Exploring the effect of photobiomodulation and gamma visual stimulation induced by 808 nm and visible LED in Alzheimer's disease mouse model.

Although photobiomodulation (PBM) and gamma visual stimulatqion (GVS) have been overwhelmingly explored in the recent time as a possible light stimulation (LS) means of Alzheimer's disease (AD) therapy, their effects have not been assessed at once. In our research, the AD mouse model was stimulated using light with various parameters [continuous wave (PBM) or 40 Hz pulsed visible LED (GVS) or 40 Hz pulsed 808 nm LED (PBM and GVS treatment)]]. The brain slices collected from the LS treated AD model mice were evaluated using (i) fluorescence microscopy to image thioflavine-S labeled amy-loid-ß (Aß) plaques (the main hallmark of AD), or (ii) two-photon excited fluorescence (TPEF) imaging of unlabeled Aß plaques, showing that the amount of Aß plaques was reduced after LS treatment. The imaging results correlated well with the results of Morris water maze (MWM) test, which demonstrated that the spatial learning and memory abilities of LS treated mice were noticeably higher than those of untreated mice. The LS effect was also assessed by in vivo nonlinear optical imaging, revealing that the cerebral amyloid angiopathy decreased spe-cifically as a result of 40 Hz pulsed 808 nm irradiation, on the contrary, the angiopathy reversed after visible 40 Hz pulsed light treatment. The obtained results provide useful reference for further optimization of the LS (PBM or GVS) parameters to achieve efficient phototherapy of AD.

Xanthene, cyanine, oxazine and BODIPY: the four pillars of the fluorophore empire for super-resolution bioimaging.

In the realm of biological research, the invention of super-resolution microscopy (SRM) has enabled the visualization of ultrafine sub-cellular structures and their functions in live cells at the nano-scale level, beyond the diffraction limit, which has opened up a new window for advanced biomedical studies to unravel the complex unknown details of physiological disorders at the sub-cellular level with unprecedented resolution and clarity. However, most of the SRM techniques are highly reliant on the personalized special photophysical features of the fluorophores. In recent times, there has been an unprecedented surge in the development of robust new fluorophore systems with personalized features for various super-resolution imaging techniques. To date, xanthene, cyanine, oxazine and BODIPY cores have been authoritatively utilized as the basic fluorophore units in most of the small-molecule-based organic fluorescent probe designing strategies for SRM owing to their excellent photophysical characteristics and easy synthetic acquiescence. Since the future of next-generation SRM studies will be decided by the availability of advanced fluorescent probes and these four fluorescent building blocks will play an important role in progressive new fluorophore design, there is an urgent need to review the recent advancements in designing fluorophores for different SRM methods based on these fluorescent dye cores. This review article not only includes a comprehensive discussion about the recent developments in designing fluorescent probes for various SRM techniques based on these four important fluorophore building blocks with special emphasis on their effective integration into live cell super-resolution bio-imaging applications but also critically evaluates the background of each of the fluorescent dye cores to highlight their merits and demerits towards developing newer fluorescent probes for SRM.

Recent advances in plasmon-enhanced luminescence for biosensing and bioimaging.

Plasmon-enhanced luminescence (PEL) is a unique photophysical phenomenon in which the interaction between luminescent moieties and metal nanostructures results in a marked luminescence enhancement. PEL offers several advantages and has been extensively used to design robust biosensing platforms for luminescence-based detection and diagnostics applications, as well as for the development of many efficient bioimaging platforms, enabling high-contrast non-invasive real-time optical imaging of biological tissues, cells, and organelles with high spatial and temporal resolution. This review summarizes recent progress in the development of various PEL-based biosensors and bioimaging platforms for diverse biological and biomedical applications. Specifically, we comprehensively assessed rationally designed PEL-based biosensors that can efficiently detect biomarkers (proteins and nucleic acids) in point-of-care tests, highlighting significant improvements in the sensing performance upon the integration of PEL. In addition to discussing the merits and demerits of recently developed PEL-based biosensors on substrates or in solutions, we include a brief discussion on integrating PEL-based biosensing platforms into microfluidic devices as a promising multi-responsive detection method. The review also presents comprehensive details about the recent advances in the development of various PEL-based multi-functional (passive targeting, active targeting, and stimuli-responsive) bioimaging probes, highlighting the scope of future improvements in devising robust PEL-based nanosystems to achieve more effective diagnostic and therapeutic insights by enabling imaging-guided therapy.

