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Photobleaching of organic fluorophores: Quantitative characterization, mechanisms, protection
Abstract
Photochemical stability is one of the most important parameters that determine the usefulness of organic dyes in different applications. This Review addresses key factors that determine the dye photostability. It is shown that photodegradation can follow different oxygen-dependent and oxygen-independent mechanisms and may involve both 1S1 - 3T1 and higher-energy 1Sn - 3Tn excited states. Their involvement and contribution depends on dye structure, medium conditions, irradiation power. Fluorescein, rhodamine, BODIPY and cyanine dyes, as well as conjugated polymers are discussed as selected examples illustrating photobleaching mechanisms. The strategies for modulating and improving the photostability are overviewed. They include the improvement of fluorophore design, particularly by attaching protective and anti-fading groups, creating proper medium conditions in liquid, solid and nanoscale environments. The special conditions for biological labeling, sensing and imaging are outlined.
... Excessive ROS shifts the homeostasis of the cell cytoplasmic environment' leading to organelle damage like mitochondrial fragmentation due to compromised membrane potential and integrity, among others (13,35,(43)(44)(45). While helpful and versatile, fluorescence microscopy illumination entails an additional source of ROS formation (46). When excited, fluorophores undergo autocatalysis through dioxygen, releasing hydrogen peroxide and consequently degrading -a process commonly known as photobleaching (47). ...
... When excited, fluorophores undergo autocatalysis through dioxygen, releasing hydrogen peroxide and consequently degrading -a process commonly known as photobleaching (47). Photobleaching produces ROS similar to other biomolecules, intensifying phototoxic effects (46). However, despite the interrelation between photobleaching and phototoxicity (20), these phenomena exhibit distinct features and can occur independently. ...
... However, despite the interrelation between photobleaching and phototoxicity (20), these phenomena exhibit distinct features and can occur independently. Namely, identical oxygen radical compounds originate from photobleaching and direct fluorescence excitation light interactions with other cellular components (46,47). Suggesting that a reduction of photobleaching does not necessarily imply a decrease in phototoxicity, and the other way around. ...
Fluorescence microscopy, widely used in the study of living cells, tissues, and organisms, often faces the challenge of photodamage. This is primarily caused by the interaction between light and biochemical components during the imaging process, leading to compromised accuracy and reliability of biological results. Methods necessitating extended high-intensity illumination, such as super-resolution microscopy or thick sample imaging, are particularly susceptible to this issue. As part of the solution to these problems, advanced imaging approaches involving artificial intelligence (AI) have been developed. Here we underscore the necessity of establishing constraints to maintain light-induced damage at levels that permit cells to sustain their live behaviour. From this perspective, data-driven live-cell imaging bears significant potential in aiding the development of AI-enhanced photodamage-aware microscopy. These technologies could streamline precise observations of natural biological dynamics while minimising phototoxicity risks.
... In this sense, it is noteworthy that the low photostability of other dyes is characterized by high emission efficiencies (4l and mainly 4i, both sharing 8-anisole). It has been reported that the 8-position is involved in the photobleaching mechanism, and its substitution (for instance with phenyl) is recommended to enhance photostability [42,43]. However, it seems that the parafunctionalization of this ring with the electron donor methoxy is detrimental in terms of photostability. ...
... In this sense, it is noteworthy that the low photostability of other dyes is characterized by high emission efficiencies (4l and mainly 4i, both sharing 8-anisole). It has been reported that the 8-position is involved in the photobleaching mechanism, and its substitution (for instance with phenyl) is recommended to enhance photostability [42,43]. However, it seems that the para-functionalization of this ring with the electron donor methoxy is detrimental in terms of photostability. ...
Herein, we report the synthetic access to a set of π-extended BODIPYs featuring a penta-arylated (phenyl and/or thiophene) dipyrrin framework. We take advantage of the full chemoselective control of 8-methylthio-2,3,5,6-tetrabromoBODIPY when we conduct the Liebeskind–Srogl cross-coupling (LSCC) to functionalize exclusively the meso-position, followed by the tetra-Suzuki reaction to arylate the halogenated sites. All these laser dyes display absorption and emission bands in the red edge of the visible spectrum reaching the near-infrared with thiophene functionalization. The emission efficiency, both fluorescence and laser, of the polyphenylBODIPYs can be enhanced upon decoration of the peripheral phenyls with electron donor/acceptor groups at para positions. Alternatively, the polythiopheneBODIPYs show an astonishing laser performance despite the charge transfer character of the emitting state. Therefore, these BODIPYs are suitable as a palette of stable and bright laser sources covering the spectral region from 610 nm to 750 nm.
... The immune system's activity in SLE leads to the formation and deposition of immune complexes in the kidneys, triggering inflammation and damage that, without precise and early intervention, often progresses to LN with poor prognostic outcomes [1]. The variability in the progression and presentation of LN among patients without the need for specialized equipment and is unaffected by photobleaching, a common issue in fluorescence detection systems [23]. This makes it more suitable for low-resource environments and home-care settings where complex equipment may not be readily available. ...
To improve the efficiency and patient coverage of the current healthcare system, user-friendly novel homecare devices are urgently needed. In this work, we developed a smartphone-based analyzing and reporting system (SBARS) for biomarker detection in lupus nephritis (LN). This system offers a cost-effective alternative to traditional, expensive large equipment in signal detection and quantification. This innovative approach involves using a portable and affordable microscopic reader to capture biomarker signals. Through smartphone-based image processing techniques, the intensity of each biomarker signal is analyzed. This system exhibited comparable performance to a commercial Genepix scanner in the detection of two potential novel biomarkers of LN, VISG4 and TNFRSF1b. Importantly, this smartphone-based analyzing and reporting system allows for discriminating LN patients with active renal disease from healthy controls with the area-under-the-curve (AUC) value = 0.9 for TNFRSF1b and 1.0 for VSIG4, respectively, indicating high predictive accuracy.
... Photochemical stability is one of the most important parameters determining the usefulness of organic dyes in different applications, such as PDT [53]. Photobleaching of MB in aqueous solutions is a photodynamic process in which active oxygen species (ROS and singlet oxygen 1 O 2 ) generated during MB illumination can attack the sensitizer itself, leading to the so-called self-sensitized photo-oxidation [54]. ...
Photodynamic therapy (PDT) is a therapeutic option for cancer, in which photosensitizer (PS) drugs, light, and molecular oxygen generate reactive oxygen species (ROS) and induce cell death. First- and second-generation PSs presented with problems that hindered their efficacy, including low solubility. Thus, second-generation PSs loaded into nanocarriers were produced to enhance their cellular uptake and therapeutic efficacy. Among other compounds investigated, the dye methylene blue (MB) showed potential as a PS, and its photodynamic activity in tumor cells was reported even in its nanocarrier-delivered form, including liposomes. Here, we prepared polydopamine (PDA)-coated liposomes and efficiently adsorbed MB onto their surface. lipoPDA@MB vesicles were first physico-chemically characterized and studies on their light stability and on the in vitro release of MB were performed. Photodynamic effects were then assessed on a panel of 2D- and 3D-cultured cancer cell lines, comparing the results with those obtained using free MB. lipoPDA@MB uptake, type of cell death induced, and ability to generate ROS were also investigated. Our results show that lipoPDA@MB possesses higher photodynamic potency compared to MB in both 2D and 3D cell models, probably thanks to its higher uptake, ROS production, and apoptotic cell death induction. Therefore, lipoPDA@MB appears as an efficient drug delivery system for MB-based PDT.
... Notably, fluorophores in water photobleach after roughly (10 4 -10 6 ) absorption and emission cycles. [25][26][27][28] At lower flow rates, each dye molecule will spend more time traversing the excitation beam, thus being exposed to more photons and increasing its probability of photobleaching. In Refs. ...
Accuracy and temporal resolution of flow meters are often unacceptable below the microliter per minute scale, limiting their ability to evaluate the real-time performance of many microfluidic devices. For conventional flow meters, this problem arises from uncertainties that depend on physical effects, such as evaporation, whose relative impacts scale inversely with flow rate. More advanced techniques that can measure nanoliter per minute flows are often not dynamic and require specialized equipment. Herein, we report on new experimental and theoretical results that overcome both limitations using an optofluidic flow meter. Previously, we showed that this device can measure flow rates as low as 1 nl/min with roughly 5% relative uncertainty by leveraging the photobleaching rate of a fluorescent dye. We now extend that work by determining the flow meter's relaxation time over a wide range of flow rates and incident irradiances. Using a simplified analytical model, we deduce that this time constant arises from the interplay between the photobleaching rate and transit time of the dye through the optical interrogation region. This motivates us to consider a more general model of the device, which, surprisingly, implies that all time constants are related by a simple scaling relationship depending only on the flow rate and optical irradiance. We experimentally validate this relationship to within 5% uncertainty down to 1 nl/min. Additionally, we measure a relaxation time of the flow meter on the order of 100 ms for 1 nl/min flows, demonstrating the ability to make dynamic measurements of small flows with unprecedented accuracy.
... Higher-intensity UV and blue excitation light can also directly damage DNA by producing thymine dimers (Zhang et al., 2022b). Additionally, fluorophores photobleach via ROS generation upon light exposure (Demchenko, 2020). Although interrelated, photobleaching and photodamage are distinct and can occur independently (Ludvikova et al., 2023). ...
Fluorescence microscopy is essential for studying living cells, tissues and organisms. However, the fluorescent light that switches on fluorescent molecules also harms the samples, jeopardizing the validity of results – particularly in techniques such as super-resolution microscopy, which demands extended illumination. Artificial intelligence (AI)-enabled software capable of denoising, image restoration, temporal interpolation or cross-modal style transfer has great potential to rescue live imaging data and limit photodamage. Yet we believe the focus should be on maintaining light-induced damage at levels that preserve natural cell behaviour. In this Opinion piece, we argue that a shift in role for AIs is needed – AI should be used to extract rich insights from gentle imaging rather than recover compromised data from harsh illumination. Although AI can enhance imaging, our ultimate goal should be to uncover biological truths, not just retrieve data. It is essential to prioritize minimizing photodamage over merely pushing technical limits. Our approach is aimed towards gentle acquisition and observation of undisturbed living systems, aligning with the essence of live-cell fluorescence microscopy.
... Stability against oxygen is of great importance in the practical applications of organic semiconductor materials, considering the functional groups with high photoelectric activity are particularly sensitive to oxygen when excited by light and electricity. [1][2] The adverse effects of unstable organic semiconductors as shortened service lifetime, deteriorative mechanical property and optoelectronic performance are ubiquitous in various single-molecular and hybrid systems which must work under ambient conditions, including integrated photovoltaic, [3][4] fluorescence imaging, [5][6][7] organic light emitting diode (OLED), [8] and organic field-effect transistors (OFET). [9] To inhibit the degradation, several kinds of additives such as light screeners/absorbers, excited-state quenchers, antioxidants, radical scavengers have been developed according to different stabilization mechanisms. ...
