Excitation Of Fluorophores Research Articles

Gentle Rhodamines for Live-Cell Fluorescence Microscopy.

Rhodamines have been continuously optimized in brightness, biocompatibility, and color to fulfill the demands of modern bioimaging. However, the problem of phototoxicity caused by the excited fluorophore under long-term illumination has been largely neglected, hampering their use in time-lapse imaging. Here we introduce cyclooctatetraene (COT) conjugated rhodamines that span the visible spectrum and exhibit significantly reduced phototoxicity. We identified a general strategy for the generation of Gentle Rhodamines, which preserved their outstanding spectroscopic properties and cell permeability while showing an efficient reduction of singlet-oxygen formation and diminished cellular photodamage. Paradoxically, their photobleaching kinetics do not go hand in hand with reduced phototoxicity. By combining COT-conjugated spirocyclization motifs with targeting moieties, these Gentle Rhodamines compose a toolkit for time-lapse imaging of mitochondria, DNA, and actin, and synergize with covalent and exchangeable HaloTag labeling of cellular proteins with less photodamage than their commonly used precursors. Taken together, the Gentle Rhodamines generally offer alleviated phototoxicity and allow advanced video recording applications, including voltage imaging.

Spectrofluorimetric Method for Simultaneous Determination of Trimethoprim and Sulfamethoxazole with <i>O</i>-phthaladehyde Reagent by H-point Standard Addition Method

Simultaneous spectrofluorometric method described for the determination of trimethoprim (TMP) and sulfamethoxazole (SMZ) in pure and pharmaceutical preparations using H-point standard addition method (HPSAM) according to the reaction of nanograms of drugs with O-phthaladehyde (OPA) reagent to forms highly fluorescence compounds. The formed fluorophore excitation and emission at 342 and 458 nm, respectively, for OPA-TMP compound, at 424 and 508 nm, respectively, for OPA-SMZ compound under basic condition (pH 9.8) in the presence of 2-mercabtoethanol. A simple and accurate HPSAM is reported to resolve the overlapping in the fluorescence spectrum of these two drugs without prior separation of samples. The linear range was 100–1200 ng/mL for TMP and 100–1100 ng/mL for SMZ. The LOD and LOQ were 16.64 and 36.80 ng/mL, as well as 15.76 and 33.88 ng/mL for TMP and SMZ, respectively. The relative standard deviations and recovery percentages were 0.641% and 101.29% for TMP as well as 0.558% and 100.96% for SMZ, respectively. The procedure has been applied successfully in various pharmaceutical preparations. It was discovered that the experimental F- and t-values at a 95% confidence level were no higher than the theoretical values, showing that the HPSAM method is accurate and valid.

Image response-assisted volumetric reconstruction for simultaneous multi-color light-field microscopy

Light-field microscopy enables real-time volumetric imaging, offering substantial advantages for real-time fluorescence imaging. However, when applied to multi-color imaging, positional cross-talk between different fluorescent signals in the object space reduces reconstruction accuracy. Additionally, imaging each color through sequential excitation of fluorophores significantly compromises imaging speed. In this paper, an image response-assisted volumetric reconstruction method that unmixes multi-color fluorescence light-field images using pixel support derived from the light-field imaging response is proposed. This approach enables simultaneous multi-color imaging with significantly improved volumetric reconstruction accuracy. The correctness and effectiveness of the proposed method are validated through both simulations and experiments. The root-mean-square-error of multi-color volumetric reconstruction is reduced by 37.87 % on average compared with the simultaneous multi-color reconstruction methods obtained by simply combining single-pixel spectrum conversion methods and linear unmixing method in rapid-moving micro-particle observation, showcasing high accuracy simultaneous multi-color imaging performance. Volumetric imaging of motor neurons and whole-body cells of live dual-color zebrafish larvae at 20 Hz demonstrates the ability to be applied to real biomedical imaging.

