE-STED) and fluorescence correlation spectroscopy (FCS) is discussed. These studies demonstrate that ATTO 565 plays a crucial role in areas such as biological imaging and single-molecule localization, thus warranting further in-depth investigations. Finally, we present some prospects and concepts for the future applications of ATTO 565 in the fields of biocompatibility and metal ion detection. This review does not include theoretical calculations for the ATTO 565 molecule. Full article

Surface enhanced fluorescence (SEF) based on noble metal nanoparticles is an effective means to achieve high sensitivity in fluorescence detection. Currently, the physical mechanism behind enhanced fluorescence is not fully understood. This paper measures the fluorescence signals of Dihydroporphyrin f methyl ether (CPD4) under both single-photon and two-photon excitation based on submicrometer silver particles with rough morphologies, achieving enhancement factors of 34 and 45 times, respectively. On this basis, by combining the radiative field characteristics produced by the silver particles, a stimulated radiation model of molecules is established to elucidate the changes in the molecular photophysical process when influenced by silver particles. Moreover, the fluorescence lifetime of the molecules was measured, showing that the presence of silver particles induces an increase in the molecular radiative decay rate, causing the fluorescence lifetime to decay from 3.8 ns to 3 ns. The results indicate that the fluorescence enhancement primarily originates from the submicrometer silver particles’ enhancement effect on the excitation light. Additionally, the fluorescence signal emitted by the molecules couples with the silver particles, causing the local surface plasmon resonances generated by the silver particles to also emit light signals of the same frequency. Under the combined effect, the fluorescence of the molecules is significantly enhanced. The findings provide a theoretical foundation for understanding the fluorescence enhancement mechanism of silver particles, adjusting the enhancement effect, and developing enhanced fluorescence detection devices based on submicrometer silver particles, holding significant practical importance. Full article

Fluorescence lifetime imaging (FLIM) and confocal fluorescence studies of a porphyrin-based photosensitiser (meso-tetraphenylporphine disulfonate: TPPS_2a) were evaluated in 2D monolayer cultures and 3D compressed collagen constructs of a human ovarian cancer cell line (HEY). TPPS_2a is known to be an effective model photosensitiser for both Photodynamic Therapy (PDT) and Photochemical Internalisation (PCI). This microspectrofluorimetric study aimed firstly to investigate the uptake and subcellular localisation of TPPS_2a, and evaluate the photo-oxidative mechanism using reactive oxygen species (ROS) and lipid peroxidation probes combined with appropriate ROS scavengers. Light-induced intracellular redistribution of TPPS_2a was observed, consistent with rupture of endolysosomes where the porphyrin localises. Using the same range of light doses, time-lapse confocal imaging permitted observation of PDT-induced generation of ROS in both 2D and 3D cancer models using fluorescence-based ROS together with specific ROS inhibitors. In addition, the use of red light excitation of the photosensitiser to minimise auto-oxidation of the probes was investigated. In the second part of the study, the photophysical properties of TPPS_2a in cells were studied using a time-domain FLIM system with time-correlated single photon counting detection. Owing to the high sensitivity and spatial resolution of this system, we acquired FLIM images that enabled the fluorescence lifetime determination of the porphyrin within the endolysosomal vesicles. Changes in the lifetime dynamics upon prolonged illumination were revealed as the vesicles degraded within the cells. Full article

The intrinsic fluorescence of bacterial samples has a proven potential for label-free bacterial characterization, monitoring bacterial metabolic functions, and as a mechanism for tracking the transport of relevant components through vesicles. The reduced scattering and axial confinement of the excitation offered by multiphoton imaging can be used to overcome some of the limitations of single-photon excitation (e.g., scattering and out-of-plane photobleaching) to the imaging of bacterial communities. In this work, we demonstrate in vivo multi-photon microscopy imaging of Streptomyces bacterial communities, based on the excitation of blue endogenous fluorophores, using an ultrafast Yb-fiber laser amplifier. Its parameters, such as the pulse energy, duration, wavelength, and repetition rate, enable in vivo multicolor imaging with a single source through the simultaneous two- and three-photon excitation of different fluorophores. Three-photon excitation at 1040 nm allows fluorophores with blue and green emission spectra to be addressed (and their corresponding ultraviolet and blue single-photon excitation wavelengths, respectively), and two-photon excitation at the same wavelength allows fluorophores with yellow, orange, or red emission spectra to be addressed (and their corresponding green, yellow, and orange single-photon excitation wavelengths). We demonstrate that three-photon excitation allows imaging over a depth range of more than 6 effective attenuation lengths to take place, corresponding to an 800 micrometer depth of imaging, in samples with a high density of fluorescent structures. Full article