Stability against oxygen is an important factor affecting the performance of organic semiconductor devices. Improving photooxidation stability can prolong the service life of the device and maintain the mechanical and photoelectric properties of the device. Generally, various encapsulation methods from molecular structure to macroscopic device level are used to improve photooxidation stability. Here, we adopted a crystallization strategy to allow 14H-spiro[dibenzo[c,h]a-cridine-7,9′-uorene] (SFDBA) to pack tightly to resist fluo-rescence decay caused by oxidation. In this case, the inert group of SFDBA acts as a “steric armor”, protecting the photosensitive group from being attacked by oxygen. Therefore, compared with the fluorescence quenching of SFDBA powder under two hours of sunlight, SFDBA crystal can maintain its fluorescence emission for more than eight hours under the same conditions. Furthermore, the photolu-minescence quantum yields (PLQYs) of the crystalline film is 327.37 % higher than that of the amorphous film. It shows that the crystal-lization strategy is an effective method to resist oxidation.
... In addition, the polymeric network can improve the photostability of fluorophores by providing efficient screening of fluorophores from oxygen and radical attack. 11 Poly(N-isopropylacrylamide) (pNIPAm) is a thermoresponsive polymer that has been investigated extensively over the past few decades. 1 Linear pNIPAm exhibits a lower critical solution temperature (LCST) at 32°C, where it undergoes a reversible coil (extended) to globule (collapsed) transition. 12 That is, when the solution temperature is below the LCST, the pNIPAm chain exists as a "solvated" random coil, and transitions to a "desolvated" globule as the temperature exceeds the LCST. ...
Fluorescent poly(N‐isopropylacrylamide‐ co ‐Nile blue) (pNIPAm‐ co ‐NB) microgels were synthesized that exhibited fluorescence intensity changes in a water temperature‐dependent fashion. NB is well known to exhibit fluorescence intensity that depends on the hydrophobicity of the environment, while pNIPAm‐based microgels are well known to transition from swollen (hydrophilic) to collapsed (relatively hydrophobic) at temperatures greater than 32 °C; hence, we attribute the above behavior to the hydrophobicity changes of the microgels with increasing temperature. This phenomenon is ultimately due to NB dimers (relatively quenched fluorescence) being broken in the hydrophobic environment of the microgels leading to relatively enhanced fluorescence. We went on to show that the introduction of cucurbit[7]uril (CB[7]) into the pNIPAm‐ co ‐NB microgels enhanced their fluorescence allowing them to be used for polyamine (e.g., spermine [SPM]) detection. Specifically, CB[7] forms a host–guest interaction with NB in the microgels, which prevents NB dimerization and enhances their fluorescence. When SPM is present, it forms a host–guest complex that is favored over the CB[7]‐NB host–guest interaction, which frees the NB for dimerization and leads to fluorescence quenching. As a result, we could generate an SPM sensor capable of SPM detection down to ~0.5 µmol/L in complicated matrixes such as serum and urine.
... Nevertheless, obtaining high-resolution 3D reconstructions of neural organoids to observe fine structures like neurons is a labor-intensive task, with imaging taking several hours and the sharpness of images fading as acquisition goes deeper into the layers [10]. Additionally, the presence of strong cross-talk among neighboring layers further complicates the reconstruction process， and longer and more intensive laser beam exposure leads to photobleaching of fluorophores [11], resulting in a reduced signal-to-noise ratio. However, there is a lack of active research on enhancing resolution in the z-axis (depth) through post-processing methods. ...
... [1][2][3] Rapid development over the last decade has provided access to a wide range of reactions previously only achieved through the use of classic catalysts. [4][5][6][7][8][9][10] However, homogeneous and heterogeneous photocatalysts suffer from significant intrinsic drawbacks, [11][12][13][14][15] including photobleaching, a lack of easy recycling possibilities, limited mass transport to the active center, or poor light absorption. [16][17][18][19][20] An ideal photocatalyst would possess the benefits of both homogeneous and heterogeneous systems, achieving high efficiency while allowing the alteration of materials properties. ...
The copolymerization of photocatalytic moieties into a polymeric material has emerged as a new development platform for heterogeneous photocatalysts. Incorporating small molecule photocatalysts into polymeric structures has created a new class of heterogeneous photocatalysts. However, the effect and interaction of the comonomer on the photocatalyst have been mostly ignored and little is known about the influence of classical polymer composition on photocatalytic efficiency. Here a vinyl functionalized benzothiadiazole photocatalyst was copolymerized with three dramatically different monomers, methyl methacrylate, styrene, and acrylonitrile, via free radical polymerization, to investigate the effect of the comonomer choice on the photophysical properties and photocatalytic efficiency.
... The margin of error grows larger in research where quantitative analysis is important. Immunostaining has several potential sources of inaccuracy, such as the background noise present in a fluorescence microscope 6 , non-specific binding of the antibodies 7 , variability in staining intensity due to experimental conditions 8 , and the potential for photobleaching of fluorescent dyes, all of which must be considered and addressed 9 . ...
Immunocytochemical staining of microorganisms and cells has long been a popular method for examining their specific subcellular structures in greater detail. Recently, generative networks have emerged as an alternative to traditional immunostaining techniques. These networks infer fluorescence signatures from various imaging modalities and then virtually apply staining to the images in a digital environment. In numerous studies, virtual staining models have been trained on histopathology slides or intricate subcellular structures to enhance their accuracy and applicability. Despite the advancements in virtual staining technology, utilizing this method for quantitative analysis of microscopic images still poses a significant challenge. To address this issue, we propose a straightforward and automated approach for pixel-wise image-to-image translation. Our primary objective in this research is to leverage advanced virtual staining techniques to accurately measure the DNA fragmentation index in unstained sperm images. This not only offers a non-invasive approach to gauging sperm quality, but also paves the way for streamlined and efficient analyses without the constraints and potential biases introduced by traditional staining processes. This novel approach takes into account the limitations of conventional techniques and incorporates improvements to bolster the reliability of the virtual staining process. To further refine the results, we discuss various denoising techniques that can be employed to reduce the impact of background noise on the digital images. Additionally, we present a pixel-wise image matching algorithm designed to minimize the error caused by background noise and to prevent the introduction of bias into the analysis. By combining these approaches, we aim to develop a more effective and reliable method for quantitative analysis of virtually stained microscopic images, ultimately enhancing the study of microorganisms and cells at the subcellular level.
... 69−71 This effect can be accounted for by considering that the reactive species acting as mediators for the photodegradative reactions can destructively interact with the external matrix before reaching the fluorophores embedded within, differently from solvent molecules or other surrounding chemical moieties. 72 A few hundred micrometers thick film can be obtained by drop-casting a concentrated solution of NR-CDs in PVA onto a quartz substrate. As shown in Figure 4c, the nanocomposite film is able to convert the blue emission of a LED source into orange light, allowing for a proper extinction of the incident radiation through the sample volume and demonstrating the potentiality of NR-CDs to act as color converters for LEDs. ...
Carbon dots are carbon-based nanoparticles renowned for their intense light-emitting capabilities covering the whole visible light range. Achieving carbon dots emitting in the red region with high efficiency is extremely relevant due to their huge potential in biological applications and in optoelectronics. Currently, photoluminescence in such an energy interval is often associated with polyheterocyclic molecular domains forming during the synthesis that, however, present low emission efficiency and issues in controlling the optical features. Here, we overcome these problems by solvothermally synthesizing carbon dots starting from Neutral Red, a common red-emitting dye, as a molecular precursor. As a result of the synthesis, such molecular fluorophore is incorporated into a carbonaceous core while retaining its original optical properties. The obtained nanoparticles are highly luminescent in the red region, with a quantum yield comparable to that of the starting dye. Most importantly, the nanoparticle carbogenic matrix protects the Neutral Red molecules from photobleaching under ultraviolet excitation while preventing aggregation-induced quenching, thus allowing solid-state emission. These advantages have been exploited to develop a fluorescence-based color conversion layer by fabricating polymer-based highly concentrated solid-state carbon dot nanocomposites. Finally, the dye-based carbon dots demonstrate both stable Fabry–Perot lasing and efficient random lasing emission in the red region.
... 26 Conversely, it renders applications in synthesis via photosensitization inherently challenging, because of photosensitizer selfcatalyzed photobleaching. [27][28][29][30] This effect can however be mitigated by increasing the PS loading (typically ranging from 1 to 10 mol%) which comes at the cost of an increased absorption and thus lower light penetration into the reaction vessel. Adding to this, the relative instability of common PSs generates a large variation of kinetic profiles, which in turn engenders numerous scale-up difficulties. ...
... Early detection of numerous diseases, particularly glioblastoma, the most aggressive brain tumor, is a critical aspect in clinical practice; with the help of diagnostics, the five-year survival rate for malignant brain and other central nervous system tumors in the United States has risen to 35.7% [1,2]. Unlike traditional organic small molecules that possess deficiencies such as poor solubility as well as photo-instability [3,4], carbon nanodots (zerodimensional nanoparticles) with a sp 2 -and sp 3 -hybridized carbon skeleton offer resistance to photobleaching and superior aqueous dispersibility, making them new contributors to medical imaging [5][6][7][8]. Importantly, the emission wavelength of carbon nanodots can be well-regulated into the near-infrared or red region through heteroatom-doping and creating surface defects [9][10][11][12]. ...
Carbon nanodots present resistance to photobleaching, bright photoluminescence, and superior biocompatibility, making them highly promising for bioimaging applications. Herein, nanoprobes were caged with four-armed oligomers and subsequently modified with a novel DBCO–PEG-modified retro-enantio peptide ligand reL57, enhancing cellular uptake into U87MG glioma cells highly expressing low-density lipoprotein receptor-related protein 1 (LRP1). A key point in the development of the oligomers was the incorporation of ε-amino-linked lysines instead of standard α-amino-linked lysines, which considerably extended the contour length per monomer. The four-armed oligomer 1696 was identified as the best performer, spanning a contour length of ~8.42 nm for each arm, and was based on an altering motive of two cationic ε-amidated lysine tripeptides and two tyrosine tripeptides for electrostatic and aromatic stabilization of the resulting formulations, cysteines for disulfide-based caging, and N-terminal azidolysines for click-modification. This work highlights that well-designed four-armed oligomers can be used for noncovalent coating and covalent caging of nanoprobes, and click modification using a novel LRP1-directed peptide ligand facilitates delivery into receptor-expressing target cells.
... It was found that DPA may slowly decompose upon irradiation even in the absence of porphyrin in the medium (Fig. 3). This peculiarity may arise due to photolysis process [44] or due to generation of 1 O 2 by the trap itself. The latter hypothesis is supported by a fact that structurally similar compound -9,10-dibutoxyanthraceneis able to generate 1 O 2 upon irradiation [37]. ...
