Methods and Applications in Fluorescence

Quantum dots - polymer films as the reversible luminescent wide range temperature sensors
Sagdeev DO and Galyametdinov YG
The paper considers the temperature sensitivity of luminescence of three polymer types films: polymethyl methacrylate, polystyrene and polylactide doped with core-shell CdSe/CdS/ZnS and CdMnS/ZnS quantum dots. Films with uniform distribution of quantum dots in the polymer matrix were obtained by the spin-coating method. The influence of the quantum dots and polymer type, as well as their mutual content in the composite, on the thermal sensitivity and thermal stability of the films is considered. The thermal stability of the obtained composites was studied during multiple heating-cooling cycles to 100 and 175 °C, and the conditions for obtaining reusable reversible luminescent thermal sensors were established.
Microwave synthesis of molybdenum disulfide quantum dots and the application in bilirubin sensing
Pang S, Chang X, Xu L and Wu J
Molybdenum disulfide quantum dots (MoS2 QDs) is a new type of graphite like nanomaterial, which exhibited well chemical stability, unique fluorescence characteristics, and excellent biocompatibility. The conventional hydrothermal synthesis of MoS2 generally requires a long-term reaction at high temperature and high pressure. Herein, we have developed a simple and fast MoS2 QDs synthesis scheme using microwave heating, and further modified the surface of MoS2 QDs using 3-aminophenylboronic acid. The 3- aminophenylboronic acid modified MoS2 QDs (B-MoS2 QDs) were further coated by a zinc-based metal-organic backbone (ZIF-8) in a solution containing zinc ions and 2-methylimidazolium. The constructed nanohybrid B-MoS2@ZIF-8 were successfully applied to the visualization and rapid detection of bilirubin based on the ratiometric fluorescence changes. The linear range for bilirubin detection is 0.2-75 μmol•L-1, and detection limit is 0.017 μmol•L-1.
Cell viability assessment by using GelRed/SYTO 9-based double staining
Zheng Y, Sun J, Li X, Qiu H, Liao L and He S
Cell viability assessment plays a crucial role in biological research, pharmaceutical development, and toxicological identification. Here, we used GelRed, a sensitive and safer nucleic acid dye, to selectively label dead cells with red fluorescence (FL) thus distinguishing dead cells from live ones. Further more, the combined use of GelRed and SYTO 9 (another nucleic acid dye) enabled the clear differentiation in FL spectra between the two physiological statuses. The GelRed and SYTO 9 concentrations were optimized to obtain the highest FL ratio of dead to live cells. The GelRed/SYTO 9-based double staining could quantify the cell viability through flow cytometry analysis, with a good correlation between the detected and theoretical dead cell ratios. Compared with traditional prodium iodide (PI) staining, the GelRed/SYTO 9-based double staining showed high accuracy in quantifying dead cell of low levels. The as-developed staining method could be used in biomedical research to accurately measure the cytotoxic effect of various substances in living cells.
Microwave synthesis of molybdenum disulfide quantum dots and the application in bilirubin sensing
Pang S, Chang X, Xu L and Wu J
Molybdenum disulfide quantum dots (MoSQDs) is a new type of graphite like nanomaterial, which exhibited well chemical stability, unique fluorescence characteristics, and excellent biocompatibility. The conventional hydrothermal synthesis of MoSgenerally requires a long-term reaction at high temperature and high pressure. Herein, we have developed a simple and fast MoSQDs synthesis scheme using microwave heating, and further modified the surface of MoSQDs using 3-aminophenylboronic acid. The 3- aminophenylboronic acid modified MoSQDs (B-MoSQDs) were further coated by a zinc-based metal-organic backbone (ZIF-8) in a solution containing zinc ions and 2-methylimidazolium. The constructed nanohybrid B-MoS@ZIF-8 were successfully applied to the visualization and rapid detection of bilirubin based on the ratiometric fluorescence changes. The linear range for bilirubin detection is 0.2-75 μmol·l, and detection limit is 0.017 μmol·l.