Dinitrophenyl and NBD platforms based fluorescent sensors for nanomolar detection of zinc ions: Synthesis, zinc ions sensing and DFT studies

The design and synthesis of two new fluorescent probes based on dinitrophenyl (1) and 7-nitrobenz-2-oxa-1,3-diazole (2) platforms is reported. Both probes, containing bis(pyridin-2-ylmethyl)amine as a receptor unit can efficiently detect zin ions (Zn2+) in aqueous solution, with limit of detections of 124 nM and 1.06 µM, respectively. Probe 1 and 2 led to 3-fold and 2.4-fold fluorescent enhancements, respectively, with the addition of Zn2+ which suggested that working mechanism of these probes is based on the inhibition of the photoinduced electron transfer from the electron donating receptor towards the excited fluorophore. Studying binding stoichiometry by job’s plot suggested 1:1 complexation between the probe and Zn2+. Moreover, the formation of probes-Zn2+ complexes was validated by both FT-IR and 1H NMR wherein marked changes in the absorption frequencies as well as distinct downfield shifts in the resonance peaks of pyridine function along with the CH2 group linked directly to the tertiary nitrogen of the DPA unit were observed. Density functional theory (DFT) calculations suggested that the electronic density of the HOMO in 1 was concentrated on the linker between DPA and dinitrophenyl, whereas the density of the LUMO was concentrated on dinitrophenyl moiety. Upon Zn2+ binding, the electronic density in HOMO and LUMO become more delocalized. However, in case of probe 2 the delocalization of electronic density does not appreciably occur. Based on DFT simulations, both sensors form thermodynamic stable complexes with Zn2+ ions. However, sensor 2 exhibited slightly better binding preference toward Zn2+ as compared to 1. The PET based probes exhibited good selectivity to detect Zn2+ in the presence of a variety of competing metal cations. These sensors have higher sensitivity and would be able to detect chronic level of Zn2+ in the freshwater (>1.84 μM) as permitted by the US EPA.

A Water‐Soluble Fluorescent Probe Derived from Pyridine‐2,6‐Dicarboxylic Acid: Synthesis, Interaction with Ions, and Two‐Photon Microscopy

AbstractTwo‐photon excited fluorescence is a process in which the excited fluorophore relaxes and emits fluorescence after two‐photon absorption. Combined with appropriate two‐photon fluorescent probes, two‐photon microscopy enables observation deep inside tissue without damage. Former reports showed that the two‐photon absorption cross sections of donor–acceptor (D–A) dipoles, as well as D‐π‐D and D–A–D quadrupoles can be strengthened by using strong D–A groups. In this study, a novel water‐soluble fluorescent probe with D‐π‐A structure containing pyridine‐2,6‐dicarboxylic acid unit was synthesized. The maximum excitation wavelength and emission wavelength of the probe in aqueous solution were 328 nm and 471 nm, respectively. The fluorescence emitted by the probe can be quenched by various metal cations. The pyridyl N and carboxyl O in the probe formed chelates with Cu2+, which resulted in a fluorescence quenching response, and showed a linear relationship between fluorescent intensity and Cu2+ concentration with wide linear range and low limit of detection. CCK‐8 tests showed the probe had no obvious toxicity to 4T1 cells in the range of 0.4–50 μmol L−1, and the cell survival rate was above 86 %. The probe had a large two‐photon absorption cross section ranging from 680 nm to 820 nm. The two‐photon absorption cross section reached 100 GM at 760 nm of excitation wavelength. By means of microscopic imaging of 4T1 cells stained with the probe, one‐photon fluorescence represented blue emission, while two‐photon fluorescence represented bright green emission. The probe was proved to have good cell permeability, and preferred to target nucleus, showing application potential in two‐photon fluorescence imaging technology.