The investigation of the activity of photosensitizers is a multivariate challenge for their further application in photocatalysis. Porphyrins are attractive photoactive molecules for chemical and biochemical applications, while their observed photocatalytic efficiency is a function of a variety of characteristics. In the present work the influence of porphyrin substituents on their photoactivity and photostability along with the solvent effect were studied and discussed in details. Series of experiments with 9,10-diphenylanthracene (DPA) as a selective 1O2 trap allowed to develop the approach for direct measurement of [1O2] and quantitatively evaluate the influence of various factors onto it. The processes of 1O2 relaxation, as well as its chemical and physical quenching were separated and analyzed individually. A kinetic scheme for the photosensitization process was treated with steady-state approximation to develop a mathematical apparatus, where a non-linear dependence between photosensitizer and singlet oxygen concentration was found. The general character of the derived non-linear dependence was proved experimentally for each studied porphyrin. Physical and chemical quenching rate constants of the studied porphyrins were calculated. DFT calculations allowed to find an empirical correlation between HOMO energies and observed rate constants. A plausible reaction mechanism between TPP and 1O2 was examined with Fast-NEB-TS method.
... Nanomaterials are generally categorized as nanoparticles, quantum dots, carbon-related materials, nanogels, and dendrimers [1,2]. Fluorescent nanoparticles contain several advantages, such as high signal-to-noise ratio, excellent spatial resolution, high stability, and ease of implementation that have demonstrated high potential for their use in biomedical applications [3][4][5][6][7]. Fluorescent nanoparticles include fluorescent proteins, polymer/dye-based nanoparticles, fluorophores, hybrid particles, quantum dots, organic dye molecules [1,2], etc. ...
... A significant obstacle to the use of organic dyes in fluorescence microscopy is photobleaching. It is currently believed that the main mechanism of photobleaching is a photochemical reaction with singlet oxygen [34]. From this point of view, the inclusion of fluorescent dyes in a silica matrix should increase their photostability. ...
The development of a simple and reproducible method for the synthesis of monodisperse silica particles is of considerable interest from the point of view of their numerous applications in photonics, biosensing, and biomedicine. When using the well-known Stober method, there is a continuous formation and growth of seeds, which leads to the synthesis of polydisperse colloids. In this work, we used the method of successive growth of silica particles obtained by hydrolytic condensation of tetraethylorthosilicate in an alcoholic-aqueous medium using an alkaline catalyst. It is shown that this technique makes it possible to obtain colloids with a particle size from 50 nm to 3 μm and a standard deviation of less than 5%. An additional advantage of the developed method of stepwise growth is the possibility to include fluorophores and SERS tags into the silica matrix.
... Photobleaching of organic fluorophores may occur through different bimolecular reactions with other species as dissolved oxygen or solvent molecules. [40] Unlike organic fluorophores in solvents, silver clusters are immobilized in a glass host and isolated from the atmosphere. We suggest that the mechanism of the observed photobleaching is the photoactivated electron transfer [Eq. ...
Inorganic glasses doped with luminescent silver clusters are promising materials for photonic applications as white light generation, optical data storage, and spectral conversion. This work reports the photostability study of luminescent silver clusters dispersed in silica‐based glass under continuous ultraviolet irradiation. The photobleaching process model is proposed and the quantum yield of photobleaching is derived from the experimental data. The proposed mechanism of photobleaching is photoionization of silver clusters. Degradation of cluster luminescence is reversible and restores after the heat treatment, indicating the possibility to release trapped electrons and return the initial charge state of clusters. The effect of heat treatment temperature on the luminescence restoration is studied, the amount of restored luminescent clusters depends linearly on the heat treatment temperature.
... To relieve the constraints imposed by the clearing liquid, preparation of transparent tissues in a solid, liquid-free condition provides an attractive strategy to fundamentally change the current setting for 3D tissue imaging. In the solid state, an extra advantage is that the firm and stable environment hinders chemical reactions (e.g., photobleaching) by limiting the diffusional contact between the indicator dye and reactive species (e.g., oxygen) 11,12 , thereby improving the shelf life and photochemical stability of the fluorescently labeled specimens. The photostability is particularly important in high-resolution 3D imaging (e.g., super-resolution microscopy) due to the prolonged laser exposure time under the high-power objective 13 . ...
Optical clearing with high-refractive-index (high-n) reagents is essential for 3D tissue imaging. However, the current liquid-based clearing condition and dye environment suffer from solvent evaporation and photobleaching, causing difficulties in maintaining the tissue optical and fluorescent features. Here, using the Gladstone-Dale equation [(n−1)/density=constant] as a design concept, we develop a solid (solvent-free) high-n acrylamide-based copolymer to embed mouse and human tissues for clearing and imaging. In the solid state, the fluorescent dye-labeled tissue matrices are filled and packed with the high-n copolymer, minimizing scattering in in-depth imaging and dye fading. This transparent, liquid-free condition provides a friendly tissue and cellular environment to facilitate high/super-resolution 3D imaging, preservation, transfer, and sharing among laboratories to investigate the morphologies of interest in experimental and clinical conditions.
Room‐temperature phosphorescent materials, renowned for their long luminescence lifetimes, have garnered significant attention in the field of optical materials. However, the challenges posed by thermally induced quenching have significantly hindered the advancement of luminescence efficiency and stability. In this study, thermally enhanced phosphorescent carbon nanodots (CND) are developed by incorporating them into fiber matrices. Remarkably, the phosphorescence lifetime of the thermally enhanced CND exhibits a twofold enhancement, increasing from 326 to 753 ms, while the phosphorescence intensity experienced a tenfold enhancement, increasing from 25 to 245 as the temperature increased to 373 K. Rigid fiber matrices can effectively suppress the non‐radiative transition rate of triplet excitons, while high temperatures can desorb oxygen adsorbed on the surface of the CND, disrupting the interaction between the CND and oxygen. Consequently, a thermally enhanced phosphorescence is obtained. In addition, benefiting from the thermally enhanced phosphorescence property of CND, a warning indicator with an anti‐counterfeiting function for monitoring cold‐chain logistics is demonstrated based on CND.
Strong exciton-photon coupling offers an effective path for polariton-mediated long-distance coherent energy transfer (ET) between excitonic states. Here, we demonstrate strong coupling between excitons in WS2 monolayers, MoS2 bilayer, and photons in a tunable optical microcavity at room temperature. Full quantum dynamics simulations based on experimental parameters show that the demonstrated system provides an efficient and adjustable platform for ultrafast polariton-assisted ET between the excitons in two-dimensional materials when the separation between them exceeds 1µm.
The development of a simple and reproducible method for the synthesis of monodisperse silica particles is of considerable interest from the point of view of their numerous applications in photonics, biosensing, and biomedicine. When using the well-known Stober method, there is a continuous formation and growth of seeds, which leads to the synthesis of polydisperse colloids. In this work, we used the method of successive growth of silica particles obtained by hydrolytic condensation of tetraethylorthosilicate in an alcoholic-aqueous medium using an alkaline catalyst. It is shown that this technique makes it possible to obtain colloids with a particle size from 50 nm to 3 μm and a standard deviation of less than 5%. An additional advantage of the developed method of stepwise growth is the possibility to include fluorophores and SERS tags into the silica matrix.
Macrophage polarization in neurotoxic (M1) or neuroprotective (M2) phenotypes is known to play a significant role in neuropathic pain, but its behavioral dynamics and underlying mechanism remain largely unknown. Two‐photon excitation microscopy (2PEM) is a promising functional imaging tool for investigating the mechanism of cellular behavior, as using near‐infrared excitation wavelengths is less subjected to light scattering. However, the higher‐order photobleaching effect in 2PEM can seriously hamper its applications to long‐term live‐cell studies. Here, we demonstrate a GHz femtosecond (fs) 2PEM that enables hours‐long live‐cell imaging of macrophage behavior with reduced higher‐order photobleaching effect—by leveraging the repetition rate of fs pulses according to the fluorescence lifetime of fluorophores. Using this new functional 2PEM platform, we measure the polarization characteristics of macrophages, especially the long‐term cellular behavior in efferocytosis, unveiling the dynamic mechanism of neuroprotective macrophage polarization in neuropathic pain. These efforts can create new opportunities for understanding long‐term cellular dynamic behavior in neuropathic pain, as well as other neurobiological problems, and thus dissecting the underlying complex pathogenesis.
Random copolymers of styrene and substituted styrenes bearing arylamino substituents as fluorophore units have been obtained. Their photophysical properties have been investigated by measuring absorption and emission spectra as in solutions as solid-state. All copolymers proved to possess absolute quantum yields up to 0.39 in solution and up to 0.05 in solid-state, depending on their fluorophore substituents. Fluorescence studies have shown that these copolymers show a highly sensitive response towards a diversity of nitroaro-matic compounds, both in solutions and in a vapor phase. The detection limits for these compounds towards model nitroaromatic explosives in dichloro-methane solution proved to be in the range from 10 À6 to 10 À7 mol/L. The fluorescent materials prepared by electrospinning of synthesized copolymers have been evaluated as sensor materials for detecting nitrobenzene vapor for our handmade sniffer with detection limits of 0.5 ppm during 100-s exposure to the vapor. K E Y W O R D S detection of nitroaromatic explosives, fluorescence chemosensors, fluorescence quenching, pendant copolymers
Comprehensive Summary
Stability against oxygen is an important factor affecting the performance of organic semiconductor devices. Improving photooxidation stability can prolong the service life of the device and maintain the mechanical and photoelectric properties of the device. Generally, various encapsulation methods from molecular structure to macroscopic device level are used to improve photooxidation stability. Here, we adopted a crystallization strategy to allow 14 H ‐spiro[dibenzo[ c , h ]acridine‐7,9′‐uorene] (SFDBA) to pack tightly to resist fluorescence decay caused by oxidation. In this case, the inert group of SFDBA acts as a “steric armor”, protecting the photosensitive group from being attacked by oxygen. Therefore, compared with the fluorescence quenching of SFDBA powder under 2 h of sunlight, SFDBA crystal can maintain its fluorescence emission for more than 8 h under the same conditions. Furthermore, the photoluminescence quantum yields (PLQYs) of the crystalline film is 327% higher than that of the amorphous film. It shows that the crystallization strategy is an effective method to resist oxidation.
Evaluation of glucose in cancerous tumours acts as a hall mark for cancer progression and thus serves as a major interest in these days. Here, we have developed a biocompatible, surface enhanced Raman spectroscopy-based glucose Nano Particle Sensor (SERS-gNPS) to measure glucose dynamically in 3D Co-cultured Colon Cancer Tumour Spheroids (3D-CCTS). 3D-CCTS were produced using polydimethylsiloxane (PDMS) based µ-well array chip. SERS-gNPS was fabricated by conjugating 4-Mercaptophenylboronic acid (4-MPBA) to 50 nm silver nanoparticles (AgNP) and used under confocal Raman spectroscopy to measure glucose. The calibration curve shows direct relationship between Raman peak intensity at 1078 cm-1 with increasing glucose levels. The sensor has a limit of detection of 0.1 mM and found to be linear in the physiological range (1 mM-10 mM) of glucose with an average standard deviation of ± 5%. SERS-gNPS was applied for continuous glucose measurement in an in-vitro 3D-CCTS and 3D Mono-cultured fibroblast Spheroids (3D-MS). The results revealed that the 3D-CCTS have more prominent glucose gradient (~1 mM) as compared to 3D-MS (~0.4 mM) from core to peripheral regions at 3 days to 5 days of culture. SERS-gNPS provides a platform for accurate and real-time measurement of glucose, and offers an enhanced understanding of glucose distribution in the tumour spheroids.