Spectral properties of 4-methylumbelliferone in PVA films; long-lived room temperature phosphorescence
Lee B, Alexander E, Pham D, Gagoś M, Matwijczuk A, Gryczynski Z and Gryczynski I
We studied absorption and fluorescence as well as room temperature phosphorescence (RTP) of 4-methylumbelliferone (4MU) in poly (vinyl alcohol) (PVA) films. We focused our study on the long-wavelength basic form of 4MU with absorption centered at 375 nm. The strong fluorescence with a quantum yield of above 70% appears at ∼430 nm. The fluorescence anisotropy of 4MU-doped PVA film is high, reaching a value of about 0.3. The emission with gated detection shows a broad phosphorescence spectrum with a peak at ∼510 nm and a residual delayed fluorescence at 430 nm. The excitation spectra for fluorescence and phosphorescence roughly follows 4MU absorption. The phosphorescence lifetime of 4MU is remarkably long, almost 3 s. 4MU excitation and emission phosphorescence anisotropies are low, very close to zero.
Editorial: MAF journal and conference build together
Birch DJS, Levitus M and Mély Y
Surface plasmon enhanced auto-fluorescence and Raman spectroscopy for low-level detection of biological pathogens
Panghal A, Das S, Thapa P, Meenakshi M, Kumar A, Nagpal P, Dubey SK, Perumal V and Mehta DS
The current culture-based bacterial detection technique is time-consuming and requires an extended sample preparation methodology. We propose the potential of surface-enhanced Raman spectroscopy (SERS) and surface plasmon-enhanced auto-fluorescence spectroscopy (SPEAS) for the label-free identification and quantification of bacterial pathogens at low concentrations collecting its unique auto-fluorescence and Raman signatures utilising highly anisotropic three-dimensional nanostructures of silver nano dendrites (Ag-NDs). The SERS data facilitates qualitative bacterial identification using the spectral features coming from the bacterial cell wall compound, and the SPEAS data was utilised to gain unique auto-fluorescence spectra present on the bacterial cell wall with enhanced quantification. The enhancement of Raman and auto-fluorescence signals of Ag-NDs were first evaluated using rhodamine 6g (R6G) as a probe molecule that exhibits a significant enhancement of 106 and limit of detection (LOD) of 10-12 M for SERS and 15 fold intensity enhancement and LOD of 10-15 M for SPEAS measurements. Further, the SERS and SPEAS measurements of bacterial pathogens, such as Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus), using the Ag-NDs were recorded, and the results exhibit high auto-fluorescence and Raman signal intensity for both the samples up to 100 cfu/ml for both modalities. The significant photon count and distinct emission range in SPEAS measurements of bacteria enables accurate quantification. Therefore, the comprehensive investigation of plasmonic enhancement of Ag-NDs for SPEAS and SERS techniques provides complementary information about molecules to enable accurate and quick identification and quantification of pathogens.
Naphthylated LEGO-lipophosphonoxin antibiotics used as a fluorescent tool for the observation of target membrane perturbations preceding its disruption
Dolejšová T, Lišková P, Sahatsapan N, Mojr V, Pohl R, Brzobohatá H, Dugić M, Křížek T, Cwiklik L, Mikušová G, Rejman D and Fišer R
Linker-Evolved-Group-Optimized-Lipophosphonoxins (LEGO-LPPO) are small synthetic modular peptidomimetics with promising antimicrobial activity. The LEGO-LPPO mechanism of antibacterial action has been determined to be the depolarization and disruption of bacterial membranes. Their modular nature is advantageous for fine tuning their biological properties. In order to optimize the structure of LEGO-LPPO even further, it is important to understand the interaction of LEGO-LPPO with bacterial membranes at the molecular level. In this work, we present the synthesis of five LEGO-LPPO (designated as_naph2-4-G to_naph2-4-G) molecules bearing fluorescent naphtylethyl moieties and their usage in the study of LEGO-LPPO behaviour in the membrane. Our goal was to characterize fluorescently labelled LEGO-LPPO under conditions that do not completely disrupt the membrane, mostly in the form of membrane-bound monomers. We observed the intramolecular interactions of hydrophobic modules of_naph2-4-G in the buffer by detecting dynamic naphthyl excimers and their disappearance after_naph2-4-G bind into the membranes. In the membrane, the molecule_naph2-4-G slightly affects the membrane fluidity of DOPG membranes above the phase transition. The naphthyl fluorophore itself has fast and almost unrestricted rotation around ethylene linking groups (= 0.010), which indicates a considerable chaotropic effect of the hydrophobic modules of_naph2-4-G at the given depth of the membrane._naph2-4-G proved to be a useful model for observing the interaction of LEGO-LPPO antibiotics with the phospholipid bilayer enabling us to decipher its effects on membrane state and dynamics; its binding and penetration into the membrane, its structure and the particular depth that it occupies.
