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We present optical studies of two different size distributions of silver triangular nanoprisms, one with a dipole resonance at 520 nm and the other with a dipole resonance at 650 nm, placed in different media. Significant wavelength-dependent depolarization of scattered light from the silver nanoprisms suspended in water indicates strong interference of multiple surface plasmon resonant modes in the same particle. We use this depolarization as a probe of light scattering by the nanoprisms in a lipid solution due to the rejection of a polarized background scattering. Also, the silver nanoprisms were embedded in a polyvinyl alcohol polymer matrix and oriented by stretching the polymer/nanoprism nanocomposite films. We observe significantly increased linear dichroism in the region associated with the plasmonic in-plane dipole mode upon stretching. Additionally, there is a weaker linear dichroism in the region associated with out-of-plane modes, which vanish in the extinction spectrum of the stretched nanocomposite film. [ABSTRACT FROM AUTHOR]

This work reports the surface functionalization of polymeric PLGA nanoparticles by non-covalent insertion of a homo-bifunctional chemical crosslinker, bis(sulfosuccinimidyl) suberate (BS3) for targeted cancer therapy. We dissolved BS3 in aqueous solution of PVA during formulation of nanoparticles by a modified solid/oil/water emulsion solvent evaporation method. The non-covalent insertion of BS3 was confirmed by Fourier transform infrared (FTIR) spectroscopy. Curcumin and annexin A2 were used as a model drug and a cell specific target, respectively. Nanoparticles were characterized for particle size, zeta potential and surface morphology. The qualitative assessment of antibody attachment was performed by transmission electron microscopy (TEM) as well as confocal microscopy. The optimized formulation showed antibody attachment of 86%. However, antibody attachment was abolished upon blocking the functional groups of BS3. The availability of functional antibodies was evaluated by the presence of a light chain fraction after gel electrophoresis. We further evaluated the in vitro release kinetics of curcumin from antibody coated and uncoated nanoparticles. The release of curcumin is enhanced upon antibody attachment and followed an anomalous release pattern. We also observed that the cellular uptake of nanoparticles was significantly higher in annexin A2 positive cells than in negative cells. Therefore, these results demonstrate the potential use of this method for functionalization as well as to deliver chemotherapeutic agents for treating cancer. [ABSTRACT FROM AUTHOR]

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., (Creative Commons Attribution license.)

We studied spectral properties of 1,N 2 -etheno-2-aminopurine after immobilization in poly (vinyl alcohol) films. The absorption spectrum of 1,N 2 -ε2APu consists of two peaks centered at 300 and 370 nm, and the fluorescence spectrum has maximum at about 460 nm. The fluorescence quantum efficiency is 62%. The fluorescence anisotropy reaches a value of 0.3 at longer wavelengths, while it is low at shorter wavelengths (corresponding to the second single excited state). The 1,N 2 -ε2APu has a relatively long fluorescence lifetime of about 16 ns and a noticeable room temperature phosphorescence with a lifetime of about 220 ms. A broad phosphorescence emission band (425-675 nm) is centered at about 530 nm and markedly overlaps with fluorescence at shorter wavelengths. Surprisingly, the phosphorescence excitation spectrum of 1,N 2 -ε2APu-doped poly (vinyl alcohol) film differs from the absorption and fluorescence excitation spectra. The strongest room temperature phosphorescence excitation is about 335 nm. At longer excitation wavelengths, above 450 nm, where fluorescence cannot be excited, a triplet excitation is still possible. The 1,N 2 -ε2APu phosphorescence anisotropy spectra confirm direct triplet state excitation. The ability to excite molecules at long wavelengths can find applications in the study of biological molecules that are unstable when excited at high energies., (© 2024 The Author(s). Luminescence published by John Wiley & Sons Ltd.)

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 for in situ chemical and biological species analysis in both industrial and biomedical in vitro / in vivo 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., (Creative Commons Attribution license.)

Cytotoxic activity has been reported for the xanthone α-mangostin (AMN) against Glioblastoma multiforme (GBM), an aggressive malignant brain cancer with a poor prognosis. Recognizing that AMN's high degree of hydrophobicity is likely to limit its systemic administration, we formulated AMN using reconstituted high-density lipoprotein (rHDL) nanoparticles. The photophysical characteristics of the formulation, including fluorescence lifetime and steady-state anisotropy, indicated that AMN was successfully incorporated into the rHDL nanoparticles. To our knowledge, this is the first report on the fluorescent characteristics of AMN with an HDL-based drug carrier. Cytotoxicity studies in a 2D culture and 3D spheroid model of LN-229 GBM cells and normal human astrocytes showed an enhanced therapeutic index with the rHDL-AMN formulation compared to the unincorporated AMN and Temozolomide, a standard GBM chemotherapy agent. Furthermore, treatment with the rHDL-AMN facilitated a dose-dependent upregulation of autophagy and reactive oxygen species generation to a greater extent in LN-229 cells compared to astrocytes, indicating the reduced off-target toxicity of this novel formulation. These studies indicate the potential therapeutic benefits to GBM patients via selective targeting using the rHDL-AMN formulation.