Neural regulation of hepatic metabolism has long been recognized. However, the detailed afferent and efferent innervation of the human liver has not been systematically characterized. This is largely due to the liver's high lipid and pigment contents, causing false negative (light scattering and absorption) and false positive (autofluorescence) results in in-depth fluorescence imaging. Here, to avoid the artifacts in 3D liver neurohistology, we embed the bleached human liver in the high-refractive-index polymer for tissue clearing and antifade 3D/Airyscan super-resolution imaging. Importantly, using the paired substance P (SP, sensory marker) and PGP9.5 (pan-neuronal marker) labeling, we detect the sensory nerves in the portal space, featuring the SP ⁺ varicosities in the PGP9.5 ⁺ nerve bundles/fibers, confirming the afferent liver innervation. Also, using the tyrosine hydroxylase (TH, sympathetic marker) labeling, we identify: (1) condensed TH ⁺ sympathetic nerves in the portal space, (2) extension of sympathetic nerves from the portal to the intralobular space, in which the TH ⁺ nerve density is 2.6±0.7-fold higher than that of the intralobular space in the human pancreas, and (3) the TH ⁺ nerve fibers and varicosities contacting the ballooning cells, implicating potential sympathetic influence on hepatocytes with macrovesicular fatty change. Finally, using the vesicular acetylcholine transporter (VAChT, parasympathetic marker), PGP9.5, and CK19 (epithelial marker) labeling with panoramic-to-Airyscan super-resolution imaging, we detect and confirm the parasympathetic innervation of the septal bile duct. Overall, our labeling and 3D/Airyscan imaging approach reveal the hepatic sensory (afferent) and sympathetic and parasympathetic (efferent) innervation, establishing a clinically related setting for high-resolution 3D liver neurohistology.
Large-scale functional networks have been characterized in both rodent and human brains, typically by analyzing fMRI-BOLD signals. However, the relationship between fMRI-BOLD and underlying neural activity is complex and incompletely understood, which poses challenges to interpreting network organization obtained using this technique. Additionally, most work has assumed a disjoint functional network organization (i.e., brain regions belong to one and only one network). Here, we employ wide-field Ca²⁺ imaging simultaneously with fMRI-BOLD in mice expressing GCaMP6f in excitatory neurons. We determine cortical networks discovered by each modality using a mixed-membership algorithm to test the hypothesis that functional networks exhibit overlapping organization. We find that there is considerable network overlap (both modalities) in addition to disjoint organization. Our results show that multiple BOLD networks are detected via Ca²⁺ signals, and networks determined by low-frequency Ca²⁺ signals are only modestly more similar to BOLD networks. In addition, the principal gradient of functional connectivity is nearly identical for BOLD and Ca²⁺ signals. Despite similarities, important differences are also detected across modalities, such as in measures of functional connectivity strength and diversity. In conclusion, Ca²⁺ imaging uncovers overlapping functional cortical organization in the mouse that reflects several, but not all, properties observed with fMRI-BOLD signals.
Photoacids are organic molecules that release protons under illumination, providing spatiotemporal control of the pH. Such light-driven pH switches offer the ability to cyclically alter the pH of the medium and are highly attractive for a wide variety of applications, including CO2 capture. Although photoacids such as protonated merocyanine can enable fully reversible pH cycling in water, they have a limited chemical stability against hydrolysis (<24 h). Moreover, these photoacids have low solubility, which limits the pH-switching ability in a buffered solution such as dissolved CO2. In this work, we introduce a simple pathway to dramatically increase stability and solubility of photoacids by tuning their solvation environment in binary solvent mixtures. We show that a preferential solvation of merocyanine by aprotic solvent molecules results in a 60% increase in pH modulation magnitude when compared to the behavior in pure water and can withstand stable cycling for >350 h. Our results suggest that a very high stability of merocyanine photoacids can be achieved in the right solvent mixtures, offering a way to bypass complex structural modifications of photoacid molecules and serving as the key milestone toward their application in a photodriven CO2 capture process.
A DNA strand can encapsulate a silver molecule to create a nanoscale, aqueous stable chromophore. A protected cluster that strongly fluoresces can also be weakly photolabile, and we describe the laser-driven photochemistry of the green fluorophore C4AC4TC3GT4/Ag10⁶⁺. The embedded cluster is selectively photoexcited at 490 nm and then bleached, and we describe how the efficiency, products, and route of this photochemical reaction are controlled by the DNA cage. With irradiation at 496.5 nm, the cluster absorption progressively drops to give a photodestruction quantum yield of 1.5 (±0.2) × 10–4, ∼10³× less efficient than fluorescence. A new λabs = 335 nm chromophore develops because the precursor with 4 Ag⁰ is converted into a group of clusters with 2 Ag⁰ – Ag6⁴⁺, Ag7⁵⁺, Ag8⁶⁺, and Ag9⁷⁺. The 4–7 Ag⁺ in this series are chemically distinct from the 2 Ag⁰ because they are selectively etched by iodide. This halide precipitates silver to favor only the smallest Ag6⁴⁺ cluster, but the larger clusters re-develop when the precipitated Ag⁺ ions are replenished. DNA-bound Ag10⁶⁺ decomposes because it is electronically excited and then reacts with oxygen. This two-step process may be state-specific because O2 quenches the red luminescence from Ag10⁶⁺. However, the rate constant of 2.3 (±0.2) × 10⁶ M–1 s–1 is relatively small, which suggests that the surrounding DNA matrix hinders O2 diffusion. On the basis of analogous photoproducts with methylene blue, we propose that a reactive oxygen species is produced and then oxidizes Ag10⁶⁺ to leave behind a loose Ag⁺-DNA skeleton. These findings underscore the ability of DNA scaffolds to not only tune the spectra but also guide the reactions of their molecular silver adducts.
Single particle tracking (SPT) combined with total internal reflection fluorescence microscopy (TIRFm) is an outstanding approach to decipher mechanisms on the cell membrane at the nanoscale. Multicolor configurations, needed to investigate interactions, are still hindered by several challenges. This work systematically and quantitatively analyzes the impact of necessary elements of SPT‐TIRFm setups on the signal‐to‐noise ratio (SNR), which must be optimized especially in dynamic studies needing minimally invasive dyes for biomolecule labeling. Autofluorescence originating from commonly used optical glass results in the dominant limiting factor in TIRFm, and a cover glass material is tested yielding significant SNR improvements in multichannel TIRFm. Moreover, methodologies are optimized for reducing fluorophore photobleaching in multicolor implementations requiring simultaneous stabilization of multiple dyes. The developed strategies are applied to the fast p75NTR receptors labeled by two fluorophores on the membrane of living cells, achieving reliable, simultaneous two‐color SPT, contrary to configurations using standard cover glasses. This work highlights the importance of optical materials suitable for microscopy and with reduced autofluorescence for increasing sensitivity toward ultimate spatiotemporal resolutions. In particular, the present protocols can pave the way for multicolor super‐resolved localization and tracking of single molecules by TIRFm, greatly expanding the potential of SPT.
This study presents a novel approach for fluorescence-based spermine sensing, using a laboratory synthesized distyryl BODIPY dye in combination with a widely available and inexpensive anionic surfactant, SDS. The resulting...
Nanomedicine presents exciting new opportunities for the detection and treatment of cancer. Current cancer imaging methods and treatment approaches in clinics frequently fall short of entirely curing cancer and can have severe side effects. Theranostic nanoparticles, however, have the potential to revolutionize effective cancer treatment and early cancer detection. The objective of this study is to show how magnetic iron oxide nanoparticles and photoluminescent gold nanoclusters (MN-AuNCs) may be combined effectively to produce bimodal imaging nanoparticles that possess magnetic and optical properties and can be used for both magnetic resonance imaging and optical biopsy. These findings demonstrate that MN-AuNCs, when exposed to visible light, also have the capability to produce singlet oxygen, which is necessary for photodynamic therapy of cancer. In addition, it shows that they are non-toxic, accumulate inside the cells, and cause cell death during exposure to visible light. The creation of these MN-AuNCs offers a novel remedy for the current shortcomings in cancer diagnosis and treatment. Since they have both therapeutic and imaging capabilities, MN-AuNCs have the potential to improve patient outcomes while lowering the risk of negative side effects.
Waste-to-Energy (WTE) facilities incinerate ∼11% (∼ 222 Mt) of global solid waste, generating bottom and fly ashes. Landfilling these ashes is costly, and risks releasing contaminants into the environment. Instead, using WTE ashes in secondary industrial applications can circumvent such environmental risks. However, their secondary use is restricted by their inconsistent mineralogy, which may vary due to fluctuating waste composition and combustion conditions. Therefore, there is a need for rapid and reliable monitoring of WTE fly ash mineralogy. Here, we evaluate the employment of Raman spectroscopy for that purpose. Our initial investigation of 12 unique WTE fly ashes resulted in excessive fluorescence, rendering key Raman peaks obscure. To address this issue, we report that a mere 2 min of photobleaching can significantly reduce this fluorescence, facilitating the detection of calcite, calcium sulfate, zincite, and carbon - phases previously undetectable in original spectra. These results show the potential of Raman spectroscopy for rapid monitoring of WTE fly ash mineralogy, which could be beneficial in diverting these ashes from landfill.
Heterogeneous photocatalysis combines the benefits of light‐mediated chemistry with that of a catalytic platform that facilitates re‐use of (often expensive) photocatalysts. This provides significant opportunities towards more economical, sustainable, safe, and user‐friendly chemical syntheses of both small and macromolecular compounds. This contribution outlines recent developments in the design of heterogenous photocatalysts and their use to mediate polymerizations. We outline four classes of heterogeneous photocatalysts in detail: Nanoparticles, conjugated and non‐conjugated polymer networks, metal‐organic frameworks (MOFs), and functionalized solid supports.
Stimulated Raman Scattering microscopy is an important imaging technique. Its broader application, however, is hampered by its comparatively low sensitivity. Using organic fluorophores, it has recently been demonstrated that, similar to spontaneous Raman microscopy, the sensitivity of stimulated Raman microscopy is increased by orders of magnitudes if electronic preresonances are exploited. In this Article, we show that this approach also works with low quantum yield chromophores. We investigate the relevant photophysics and discuss the background arising from preresonant excitation conditions. Applications of preresonant stimulated Raman scattering microscopy for the imaging of weakly fluorescing labels in fixed and live cells are demonstrated.
Viscosity sensitive fluorophores termed 'molecular rotors' represent a convenient and quantitative tool for measuring intracellular viscosity via Fluorescence Lifetime Imaging Microscopy (FLIM). We compare the FLIM performance of two BODIPY-based molecular rotors bound to HaloTag protein expressed in different subcellular locations. While both rotors are able to penetrate live cells and specifically label the desired intracellular location, we found that the rotor with a longer HaloTag protein recognition motif was significantly affected by photo-induced damage when bound to the HaloTag protein, while the other dye showed no changes upon irradiation. Molecular dynamics modelling indicates that the irradiation-induced electron transfer between the BODIPY moiety and the HaloTag protein is a plausible explanation for these photostability issues. Our results demonstrate that binding to the targeted protein may significantly alter the photophysical behaviour of a fluorescent probe and therefore its thorough characterisation in the protein bound form is essential prior to any in vitro and in cellulo applications.