Heavy-atom-free photosensitizers are envisioned as the next generation of photoactive molecules for photo-theragnosis. In this approach, and after suitable irradiation, a single molecular scaffold is able to visualize and kill tumour cells by fluorescence signalling and photodynamic therapy (PDT), respectively, with minimal side effects. In this regard, BODIPY-based orthogonal dimers have irrupted as suitable candidates for this aim. Herein, we analyse the photophysical properties of a set of formyl-functionalized BODIPY dimers to ascertain their suitability as fluorescent photosensitizers. The conducted computationally aided spectroscopic study determined that the fluorescence/singlet oxygen generation dual performance of these valuable BODIPY dimers not only depends on the BODIPY-BODIPY linkage and the steric hindrance around it, but also can be modulated by proper formyl functionalization at specific chromophoric positions. Thus, we propose regioselective formylation as an effective tool to modulate such a delicate photonic balance in BODIPY-based dimeric photosensitizers. The taming of the excited-state dynamics, in particular intramolecular charge transfer as the key underlying process mediating fluorescence deactivation vs. intersystem crossing increasing, could serve to increase fluorescence for brighter bioimaging, enhance the generation of singlet oxygen for killing activity, or balance both for photo-theragnosis. Full article

The search for fluorescent proteins with large two-photon absorption (TPA) cross-sections and improved brightness is required for their efficient use in bioimaging. Here, we explored the impact of a single-point mutation close to the anionic form of the GFP chromophore on its TPA activity. We considered the lowest-energy transition of EGFP and its modification EGFP T203I. We focused on a methodology for obtaining reliable TPA cross-sections for mutated proteins, based on conformational sampling using molecular dynamics simulations and a high-level XMCQDPT2-based QM/MM approach. We also studied the numerical convergence of the sum-over-states formalism and provide direct evidence for the applicability of the two-level model for calculating TPA cross-sections in EGFP. The calculated values were found to be very sensitive to changes in the permanent dipole moments between the ground and excited states and highly tunable by internal electric field of the protein environment. In the case of the GFP chromophore anion, even a single hydrogen bond was shown to be capable of drastically increasing the TPA cross-section. Such high tunability of the nonlinear photophysical properties of the chromophore anions can be used for the rational design of brighter fluorescent proteins for bioimaging using two-photon laser scanning microscopy. Full article

Upconversion Nanoparticles (UCNPs) have attracted exceptional attention due to their great potential in high-contrast, free-background biofluorescence deep tissue imaging and quantum sensing. Most of these interesting studies have been performed using an ensemble of UCNPs as fluorescent probes in bioapplications. Here, we report a synthesis of small and efficient YLiF₄:Yb,Er UCNPs for single-particle imaging as well as sensitive optical temperature sensing. The reported particles demonstrated a bright and photostable upconversion emission at a single particle level under a low laser intensity excitation of 20 W/cm². Furthermore, the synthesized UCNPs were tested and compared to the commonly used two-photon excitation QDs and organic dyes and showed a nine times better performance at a single particle level under the same experimental conditions. In addition, the synthesized UCNPs demonstrated sensitive optical temperature sensing at a single particle level within the biological temperature range. The good optical properties of single YLiF₄:Yb,Er UCNPs open an avenue for small and efficient fluorescent markers in imaging and sensing applications. Full article