We studied the spectral properties of 4'-6-diamidino-2-phenylindole (DAPI) in poly (vinyl alcohol) (PVA) films. Absorption and fluorescence spectra, emission and excitation spectra, quantum yield, and fluorescence lifetime have been characterized. An efficient room temperature phosphorescence (RTP) of DAPI has been observed with UV and blue light excitations. A few hundred millisecond phosphorescence lifetime enables a gated detection with sufficient background reduction. We found the phosphorescent Quantum Yield of DAPI in PVA Film to be 0.0009., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

We studied the effect of annealing on the luminescence of Coumarin 106 (C106) in poly (vinyl alcohol) films (PVA films). The samples and reference polymer films were treated at temperatures between 100 °C and 150 °C (212 F and 302 F) for various times. After cooling and smoothing, the samples and references were measured at room temperature. We observed that the PVA polymer (reference films) changes its optical properties with annealing at higher temperatures, affecting the baselines in absorption and the backgrounds in emission measurements. This requires precise background subtractions and control of the signal-to-noise ratio. Whereas the fluorescence intensity of C106 in PVA films modestly decreases with annealing, the phosphorescence depends dramatically and progressively increases by many folds. The fluorescence quantum yields and lifetimes decrease with the annealing, which suggests an increase in the non-radiative processes in the singlet excited state S 1 . The increase in the phosphorescence intensities results from increased intersystem crossing (ISC), which also decreases fluorescence. We also studied the effect of annealing on phosphorescence with the directly excited triplet state of C106. In this case, two processes are affected by annealing, S 0 →T 1 absorption and T 1 →S 0 phosphorescence. The long-wavelength excitation (475 nm) avoids PVA polymer excitation. The phosphorescence lifetime decreases with annealing while the phosphorescence intensity increases. These changes suggest that the radiative rate of T 1 → S 0 increases with annealing., (Creative Commons Attribution license.)

A novel approach is presented that increases sensitivity and specificity for detecting minimal traces of DNA in liquid and on solid samples. Förster Resonance Energy Transfer (FRET) from YOYO to Ethidium Bromide (EtBr) substantially increases the signal from DNA-bound EtBr highly enhancing sensitivity and specificity for DNA detection. The long fluorescence lifetime of the EtBr acceptor, when bound to DNA, allows for multi-pulse pumping with time gated (MPPTG) detection, which highly increases the detectable signal of DNA-bound EtBr. A straightforward spectra/image subtraction eliminates sample background and allows for a huge increase in the overall detection sensitivity. Using a combination of FRET and MPPTG detection an amount as small as 10 pg of DNA in a microliter sample can be detected without any additional sample purification/manipulation or use of amplification technologies. This amount of DNA is comparable to the DNA content of a one to two human cells. Such a detection method based on simple optics opens the potential for robust, highly sensitive DNA detection/imaging in the field, quick evaluation/sorting (i.e., triaging) of collected DNA samples, and can support various diagnostic assays., (© 2023. European Biophysical Societies' Association.)

Phosphorescence emission at room temperature has been observed from 2-Aminopyridyne (2APi) embedded in poly (vinyl alcohol) (PVA) films. The gated emission with UV excitation at 305 nm results in a residual delayed fluorescence at around 350 nm and a broad phosphorescence spectrum with a maximum of around 500 nm. The phosphorescence excitation spectrum of 2APi - doped PVA film differs from the absorption spectrum in the long-wavelength part, showing a band at about 400-450 nm. The phosphorescence spectrum measured with a blue (420 nm) excitation closely resembles the spectrum measured with 305 nm excitation. Whereas the phosphorescence anisotropy measured with UV excitation is low and negative, with the blue excitation, the anisotropy is high and positive. The phosphorescence lifetimes (a fraction of a millisecond) are similar for UV and blue excitations. Both phosphorescence emissions with either UV or blue excitation strongly depend on temperature., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Phosphorescence emission of 5,6-Benzoquinoline embedded in poly (vinyl alcohol) film has been studied at room temperature. A strong green long-lived emission was observed in films doped with 5,6-Benzoquinoline while illuminated on a UV plate. A broad phosphorescence emission spectrum is centered at about 500 nm. The phosphorescence excitation spectrum follows the absorption spectrum of 5,6-Benzoquinoline, except for a long-wavelength part, which is well beyond the absorption band. This long-wavelength part of the absorption spectrum is responsible for the forbidden S 0 -T 1 transition. The excitation at 430 nm resulted in the long-lived emission with a spectrum similar to the phosphorescence spectrum obtained with UV excitation within the absorption of 5,6-Benzoquinoline. The phosphorescence anisotropy obtained with a direct S 0 -T 1 excitation is positive, while the UV excitation is negative. In contrast to fluorescence, the phosphorescence intensity strongly depends on temperature. Phosphorescence lifetimes with UV and long-wavelength excitation are similar, with a mean value of about 0.5 s., (© 2023 IOP Publishing Ltd.)