Detection of antimicrobial-induced survival/dead bacteria via mEos4b photoconversion: a preliminary study
Yilmaz I, Demir H, Tureyen AE and Ozbek T
The escalating prevalence of hospital-acquired infections poses a critical challenge for healthcare systems worldwide. Effective management requires rapid identification of pathogens and their antibiotic resistance profiles. In this study, we utilized the photoconvertible mEos4b protein, which transitions from green to red fluorescence upon blue light exposure, to distinguish live from dead bacteria. The mEos4b gene was cloned into a prokaryotic vector and expressed inBL21. The Minimum Inhibitory Concentration (MIC) of the transgenic bacteria was determined for five antibiotics, followed by a post-antibiotic effect assessment over a two-hour exposure period. The optimal photoconversion time for mEos4b was established as 90 s, and confocal microscopy was used to visualize live (green) and dead (red) cells post-exposure. The mEos4b-TR system proved highly specific, accurately distinguishing live and dead bacteria without producing false positives, even in control groups, which is a common issue in commercial live-dead kits. By relying on cellular metabolic activity rather than dyes, this system minimizes nonspecific interactions and contamination, making it more reliable than traditional methods prone to false readings. These results highlight the potential of the mEos4b-TR system as a superior alternative for rapid, precise bacterial viability assessments, particularly in determining antibiotic susceptibility. This preliminary study demonstrates the system's differentiation of viable and non-viable cells, suggesting its potential application in future studies involving novel antibacterial agents to refine antibiotic sensitivity testing.
Effect of Mndoping and DDAB-assisted postpassivation on the structural and optical properties of CsPb(Cl/Br)halide perovskite nanocrystals
Dubey C, Yadav A, Kachhap S, Singh SK, Gupta G, Singh SP and Singh AK
Cesium lead halide perovskite (CsPbX; X = Cl, Br, I) nanocrystals showing intense band-edge emission and high photoluminescence quantum yield are known to be a potential candidate for application in optoelectronic devices. However, controlling toxicity due to the presence of Pbin lead-based halide perovskites is a major challenge for the environment that needs to be tackled cautiously. In this work, we have partially replaced Pbwith Mnions in the CsPb(Cl/Br)nanocrystals and investigated their impact on the structural and optical properties. The Rietveld refinement shows that CsPbClBr nanocrystals possess a cubic crystal structure with3̅space group, the Mndoping results in the contraction of the unit cell. The CsPb(Cl/Br): Mn nanocrystals show a substantial change in the optical properties with an additional emission band at ∼588 nm through a d-d transition, changing the emission color from blue to pink. Here, a didodecyldimethylammonium bromide (DDAB) ligand that triggers both anion and ligand exchange in the CsPb(Cl/Br): Mn nanocrystals have been used to regulate the exchange reaction and tune the emission color of halide perovskites by changing the peak position and the PL intensities of band-edge and Mndefect states. We have also shown that oleic acid helps in the desorption of oleylamine capping from the CsPb(Cl/Br): Mn nanocrystal surfaces and DDAB, resulting in the substitution of Clwith Bras well as provides capping with shorter branched length ligand which led to increase in the overall PL intensity by many folds.