Channel waveguides with diffraction gratings at their input and output for light injection and extraction, respectively, constitute the key components for applications in integrated optics and photonics. Here, we report for the first time on such fluorescent micro-structured architecture entirely elaborated on glass by sol-gel processing. This architecture particularly takes advantage of a high-refractive index and transparent titanium oxide-based, sol-gel photoresist that can be imprinted through a single photolithography step. This resist enabled us to photo-imprint the input and output gratings on a photo-imprinted channel waveguide doped with a ruthenium complex fluorophore (Rudpp). In this paper, the elaboration conditions and optical characterizations of derived architectures are presented and discussed with respect to optical simulations. We firstly show how the optimization of a two-step deposition/insolation sol-gel procedure leads to reproducible and uniform grating/waveguide architectures elaborated on rather large dimensions. Then, we show how this reproducibility and uniformity govern the reliability of fluorescence measurements in waveguiding configuration. These measurements demonstrate that: (i) our sol-gel architecture is well adapted to the efficient channel–waveguide/diffraction grating coupling at the Rudpp excitation and emission wavelengths; (ii) it enables an efficient propagation of the emission signal in the core of the waveguide allowing its photo-detection after extraction through the output grating; and (iii) it is affected by very reduced parasitic mechanisms, such as propagation losses and photobleaching features. This work constitutes a promising preliminary step toward the integration of our architecture in a microfluidic platform for further fluorescence measurements in liquid medium and waveguiding configuration. Full article

Molecular probes based on the excited-state intramolecular proton-transfer (ESIPT) mechanism have emerged to be attractive candidates for various applications. Although the steady-state fluorescence mechanisms of these ESIPT-based probes have been reported extensively, less information is available about the fluorescence lifetime characteristics of newly developed NIR-emitting dyes. In this study, four NIR-emitting ESIPT dyes with different cyanine terminal groups were investigated to evaluate their fluorescence lifetime characteristics in a polar aprotic solvent such as CH₂Cl₂. By using the time-correlated single-photon counting (TCSPC) method, these ESIPT-based dyes revealed a two-component exponential decay (τ₁ and τ₂) in about 2–4 nanoseconds (ns). These two components could be related to the excited keto tautomers. With the aid of model compounds (5 and 6) and low-temperature fluorescence spectroscopy (at −189 ℃), this study identified the intramolecular charge transfer (ICT) as one of the major factors that influenced the τ values. The results of this study also revealed that both fluorescence lifetimes and fractional contributions of each component were significantly affected by the probe structures. Full article

The fluorescence anisotropy of photosensitizers Radachlorin and chlorin e6 was studied using the time-resolved single photon-counting technique under one- and two-photon excitation within the Soret absorption band. A very small negative anisotropy was observed in both photosensitizers under one-photon excitation in the vicinity of the absorption maximum within the wavelength range of 395–405 nm. Meanwhile, two-photon excitation of the photosensitizers in the same spectral range demonstrated high fluorescence anisotropy with the maximum value of about 0.43. The drastic difference of the fluorescence anisotropy parameters at one- and two-photon excitation modes was suggested to be due to the different symmetries of one- and two-photon absorption tensors when two-photon absorption tensor components have comparable values. The variation of excitation wavelengths in the spectral range of 375–425 nm demonstrated nonlinear wavelength dependence of anisotropy of both Radachlorin and chlorin e6, with opposite tendencies at one- and two-photon excitation. The data obtained suggest that one-photon excitation at about 405 nm often utilized in FLIM experiments is not sensitive to fluorescence anisotropy in Radachlorin and chlorin e6 and therefore cannot be used for the determination of anisotropy/rotational diffusion time in these molecules. Meanwhile, two-photon excitation can provide high fluorescence anisotropy and accurate determination of the rotational diffusion time. At the same time, one-photon excitation at about 405 nm can be used for the accurate evaluation of fluorescence lifetimes within the standard FLIM schematic where fluorescence polarization is not taken into account. Full article