Optical biomedical imaging and diagnostics is a rapidly growing field that provides both structural and functional information with uses ranging from fundamental to practical clinical applications. Nevertheless, imaging/visualizing fluorescence objects with high spatial resolution in a highly scattering and emissive biological medium continues to be a significant challenge. A fundamental limiting factor for imaging technologies is the signal-to-background ratio (SBR). For a long time to improve the SBR, we tried to improve the brightness of fluorescence probes. Many novel fluorophores with improved brightness (almost reaching the theoretical limit), redshifted emission, highly improved photostability, and biocompatibility greatly helped advance fluorescence detection and imaging. However, autofluorescence, scattering of excitation light, and Raman scattering remain fundamental limiting problems that drastically limit detection sensitivity. Similarly, significant efforts were focused on reducing the background. High-quality sample purification eliminates the majority of autofluorescence background and in a limited confocal volume allows detection to reach the ultimate sensitivity to a single molecule. However, detection and imaging in physiological conditions does not allow for any sample (cells or tissue) purification, forcing us to face a fundamental limitation. A significant improvement in limiting background can be achieved when fluorophores with a long fluorescence lifetime are used, and time-gated detection is applied. However, all long-lived fluorophores present low brightness, limiting the potential improvement. We recently proposed to utilize multipulse excitation (burst of pulses) to enhance the relative signal of long-lived fluorophores and significantly improve the SBR. Herein, we present results obtained with multipulse excitation and compare them with standard single-pulse excitation. Subtraction of images obtained with a single pulse from those obtained with pulse burst (differential image) highly limits background and instrumental noise resulting in more specific/sensitive detection and allows to achieve greater imaging depth in highly scattering media, including skin and tissue.

Excitation and emission (observation) conditions heavily impact fluorescence measurements. Both observed spectra and intensity decays (fluorescence lifetimes), when incorrectly measured, may lead to incorrect data interpretations. In this report, we discuss the role of observation conditions in steady-state and time-resolved (lifetime) fluorescence measurements. We demonstrate the importance of the correction for uneven transmissions of vertical and horizontal polarizations of emission light through the detection system. The necessity of using so-called total fluorescence intensity or intensity measured under magic angle (MA) conditions has been demonstrated for both steady-state and time-resolved fluorescence measurements. The dependence of lifetime measurements on observation (emission) wavelengths is also discussed. Two fluorophores, rhodamine 6G (R6G) and 4,4 Dimethylamino-cyano stilbene (DCS) in two solvents - ethanol and glycerol have been used in order to cover a broad range of dye polarities and solvent viscosities., (© 2022 IOP Publishing Ltd.)

This article presents a novel approach to increase the detection sensitivity of trace amounts of DNA in a sample by employing Förster resonance energy transfer (FRET) between intercalating dyes. Two intercalators that present efficient FRET were used to enhance sensitivity and improve specificity in detecting minute amounts of DNA. Comparison of steady-state acceptor emission spectra with and without the donor allows for simple and specific detection of DNA (acceptor bound to DNA) down to 100 pg/μL. When utilizing as an acceptor a dye with a significantly longer lifetime (e.g., ethidium bromide bound to DNA), multipulse pumping and time-gated detection enable imaging/visualization of picograms of DNA present in a microliter of an unprocessed sample or DNA collected on a swab or other substrate materials.

We studied room temperature phosphorescence of tryptophan (TRP) embedded in poly (vinyl alcohol) films. With UV (285 nm) excitation, the phosphorescence spectrum of tryptophan appears at about 460 nm. We also observed the TRP phosphorescence with blue light excitation at 410 nm, well outside of the S 0 →S 1 absorption. This excitation reaches the triplet state of tryptophan directly without the involvement of the singlet excited state. The phosphorescence lifetime of tryptophan is in the sub-millisecond range. The long-wavelength direct excitation to the triplet state results in high phosphorescence anisotropy which can be useful in macromolecule dynamics study via time-resolved phosphorescence., (© 2022 IOP Publishing Ltd.)

This work addresses the issue of dark states formation in QDs by cooperative excitonic and intrinsic defect-assisted radiative transitions. Here we refer to the observed blinking as D-type to distinguish it from purely excitonic types. It is shown experimentally that defect-assisted radiative relaxations in a single I-III-VI QD result in atypical blinking characteristics that cannot be explained on the basis of charged exciton models. In addition to the excitonic channel, it has been proposed that defect-assisted kinetics can also form blinking patterns. Two conditions for the formation of dark states have been identified which are related to correlation and competition when considering photons emitted from bright defects. Two transition schemes have therefore been proposed. The first transition scheme includes time-correlated trapping of more than one electron at a single trap centre. This is used to simulate variations in the defect's charge state and switching between radiative/nonradiative transitions. The latter scheme, on the other hand, involves uncorrelated trapping and radiative relaxations from two different types of defects (competition). Both schemes are seen to play an equal role in radiative processes in I-III-VI QDs. Considered together, the proposed models can reflect the experimental data with very good accuracy, providing a better understanding of the underlying physics. An important implication of these schemes is that dark states formation doesn't have to be limited to mechanisms that involve charged excitons, and it may also be observed for independent defect assisted kinetics. This is especially valid for highly defected or multinary QDs.