Long-Term Repeatable In Vivo Monitoring of Amyloid-ß Plaques and Vessels in Alzheimer's Disease Mouse Model with Combined TPEF/CARS Microscopy.

Long-term, repeatable monitoring of the appearance and progress of Alzheimer's disease (AD) in real time can be extremely beneficial to acquire highly reliable diagnostic insights, which is crucial for devising apt strategies towards effective AD treatment. Herein, we present an optimized innovative cranial window imaging method for the long-term repeatable imaging of amyloid-ß (Aß) plaques and vessels in an AD mouse model. Basically, two-photon excitation fluorescence (TPEF) microscopy was used to monitor the fluorescently labeled Aß plaques, whereas the label-free blood vessels were studied using coherent anti-Stokes Raman scattering (CARS) microscopy in the live in vivo AD mouse model. It was possible to clearly observe the Aß deposition and vascular structure in the target cortex localization for 31 weeks in the AD mouse model using this method. The combined TPEF/CARS imaging studies were also instrumental in realizing the relationship between the tendency of Aß deposition and ageing. Essentially, the progression of cerebral amyloid angiopathy (CAA) in the AD mouse model was quantitatively characterized, which revealed that the proportion Aß deposition in the unit vessel can increase from 13.63% to 28.80% upon increasing the age of mice from 8 months old to 14 months old. The proposed imaging method provided an efficient, safe, repeatable platform with simple target localization aptitude towards monitoring the brain tissues, which is an integral part of studying any brain-related physiological or disease conditions to extract crucial structural and functional information.

Improving the performance of rapid lifetime determination for wide-field time-gated imaging in live cells.

In biological research, rapid wide-field fluorescence lifetime imaging has become an important imaging tool. However, the biological samples with weak fluorescence signals and lower sensitivity often suffer from very low precision in lifetime determinations which restricts its widespread utilization in many bioimaging applications. To address this issue, a method is presented in this paper to substantially enhance the precision of rapid lifetime determination (RLD). It expedites the discrimination of fluorescence lifetimes, even for the weak signals coming from the cells, stained with long-lived biocompatible AIS/ZnS QDs. The proposed method works in two phases. The first phase deals with the systematic noise analysis based on the signal and contrast of the images in a time-gated imaging system, wherein acquiring the high-quality imaging data through optimization of hardware parameters improves the overall system performance. In the second phase, the chosen images are treated using total variation denoising method combined with the Max/Min filtering method for extracting the region of interest to reconstruct the intensity images for RLD. We performed several experiments on live cells to demonstrate the improvements in imaging performance by the systematic optimizations and data treatment. Obtained results demonstrated a great enhancement in signal-to-noise and contrast-to-noise ratios beside witnessing an obvious improvement in RLD for weak signals. This approach can be used not only to improve the quality of time-gated imaging data but also for efficient fluorescence lifetime imaging of live biological samples without compromising imaging speed and light exposure.

Synergistic photobiomodulation with 808-nm and 1064-nm lasers to reduce the ß-amyloid neurotoxicity in the in vitro Alzheimer's disease models.

Background: In Alzheimer's disease (AD), the deposition of ß-amyloid (Aß) plaques is closely associated with the neuronal apoptosis and activation of microglia, which may result in the functional impairment of neurons through pro-inflammation and over-pruning of the neurons. Photobiomodulation (PBM) is a non-invasive therapeutic approach without any conspicuous side effect, which has shown promising attributes in the treatment of chronic brain diseases such as AD by reducing the Aß burden. However, neither the optimal parameters for PBM treatment nor its exact role in modulating the microglial functions/activities has been conclusively established yet. Methods: An inflammatory stimulation model of Alzheimer's disease (AD) was set up by activating microglia and neuroblastoma with fibrosis ß-amyloid (fAß) in a transwell insert system. SH-SY5Y neuroblastoma cells and BV2 microglial cells were irradiated with the 808- and 1,064-nm lasers, respectively (a power density of 50 mW/cm2 and a dose of 10 J/cm2) to study the PBM activity. The amount of labeled fAß phagocytosed by microglia was considered to assess the microglial phagocytosis. A PBM-induced neuroprotective study was conducted with the AD model under different laser parameters to realize the optimal condition. Microglial phenotype, microglial secretions of the pro-inflammatory and anti-inflammatory factors, and the intracellular Ca2+ levels in microglia were studied in detail to understand the structural and functional changes occurring in the microglial cells of AD model upon PBM treatment. Conclusion: A synergistic PBM effect (with the 808- and 1,064-nm lasers) effectively inhibited the fAß-induced neurotoxicity of neuroblastoma by promoting the viability of neuroblastoma and regulating the intracellular Ca2+ levels of microglia. Moreover, the downregulation of Ca2+ led to microglial polarization with an M2 phenotype, which promotes the fAß phagocytosis, and resulted in the upregulated expression of anti-inflammatory factors and downregulated expression of inflammatory factors.

