Traditional Fluorescence Research Articles

Virus morphology: Insights from super-resolution fluorescence microscopy.

As epitomised by the COVID-19 pandemic, diseases caused by viruses are one of the greatest health and economic burdens to human society. Viruses are ‘nanostructures’, and their small size (typically less than 200 nm in diameter) can make it challenging to obtain images of their morphology and structure. Recent advances in fluorescence microscopy have given rise to super-resolution techniques, which have enabled the structure of viruses to be visualised directly at a resolution in the order of 20 nm. This mini-review discusses how recent state-of-the-art super-resolution imaging technologies are providing new nanoscale insights into virus structure.

Recent advances in fluorescence anisotropy/polarization signal amplification.

Fluorescence anisotropy/polarization is an attractive and versatile technique based on molecular rotation in biochemical/biophysical systems. Traditional fluorescence anisotropy/polarization assays showed relatively low sensitivity for molecule detection, because widespread molecular masses are too small to produce detectable changes in fluorescence anisotropy/polarization value. In this review, we discuss in detail how the potential of fluorescence anisotropy/polarization signal approach considerably expanded through the implementation of mass amplification, recycle the target amplification, fluorescence probes structure-switching amplification, resonance energy transfer amplification, and provide perspectives at future directions and applications.

Multifunctional AIEgen-based luminescent metal–organic frameworks with coordination-induced emission for chemical sensing

Aggregation-induced emission luminogens (AIEgens) offer a timely remedy to tackle aggregation-caused quenching of traditional organic fluorescence probes.

Microvasculature Features of Vogt-Koyanagi-Harada Disease Revealed by Widefield Swept-Source Optical Coherence Tomography Angiography.

Background: Vogt-Koyanagi-Harada (VKH) disease is a multisystem autoimmune disorder which could induce bilateral panuveitis involving the posterior pole and peripheral fundus. Optical coherence tomography angiography (OCTA) provides several advantages over traditional fluorescence angiography for revealing pathological abnormalities of the retinal vasculature. Until recently, however, the OCTA field of view (FOV) was limited to 6 × 6 mm2 scans.Purpose: This study examined retinal vasculature and choriocapillaris abnormalities across multiple regions of the retina (15 × 9 mm2 wide field, macular, peripapillary regions) among acute and convalescent VKH patients using a novel widefield swept-source OCTA (WSS-OCTA) device and assessed correlations between imaging features and best-corrected visual acuity (BCVA).Methods: Twenty eyes of 13 VHK disease patients in the acute phase, 30 eyes of 17 patients in the convalescent phase, and 30 eyes of 15 healthy controls (HCs) were included in this study. Vascular length density (VLD) in superficial and deep vascular plexuses (SVP, DVP), vascular perfusion density (VPD) in SVP, DVP, and choriocapillaris (CC), and flow voids (FV) in CC were measured across multiple retinal regions via WSS-OCTA (PLEX Elite 9000, Carl Zeiss Meditec Inc., USA) using the 15 × 9 mm2 scan pattern centered on the fovea and quantified by ImageJ.Results: Compared to HCs, acute phase VKH patients exhibited significantly reduced SVP-VLD, SVP-VPD, and CC-VPD across multiple retinal regions (all p < 0.01). Notably, the FV area was more extensive in VKH patients, especially those in the acute phase (p < 0.01). These changes were reversed in the convalescent phase. Stepwise multiple linear regression analysis demonstrated that macular DVP-VLD and macular CC-VPD were the best predictive factors for BCVA in the acute and convalescent VKH groups.Conclusion: The wider field of SS-OCAT provides more comprehensive and detailed images of the microvasculature abnormalities characterizing VKH disease. The quantifiable and layer-specific information from OCTA allows for the identification of sensitive and specific imaging markers for prognosis and treatment guidance, highlighting WSS-OCTA as a promising modality for the clinical management of VKH disease.

Super-Resolution Imaging With Lanthanide Luminescent Nanocrystals: Progress and Prospect.

Stimulated emission depletion (STED) nanoscopy has overcome a serious diffraction barrier on the optical resolution and facilitated new discoveries on detailed nanostructures in cell biology. Traditional fluorescence probes employed in the super-resolution imaging approach include organic dyes and fluorescent proteins. However, some limitations of these probes, such as photobleaching, short emission wavelengths, and high saturation intensity, still hamper the promotion of optical resolution and bio-applications. Recently, lanthanide luminescent probes with unique optical properties of non-photobleaching and sharp emissions have been applied in super-resolution imaging. In this mini-review, we will introduce several different mechanisms for lanthanide ions to achieve super-resolution imaging based on an STED-like setup. Then, several lanthanide ions used in super-resolution imaging will be described in detail and discussed. Last but not least, we will emphasize the future challenges and outlooks in hope of advancing the next-generation lanthanide fluorescent probes for super-resolution optical imaging.

Collagen Glycation Detected by Its Intrinsic Fluorescence.

Collagen’s long half-life (in skin approximately 10 years) makes this protein highly susceptible to glycation and formation of the advanced glycation end products (AGEs). Accumulation of cross-linking AGEs in the skin collagen has several detrimental effects; thus, the opportunity for non-invasive monitoring of skin glycation is essential, especially for diabetic patients. In this paper, we report using the time-resolved intrinsic fluorescence of collagen as a biomarker of its glycation. Contrary to the traditional fluorescence intensity decay measurement at the arbitrarily selected excitation and detection wavelengths, we conducted systematic wavelength- and time-resolved measurements to achieve time-resolved emission spectra. Changes in the intrinsic fluorescence kinetics, caused by both collagen aggregation and glycation, have been detected.