This report presents a novel approach for detecting and visualizing small to trace amounts of DNA in a sample. By utilizing both the change in emission spectrum and change in fluorescence lifetime, there is a significant increase in detection sensitivity allowing for the imaging/visualizing of a picograms amount of DNA in a microliters volume. As in the previous reports, one of the oldest DNA intercalators, Ethidium Bromide (EtBr), is employed as a model system. With this new approach, it is feasible to visualize just a few hundred picograms of DNA without the need for prior DNA amplification. The sensitivity can later be largely improved by using an intercalator that exhibits a higher affinity to DNA and a larger fluorescence change upon binding to DNA ( e.g. , ethidium homodimer, YOYO, or Diamond nucleic acid dyes).

Fluorescence is an established technology for studying molecular processes and molecular interactions. More recently fluorescence became a leading method for detection, sensing, medical diagnostics, biotechnology, imaging, DNA analysis, and gene expression. Consequently, precise and accurate measurements in various conditions have become more critical for proper result interpretations. Previously, in Part 1, we discussed inner filter effect type I, which is a consequence of the instrumental geometrical sensitivity factor and absorption of the excitation. In this part, we analyze inner filter effect type II and discuss the practical consequences for fluorescence measurements in samples of high optical density (absorbance/scattering). We consider both the standard square and front-face experimental configurations, discuss experimental approaches to limit/mitigate the effect and discuss methods for correcting and interpreting experimental results., (© 2021 IOP Publishing Ltd.)

In this report, a simple and practical procedure is proposed for DNA localization on a solid matrix e.g., a collection swab. The approach is straightforward and employs spectrum decomposition using a model DNA intercalator Ethidium Bromide (EtBr). The proposed approach can detect picograms of DNA in solution and nanograms of DNA on solid surfaces (swabs) without the need for PCR amplification. The proposed technology offers the possibility for developing an inexpensive, sensitive, rapid, and practical method for localizing and recovering DNA deposited on collection swabs during routine DNA screening. Improved detection of low DNA concentrations is needed and, if feasible, will allow for better decision making in clinical medicine, biological and environmental research, and human identification in forensic investigations.

We studied the luminescence properties of indole in poly (vinyl alcohol) (PVA) film. The indole molecules are effectively immobilized in this polymer film and display both fluorescence and phosphorescence emission at room temperature. We noticed that the phosphorescence of indole in PVA film can be effectively excited at a longer wavelength than its typical singlet to triplet population route involving intersystem crossing. The maximum of the phosphorescence excitation is about 410 nm which corresponds to the energy of indole's triplet state. Interestingly, the phosphorescence anisotropy excited with the longer wavelength (405 nm) is positive and reaches a value of about 0.25 in contrast to the phosphorescence anisotropy excited within the indole singlet absorption spectrum (290 nm), which is negative. Very different temperature dependences have been observed for fluorescence and phosphorescence of indole in PVA film. While fluorescence depends minimally, the phosphorescence decreases with temperature dramatically. The fluorescence lifetime was measured to be a single component 4.78 ns while the intensity weighted average phosphorescence lifetime with 290 nm and 405 nm excitations were 6.57 and 5.62 ms, respectively. We believe that the possibility of the excitation of indole phosphorescence in the blue region of visible light and its high anisotropy opens a new avenue for future protein studies., Competing Interests: Declaration of Competing Interest The authors declare no competing financial interests., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Fluorescence technologies have been the preferred method for detection, analytical sensing, medical diagnostics, biotechnology, imaging, and gene expression for many years. Fluorescence becomes essential for studying molecular processes with high specificity and sensitivity through a variety of biological processes. A significant problem for practical fluorescence applications is the apparent non-linearity of the fluorescence intensity resulting from inner-filter effects, sample scattering, and absorption of intrinsic components of biological samples. Sample absorption can lead to the primary inner filter effect (Type I inner filter effect) and is the first factor that should be considered. This is a relatively simple factor to be controlled in any fluorescence experiment. However, many previous approaches have given only approximate experimental methods for correcting the deviation from expected results. In this part we are discussing the origin of the primary inner filter effect and presenting a universal approach for correcting the fluorescence intensity signal in the full absorption range. Importantly, we present direct experimental results of how the correction works. One considers problems emerging from varying absorption across its absorption spectrum for all fluorophores. We use Rhodamine 800 and demonstrate how to properly correct the excitation spectra in a broad wavelength range. Second is the effect of an inert absorber that attenuates the intensity of the excitation beam as it travels through the cuvette, which leads to a significant deviation of observed results. As an example, we are presenting fluorescence quenching of a tryptophan analog, NATA, by acrylamide and we show how properly corrected results compare to the initial erroneous results. The procedure is generic and applies to many other applications like quantum yield determination, tissue/blood absorption, or acceptor absorption in FRET experiments.