High-throughput fluorescence lifetime imaging flow cytometry

Flow cytometry is a vital tool in biomedical research and laboratory medicine. However, its accuracy is often compromised by undesired fluctuations in fluorescence intensity. While fluorescence lifetime imaging microscopy (FLIM) bypasses this challenge as fluorescence lifetime remains unaffected by such fluctuations, the full integration of FLIM into flow cytometry has yet to be demonstrated due to speed limitations. Here we overcome the speed limitations in FLIM, thereby enabling high-throughput FLIM flow cytometry at a high rate of over 10,000 cells per second. This is made possible by using dual intensity-modulated continuous-wave beam arrays with complementary modulation frequency pairs for fluorophore excitation and acquiring fluorescence lifetime images of rapidly flowing cells. Moreover, our FLIM system distinguishes subpopulations in male rat glioma and captures dynamic changes in the cell nucleus induced by an anti-cancer drug. FLIM flow cytometry significantly enhances cellular analysis capabilities, providing detailed insights into cellular functions, interactions, and environments.

RPA-CRISPR/Cas12a-mediated isothermal amplification for rapid detection of Phytopythium helicoides.

Phytopythium helicoides, which belongs to the algae (Chromista), Oomycota, Pythiales, Pythiaceae and Phytophthora, is a quarantine pathogen that causes brown rot of fruits, stem rot and root rot, along withother symptoms that can damage severaltreespecies in urban landscaping. Therefore, disease management requires rapid and accurate diagnosis. The present study used recombinase polymerase amplification (RPA) in conjunction with the CRISPR/Cas12a system to identifyP. helicoides. The test exhibited high specificity and sensitivity and could detect10 pg.µL-1 of P. helicoides genomic DNA at 37 ℃ within 20 minutes. The test results were visible by excitation of fluorophores by blue light. This groundbreaking testis able to detect P.helicoides in artificially inoculated Rhododendron leaves. The RPA-CRISPR/Cas12a detection assay developed in this study is characterizedby its sensitivity, efficiency, and convenience. Early detection and control of P. helicoides is crucial for the protection of urban green coverspecies.

Mechanisms of formation of heteroassociates of ATP with ag+ ions

Emission and excitation spectra of fluorescence (FL) were studied for aqueous solutions of ATP with AgNO3 salt at different concentrations of silver nitride and temperatures. It is shown that with increasing Ag+ content and temperature, a complex nature of FL quenching occurs due to the formation of ATP - Ag+ complexes. This quenching occurs when the position of the emission bands and FL excitation of the ATP fluorophore is preserved, and the quenching corresponds to the Stern-Volmer linear dependence. At the same time, the values of KSV and kq and their dependence on the temperature indicate a static quenching mechanism. The binding parameter n indicates that the metal–ligand complexes have a stoichiometric ratio of 1:1, and KA decreases with increasing temperature. In the heteroassociates, energy is transferred by the FRET mechanism, and binding of components is mainly realized due to electrostatic and van der Waals interaction.

Fluorophore-Induced Plasmonic Current Generation from Copper Nanoparticle Films.

We describe the process of generating a fluorophore-induced plasmonic current (FIPC) from copper nanoparticle films. Previous work and the literature have shown that excited near-field fluorophores are able to plasmonically couple with metal nanoparticle films (MNFs), inducing surface plasmons in the films. These induced surface plasmons are then in turn able to generate a directly measurable electrical current across the film. These generated currents have been quantified and detected in noble metal films, such as those made from Ag and Au, but due to the cost of such films, there has been a push to use lower cost materials for FIPC. Previous work has detailed the use of gold, silver, and aluminum films for these purposes, and in this paper, we will subsequently examine the ability of thermally deposited copper films to generate FIPC when in close proximity to excited near-field fluorophores. We report the effects of copper film thickness, the effects of light polarization and solution conductance, and the effects of metal-enhanced fluorescence (MEF) emission on the generation of plasmonic current.

Stretchable Photonic Crystal-Assisted Glycoprotein Identification for Ovarian Cancer Diagnosis.