Pterins are an inseparable part of living organisms. Pterins participate in metabolic reactions mostly as tetrahydropterins. Dihydropterins are usually intermediates of these reactions, whereas oxidized pterins can be biomarkers of diseases. In this review, we analyze the available data on the quantum chemistry of unconjugated pterins as well as their photonics. This gives a comprehensive overview about the electronic structure of pterins and offers some benefits for biomedicine applications: (1) one can affect the enzymatic reactions of aromatic amino acid hydroxylases, NO synthases, and alkylglycerol monooxygenase through UV irradiation of H₄pterins since UV provokes electron donor reactions of H₄pterins; (2) the emission properties of H₂pterins and oxidized pterins can be used in fluorescence diagnostics; (3) two-photon absorption (TPA) should be used in such pterin-related infrared therapy because single-photon absorption in the UV range is inefficient and scatters in vivo; (4) one can affect pathogen organisms through TPA excitation of H₄pterin cofactors, such as the molybdenum cofactor, leading to its detachment from proteins and subsequent oxidation; (5) metal nanostructures can be used for the UV-vis, fluorescence, and Raman spectroscopy detection of pterin biomarkers. Therefore, we investigated both the biochemistry and physical chemistry of pterins and suggested some potential prospects for pterin-related biomedicine. Full article

Two-photon excitation fluorescence laser-scanning microscopy is the preferred method for studying dynamic processes in living organ models or even in living organisms. Thanks to near-infrared and infrared excitation, it is possible to penetrate deep into the tissue, reaching areas of interest relevant to life sciences and biomedicine. In those imaging experiments, two-photon excitation spectra are needed to select the optimal laser wavelength to excite as many fluorophores as possible simultaneously in the sample under consideration. The more fluorophores that can be excited, and the more cell populations that can be studied, the better access to their arrangement and interaction can be reached in complex systems such as immunological organs. However, for many fluorophores, the two-photon excitation properties are poorly predicted from the single-photon spectra and are not yet available, in the literature or databases. Here, we present the broad excitation range (760 nm to 1300 nm) of photon-flux-normalized two-photon spectra of several fluorescent proteins in their cellular environment. This includes the following fluorescent proteins spanning from the cyan to the infrared part of the spectrum: mCerulean3, mTurquoise2, mT-Sapphire, Clover, mKusabiraOrange2, mOrange2, LSS-mOrange, mRuby2, mBeRFP, mCardinal, iRFP670, NirFP, and iRFP720. Full article

Scan-free grazing-emission X-ray fluorescence spectroscopy (GEXRF) is an established technique for the investigation of the elemental depth-profiles of various samples. Recently it has been applied to investigating structured nanosamples in the tender X-ray range. However, lighter elements such as oxygen, nitrogen or carbon cannot be efficiently investigated in this energy range, because of the ineffective excitation. Moreover, common CCD detectors are not able to discriminate between fluorescence lines below 1 keV. Oxygen and nitrogen are important components of insulation and passivation layers, for example, in silicon oxide or silicon nitride. In this work, scan-free GEXRF is applied in proof-of-concept measurements for the investigation of lateral ordered 2D nanostructures in the soft X-ray range. The sample investigated is a ${\mathrm{Si}}_3{\mathrm{N}}_4$ lamellar grating, which represents 2D periodic nanostructures as used in the semiconductor industry. The emerging two-dimensional fluorescence patterns are recorded with a CMOS detector. To this end, energy-dispersive spectra are obtained via single-photon event evaluation. In this way, spatial and therefore angular information is obtained, while discrimination between different photon energies is enabled. The results are compared to calculations of the sample model performed by a Maxwell solver based on the finite-elements method. A first measurement is carried out at the UE56-2 PGM-2 beamline at the BESSY II synchrotron radiation facility to demonstrate the feasibility of the method in the soft X-ray range. Furthermore, a laser-produced plasma source (LPP) is utilized to investigate the feasibility of this technique in the laboratory. The results from the BESSY II measurements are in good agreement with the simulations and prove the applicability of scan-free GEXRF in the soft X-ray range for quality control and process engineering of 2D nanostructures. The LPP results illustrate the chances and challenges concerning a transfer of the methodology to the laboratory. Full article