Reconstituted high-density lipoprotein (HDL) containing apolipoprotein A-I (Apo A-I) mimics the structure and function of endogenous (human plasma) HDL due to its function and potential therapeutic utility in atherosclerosis, cancer, neurodegenerative diseases, and inflammatory diseases. Recently, a new class of HDL mimetics has emerged, involving peptides with amino acid sequences that simulate the the primary structure of the amphipathic alpha helices within the Apo A-I protein. The findings reported in this communication were obtained using a similar amphiphilic peptide (modified via conjugation of a myristic acid residue at the amino terminal aspartic acid) that self-assembles (by itself) into nanoparticles while retaining the key features of endogenous HDL. The studies presented here involve the macromolecular assembly of the myristic acid conjugated peptide (MYR-5A) into nanomicellar structures and its characterization via steady-state and time-resolved fluorescence spectroscopy. The structural differences between the free peptide (5A) and MYR-5A conjugate were also probed, using tryptophan fluorescence, Fӧrster resonance energy transfer (FRET), dynamic light scattering, and gel exclusion chromatography. To our knowledge, this is the first report of a lipoprotein assembly generated from a single ingredient and without a separate lipid component. The therapeutic utility of these nanoparticles (due to their capablity to incorporate a wide range of drugs into their core region for targeted delivery) was also investigated by probing the role of the scavenger receptor type B1 in this process. SIGNIFICANCE STATEMENT: Although lipoproteins have been considered as effective drug delivery agents, none of these nanoformulations has entered clinical trials to date. A major challenge to advancing lipoprotein-based formulations to the clinic has been the availability of a cost-effective protein or peptide constituent, needed for the assembly of the drug/lipoprotein nanocomplexes. This report of a robust, spontaneously assembling drug transport system from a single component could provide the template for a superior, targeted drug delivery strategy for therapeutics of cancer and other diseases (Counsell and Pohland, 1982)., (Copyright © 2020 by The American Society for Pharmacology and Experimental Therapeutics.)

We report the spectral properties of 2-Phenylindole (2PI) embedded in rigid poly (vinyl alcohol) (PVA) film. The 2PI in PVA film shows relatively strong and structured fluorescence with a maximum at 370 nm and surprisingly strong room temperature phosphorescence with an emission maximum of about 500 nm. The dye is highly immobilized in the polymer matrix, thus presenting high fluorescence anisotropy in an isotropic film of about 0.3 at room temperature. The 2-Phenylindole phosphorescence excited in the usual way through the electronic singlet state excitation (S 0  → S 1 absorption) results in a very low, near zero anisotropy. We now report that we can directly excite the dye to the triplet state T 1 and observe high phosphorescence anisotropy similar to the fluorescence anisotropy. The extinction coefficient for S 0  → T 1 absorption in the PVA matrix is unusually high- only about 3 orders of magnitude lower than S 0  → S 1 absorption. We consider this direct excitation to indole's triplet state a very significant finding that may lead to many practical applications. The unusually long-wavelength of excitation around 400 nm, much above typical UV absorption, results in a high phosphorescence anisotropy. This provides a new way to study rotational motion of larger biological objects in the microsecond time scale not accessible through typical fluorescence studies.

Intrinsic emission from typical filters can unexpectedly contribute to the total measured signal in a fluorescence system. This emission becomes even more problematic for in-line geometry measurements where the excitation light can directly excite the emission filter. Potassium dichromate has minimal intrinsic fluorescence even with ultra-violet (UV) excitation. We show that a liquid sample can be tuned for its transmission properties by adding a base to the solution. This makes it attractive for use as an optical filter. In addition, when embedded in poly (vinyl alcohol) (PVA) film, potassium dichromate is even less fluorescent. These films can be cut and molded into practically any size or shape. Thus non-fluorescent, modular and cheap filters composed of potassium dichromate embedded in a PVA film is proposed to eliminate this unwanted emission and is suitable for a wide variety of devices and applications.

The overexpression of multidrug efflux pumps is an important mechanism of clinical resistance in Gram-negative bacteria. Recently, four small molecules were discovered that inhibit efflux in Escherichia coli and interact with the AcrAB-TolC efflux pump component AcrA. However, the binding site(s) for these molecules was not determined. Here, we combine ensemble docking and molecular dynamics simulations with tryptophan fluorescence spectroscopy, site-directed mutagenesis, and antibiotic susceptibility assays to probe binding sites and effects of binding of these molecules. We conclude that clorobiocin and SLU-258 likely bind at a site located between the lipoyl and β-barrel domains of AcrA., (Copyright © 2019 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

Fluorescence signal enhancement induced by the binding of intercalators to DNA has been broadly utilized in various DNA detection methods. In most instances the increase in fluorescence intensity is associated with a concomitant increase of fluorescence lifetime. This increase of the fluorescence lifetime presents an additional opportunity to increase detection sensitivity. In this paper, we present a new approach to significantly enhance the sensitivity in detecting minute DNA concentrations. The approach is based on simultaneous use of time-gated detection and multi-pulse pumping. By using a calibrated burst of short pulses we greatly enhance the contribution of long-lived fluorescence species, thus enabling easy time-gated detection. Using a classic DNA intercalator - Ethidium Bromide (EtBr) - as an example with our novel multi-pulse pumping and time-gated detection technique, we were able to increase detection sensitivity over 70-fold with only 3 pulse excitation. This approach is generic and can be used with any analytical probe (exhibiting about 10 times change in lifetime) that shows an increase in fluorescence signal and fluorescence lifetime upon binding to a target.