Super-resolution Microscopy for Biological Imaging.

Studying the ultra-fine structures and functions of the subcellular organelles and exploring the dynamic biological events in depth are the key issues in contemporary biological research. Fluorescence bio-imaging has been used to study cell biology for decades. However, the structures and functions of the subcellular organelles which fall under the diffraction limit are still not explored fully at a nanoscale level. Several super-resolution microscopy (SRM) techniques have been devised over the years which can be utilized to overcome diffraction limit. These techniques have opened a new window in biological research. However, SRM methods are highly vulnerable to the lack of appropriate fluorophores and other sophisticated technical considerations. Therefore, this chapter briefly summarizes the basic principles of various SRM methods which have been frequently utilized in biological imaging. The chapter not only gives an overview of the technical advantages and drawbacks about using different SRM techniques for bio-imaging applications but also briefly articulates the nitty-gritties of selecting a proper fluorescent probe for a specific SRM experiment with biological samples.

AIE-active two-photon fluorescent nanoprobe with NIR-II light excitability for highly efficient deep brain vasculature imaging.

Aggregation induced emission (AIE)-active bright two-photon fluorescent probes with second near-infrared (NIR-II) light excitability can be used for efficient brain bioimaging studies, wherein the fabrication of water-dispersible nanoparticles by encapsulating the hydrophobic probes with amphiphilic polymer holds the key to ensuring biocompatibility and in vivo adaptability. However, barely any study has evaluated the structural requirements that can substantially affect the water-dispersible nanoparticle formation ability of an organic AIE-active dye with amphiphilic polymers. The present study systematically assessed the structural dependency of a well-known acrylonitrile based AIE system/fluorogenic core upon the formation of water-dispersible nanoparticles and elucidated how the structural modifications can impact the in vivo two-photon imaging. Methods: A total of four acrylonitrile-based aggregation induced emission (AIE)-active two-photon (TP) fluorescent probes (AIETP, AIETP C1, AIETP C2 and AIETP C3) have been judiciously designed and synthesized with structural variations to realize how the structural alterations could substantially influence the water-dispersible nanoparticle formation ability (with amphiphilic polymers) and photo-stability to impact the in vivo imaging. Results: It has been found that the incorporation of the phenyl-thiazole unit in AIETP, AIETP C2 and AIETP C3 facilitated the formation of water-dispersible nanoparticles (NPs) with amphiphilic polymers (Pluronic F127) whereas the presence of only phenyl moiety instead in AIETP C1 could not meet the suitable condition to form the NPs with good aqueous dispersibility. Rationally designed AIETP NPs that exhibited higher brightness, improved photostability and good two-photon absorption cross section was successfully employed for in vivo brain vasculature imaging. Conclusions: Robust noninvasive 2D and 3D two-photon (NIR-II light, 1040 nm) brain vasculature imaging with beneficial attributes such as outstanding penetration depth (800 µm) and exceptional spatial resolution (1.92 µm), were achieved by utilizing AIETP NPs in this study.

Solo Smart Fluorogenic Probe for Potential Cancer Diagnosis and Tracking in Vivo Tumorous Lymphatic Systems via Distinct Emission Signals.

A versatile twisted-intramolecular-charge-transfer (TICT)-based near-infrared (NIR) fluorescent probe (L) has been judiciously designed and synthesized that could be utilized for potential cancer diagnosis and to track lymph node(s) in mice through distinct emission signals. Essentially, the probe rendered the capability to preferentially recognize the cancer cells over the noncancer cells by polarity-guided lipid droplet specific differential bioimaging (in green emission channel) studies. The probe also exhibited selective turn-on fluorescence response toward HSA/BSA in physiological media (aqueous PBS buffer; pH 7.4) at far-red/NIR regions, because of the 1:1 chelation between the probe and HSA/BSA. Therefore, the fluorescent probe was then maneuvered to track the draining lymphatic system and sentinel lymph node in tumor mice model by fluorescence imaging (NIR/deep-red channel), wherein the accumulated albumin protein in the draining tumor lymphatic system facilitated the in situ formation of the fluorescent albumin-L complex.

Redefining the photo-stability of common fluorophores with triplet state quenchers: mechanistic insights and recent updates.