Methylene Blue has a long history as a photochemical reagent and is known to undergo photofading on exposure to visible light in aqueous solution. Under aerobic conditions, photobleaching occurs by way of a two‐step process involving intermediary formation of singlet oxygen. The first step is ascribed to regio‐selective addition of singlet oxygen within the precursor complex. This geminate reaction ultimately leads to formation of the leuco‐dye via a slower second step. Urea forms a weak ground‐state complex with Methylene Blue which affects the optical properties of the dye but is not evident by NMR spectroscopy. This complex is weakly fluorescent and undergoes intersystem crossing to the triplet manifold. The presence of urea decreases the rate of photobleaching of the dye and, at high concentrations of urea, the bleaching kinetics are consistent with an equilibrium mixture of complexed and free dye. The complexed dye does not bleach on the timescale of the experiment. Such protection might arise from urea blocking access to the site where geminate addition of O2 takes place.
In this communication, the synthesis of three unknown polyfluorinated cyanine dyes and their application as selective markers for mitochondria are presented. By incorporating fluorous side chains into cyanine dyes, their remarkable photophysical properties were enhanced. To investigate their biological application, several different cell lines were incubated with the synthesized cyanine dyes. It was discovered that the presented dyes can be utilized for selective near‐infrared‐light (NIR) staining of mitochondria, with very low cytotoxicity determined by MTT assay. This is the first time that polyfluorinated cyanine fluorophores are presented as selective markers for mitochondria. Due to the versatile applications of polyfluorinated fluorophores in bioimaging and materials science, it is expected that the presented fluorophores will be stimulating for the scientific community.
One of the most important drawback of organic dyes is their low photo-stability which reduces possibility of their commercial utilization. In this article we employ the strategy of dye re-crystallization from oversaturated matrix in order to enhance material’s durability. One of the main advantages of perylene derivative is ability to form emissive j-aggregates, good miscibility and incorporation into liquid crystalline matrix. Investigation of perylene-based dye and LC matrix brought as the result very efficient light amplification modulation by applied external electric field. In our article we show that Stimulated Emission (STE) is possible to achieve from perylene-derivative based system, at typical fluence thresholds for laser dyes: 3.9 mJ/cm2. Moreover, presented system proves ultra-high photostability, showing lack of STE reduction even after 12 000 excitation laser pulses. Furthermore, we proved the possibility of light emission intensity control using external electric field.
A major challenge in single-molecule imaging is tracking the dynamics of proteins or complexes for long periods of time in the dense environments found in living cells. Here, we introduce the concept of using FRET to enhance the photophysical properties of photo-modulatable (PM) fluorophores commonly used in such studies. By developing novel single-molecule FRET pairs, consisting of a PM donor fluorophore (either mEos3.2 or PA-JF549) next to a photostable acceptor dye JF646, we demonstrate that FRET competes with normal photobleaching kinetic pathways to increase the photostability of both donor fluorophores. This effect was further enhanced using a triplet-state quencher. Our approach allows us to significantly improve single-molecule tracking of chromatin-binding proteins in live mammalian cells. In addition, it provides a novel way to track the localization and dynamics of protein complexes by labeling one protein with the PM donor and its interaction partner with the acceptor dye.
Two Chromophore-Quencher Conjugates (CQCs) have been synthesized by covalent attachment of the anti-oxidant dibutylated-hydroxytoluene (BHT) to a pyrrole-BF2 chromophore (BOPHY) in an effort to protect the latter against photofading. In fluid solution, light-induced intramolecular charge transfer is favoured in polar solvents and helps to inhibit photo-bleaching of the chromophore. The rate of photo-fading, which scales with the number of BHT residues, is zero-order in polar solvents but shows a linear dependence on the number of absorbed photons. The zero-order rate constant shows an inverse correlation with the fluorescence quantum yield measured in the same solvent. Photo-bleaching in benzonitrile involves autocatalysis while reaction in cyclohexane shows an unexpected stoichiometry. NMR spectroscopy indicates initial damage takes place at the BHT unit and allows identification of a reactive hydroperoxide as being the primary product. In the presence of an adventitious substrate, this hydroperoxide is a photocatalyst for amide formation under mild conditions.
In this study, two novel boron dipyrromethene-based photosensitizers (BDP3andBDP6) substituted with three or six trifluoromethyl groups have been synthesized and characterized with various spectroscopic methods, and their photo-physical, photo-chemical, and photo-biological properties have also been explored. The two photosensitizers are highly soluble and remain nonaggregated in N,N-dimethylformamide as shown by the intense and sharp Q-band absorption. Under red light irradiation (λ = 660 nm, 1.5 J/cm²), both photosensitizers show high and comparable cytotoxicity towards HepG2 human hepatocarcinoma and HeLa human cervical carcinoma cells with IC50values of 0.42-0.49 μM. The high photocytotoxicity ofBDP3andBDP6can be due to their high cellular uptake and low aggregation tendency in biological media, which result in a high efficiency to generate reactive oxygen species inside the cells. Confocal laser fluorescence microscopic studies indicate that they have superior selective affinities to the mitochondria and lysosomes of HepG2 and HeLa cells. The results show that these two trifluoromethyl boron dipyrromethene derivatives are potential anticancer agents for photodynamic therapy.
Intermixed light-matter quasiparticles - polaritons - possess unique optical properties owned to their compositional nature. These intriguing hybrid states have been extensively studied over the past decades in a wide range of realizations aiming at both basic science and emerging applications. However, recently it has been demonstrated that not only optical, but also material-related properties, such as chemical reactivity and charge transport, may be significantly altered in the strong coupling regime of light-matter interactions. Here, we show that a nanoscale system, comprised of a plasmonic nanoprism strongly coupled to excitons in J-aggregated form of organic chromophores, experiences modified excited state dynamics and therefore modified photo-chemical reactivity. Our experimental results reveal that photobleaching, one of the most fundamental photochemical reactions, can be effectively controlled and suppressed by the degree of plasmon-exciton coupling and detuning. In particular, we observe a 100-fold stabilization of organic dyes for the red-detuned nanoparticles. Our findings contribute to understanding of photochemical properties in the strong coupling regime and may find important implications for the performance and improved stability of optical devices incorporating organic dyes.
In modern biotechnological science, there is a need for visualization of objects under study at the levels of cells, organelles, and individual molecules. Prominent among imaging methods are the methods based on the detection of fluorescence from the fluorophores with which objects under study are labeled. Fluorescent proteins (FPs) are very popular as genetically encoded fluorescent labels for lifetime imaging of target structures and processes in living systems. One of the key characteristics of FPs is their photostability, i.e., their resistance to photochemical reactions that quench the fluorescence signal. This review describes the currently known molecular mechanisms underlying photobleaching and the methods used to improve the photostability of fluorescent proteins.
Erythrosine, a popular food dye, undergoes fast O2-sensitive bleaching in water when subjected to visible light illumi-nation. In dilute solution, Erythrosine undergoes photo-bleaching via first-order kinetics, where the rate of bleaching depends critically on the rate of photon absorption and on the concentration of dissolved oxygen. Kinetic studies indi-cate that this inherent bleaching is augmented by self-catalysis at higher concentrations of Erythrosine and on long exposure times. Under the conditions used, bleaching occurs by way of geminate attack of singlet molecular oxygen on the chromophore. Despite the complexity of the overall photo-bleaching process, the rate constants associated with both inherent and self-catalytic bleaching reactions follow Arrhenius-type behavior, allowing the activation parame-ters to be resolved. Bleaching remains reasonably efficient in the solid state, especially if the sample is damp, and pro-vides a convenient means by which to construct a simple chemical actinometer.
Imaging lipid organization in cell membranes requires advanced fluorescent probes. Here, we show that a recently synthesized push-pull pyrene (PA), similarly to popular probe Laurdan, changes the emission maximum as a function of lipid order, but outperforms it by spectroscopic properties. In addition to red-shifted absorption compatible with common 405 nm diode laser, PA shows higher brightness and much higher photostability than Laurdan in apolar membrane environments. Moreover, PA is compatible with two-photon excitation at wavelengths >800 nm, which was successfully used for ratiometric imaging of coexisting liquid ordered and disordered phases in giant unilamellar vesicles. Fluorescence confocal microscopy in Hela cells revealed that PA efficiently stains the plasma membrane and the intracellular membranes at >20-fold lower concentrations, as compared to Laurdan. Finally, ratiometric imaging using PA reveals variation of lipid order within different cellular compartments: plasma membranes are close to liquid ordered phase of model membranes composed of sphingomyelin and cholesterol, while intracellular membranes are much less ordered, matching well membranes composed of unsaturated phospholipids without cholesterol. These differences in the lipid order were confirmed by fluorescence lifetime imaging (FLIM) at the blue edge of PA emission band. PA probe constitutes thus a new powerful tool for biomembrane research.
Intramolecular photostabilization via triple-state quenching was recently revived as a tool to impart synthetic organic fluorophores with 'self-healing' properties. To date, utilization of such fluorophore derivatives is rare due to their elaborate multi-step synthesis. Here we present a general strategy to covalently link a synthetic organic fluorophore simultaneously to a photostabilizer and biomolecular target via unnatural amino acids. The modular approach uses commercially available starting materials and simple chemical transformations. The resulting photostabilizer-dye conjugates are based on rhodamines, carbopyronines and cyanines with excellent photophysical properties, that is, high photostability and minimal signal fluctuations. Their versatile use is demonstrated by single-step labelling of DNA, antibodies and proteins, as well as applications in single-molecule and super-resolution fluorescence microscopy. We are convinced that the presented scaffolding strategy and the improved characteristics of the conjugates in applications will trigger the broader use of intramolecular photostabilization and help to emerge this approach as a new gold standard.
Bright, long-lasting and non-phototoxic organic fluorophores are essential to the continued advancement of biological imaging. Traditional approaches towards achieving photostability, such as the removal of molecular oxygen and the use of small-molecule additives in solution, suffer from potentially toxic side effects, particularly in the context of living cells. The direct conjugation of small-molecule triplet state quenchers, such as cyclooctatetraene (COT), to organic fluorophores has the potential to bypass these issues by restoring reactive fluorophore triplet states to the ground state through intra-molecular triplet energy transfer. Such methods have enabled marked improvement in cyanine fluorophore photostability spanning the visible spectrum. However, the generality of this strategy to chemically and structurally diverse fluorophore species has yet to be examined. Here, we show that the proximal linkage of COT increases the photon yield of a diverse range of organic fluorophores widely used in biological imaging applications, demonstrating that the intra-molecular triplet energy transfer mechanism is a potentially general approach for improving organic fluorophore performance and photostability.