Combiningautofluorescence intensity and F680/F750 ratio to discriminate responses to environmental stressors
Navickaite A, Pupkis V, Kalnaityte-Vengeliene A, Lapeikaite I, Kisnieriene V and Bagdonas S
Detection of autofluorescence parameters is a useful approach to gain insight into the physiological state of plants and algae, but the effect of reabsorption hinders unambiguous interpretation ofdata. The exceptional morphological features ofmade it possible to measure autofluorescence spectra along single internodal cells and estimate relative changes in autofluorescence intensity in selected spectral regions at room temperatures, avoiding the problems associated with thick or optically dense samples. The response of algal cells to controlled white light and DCMU herbicide was analyzed by monitoring changes in peak FL intensity at 680 nm and in F680/F750 ratio. Determining the association between the selected spectral FL parameters revealed an exponential relationship, which provides a quantitative description of photoinduced changes. The ability to discern the effect of DCMU not only in the autofluorescence spectra of dark-adapted cells, but also in the case of light-adapted cells, and even after certain doses of excess light, suggests that the proposed autofluorescence analysis ofmay be useful for detecting external stressors in the field.
Fiber-optics based fluorescence detection. Part I: Basic concepts
Lee B, Ceresa L, Pham D, Kimball J, Alexander E, Ye X, Gryczynski I and Gryczynski Z
Continuous in-line detection and process monitoring are essential for industrial, analytical, and biomedical applications. Lightweight, highly flexible, and low-cost fiber optics enable the construction of compact and robust hand-held devices forchemical and biological species analysis in both industrial and biomedical/detection. Despite the broad range of fiber-optic based applications, we lack a good understanding of the parameters that govern the efficiency of light collection or the sensitivity of detection. Consequently, comparing samples of different optical density and/or geometry becomes challenging and can lead to misinterpretation of results; especially when we lack the approaches necessary to correct the detected signal (spectra) for artifacts such as inner-filter effect or scattering. Hence, in this work, we discuss factors affecting the signal detected by the fiber optic in the bare and lens-coupled flat-tipped configurations that lead to signal/spectral distortions. We also present a simple generic model describing the excitation profile and emission collection efficiency that we verify with experimental data. Understanding the principles governing the signal collected by the fiber will provide rationales for correcting the measured emission spectra and recovering the true emission profile of optically dense samples.
Effect of molecular crowders on ligand binding kinetics with G-quadruplex DNA probed by fluorescence correlation spectroscopy
Alam P, Clovis NS, Chand AK, Khan MF and Sen S
Guanine-rich single-stranded DNA folds into G-quadruplex DNA (GqDNA) structures, which play crucial roles in various biological processes. These structures are also promising targets for ligands, potentially inducing antitumor effects. While thermodynamic parameters of ligand/DNA interactions are well-studied, the kinetics of ligand interaction with GqDNA, particularly in cell-like crowded environments, remain less explored. In this study, we investigate the impact of molecular crowding agents (glucose, sucrose, and ficoll 70) at physiologically relevant concentrations (20% w/v) on the association and dissociation rates of the benzophenoxazine-core based ligand, cresyl violet (CV), with human telomeric antiparallel-GqDNA. We utilized fluorescence correlation spectroscopy (FCS) along with other techniques. Our findings reveal that crowding agents decrease the binding affinity of CV to GqDNA, with the most significant effect-a nearly three-fold decrease-observed with ficoll 70. FCS measurements indicate that this decrease is primarily due to a viscosity-induced slowdown of ligand association in the crowded environment. Interestingly, dissociation rates remain largely unaffected by smaller crowders, with only small effect observed in presence of ficoll 70 due to direct but weak interaction between the ligand and ficoll. These results along with previously reported data provide valuable insights into ligand/GqDNA interactions in cellular contexts, suggesting a conserved mechanism of saccharide crowder influence, regardless of variations in GqDNA structure and ligand binding mode. This underscores the importance of considering crowding effects in the design and development of GqDNA-targeted drugs for potential cancer treatment.