Total internal reflection microscopy (TIRF) has been a powerful tool in biological research. The most valuable feature of the method has been the ability to image 100- to 200-nm-thick layer of cell features adjacent to a coverslip, such as membrane lipids, membrane receptors, and structures proximal-to-basal membranes. Here, we demonstrate an alternative method of imaging thin-layer proximal-to-basal membranes by placing a sample on a high refractive index coverslip covered by a thin layer of gold. The sample is illuminated using the Kretschmann method (i.e., from the top to an aqueous medium). Fluorophores that are close to the metal surface induce surface plasmons in the metal film. Fluorescence from fluorophores near the metal surface couple with surface plasmons allowing them to penetrate the metal surface and emerge at a surface plasmon coupled emission angle. The thickness of the detection layer is further reduced in comparison with TIRF by metal quenching of fluorophores at a close proximity (below 10 nm) to a surface. Fluorescence is collected by a high NA objective and imaged by EMCCD or converted to a signal by avalanche photodiode fed by a single-mode optical fiber inserted in the conjugate image plane of the objective. The system avoids complications of through-the-objective TIRF associated with shared excitation and emission light path, has thin collection thickness, produces excellent background rejection, and is an effective method to study molecular motion.

Abnormalities of mucus viscosity play a critical role in the pathogenesis of several respiratory diseases, including cystic fibrosis. Currently, there are no approaches to assess the rheological properties of mucin granule matrices in live cells. This is the first example of the use of a molecular rotor, a BODIPY dye, to quantitatively visualize the viscosity of intragranular mucin matrices in a large population of individual granules in differentiated primary bronchial epithelial cells using fluorescence lifetime imaging microscopy.

Styryl dyes, specifically LDS group dyes, are known solvatochromic and electrochromic probes for monitoring mitochondrial potential in cellular environments. However, the ability of these dyes to respond to fluctuations in viscosity, pH and temperature has not been established. In this study, we demonstrated that LDS 798 (also known as Styryl-11) can sense environmental viscosity (via fluorescence lifetime changes) as well as pH changes (ratiometric intensity change) in the absence of polarity variations. Polarity changes can be probed by spectral changes using LDS 798. Therefore, all properties of the media should be considered, when these types of dyes are used as electrochromic/solvatochromic sensors in cellular environments.

The systemic circulation offers larger resistance to the blood flow than the pulmonary system. Consequently, the left ventricle (LV) must pump blood with more force than the right ventricle (RV). The question arises whether the stronger pumping action of the LV is due to a more efficient action of left ventricular myosin, or whether it is due to the morphological differences between ventricles. Such a question cannot be answered by studying the entire ventricles or myocytes because any observed differences would be wiped out by averaging the information obtained from trillions of myosin molecules present in a ventricle or myocyte. We therefore searched for the differences between single myosin molecules of the LV and RV of failing hearts In-situ . We show that the parameters that define the mechanical characteristics of working myosin (kinetic rates and the distribution of spatial orientation of myosin lever arm) were the same in both ventricles. These results suggest that there is no difference in the way myosin interacts with thin filaments in myocytes of failing hearts, and suggests that the difference in pumping efficiencies are caused by interactions between muscle proteins other than myosin or that they are purely morphological.

We studied steady-state and time-resolved fluorescence properties of an anticancer drug Doxorubicin in a saline buffer and poly-vinyl alcohol (PVA) film. Absorption of Doxorubicin, located at blue-green spectral region, allows a convenient excitation with visible light emitting diodes or laser diodes. Emission of Doxorubicin with maximum near 600nm can be easily detected with photomultipliers and CCD cameras. Both, absorption and fluorescence spectra in polymeric matrix show more pronounced vibronic structures than in solution. Also, the steady-state anisotropy in the polymer film is significantly higher than in the saline solution. In PVA film the fluorescence anisotropy is about 0.30 whereas in the saline buffer only 0.07. Quantum efficiencies of Doxorubicin were compared to a known standard Rhodamine 101 which has fluorescence emission in a similar spectral region. The quantum yield of Doxorubicin in PVA film is more than 10% and about twice higher than in the saline solution. Similarly, the lifetime of doxorubicin in PVA film is about 2ns whereas in the saline solution only about 1ns. The fluorescence anisotropy decays show that Doxorubicin molecules are freely rotating in the saline buffer with a correlation time of about 290ps, and are almost completely immobilized in the PVA film. The spectroscopic investigations presented in this manuscript are important, as they provide answers to changes in molecular properties of Doxorubicin depending changes in the local environment, which is useful when synthesizing nanoparticles for Doxorubicin entrapment., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Graphene possesses a number of advantageous properties, however, does not exhibit optical emission, which limits its use in optoelectronics. Unlike graphene, its functional derivative, graphene oxide (GO) exhibits fluorescence emission throughout the visible. Here, we focus on controlled methods for tuning the optical properties of GO. We introduce ozone treatment of reduced graphene oxide (RGO) in order to controllably transform it from non-emissive graphene-like material into GO with a specific fluorescence emission response. Solution-based treatment of RGO for 5-45 min with ∼1.2 g l -1 ozone/oxygen gas mixture yields a drastic color change, bleaching of the absorption in the visible and the stepwise increase in fluorescence intensity and lifetime. This is attributed to the introduction of oxygen-containing functional groups to RGO graphitic platform as detected by the infrared spectroscopy. A reverse process: controllable quenching of this fluorescence is achieved by the thermal treatment of GO in aqueous suspension up to 90 °C. This methodology allows for the wide range alteration of GO optical properties starting from the dark-colored non-emissive RGO material up to nearly transparent highly ozone-oxidized GO showing substantial fluorescence emission. The size of the GO flakes is concomitantly altered by oxidation-induced scission. Semi-empirical PM3 theoretical calculations on HyperChem models are utilized to explore the origins of optical response from GO. Two models are considered, attributing the induced emission either to the localized states produced by oxygen-containing addends or the islands of graphitic carbon enclosed by such addends. Band gap values calculated from the models are in the agreement with experimentally observed transition peak maxima. The controllable variation of GO optical properties in aqueous suspension by ozone and thermal treatments shown in this work provides a route to tune its optical response for particular optoelectronics or biomedical applications.