Photonic crystals with specific wavelengths can realize surface-enhanced excitation and emission intensities of fluorophores and enhance the fluorescence signals of fluorescent molecules. Herein, stretchable photonic crystals with good mechanochromic properties provide continuously adjustable forbidden wavelengths by stretching to change the lattice spacing, with reflectance peaks blue-shifted up to 110 nm to match indicators of different wavelengths and produce differentiated optical enhancement effects. Glycoproteins are significantly identified as clinical markers. However, the wide participation of glycoproteins in various life processes poses enormous complexity and critical challenges for rapid, facile, high-throughput, and accurate clinical analysis or health assessment. In this work, we proposed a stretchable photonic crystal-assisted glycoprotein identification approach for early ovarian cancer diagnosis. Stretchable photonic crystals can provide rich optical information to efficiently identify glycoproteins in complex matrices. A double-indicator fluorescence sensor was designed to respond to the protein trunk and oligosaccharide segment of glycoproteins separately for improved recognition accuracy. Seven typical glycoproteins could be discriminated from proteins, saccharides, or mixture interferents. Clinical ovarian cancer samples for early, intermediate, and advanced ovarian cancer and healthy subjects were verified with 100% accuracy. This strategy of stretchable photonic crystal-assisted glycoprotein identification provides an effective method for accurate, rapid ovarian cancer diagnosis and timely clinical treatment.

A cost-effective approach to measurements of fluorophore temperature sensitivity and temperature change with reasonable accuracy

The demand for measuring fluorophore temperature sensitivity and temperature change in chemical or biological samples has spurred the search for effective methods. While infrared (IR) light-based thermal devices are popular, they are limited to surface temperature measurement. Fluorescence-based thermometry, which utilizes intensity, lifetime, polarization, and spectrum change, provides the temperature information directly from the samples and can have high temporal and spatial resolution. However, measuring fluorescence can be tricky and expensive. A cost-effective approach to achieving reasonable accuracy is highly desired. This study introduces such an approach, employing a light-emitting diode (LED) for fluorophore excitation and a laser diode (LD) for sample heating, with a phone camera recording fluorescence changes. A data processing method converts the video into digital data, processed through digital filters. Utilizing a small-volume cuvette enhances heating efficiency. This study serves as a practical guide for inexperienced individuals, including students, instructors, and researchers, facilitating entry into the field and navigating the complexities of fluorescence-based thermometry.

Applications of multiphoton microscopy in imaging cerebral and retinal organoids.

Cerebral organoids, self-organizing structures with increased cellular diversity and longevity, have addressed shortcomings in mimicking human brain complexity and architecture. However, imaging intact organoids poses challenges due to size, cellular density, and light-scattering properties. Traditional one-photon microscopy faces limitations in resolution and contrast, especially for deep regions. Here, we first discuss the fundamentals of multiphoton microscopy (MPM) as a promising alternative, leveraging non-linear fluorophore excitation and longer wavelengths for improved imaging of live cerebral organoids. Then, we review recent applications of MPM in studying morphogenesis and differentiation, emphasizing its potential for overcoming limitations associated with other imaging techniques. Furthermore, our paper underscores the crucial role of cerebral organoids in providing insights into human-specific neurodevelopmental processes and neurological disorders, addressing the scarcity of human brain tissue for translational neuroscience. Ultimately, we envision using multimodal multiphoton microscopy for longitudinal imaging of intact cerebral organoids, propelling advancements in our understanding of neurodevelopment and related disorders.

Simultaneous Two- and Three-Photon Deep Imaging of Autofluorescence in Bacterial Communities.

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

Structure-activity relationships of aniline-based squaraines for distinguishable staining and bright two-photon fluorescence bioimaging in plant cells.