Multidimensional fluorescence spectroscopy of wine using synchronous excitation/emission matrices and time-resolved fluorescence interferometric detection
Mori S, Hall CR, Bradley SJ and Smith TA
Wines are complex mixtures of chemical compounds with broad and overlapping absorption and emission spectral features in the UV and visible spectral regions, making them challenging to study with conventional optical spectroscopic techniques. Multidimensional fluorescence spectroscopies correlate fluorescence spectra with other degrees of freedom, and have proven useful for studying complex molecular systems, offering a pathway for the analysis of wines utilising their inherent fluorescence. Here we employ steady-state excitation-emission matrix (EEM) and time-resolved fluorescence spectral measurements to investigate representative commercial white and red wine samples and a fluorescent 'model' wine base. Combining these multidimensional measurement methods provides information on the emission characteristics of the components that wines contain. This investigation illustrates the potential for multidimensional fluorescence techniques as diagnostic tools for the wine industry.
Influence of Yb/Hocodoping on optical and thermal properties of TeO-ZnO glass
Azam M, Rai VK, Khan S and Singh K
This paper reports the effect of incorporation of Ybions on the frequency downconversion luminescence and thermal properties of triply ionised Hodoped zinc tellurite (TZ) glasses. The photoluminescence spectra of both the Ho/Ybdoped and codoped glasses have been recorded and observed a green emission band corresponding to theF,S→I(∼550 nm) transition upon various excitations. In the downconversion (DC) emission process, the back energy transfer (BET) mechanism from Hoions to Ybions has also been explored. The colour emitted in the downconversion process is found to be non-tunable at different excitations. Thus, the Ho:TZ glass can be utilised for non-colour tunable optical devices under various UV excitations. Also the glass transition (T) and crystallisation (T) temperatures have been measured for both the doped and codoped glasses and found to be increased in the codoped glass. The singly Hoions doped TZ glass shows better optical downconversion and glass forming ability.
A simple light path modifying device to reduce scattering in front-face fluorescence spectra
Peters FB and Rapp AO
This technical note presents a device to diminish scattering signal in front-face fluorescence spectra while obtaining fluorescence signal. The beam path in a commercial fluorescence spectrometer was modified by two deflecting mirrors, leading reflections away from the sensor. This light path modifying (LPM) device was tested with two fluid and three solid substances, where the scattering-to-fluorescence ratio improved by a factor of 1.7 to 7.6. The spectra obtained with the LPM were much clearer, and distortion of the fluorescence peaks was avoided. Scans of quinine sulphate complied well with reference spectra.
Bright ESIPT emission from 2,6-di(thiazol/oxazol/imidazol-2-yl)phenol derivatives in solution, aggregation and solid states
Chen P, Niu Z and Wang E
Most luminophores often suffer from the problem of aggregation-caused quenching (ACQ) or fluorescence disappearance in dilute solution. It is significant to bridge the gap between ACQ and AIE. In this work, a facile but effective strategy was proposed for the fabrication of always-on luminophores based on the excited state intramolecular proton transfer (ESIPT) mechanism, and six luminophores emitting bright fluorescence in solution, aggregation and solid states were synthesized from 5-tert-butyl-2-hydroxyisophthalaldehyde. All these ESIPT systems show only keto emission owing to their congested structures which block the breakage of intramolecular hydrogen bond (O-H⋯N) by solvation, and subsequently make enol emission impossible. Three of these luminophores are prone to convert into the corresponding phenolate anions emitting blue-shifted emission, which enable them to sense pH variation in the weakly basic range. Furthermore, white-light emission was achieved by combining two of them which show complementary-color fluorescence, and one of them was utilized for bioimaging of living Hela cells and the high-resolution image was obtained.