Light microscopy can offer certain advantages over electron microscopy in terms of acquiring detailed insights into 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 acquiring 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 yields, 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 considering contemporary literature reports and applications.

Exploring the potential of a urea derivative: an AIE-luminogen and its interaction with human serum albumin in aqueous medium.

A urea derivative L1 exhibits Aggregation-Induced Emission (AIE) activity in an acetonitrile-water mixed solvent. The aggregation phenomenon has been corroborated by microscopy and light scattering studies. The ligand (L1) also displays a selective turn-on fluorescence response towards human serum albumin (HSA) in 100% aqueous medium over various other comparable proteins (even bovine serum albumin (BSA)) and enzymes. The weakly emissive probe L1 showed a substantial increase in emission intensity upon binding with HSA through electrostatic interactions. The good linear relationship between the fluorescence enhancement (I/I0 - 1) and the concentration of HSA provided the scope to attain an impressive detection limit as low as 5 µg mL-1. A drug displacement experiment and molecular docking study were employed to ascertain the likely protein (HSA)-ligand binding interactions.

Twisted-Intramolecular-Charge-Transfer-Based Turn-On Fluorogenic Nanoprobe for Real-Time Detection of Serum Albumin in Physiological Conditions.

Two cyanine-based fluorescent probes, ( E)-2-(4-(diethylamino)-2-hydroxystyryl)-3-ethyl-1,1-dimethyl-1 H-benzo[ e]indol-3-ium iodide (L) and ( E)-3-ethyl-1,1-dimethyl-2-(4-nitrostyryl)-1 H-benzo[ e]indol-3-ium iodide (L1), have been designed and synthesized. Of these two probes, the twisted-intramolecular-charge-transfer (TICT)-based probe, L, can preferentially self-assemble to form nanoaggregates. L displayed a selective turn-on fluorescence response toward human and bovine serum albumin (HSA and BSA) in ∼100% aqueous PBS medium, which is noticeable with the naked eye, whereas L1 failed to sense these albumin proteins. The selective turn-on fluorescence response of L toward HSA and BSA can be attributed to the selective binding of probe L with HSA and BSA without its interfering with known drug-binding sites. The specific binding of L with HSA led to the disassembly of the self-assembled nanoaggregates of L, which was corroborated by dynamic-light-scattering (DLS) and transmission-electron-microscopy (TEM) analysis. Probe L has a limit of detection as low as ∼6.5 nM. The sensing aptitude of probe L to detect HSA in body fluid and an artificial-urine sample has been demonstrated.

Al3+ sensing through different turn-on emission signals vis-à-vis two different excitations: Applications in biological and environmental realms.

A rationally designed Schiff base chemosensor (L) could render specific detection of Al3+ ions with two distinct turn-on emission signals, separated by over 100 nm upon excitation at two different wavelengths. The utility of the probe lies in facilitating sensing in 80% aqueous medium with an emission close to 600 nm via an intramolecular charge transfer (ICT) mechanism. The biocompatible and cell permeable probe could readily sense Al3+ in live HeLa cells as well. The affinity of the probe for Al3+could be leveraged to specifically study DNA- Al3+ interaction in solution.

A ratiometric fluorogenic probe for the real-time detection of SO32- in aqueous medium: application in a cellulose paper based device and potential to sense SO32- in mitochondria.

A new water soluble and fluorogenic probe (L) that can demonstrate a specific ratiometric detection of a SO2 derivative (SO32-) in 100% aqueous medium and live cells has been designed and synthesized. The detection process can be visualized by the naked eye, as the orange-red fluorescence of L turns into a strong blue fluorescence upon interaction with SO32-. L displayed several beneficial attributes such as detection in complete aqueous medium, extremely fast response time along with high selectivity and sensitivity. The ratiometric sensing was attributed to the selective nucleophilic addition reaction of SO32- with L. The probe was further used to develop a low cost microfluidic sensor device (µPAD). The probe was biocompatible and its potential to sense SO32- in mitochondria was captured in live HeLa cells.

Fixation of atmospheric CO2 as novel carbonate-(water)2-carbonate cluster and entrapment of double sulfate within a linear tetrameric barrel of a neutral bis-urea scaffold.