The purification of industrial wastewater from dyes is becoming increasingly important since they are toxic or carcinogenic to human beings. Nanomaterials have been receiving significant attention due to their unique physical and chemical properties compared with their larger-size counterparts. The aim of the present investigation was to fabricate magnetic nanoparticles (MNPs) using a coprecipitation method, followed by coating with silver (Ag) in order to enhance the photocatalytic activity of the MNPs by loading metal onto them. The fabricated magnetic nanoparticles coated with Ag were characterised using different instruments such as a scanning electron microscope (SEM), transmission electron microscopy (TEM), energy-dispersive X-ray (EDAX) spectroscopy, and X-ray diffraction (XRD) analysis. The average size of the magnetic nanoparticles had a mean diameter of about 48 nm, and the average particle size changed to 55 nm after doping. The fabricated Ag-doped magnetic nanoparticles were used for the degradation of eosin Y under UV-lamp irradiation. The experimental results revealed that the use of fabricated magnetic nanoparticles coated with Ag can be considered as reliable methods for the removal of eosin Y since the slope of evaluation of pseudo-first-order rate constant from the slope of the plot between
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We have synthesized a new family of directly-fused bisBODIPY BBP through a key FeCl3-mediated intramolecular oxidative cyclodehydrogenation reaction and its derivatives and from the Knoevenagel reaction. These dyes display effective expansion of π-conjugation over the two BODIPYs due to their locked coplanar conformation, showing intriguing electrochemical and spectroscopic properties, such as intensive absorption/emission bands ranging from 676 to 877 nm and high photostability.
Heptamethine cyanines are important near-IR fluorophores used in many fluorescence applications. Despite this utility, these molecules are susceptible to light-promoted reactions (photobleaching) involving photochemically generated reactive oxygen species (ROS). Here, we have sought to define key chemical aspects of this nearly inescapable process. Near-IR photolysis of a model heptamethine cyanine leads to the regioselective oxidative cleavage of the cyanine polyene. We report the first quantitative analysis of the major reaction pathway following either photolysis or exposure to candidate ROS. These studies clearly indicate that only singlet oxygen ((1)O2), and not other feasible ROS, recapitulates the direct photolysis pathway. Computational studies were employed to investigate the regioselectivity of the oxidative cleavage process, and the theoretical ratio is comparable to observed experimental values. These results provide a more complete picture of heptamethine cyanine photooxidation, and provide insight for design of improved compounds for future applications.
The computationally-aided photophysical and lasing properties of a selected battery of BOPHYs are described and compared to those of related BODIPY counterparts. The present joined theoretical-experimental study helps to put into context the weaknesses and strengths of both dye families under different irradiation conditions. The chemical versatility of the BOPHY scaffold has been also comparatively explored to modulate key photonic properties towards the development of red-emitting dyes, chiroptical dyes and singlet oxygen photosensitizers. Thus, BOPHY BINOLation by fluorine substitution with enantiopure BINOLs endows the BOPHY chromophore with chiroptical activity, as supporting by the simulated circular dichroism, decreasing deeply its fluorescent response due to the promotion of fluorescence-quenching intramolecular charge transfer (ICT). Interestingly, the sole alkylation of the BOPHY core strongly modulates the promotion of ICT, allowing the generation of highly bright BINOL-based BOPHY dyes. Moreover, 3,3′-dibromoBINOLating BOPHYs can easily achieve singlet-oxygen photogeneration, owing to spin-orbit coupling mediated by heavy-atom effect feasible in view of the theoretically predicted disposition of the bromines surrounding the chromophore. From this background, we have established the master guidelines to design bright fluorophores and laser dyes, photosensitizers for singlet oxygen production and chiroptical dyes based on BOPHYs. The possibility to finely mix and balance such properties in a given molecular scaffold outstands BOPHYs as promising dyes competing with the well-settled BODIPY dyes.
Light microscopy can offer certain advantages over electron microscopy in terms of acquiring detailed insights of the biological/intra-cellular milieu. In recent years, with the development of new fluorescence imaging technologies, it has become extremely important to assess the role of designing appropriate fluorophores in procuring desired biological information without encountering any untoward hitches. Over the years, external fluorophores have been prevalently used in fluorescence microscopy and single-molecule fluorescence microscopy based studies. Photostable fluorogenic probes with high extinction coefficients and quantum yield, exhibiting minimum autofluorescence and photobleaching properties, are preferred in single-molecule microscopy as they can tolerate long-term laser exposure Therefore, the development of triplet state quenchers and/or any other suitable new strategy to ensure the photo-stability of the fluorophores during long-term live cell imaging exercises, is highly anticipated. In this feature article, various strategies for stabilizing fluorophores, including the mechanisms of TSQ-induced stabilization, have been thoroughly reviewed in light of contemporary literature reports and applications.
Chemical sensing in living systems demands optical sensors that are bright, stable, and sensitive to the rapid dynamics of chemical signaling. Lanthanide-doped upconverting nanoparticles (UCNPs) efficiently convert near infrared (NIR) light to higher energy emission and allow biological systems to be imaged with no measurable background or photobleaching, and with reduced scatter for subsurface experiments. Despite their advantages as imaging probes, UCNPs have little innate chemical sensing ability and require pairing with organic fluorophores to act as biosensors, although the design of stable UCNP-fluorophore hybrids with efficient upconverted energy transfer (UET) has remained a challenge. Here, we report Yb3+- and Er3+-doped UCNP-fluorophore conjugates with UET efficiencies up to 88%, and photostabilities 100-fold greater by UET excitation than those of the free fluorophores under direct excitation. Despite adding distance between Er3+ donors and organic acceptors, thin inert shells significantly enhance overall emission without compromising UET efficiency. This can be explained by the large increase in quantum yield of Er3+ donors at the core/shell interface and the large number of fluorophore acceptors at the surface. Sensors excited by UET show increases in photostability well beyond those reported for other methods for increasing the longevity of organic fluorophores, and those covalently attached to UCNP surface polymers show greater chemical stability than those directly coordinated to the nanocrystal surface. By conjugating other fluorescent chemosensors to UCNPs, these hybrids may be extended to a series of NIR-responsive biosensors for quantifying the dynamic chemical populations critical for cell signaling.
FRET improves the photostability of fluorophores for longer single-molecule tracking.
Various fluorescence microscopy techniques require bright NIR‐emitting fluorophores with high chemical and photostability. Herein, the significant performance improvement of phosphorus‐substituted rhodamine dyes (PORs) upon substitution at the 9‐position with a 2,6‐dimethoxyphenyl group is reported. The thus obtained dye PREX 710 was used to stain mitochondria in living cells, which allowed long‐term and three‐color imaging in the vis‐NIR range. Moreover, the high fluorescence longevity of PREX 710 allows tracking a dye‐labeled biomolecule by single‐molecule microscopy under physiological conditions. Deep imaging of blood vessels in mice brain has also been achieved using the bright NIR‐emitting PREX 710‐dextran conjugate.
In this review, the literature on the new fluorophore BOPHY is covered. This doubly boron-centered fluorophore resembles the well-studied BODIPY, but has its own characteristics, both in synthesis and spectroscopy. The general synthesis and properties of this fluorophore, the possibilities for postmodifications, the literature on aromatic ring-fused BOPHYs and boron substitution are discussed.
Air pollution from volatile organic compounds (VOCs) is one of the most important environmental hazards. Advanced oxidation processes (AOPs) with UV systems have been showing high potential for the abatement of VOCs. This work is aimed at modeling UV reactors for scaling-up AOPs from lab-scale to full-scale. The proposed model has a novel approach coupling the UV fluence rate to the photo-kinetic mechanism, for a robust understanding of the phenomena involved. The results show that the 185 nm wavelength is deeply absorbed within few centimeters by oxygen, while the 254 nm wavelength is weakly absorbed by the ozone generated in the reactor. Based on the fluence rate calculations, the reactions of ozone generation and depletion were modeled. The ozone net concentration was compared to the experimental results, for model verification. The model accurately predicts the effect of the airflow rate and reactor diameter for the tested cases. The acetaldehyde oxidation reaction was modeled using a simplified kinetic mechanism, using the experimental data of VOC conversion for a further model verification. The suggested reactor models accurately predicted the effect of airflow rate, while exhibiting limitations for the effect of different reactor diameters. Therefore, a computational fluid dynamics (CFD) investigation is needed for an accurate modeling of the VOCs oxidation reaction, implementing the developed analytical expression for reducing the computational workload. By combining the developed model with a CFD simulator, it would be possible to simulate several reactors, also at full-scale, for predicting their performance and identifying optimal configurations.
Photophysics and photostability of 12,13-dihydro-5H-indolo[3,2-c]acridine (IA), a rigid bifunctional indole derivative with proton donor/acceptor functionalities can be drastically changed by the environment. The formation of hydrogen bonds with alcohols leads to a significant decrease of the triplet formation efficiency and an increase of photostability. The photodegradation yield was found to be about two hundred times lower in methanol and 1-propanol than in n-hexane or acetonitrile. A similar effect has been reported for two indole-naphthyridines, molecules that can exist in syn and anti rotameric forms. We demonstrate that IA, which can exist only in the syn form, is more photostable in alcohols than similar, but non-rigid molecules. This additional photostability enhancement is due to elimination of a slower channel of excited state deactivation in alcohol complexes, S0←S1 internal conversion. The dominant, faster channel of S1 depopulation is the excited state double proton transfer, manifested by the presence of low energy tautomeric fluorescence.
Visible light photocatalysis has evolved over the last decade into a widely used method in organic synthesis. For many important transformations, such as cross-coupling reactions, alpha-amino functionalizations, cycloadditions, ATRA reactions, or fluorinations, photocatalytic variants have been reported. In this review, we try to compare classical and photocatalytic procedures for selected classes of reactions and highlight their advantages and limitations. In many cases, the photocatalytic reactions proceed at milder reaction conditions, typically at room temperature, and stoichiometric reagents are replaced by simple oxidants or reductants, like air oxygen or amines. This way, besides providing alternative protocols for established transformations that allow a broadening of the substrate scope, also new transformations become possible, especially by merging photocatalysis with organo- or metal catalysis. Does visible light photocatalysis make a difference in organic synthesis? The prospect to shuttle electrons back and forth to substrates and intermediates or to selectively transfer energy through a visible light absorbing photocatalyst holds the promise to improve current protocols in radical chemistry and to open up new avenues by accessing reactive species hitherto unknown.
Fluorescence dyes are based on small organic molecules have become of interest in chemical biology and widely used for cell and intracellular imaging. However, fluorescence dyes have limitations such as photo bleaching, poor photochemical stability and has a short Stokes shift. It is less valuable for long-term cell tracking strategies and has very short lifetime. In order to overcome the problems, dye-incorporated nanomaterials become of interest. Nanomaterials encapsulation provides a protection layer around the fluorescence dye which improves the stability of fluorescence dye. In this study, silica nanoparticles encapsulated with 1,1%-dioctadecyl-3,3,3%,3%-tetramethylindocarbocyanine perchlorate (Dil) was successfully synthesised by using micelle entrapment method to investigate the effect of encapsulation of nanoparticles towards the properties of fluorescent dye. The synthesised nanoparticles (SiDil) was characterised by particle size analyser, Transmission Electron Microscopy (TEM), UV-Vis spectrometer and Fluorescent spectrometer. Observation using TEM showed spherical shape of nanoparticles with 53 nm diameter. Monodispersed and well nanoparticles distribution was confirmed by low polydispersity index of 0.063 obtained by particle size analyser. Furthermore, the photoluminescence properties of the SiDil were evaluated and compared with bare Dil dye. Both SiDil and bare Dil was radiated under 200 W of Halogen lamp for 60 minutes and the absorbance intensity was measured using UV-Vis spectrometer. The result showed more stable absorbance intensity for SiDil compared to bare Dil dye, which indicated that Si nanoparticles encapsulation improved the photostability property.