An organelle-selective vision provides insights into the physiological response of plants and crops to environmental stresses in sustainable agriculture ecosystems. Biological applications often require two-photon excited fluorophores with low phototoxicity, high brightness, deep penetration, and tuneable cell entry. We obtained three aniline-based squaraines (SQs) tuned from hydrophobic to hydrophilic characteristics by modifying terminal pendant groups and substituents, and investigated their steady-state absorption and far-red-emitting fluorescence properties. The SQs exhibited two-photon absorption (2PA) ranging from 750 to 870 nm within the first biological spectral window; their structure-property relationships, corresponding to the 2PA cross sections (δ2PA), and structure differences were demonstrated. The maximum δ2PA value was ∼1220 GM at 800 nm for hydrophilic SQ3. Distinct biological staining efficiency and selective SQ bioimaging were evaluated utilizing the onion epidermal cell model. Contrary to the hydrophobic SQ1 results in the onion epidermal cell wall, amphiphilic SQ2 tagged the vacuole and nucleus and SQ3 tagged the vacuole. Distinguishable staining profiles in the roots and leaves were achieved. We believe that this study is the first to demonstrate distinct visualisation efficiency induced by the structure differences of two-photon excited SQs. Our results can help establish the versatile roles of novel near-infrared-emitting SQs in biological applications.

High NA and polarization-insensitive ultra-broadband achromatic metalens from 500 to 1050 nm for multicolor two-photon endomicroscopy imaging.

Multicolor two-photon endomicroscopy has become a highly competitive tool for functional imaging in biomedical researches. However, to make the imaging system miniature and applicable for freely behaving animal brain activity, metalenses have received much attention in compact imaging systems. For high resolution multicolor imaging and maximizing fluorescence collection, there is a challenge metalenses faced to achieve large numerical aperture (NA) and focus the NIR excitation and VIS emission lights of multiple fluorophores to the same distance simultaneously because of the limitation of the group delay range of the meta-units. In this paper, we proposed a high NA and polarization-insensitive ultra-broadband achromatic metalens specifically for achromatically focusing the excitation and emission light of multiple fluorophores commonly used in neuroscience studies. TiO2 and Si meta-unit libraries composed of heights, widths and the corresponding phase and group delay were constructed, and the optimal meta-units were selected by particle swarm optimization algorithm to engineer the dispersion of metalens in the VIS band and NIR band, respectively. Combining dispersion engineering with spatial multiplexing, the proposed metalens achieved the maximal effective NA up to 0.8 and large achromatic bandwidth ranging from 500 nm to 1050 nm, which exhibited the coefficient of variation of focal lengths was only 3.41%. The proposed achromatic metalens could successfully achromatically focus different fluorescence with any polarization, which was suitable for most fluorophores. Our results firmly establish that the proposed metalens can open the door to high resolution and minimally invasive multicolor two-photon functional imaging in intravital deep brain.

Near-infrared co-illumination of fluorescent proteins reduces photobleaching and phototoxicity

Here we present a method to reduce the photobleaching of fluorescent proteins and the associated phototoxicity. It exploits a photophysical process known as reverse intersystem crossing, which we induce by near-infrared co-illumination during fluorophore excitation. This dual illumination method reduces photobleaching effects 1.5–9.2-fold, can be easily implemented on commercial microscopes and is effective in eukaryotic and prokaryotic cells with a wide range of fluorescent proteins.

Visible-light Excited and Highly Photostable Organic Fluorophore with dual-state Emission.

Organic fluorophores with dual-state emission (DSE) are rare or difficult to observe because most of them display either aggregation-induced emission (AIE) or aggregation-caused quenching (ACQ). Amazing works have been accomplished, yet most of the DSE compounds were excited by UV light which limits their wide application in bioimaging. In this work, we achieved a visible-light excited DSE fluorophore and realized its imaging in SKOV-3 cells and zebrafish. The naphtho[2',3':4,5]imidazo[1,2-a]pyridine (NIP) core ensures its emission in dilute solution. Meanwhile, the twisted phenyl ring blocks fluorescence quenching induced by the π-π stacking and leads to the emission of the solid. The fluorescence intensity is steady even after 6h of continuous intense sunlight. More importantly, photostability of NIP in cells is much better than commercial dye (mitochondrial green).

UV generation via periodically poled MgO:LiTaO3 circular waveguide crystal and diode-based master oscillator power amplifier.