Exploring light-emitting diode pumped luminescent concentrators in solid-state laser applications
Perera HCS, Ford B, Das G, Balembois F and Sathian J
In the past, there were limited efforts to use light-emitting diodes (LEDs) for pumping solid-state lasers. However, these attempts were overshadowed by the introduction of laser diodes, which offered more favourable pumping conditions. Nevertheless, recent advancements in high-power LEDs, coupled with the utilization of luminescent concentrators (LC), have paved the way for a novel approach to pump solid-state lasers. The combination of LEDs and LC in this LED-LC system presents several advantages, including enhanced ruggedness, stability, and cost-effectiveness compared to other laser pumping methods. This review explores the various techniques employed to pump solid-state lasers using LED-LC as a pump source, along with improvements made to enhance the brightness of LEDs in this context.
Fluorescence in depth: integration of spectroscopy and imaging with Raman, IR, and CD for advanced research
Aeindartehran L, Sadri Z, Rahimi F and Alinejad T
Fluorescence spectroscopy serves as a vital technique for studying the interaction between light and fluorescent molecules. It encompasses a range of methods, each presenting unique advantages and applications. This technique finds utility in various chemical studies. This review discusses Fluorescence spectroscopy, its branches such as Time-Resolved Fluorescence Spectroscopy (TRFS) and Fluorescence Lifetime Imaging Microscopy (FLIM), and their integration with other spectroscopic methods, including Raman, Infrared (IR), and Circular Dichroism (CD) spectroscopies. By delving into these methods, we aim to provide a comprehensive understanding of the capabilities and significance of fluorescence spectroscopy in scientific research, highlighting its diverse applications and the enhanced understanding it brings when combined with other spectroscopic methods. This review looks at each technique's unique features and applications. It discusses the prospects of their combined use in advancing scientific understanding and applications across various domains.
A quinoline derivative-based supramolecular gel for fluorescence 'turn-off' detection of Feand Cu
Fu S, Wu S, Liu J, Wang J, Tian S, Zhang G, Yin F, Sun Y, Zhang P and Yang Q
In this research, we synthesized and constructed a novel gelator (named) combining quinoline and naphthalene that self-assembled in N, N-dimethylformamide (DMF) to form a stable supramolecular gel (named). Under UV light, gelexhibited extremely bright yellow fluorescence. The gelshowed excellent sensing performance for both Feand Cu, with a fluorescence 'turn-off' detection mechanism and the lowest detection limit of 7.58 × 10M and 1.51 × 10M, respectively. Scanning electron microscopy (SEM), transmission electron microscopy (TEM), Fourier transform infrared (FTIR) spectra, x-ray powder diffraction (XRD), rheological measurements, x-ray photoelectron spectroscopy (XPS), and fluorescence spectroscopy were used to characterize the gel. Theion-responsive membrane created is an excellent fluorescent writing material.
Enhanced fluorescence blinking of AF647 fluorophores in Mowiol via violet and UV light induced recovery for superior localization microscopy
Bharadwaj A, Kumar A, Mitra R, Jaganathan BG and Boruah BR
Blinking of fluorophores is essential in the context of single molecule localization-based optical super-resolution microscopy methods. To make the fluorescence molecule undergo blinking specific complex chemical mounting buffer systems, combined with suitable oxygen scavengers, and reducing agents are required. For instance to realise blinking in widely used fluorescence tags, like Alexa Fluor 647 (AF647), they are to be mounted on anti-fading buffer such as Mowiol and reducing agent such as Beta () - ME. However, the quality of the super-resolved images is decided by the total number of blinking events or in other words net duration for which the fluorescence blinking persists. In this paper we investigate how a violet and UV light induced fluorescence recovery mechanism can enhance the duration of fluorescence blinking. Our study uses AF647 dye conjugated with Phalloidin antibody in U87MG cell line mounted on Mowiol and- ME. On the basis of the investigation we optimize the intensity, at the sample plane, of fluorescence excitation laser at 638 nm and fluorescence recovery beam at 405 nm or in the UV giving the maximum possible fluorescence blinking duration. We observe that the longer blinking duration, using the optimized illumination scheme, has brought down the resolution in the super-resolved image, as given by Fourier Ring Correlation method, from 168 nm to 112 nm, while the separation between two nearby resolvable filaments has been brought down to ≤ 60 nm.