An oligopeptide: Lys-Gly-Pro-Arg-Ser-Leu-Ser-Gly-Lys-NH 2 , cleaved specifically by a matrix metalloproteinase 9 (MMP-9) at the Ser-Leu bond, was labeled on the ε-NH 2 groups of lysine with donor (5, 6 TAMRA) and acceptor (HiLyte647) dye. The donor control was a peptide labeled with 5, 6 TAMRA only on the C-terminal lysine, and the acceptor control was free HiLyte647. Following three products were studied by dissolving in 10% (w/w) poly(vinyl alcohol) and dried on glass slides forming 200 micron films. Absorption spectra of the films show full additivity of donor and acceptor absorptions. A strong Fluorescence Resonance Energy Transfer (FRET) with an efficiency of about 85% was observed in the fluorescence emission and excitation spectra. The lifetime of the donor was shorter and heterogeneous compared with the donor control.

A fluorescence lifetime imaging probe with a long lifetime was used in combination with time-gating for the detection of hyaluronidase using hyaluronic acid as the probe template. This probe was developed by heavily labeling hyaluronic acid with long lifetime azadioxatriangulenium fluorophores (ADOTA). We used this probe to image hyaluronidase produced by DU-145 prostate cancer cells.

We present optical studies of two different size distributions of silver triangular nanoprisms, one with a dipole resonance at 520 nm and the other with a dipole resonance at 650 nm, placed in different media. Significant wavelength-dependent depolarization of scattered light from the silver nanoprisms suspended in water indicates strong interference of multiple surface plasmon resonant modes in the same particle. We use this depolarization as a probe of light scattering by the nanoprisms in a lipid solution due to the rejection of a polarized background scattering. Also, the silver nanoprisms were embedded in a polyvinyl alcohol polymer matrix and oriented by stretching the polymer/nanoprism nanocomposite films. We observe significantly increased linear dichroism in the region associated with the plasmonic in-plane dipole mode upon stretching. Additionally, there is a weaker linear dichroism in the region associated with out-of-plane modes, which vanish in the extinction spectrum of the stretched nanocomposite film.

Stretching a polymer film induces a conformational change (from the twisted to planar state) in the embedded porphyrin dimer, as evidenced by steady-state and time-resolved emission spectra.

In this report, we have designed a rapid and sensitive, intensity-based ratiometric sensing as well as lifetime-based sensing probe for the detection of hyaluronidase activity. Hyaluronidase expression is known to be upregulated in various pathological conditions. We have developed a fluorescent probe by heavy labeling of hyaluronic acid with a new orange/red-emitting organic azadioxatriangulenium (ADOTA) fluorophore, which exhibits a long fluorescence lifetime (∼20 ns). The ADOTA fluorophore in water has a peak fluorescence lifetime of ∼20 ns and emission spectra centered at 560 nm. The heavily ADOTA-labeled hyaluronic acid (HA-ADOTA) shows a red shift in the peak emission wavelength (605 nm), a weak fluorescence signal, and a shorter fluorescence lifetime (∼4 ns) due to efficient self-quenching and formation of aggregates. In the presence of hyaluronidase, the brightness and fluorescence lifetime of the sample increase with a blue shift in the peak emission to its original wavelength at 560 nm. The ratio of the fluorescence intensity of the HA-ADOTA probe at 560 and 605 nm can be used as the sensing method for the detection of hyaluronidase. The cleavage of the hyaluronic acid macromolecule reduces the energy migration between ADOTA molecules, as well as the degree of self-quenching and aggregation. This probe can be efficiently used for both intensity-based ratiometric sensing as well as fluorescence lifetime-based sensing of hyaluronidase. The proposed method makes it a rapid and sensitive assay, useful for analyzing levels of hyaluronidase in relevant clinical samples like urine or plasma. Graphical Abstract Scheme showing cleavage of HA-ADOTA probe by hyaluronidase and the change in the emission spectrum of HA-ADOTA probe before and after cleavage by hyaluronidase.