Single molecule fluorescence (SMF) studies on conjugated polymers yield enhanced information on exciton dynamics and on the interplay between polymer conformation/morphology and photophysical behavior. SMF studies, however, demand good signal...
This paper provides a systematic review and analysis of different phenomena that violate a basic principle, Kasha’s rule, when applied to photochemical reactions. In contrast to the classical route of ultrafast transition to the lowest energy excited state and photochemical reaction starting therein, in some cases, these reactions proceed directly from high-energy excited states. Nowadays, this phenomenon can be observed for a number of major types of excited-state reactions: harvesting product via intersystem crossing; photoisomerizations; bond-breaking; and electron, proton, and energy transfers. We show that specific conditions for their observation are determined by kinetic factors. They should be among the fastest reactions in studied systems, competing with vibrational relaxation and radiative or nonradiative processes occurring in upper excited states. The anti-Kasha effects, which provide an important element that sheds light on the mechanisms of excited-state transformations, open new possibilities of selective control of these reactions for a variety of practical applications. Efficient utilization of excess electronic energy should enhance performance in the systems of artificial photosynthesis and photovoltaic devices. The modulation of the reporting signal by the energy of excitation of light should lead to new technologies in optical sensing and imaging.
J-aggregates are fascinating fluorescent nanomaterials formed by highly ordered assembly of organic dyes with the spectroscopic properties dramatically different from that of single or disorderly assembled dye molecules. They demonstrate very narrow red-shifted absorption and emission bands, strongly increased absorbance together with the decrease of radiative lifetime, highly polarized emission and other valuable features. The mechanisms of their electronic transitions are understood by formation of delocalized excitons already on the level of several coupled monomers. Cyanine dyes are unique in forming J-aggregates over the broad spectral range, from blue to near-IR. With the aim to inspire further developments, this Review is focused on the optical characteristics of J-aggregates in connection with the dye structures and on their diverse already realized and emerging applications.
Here we report transient absorption studies on the ground state recovery dynamics of the single–molecule fluorophore Cy3B in the presence of four different photostabilizing agents, namely β–mercaptoethanol (β–ME), Trolox (TX), n–propyl gallate (n–PG), and ascorbic acid (AA). These are triplet state quenchers that operate via photoinduced electron transfer (PeT). While quantitative gem-inate recombination was recorded following PeT for β–ME (~100%), for Trolox, n–propyl gallate, and ascorbic acid the extent of geminate recombination was >48 %, >27 % and >13 %, respectively. The results are rationalized in terms of the rates of intersystem crossing (ISC) in the newly formed geminate radical ion pairs (GRIPs). Rapid spin relaxation in the radicals formed accounts for quantitative geminate recombination with β–ME and efficient geminate recombination with TX. Our results illustrate how the interplay of PeT quenching efficiency and geminate recombination dynamics may lead to improved photostabilization strategies, critical for single–molecule fluorescence and superresolution imaging.
Covalent dimerization and iodine substitution methods were used to synthesize heavy atom free and heavy atom present BODIPY singlet oxygen photosensitizers. Both dimerization and iodization could strongly enhance the excited triplet state (T1) formation up to 95% efficiency. The mechanism for excited triplet state formation was revealed by examining UV–vis electronic absorption, steady state and time resolved visible and NIR luminescence, laser flash photolysis, singlet oxygen chemical trapping, and quantum chemical calculation. The dimerization method shows a different mechanism in T1 generation: photoinduced electron transfer (PET) from lowest lying excited singlet state (S1) followed by charge recombination, instead of the traditional intersystem crossing from S1 state. The PET mechanism makes such a photosensitizer sensitive to solvent polarity. This type of heavy atom free and PET-based photosensitizers could be important in photodynamic therapy of tumor and organic photochemistry.
As stimulated emission depletion (STED) microscopy can provide structural details of cells with an optical resolution beyond the diffraction limit, it has become an indispensable tool in cell biology. However, the intense STED laser beam usually causes rapid photobleaching of the employed fluorescent dyes, which significantly limits the utility of STED microscopy from a practical perspective. Herein we report a new design of super-photostable dye, PhoxBright 430 (PB430), comprising a fully ring-fused π-conjugated skeleton with an electron-accepting phosphole P-oxide unit. We previously developed a super-photostable dye C-Naphox by combining the phosphole unit with an electron-donating triphenylamine moiety. In PB430, removal of the amino group alters the transition type from intramolecular charge transfer character to π–π* transition character, which gives rise to intense fluorescence insensitive to molecular environment in terms of fluorescence colors and intensity, and bright fluorescence even in aqueous media. PB430 also furnishes high solubility in water, and is capable of labeling proteins with maintaining high fluorescence quantum yields. This dye exhibits outstanding resistance to photoirradiation even under the STED conditions and allows continuous acquisition of STED images. Indeed, using a PB430-conjugated antibody, we succeed in attaining a 3-D reconstruction of super-resolution STED images as well as photostability-based multicolor STED imaging of fluorescently labeled cytoskeletal structures.
Advances in fluorescence microscopy promise to unlock details of biological systems with high spatiotemporal precision. These new techniques also place a heavy demand on the ‘photon budget’—the number of photons one can extract from a sample. Improving the photostability of small molecule fluorophores using chemistry is a straightforward method for increasing the photon budget. Here, we review the (sometimes sparse) efforts to understand the mechanism of fluorophore photobleaching and recent advances to improve photostability through reducing the propensity for oxidation or through intramolecular triplet-state quenching. Our intent is to inspire a more thorough mechanistic investigation of photobleaching and the use of precise chemistry to improve fluorescent probes.
The photochemical stability of two symmetrical pyrrole-BF2 (BOPHY) dyes has been assessed under continuous exposure to white light. The parent dye, lacking conjugated substituents, is highly stable when illuminated in a nonpolar film for prolonged periods but auto-catalysis is observed such that the rate of degradation escalates with increasing exposure time. The rate of photo-fading is faster in solution and involves intermediate formation of a coloured product. The nature of the solvent exerts a small effect on the rate of bleaching. Under the same conditions, a BOPHY derivative equipped with both extended pi-conjugation and a high internal dipole moment displays a pronounced solvent effect. Here, the rate of photo-fading varies over a factor of 2,000-fold on changing the solvent from cyclohexane to toluene. Most of the kinetic data can be explained in terms of polar and neutral resonance forms but, on the basis of NMR spectroscopy, it is concluded that toluene forms a weak complex with the dye that helps protects against photochemical damage.
In this study, we engineered liposomal indocyanine green (ICG) to maximize its photothermal effects while maintaining the fluorescence intensity. Various liposomal formulations of ICG were prepared by varying the lipid composition and the molar ratio between total lipid and ICG, and their photothermal characteristics were evaluated under near-infrared irradiation. We showed that the ICG dispersity in the liposomal membrane and its physical interaction with phospholipids were the main factors determining the photothermal conversion efficiency. In phototherapeutic studies, the optimized formulation of liposomal ICG showed greater anti-cancer effects in a mouse tumor model compared with other liposomal formulations and the free form of ICG. Furthermore, we utilized liposomal ICG to visualize the metastatic lymph node around the primary tumor under fluorescence imaging guidance and ablate the lymph node with the enhanced photothermal effect, indicating the potential for selective treatment of metastatic lymph node.
This review describes the growing partnership between super-resolution imaging and plasmonics, by describing the various ways in which the two topics mutually benefit one another to enhance our understanding of the nanoscale world. First, localization-based super-resolution imaging strategies, where molecules are modulated between emissive and nonemissive states and their emission localized, are applied to plasmonic nanoparticle substrates, revealing the hidden shape of the nanoparticles while also mapping local electromagnetic field enhancements and reactivity patterns on their surface. However, these results must be interpreted carefully due to localization errors induced by the interaction between metallic substrates and single fluorophores. Second, plasmonic nanoparticles are explored as image contrast agents for both superlocalization and super-resolution imaging, offering benefits such as high photostability, large signal-to-noise, and distance-dependent spectral features but presenting challenges for localizing individual nanoparticles within a diffraction-limited spot. Finally, the use of plasmon-tailored excitation fields to achieve subdiffraction-limited spatial resolution is discussed, using localized surface plasmons and surface plasmon polaritons to create confined excitation volumes or image magnification to enhance spatial resolution.
One cationic BODIPY chromophore was synthesized and its complexation behaviour with the macrocyclic host cucurbit[7]uril (CB[7]) was studied using different spectroscopy techniques such as UV-vis absorption, steady-state and time-resolved fluorescence, (1)H NMR as well as DFT based quantum calculations. The dye showed formation of a 1 : 1 dye-CB[7] complex with improvement in the fluorescence intensity. These new results of the formation of moderate association of aqueous BODIPYs with the nontoxic host CB[7] may lead to promising applications of the dye molecule as a sensitive and efficient off-on mode fluorescent probe in chemical and biological studies.
In this Chapter the reader may not find exhaustive coverage of data on the sensing of all possible targets. This information is immense with the tendency of its exponential expansion day by day. The aim is different — to show the diversity of tasks and the diversity of possibilities for their solutions. Responding to intermolecular interactions, fluorescent dyes can recognize the medium and determine its physical parameters, such as temperature and pressure. Being incorporated into structures adsorbing gas molecules, they are able to identify the gas composition. Fluorescence sensors are also applicable in solutions, being efficient in the broadest possible range of target concentrations. Increasing the complexity of the target molecules requires adequate improvement of the binding and reporting properties of the sensors. Molecules of biological importance and especially proteins, nucleic acids and glycopolymers, are the targets, where the principles of biomolecular recognition are of great utility. Finally, we have to identify bacteria and viruses, achieving ultimate speed and ultimate sensitivity of their detection. This is also a resolvable task for fluorescence sensors.
Background: There has been a recent interest in the use of Indocyanine green (ICG) imaging. The aim of this study is to review our initial experience in liver surgery.
Methods: ICG fluorescent imaging was used in 15 patients undergoing surgical treatment of their liver tumors between 2015 and 2016. ICG imaging was initially performed, followed by intraoperative ultrasound (IOUS). Findings on fluorescence were compared with preoperative crosssectional imaging and IOUS.
Result: Sixty-two lesions were identified, with 34 located superficially and 28 deeply in the liver. While 13 patients underwent surgery for malignant liver metastases, two patients had operations for benign liver diseases. Seven patients underwent open or robotic liver resections, five laparoscopic microwave liver ablation, and three diagnostic laparoscopy. ICG identified all of the superficial lesions. IOUS identified 98% of all
lesions. The most benefit of ICG was in showing the margins of the superficial lesions in real-time and guiding surgical treatment, which was limited by IOUS.
Conclusion: This is the first North American study to evaluate the potential utility of ICG during liver surgery. Its major benefit seems to be in providing real-time feedback to the surgeon about the margins of superficial tumors for resection or ablation.