Lasers with emission wavelengths in the near-ultraviolet (UV) spectral range have been used in many applications across various fields, and the demand for these lasers has been on the rise. For example, in medicine, near-UV light has been used for fluorophore excitation. Although laser diodes emitting in this region exist, single longitudinal mode lasers emitting at 380nm with high optical power are limited. One of the solutions to this problem is the use of second harmonic generation by a non-linear crystal. In this work, single-longitudinal-mode laser emission at 380.5nm with an optical power of up to 13mW has been achieved. The emission was realized by frequency doubling using a periodically poled circular waveguide crystal of stoichiometric L i T a O 3 doped with MgO (PPMgSLT) pumped by a master oscillator power amplifier with optical power up to 5W. A distributed Bragg reflector ridge waveguide laser diode at 761nm was used as the master oscillator and a tapered amplifier as the power amplifier.

Realization of Pyreno[4,5-d]imidazole-based HLCT emissions through tuning the energy level of the charge-transfer state

Organic light-emitting diodes (OLEDs) have lately been used in tablet displays, for instance in mobile phones and televisions, and are considered to be the next generation of displays and solid-state lighting due to their light weight, high flexibility, and energy efficiency. Hybridized local and charge-transfer (HLCT) excited state fluorophores, capable of making full use of excitons by reverse intersystem crossing from high-lying triplet states to singlet states, have drawn increasing attention in OLED applications. The HLCT state can be efficiently achieved by constructing D-π-A type molecules, whose characteristics of their excited state are widely influenced by the difference of torsion angles between donor (D) and acceptor (A). Herein, pyreno[4,5-d]imidazole and triphenylamine were selected as the electron acceptor and electron donor, respectively. Based on this molecular design, two D-π-A type molecules, pTPA-PyI and mTPA-PyI were designed and synthesized with different torsion angles between D and A. In addition, the introduction of fluorine atoms in molecules could provide more and stronger charge transfer (CT) state components into the excited states of the molecule, which could facilitate the formation of HLCT states with improving exciton utilization of the molecule. As a result, pTPA-PyI with linear molecular geometry possessed HLCT properties, which could achieve a large yield of singlet exciton formation. The non-doped pTPA-PyI-based OLED demonstrated steady blue emission with a peak at 460 nm and CIE coordinates of (0.15, 0.20). The highest current efficiency was 7.7 cd A−1, the maximum brightness was 48637 cd m−2, and the external quantum efficiency (EQE) was 6.38%. When the brightness was 1000 cd m−2, the efficiency roll-off was incredibly low as 0.6%, yet the EQE of 6.34% was still achievable. Additionally, it possesses a high exciton-utilization efficiency of up to 97%. However, the locally excited (LE) and CT states of mTPA-PyI could not sufficiently hybridize due to its larger torsion angle between D and A with the smaller dipole moment and the interrupted conjugation structure of the molecule which led to high energy level of CT state. As a result, by altering the torsion angle between D and A, it is feasible to change the constituents and levels of the LE and CT states in order to actualize the HLCT feature.

FRET Based Biosensor: Principle Applications Recent Advances and Challenges.

Förster resonance energy transfer (FRET)-based biosensors are being fabricated for specific detection of biomolecules or changes in the microenvironment. FRET is a non-radiative transfer of energy from an excited donor fluorophore molecule to a nearby acceptor fluorophore molecule. In a FRET-based biosensor, the donor and acceptor molecules are typically fluorescent proteins or fluorescent nanomaterials such as quantum dots (QDs) or small molecules that are engineered to be in close proximity to each other. When the biomolecule of interest is present, it can cause a change in the distance between the donor and acceptor, leading to a change in the efficiency of FRET and a corresponding change in the fluorescence intensity of the acceptor. This change in fluorescence can be used to detect and quantify the biomolecule of interest. FRET-based biosensors have a wide range of applications, including in the fields of biochemistry, cell biology, and drug discovery. This review article provides a substantial approach on the FRET-based biosensor, principle, applications such as point-of-need diagnosis, wearable, single molecular FRET (smFRET), hard water, ions, pH, tissue-based sensors, immunosensors, and aptasensor. Recent advances such as artificial intelligence (AI) and Internet of Things (IoT) are used for this type of sensor and challenges.