Ternary AgInS2 quantum dots (QDs) have been found as promising cadmium-free, red-shifted, and tunable luminescent bio-probes with efficient Stokes and anti-Stokes excitations and luminescence lifetimes (ca. 100 ns) convenient for time resolved techniques like fluorescence life-time imaging. Although the spectral properties of the AgInS2 QDs are encouraging, the complex recombination kinetics in the QDs being still far from understood, limits their full utility. In this paper we report on a model describing the recombination pathways responsible for large deviations from the first-order decay law observed commonly in the ternary chalcogenides. The presented results were evaluated by means of individual AgInS2 QD spectroscopy aided by first principles calculations including the electronic structure and structural reconstruction of the QDs. Special attention was devoted to study the impact of the surface charge state on the excited state relaxation and effect of its passivation by Zn(2+) ion alloying. Two different blinking mechanisms related to defect-assisted charge imbalance in the QD responsible for fast non-radiative relaxation of the excited states as well as surface recharging of the QD were found as the major causes of deviations from the first-order decay law. Careful optimization of the AgInS2 QDs would help to fabricate new red-shifted and tunable fluorescent bio-probes characterized by low-toxicity, high quantum yield, long luminescence lifetime, and time stability, leading to many novel in vitro and in vivo applications based on fluorescence lifetime imaging (FLIM) and time-gated detection.

Photophysical behaviour of a novel trimeric BODIPY rotor with a high extinction coefficient is reported. Steady state and time resolved fluorescence measurements established that the trimer could be used as a viscometer for molecular solvents, membrane-like environments and several cancer cell lines.

Nanoparticles are target-specific drug delivery agents that are increasingly used in cancer therapy to enhance bioavailability and to reduce off target toxicity of anti-cancer agents. Valrubicin is an anti-cancer drug, currently approved only for vesicular bladder cancer treatment because of its poor water solubility. On the other hand, valrubicin carrying reconstituted high density lipoprotein (rHDL) nanoparticles appear ideally suited for extended applications, including systemic cancer chemotherapy. We determined selected fluorescence properties of the free (unencapsulated) drug vs. valrubicin incorporated into rHDL nanoparticles. We have found that upon encapsulation into rHDL nanoparticles the quantum yield of valrubicin fluorescence increased six fold while its fluorescence lifetime increased about 2 fold. Accordingly, these and potassium iodide (KI) quenching data suggest that upon incorporation, valrubicin is localized deep in the interior of the nanoparticle, inside the lipid matrix. Fluorescence anisotropy of the rHDL valrubicin nanoparticles was also found to be high along with extended rotational correlation time. The fluorescence of valrubicin could also be utilized to assess its distribution upon delivery to prostate cancer (PC3) cells. Overall the fluorescence properties of the rHDL: valrubicin complex reveal valuable novel characteristics of this drug delivery vehicle that may be particularly applicable when used in systemic (intravenous) therapy., (Copyright © 2015 Elsevier B.V. All rights reserved.)

In this paper, we have synthesized BSA protected gold nanoclusters (BSA Au nanocluster) and studied the effect of quencher, protein denaturant, pH and temperature on the fluorescence properties of the tryptophan molecule of the BSA Au nanocluster and native BSA. We have also studied their effect on the peak emission of BSA Au nanoclusters (650 nm). The phtophysical characterization of a newly developed fluorophore in different environments is absolutely necessary to futher develop their biomedical and analytical applications. It was observed from our experiments that the tryptophan in BSA Au nanoclusters is better shielded from the polar environment. Tryptophan in native BSA showed a red shift in its peak emission wavelength position. Tryptophan is a highly polarity sensitive dye and a minimal change in its microenvironment can be easily observed in its photophysical properties.

Contraction of muscles results from the ATP-coupled cyclic interactions of the myosin cross-bridges with actin filaments. Macroscopic parameters of contraction, such as maximum tension, speed of shortening, or ATPase activity, are unlikely to reveal differences between the wild-type and mutated (MUT) proteins when the level of transgenic protein expression is low. This is because macroscopic measurements are made on whole organs containing trillions of actin and myosin molecules. An average of the information collected from such a large assembly is bound to conceal any differences imposed by a small fraction of MUT molecules. To circumvent the averaging problem, the measurements were done on isolated ventricular myofibril (MF) in which thin filaments were sparsely labeled with a fluorescent dye. We isolated a single MF from a ventricle, oriented it vertically (to be able measure the orientation), and labeled 1 in 100,000 actin monomers with a fluorescent dye. We observed the fluorescence from a small confocal volume containing approximately three actin molecules. During the contraction of a ventricle actin constantly changes orientation (i.e., the transition moment of rigidly attached fluorophore fluctuates in time) because it is repetitively being "kicked" by myosin cross-bridges. An autocorrelation functions (ACFs) of these fluctuations are remarkably sensitive to the mutation of myosin. We examined the effects of Alanine to Threonine (A13T) mutation in the myosin regulatory light chain shown by population studies to cause hypertrophic cardiomyopathy. This is an appropriate example, because mutation is expressed at only 10% in the ventricles of transgenic mice. ACFs were either "Standard" (Std) (decaying monotonically in time) or "Non-standard" (NStd) (decaying irregularly). The sparse labeling of actin also allowed the measurement of the spatial distribution of actin molecules. Such distribution reflects the interaction of actin with myosin cross-bridges and is also remarkably sensitive to myosin mutation. The result showed that the A13T mutation caused 9% ACFs and 9% of spatial distributions of actin to be NStd, while the remaining 91% were Std, suggesting that the NStd performances were executed by the MUT myosin heads and that the Std performances were executed by non-MUT myosin heads. We conclude that the method explored in this study is a sensitive and valid test of the properties of low prevalence mutations in sarcomeric proteins.