A meta-phenylenediamine-based disubstituted bis-urea receptor L1 with electron-withdrawing 3-chloro and electron-donating 4-methylphenyl terminals has been established as a potential system to fix and efficiently capture atmospheric CO2 as air-stable entrapment of an unprecedented {CO32--(H2O)2-CO32-} cluster (complex 1a) within its tetrameric long straight pillar-like assembly entirely sealed by n-TBA cations via formation of a barrel-type architecture. L1 and its isomeric 4-bromo-3-methyl disubstituted bis-urea receptor L2 have been found to entrap similar kinds of water-free naked sulfate-sulfate double anion (complexes 1b and 2a) by cooperative binding of urea moieties inside the two pairs of the inversion-symmetric linear tetrameric barrel of L1 and L2, respectively. On the other hand, in the presence of excess halides, L1 self-assembles to form hexa-coordinated fluoride complex 1c and tetra-coordinated bromide complex 1d, while L2 self-assembles to form penta-coordinated fluoride complex 2b in the solid state via semicircular receptor architectures and non-cooperative H-bonding interactions of urea moieties.

A solo fluorogenic probe for the real-time sensing of SO3(2-) and SO4(2-)/HSO4(-) in aqueous medium and live cells by distinct turn-on emission signals.

A judiciously designed fluorogenic probe (L) rendered rapid and differential turn-on responses by exhibiting strong blue fluorescence (λem = 442 nm) for SO3(2-) and greenish-yellow fluorescence (λem = 511 nm) for SO4(2-)/HSO4(-) in 100% aqueous medium and live cells.

A multi-responsive turn-on flurogenic probe to sense Zn(2+), Cd(2+) and Pb(2+): left-right-center emission signal swing.

A versatile new fluorogenic Schiff base probe (L) has been synthesized by the reaction of quinoline-2-carbohydrazide (which acts as the chelating site) and 4-dimethylamino cinnamaldehyde (which acts as the signaling unit). L can sense three of the most biologically and environmentally important metal ions, Zn(2+), Cd(2+) and Pb(2+), among various tested metal ions through selective TURN-ON fluorescence responses in physiological pH. Interestingly, L can not only sense Zn(2+), Cd(2+) and Pb(2+) fluorometrically in physiological conditions but can also distinguish one from another by exhibiting individual intrinsic left-right-center TURN-ON emission signal swings. These selective fluorescence responses were explained by a chelation-enhanced fluorescence (CHEF) mechanism. Theoretical calculations were carried out to ascertain the preferred L-metal ion binding mode.

An aggregation-induced emission (AIE) active probe for multiple targets: a fluorescent sensor for Zn(2+) and Al(3+) & a colorimetric sensor for Cu(2+) and F(-).

A rationally designed probe L, which consists of both cation and anion binding sites, is capable of displaying interesting aggregation induced emission (AIE) properties. L not only can sense Al(3+) and Zn(2+) through selective turn-on fluorescence responses in 9 : 1 methanol-HEPES buffer (5 mM, pH 7.3; 9 : 1, v/v) medium due to metal ion triggered AIE activity, but also can distinguish them through individual emission signals. L can also detect Cu(2+) in mixed buffer medium and F(-) in acetonitrile through sharp colorimetric responses. All the sensing processes are conspicuous through the naked eye. A theoretical study strongly backed the proposed sensing mechanisms.

Colorimetric and fluorometric discrimination of geometrical isomers (maleic acid vs fumaric acid) with real-time detection of maleic acid in solution and food additives.

Heterobis imine Schiff base probe L is able to discriminate geometrical isomers (maleic acid vs fumaric acid) through sharp colorimetric as well as fluorogenic responses even conspicuous with the naked eye. Colorimetric as well as fluorogenic sensing of maleic acid among various carboxylic acids was also demonstrated in ethanol-buffer medium. Sensing behavior of L was corroborated by (1)H NMR spectra, mass spectrometry, and theoretical calculations. Subsequently sensing behavior of L was used to probe maleic acid in starch rich food samples.

A single probe to sense Al(III) colorimetrically and Cd(II) by turn-on fluorescence in physiological conditions and live cells, corroborated by X-ray crystallographic and theoretical studies.

A pyridine-2-carbohydrazide functionalized conjugated fluorophoric Schiff base ligand (L1) specifically senses Al(3+) and Cd(2+) ions through significant changes in its absorption and emission spectral behavior, respectively, in physiological conditions. The spectral changes are in the visible region of the spectrum and thus facilitate naked eye detection. Apart from the visible changes, an in-field device application was demonstrated by sensing these ions in paper strips coated with L1. The crystal structure of the L1-Cd complex provided additional insight of the metal coordination attributes of L1. Interestingly, fluorescence microscopic studies demonstrated that the ligand L1 could also be used as an effective probe in imaging experiments for the detection of intracellular Cd(2+) ions in HeLa cells, without any toxicity to these model human cells.