In recent years, photoredox catalysis has come to the forefront in organic chemistry as a powerful strategy for the activation of small molecules. In a general sense, these approaches rely on the ability of metal complexes and organic dyes to convert visible light into chemical energy by engaging in single-electron transfer with organic substrates, thereby generating reactive intermediates. In this Perspective, we highlight the unique ability of photoredox catalysis to expedite the development of completely new reaction mechanisms, with particular emphasis placed on multicatalytic strategies that enable the construction of challenging carbon-carbon and carbon-heteroatom bonds.
Highly thermal- and photo-stable NIR-absorbing heptamethine cyanine dyes were achieved with the use of fluorine-containing substituents.The prepared heptamethine cyanine dye, which has a tetrakis(pentafluorophenyl)borate as a counter anion and an N-ethyl-2,2,2-trifluoroacetamido group at the meso position, shows not only a high decomposition temperature (Tdt), but also significantly high photostability toward white LED irradiation.
Light provides a uniquely powerful stimulus to help visualize and/or perturb biological systems. The use of tissue penetrant near-IR wavelengths enables in vivo applications, however the design of molecules that function in this range remains a substantial challenge. Heptamethine cyanine fluorophores are already important tools for near-IR optical imaging. These molecules are susceptible to photobleaching through a photooxidative cleavage reaction. This review details efforts to define the mechanism of this reaction and two emerging fields closely tied to this process. In the first, efforts that slow photooxidation enable the creation of photobleaching resistant fluorophores. In the second, cyanine photooxidation has recently been employed as the cornerstone of a near-IR uncaging strategy. This review seeks to highlight the utility of mechanistic organic chemistry insights to help tailor cyanine scaffolds for new, and previously intractable, biological applications.
BODIPY laser dyes constitute a fascinating topic of research in modern photochemistry due to the large variety of options its chromophore offers, which is ready available for a multitude of synthetic routes. Indeed, in the literature one can find a huge battery of compounds based on the indacene core. The possibility of modulating the spectroscopic properties or inducing new photophysical processes by the substitution pattern of the BODIPY dyes has boosted the number of scientific and technological applications for these fluorophores. Along the following lines, I will overview the main results achieved in our laboratory with BODIPYs oriented to optoelectronic as well to biophotonic applications, stressing the more relevant photophysical issues to be considered in the design of a tailor-made BODIPY for a certain application and pointing out some of the remaining challenges.
In this article our aim is to review the supramolecularly assisted modulations in the properties of the organic chromophoric dyes, on their interactions with the macrocyclic host molecules, focusing on the possible uses of such modulated dye properties in different applications. We restrict ourselves on the modulations of two important properties of the chromophoric dyes, namely the fluorescence characteristics and the prototropic behavior, the properties that usually undergo large changes through host-guest inclusion complex formation of the dyes with the macrocyclic host molecules and show great prospects for their applications in diverse areas like sensors, catalysis, functional materials, electronic devices, pharmaceuticals, drug formulations, drug delivery, nanomedicines, and many others. To restrain the length of the article, our discussion has also been restricted to only two types of macrocyclic molecules, namely cyclodextrin ( CD) and cucurbit[n]uril (CBn) hosts, that are realized to be most promising cavitand molecules with regard to their influence in modulating the dye properties leading to their applications in the aforementioned areas. We have considered suitable examples from the dye-CD and dye-CBn systems as reported in the literature by different research groups including ours to substantiate the discussions on different aspects of the macrocyclic hosts and those on the modulations of the fluorescence and acid-base properties of the organic chromophoric dyes, bringing out their possible applications in different areas, as have been the main objectives of the present review. We strongly feel that this review article will showcase the titled theme, i. e. the supramolecularly assisted modulations in the properties of the chromophoric dyes, which can lead to different useful applications of the supramolecular host-guest systems, highlighting the immense prospects of such studies to explore the versatility of the supramolecular host-guest assemblies for their applications for the benefit of the mankind.
The electrophilic monofluorination with Selectfluor and nucleophilic trifluoromethylation with Ruppert-Prakesh reagent of the dimethyl, tetramethyl and pentamethyl boron-dipyrromethenes (BODIPY) 4, 3 and 2 are investigated. The monofluorinated dyes 7, 6 and 5 are synthesized with low yields (<30%), however the trifluoromethyl derivatives 8, 9 and 10 are obtained in moderate to high yields (~40-90%). All compounds are characterized by steady-state and time-resolved fluorescence spectroscopy, the photostability is investigated with Fluorescence Correlation Spectroscopy (FCS) and Total Internal Reflection microscopy (TIRF). Whereas monofluorination hardly affects the spectroscopic parameters of the unsubstituted ancestors but distinctly enhances the photostability, trifluoromethylation leads to a hypsochromic shift by up to 17 nm both in absorption and emission, slightly enhanced intersystem crossing and higher photostability as well. Further development of soft fluorination and trifluoromethylation methods is therefore highly desired.
Fluorescence is probably the most important optical readout mode in biological confocal microscopy, because it can be so much more sensitive and specific than absorbance or reflectance, and because it works so well with epi-illumination, which greatly simplifies scanner design. These advantages of fluorescence are critically dependent on the availability of suitable fluorophores that can either be tagged onto biological macromolecules to show their location, or whose optical properties are sensitive to the local environment. Despite the pivotal importance of good fluorophores, little is known about how to rationally design good ones. Whereas the concept of confocal microscopy is only a few decades old and nearly all the optical, electronic, and computer components to support it have been developed or redesigned in the last few years, the most popular fluorophores were developed more than a century ago (in the case of fluoresceins or rhodamines) or several billion years ago (in the case of phycobiliproteins). Moreover, whereas competition between commercial makers of confocal microscopes stimulates ardent efforts to refine the instrumentation, relatively few companies or academic scientists are interested in improving fluorophores.
For long-term performance, chemically robust materials are desired for organic solar cells (OSCs). Illuminating neat films of OSC materials in air and tracking the rate of absorption loss, or photobleaching, can quickly screen a material's photochemical stability. In this report, we photobleach neat films of OSC materials including polymers, solution-processed oligomers, solution-processed small molecules, and vacuum-deposited small molecules. Across the materials we test, we observe photobleaching rates that span 7 orders of magnitude. Furthermore, we find that the film morphology of any particular material impacts the observed photobleaching rate and that amorphous films photobleach faster than crystalline ones. In an extreme case, films of amorphous rubrene photobleach at a rate 2500 times faster than polycrystalline films. When we compare density to photobleaching rate, we find that stability increases with density. We also investigate the relationship between backbone planarity and chemical reactivity. The polymer PBDTTPD is more photostable than its more twisted and less-ordered furan derivative, PBDFTPD. Finally, we relate our work to what is known about the chemical stability of structural polymers, organic pigments, and organic light-emitting diode materials. For the highest chemical stability, planar materials that form dense, crystalline film morphologies should be designed for OSCs.
Fluorescence-based imaging techniques critically rely on bright and photostable probes for precise detection of biological molecules. Recently, a new class of multichromophoric probes based on fluorescent dendrimer nanoconjugates (FDNs) was developed for single molecule fluorescence microscopy (SMFM). FDNs are generated by covalent conjugation of multiple fluorescent dyes onto macromolecular polymeric scaffolds and show marked increases in brightness and long-term photostability relative to their single organic dye constituents. Multichromophoric probes, however, are generally known to suffer from transient fluorescence emission intensities and long excursions into dark states. To overcome these issues, photostabilizers can be added to bulk solution, though some small molecule additives may exhibit poor aqueous solubility or biological toxicity. In this work, we develop enhanced FDN derivatives by covalently linking a redox-active photostabilizer (Trolox) directly onto FDN molecular scaffolds. In one approach, multiple organic dyes (Cy5) and Trolox molecules are randomly distributed on dendritic scaffolds in tunable stoichiometric amounts, and in a second approach, Cy5 dyes are covalently linked to Trolox in a precise 1:1 stoichiometry followed by covalent attachment of Cy5-Trolox conjugates onto dendrimers. In all cases, FDN-Trolox conjugates show increases in photostability, brightness, and reduced fluctuations in transient fluorescent intensity relative to FDN probes. Bulk and single molecule photophysical data for FDN probes are compared to single self-healing dye systems such as Cy5-Trolox, and as a proof-of-principle demonstration, we use FDN-Trolox derivatives for bulk immunofluorescence imaging. Overall, our work suggests that self-healed multichromophoric systems such as FDN-Trolox probes present a useful strategy for increasing fluorescent probe photostability.
Indocyanine green (ICG) is a near-infrared optical dye approved by the Food and Drug Administration. ICG has been investigated as a simultaneous color and fluorescence-imaging tracer for the intraoperative identification of sentinel lymph nodes, but its use has recently expanded to include application as a photosensitizer for the local photodynamic/thermal treatment of identified lymph node metastases. The current study was designed to develop an ICG-loaded nanoemulsion as an effective agent for both the diagnosis and treatment of lymph node metastases. Incorporating the cationic lipid stearylamine (SA) together with ICG in the shell of nanoemulsions did not affect the loaded ICG concentration, but changed the surface charge of nanoemulsions from a negative to a positive value and improved the physical stability of nanoemulsions. Loading ICG into SA-incorporated nanoemulsions more effectively blocked the aggregation and degradation of ICG compared to loading in SA-free nanoemulsions. SA incorporation also enhanced tumor cell uptake of ICG-loaded nanoemulsions, resulting in greater cell killing upon light irradiation. After subcutaneous injection into the footpad of mice, SA-incorporated nanoemulsions increased the concentration of ICG accumulated in popliteal lymph nodes to a greater extent than SA-free nanoemulsions without affecting the kinetics of lymph node uptake of nanoemulsions. These multiple beneficial effects of incorporating SA in nanoemulsions are likely attributable to the electrostatic interaction between anionic ICG and cationic SA, as well as the change in the nanoemulsion surface charge from negative to positive. Our findings indicate that SA-incorporated nanoemulsions are promising ICG carriers for combined diagnosis and treatment of lymph node metastases.
Background:
Indocyanine green (ICG) is a dye used in medicine since the mid-1950s for a variety of applications in in cardiology, ophthalmology, and neurosurgery; however, its fluorescent properties have only recently been used in the intraoperative evaluation of tissue perfusion.
Method:
A literature review was conducted on the characterization and employment of ICG within the medical field. Historical and current context of ICG was examined while also considering implications for its future use.
Results:
ICG is a relatively nontoxic, unstable compound bound by albumin in the intravascular space until rapid clearance by the liver. It has widespread uses in hepatic, cardiac, and ophthalmologic studies, and its use in analyzing tissue perfusion and identifying sentinel lymph nodes in cancer staging is gaining popularity.
Conclusions:
ICG has myriad applications and poses low risk to the patient. Its historical uses have contributed to medical knowledge, and it is now undergoing investigation for quantifying tissue perfusion, providing targeted therapies, and intraoperative identification of neurovascular anatomy, ophthalmic structures, and sentinel lymph nodes. New applications of ICG may lead to reduction in postoperative wound-related complications, more effective ophthalmologic procedures, and better detection and treatment of cancer cells.