Comparison of Next-Generation Sequencing and Fluorescence In Situ Hybridization for Detection of Segmental Chromosomal Aberrations in Neuroblastoma.

The aim of this study was to compare next-generation sequencing (NGS) with the traditional fluorescence in situ hybridization (FISH) for detecting segmental chromosomal aberrations (SCAs) such as 1p deletion, 11q deletion and 17q gain, which are well-known predictive markers for adverse outcome in neuroblastoma. The tumor tissue obtained from 35 patients with neuroblastoma was tested by FISH and targeted NGS, which is specially designed to detect copy number alterations across the entire chromosomal region in addition to mutations in 353 cancer-related genes. All chromosomal copy number alterations were analyzed using the copy number variation plot derived from targeted NGS. FISH was performed to detect 1p deletion, 11q deletion and 17q gain. The copy numbers of 1p, 11q, and 17q obtained via NGS were correlated with those acquired via FISH. The SCAs determined by NGS were matched with those by FISH. Most 17q gain of mismatched cases detected by NGS alone showed a subsegmental gain of 17q. FISH revealed 11q deletion and 17q gain in a few tumor cells of two cases, which were not detected by NGS. NGS can be a sensitive complementary and alternative method to the conventional FISH for detecting SCAs.

Development of a Nanocluster-Based Platform for Determination of Sofosbuvir

Background: Sofosbuvir is a potent direct-acting antivirus agent that has been listed as a promising medicine for the treatment of all genotypes of hepatitis C virus. As antiviral drugs could be metabolized to their associated compounds and toxicologically and pharmacologically interfere with the parent drugs, identifying the therapeutic range of drugs would be notable. Methods: In the current study, copper nanoclusters (Cu NCs) are synthesized during the reduction of copper nitrate with hydrazine hydrate in a protected media and used as a nanoprobe for the determination of sofosbuvir in plasma samples. Herein, synchronous fluorescence spectroscopy (SFS) is used for monitoring of fluorescence variation of nanoprobe owing to the excessive benefits compared with the traditional fluorescence. Results: SFS peak of Cu NCs has appeared at 355 nm with ∆λ=80 nm which is decreased in the presence of sofosbuvir. To optimize the reaction factors, a response surface methodology is used and in the optimized conditions, a linear concentration-response plot is obtained in a range of 0.05-6.0 µg mL−1 with a limit of detection of 0.0147 µg mL−1. Conclusion: The developed method also reveals good repeatability and selectivity for sofosbuvir in plasma samples.

Aggregation‐induced emission: Red and near‐infrared organic light‐emitting diodes

AbstractRed and near‐infrared (NIR) organic light‐emitting diodes (OLED) have gained remarkable interest due to their numerous applications. However, the construction of highly emissive emitters is hampered by the energy‐gap law and aggregation‐caused quenching (ACQ) effect. Whereas, aggregation‐induced emission (AIE) materials could avoid the undesirable ACQ effect and emit bright light in aggregated state, which is one class of the most promising materials to fabricate high‐performance OLED with a high external quantum efficiency and low efficiency roll‐off. This review summarizes recent advances in red and NIR OLED with AIE property, including the traditional fluorescence, thermally activated delayed fluorescence, and hybridized local and charge transfer compounds. Meanwhile, the emphasis attention is paid to the molecular design principles, as well as the molecular structure‐photophysical characteristics. We also briefly further outlook the challenges and perspective of red and NIR AIE luminogens.

Application of organic fluorescent probe-assisted near infrared fluorescence imaging in cervical cancer diagnosis

Fluorescence imaging has been widely used in the fields of biomedicine and clinical diagnosis. Compared with traditional fluorescence imaging in the visible spectral region (400-760 nm), near-infrared (NIR, 700-1 700 nm) fluorescence imaging is more helpful to improve the signal-to-noise ratio and the sensitivity of imaging. Highly-sensitive fluorescent probes are required for high-quality fluorescence imaging, and the rapid development of nanotechnology has led to the emergence of organic dyes with excellent fluorescent properties. Among them, organic fluorescent probes with the advantages of high safety, good biocompatibility, and high optical stability, are more favorable than inorganic fluorescent probes. Therefore, NIR fluorescence imaging assisted with organic fluorescent probes can provide more structural and dynamic information of biological samples to the researchers, which becomes a hot spot in the interdisciplinary research field of optics, chemistry and biomedicine. This review summarizes the application of NIR organic fluorescent probes in cervical cancer imaging. Several typical organic fluorescent probes (such as indocyanine green, heptamethine cyanine dye, rhodamine and polymer fluorescent nanoparticles) assisted NIR fluorescence imaging and their applications in cervical cancer diagnosis were introduced, and the future development and application of these techniques were discussed.

Multimodal high-resolution embryonic imaging with light sheet fluorescence microscopy and optical coherence tomography.

A high-resolution imaging system combining optical coherence tomography (OCT) and light sheet fluorescence microscopy (LSFM) was developed. LSFM confined the excitation to only the focal plane, removing the out of plane fluorescence. This enabled imaging a murine embryo with higher speed and specificity than traditional fluorescence microscopy. OCT gives information about the structure of the embryo from the same plane illuminated by LSFM. The co-planar OCT and LSFM instrument was capable of performing co-registered functional and structural imaging of mouse embryos simultaneously.

Iodization-enhanced fluorescence and circularly polarized luminescence for dual-readout probe design

Circularly polarized luminescence (CPL) is a preferential emission from chiral systems. Introducing CPL to traditional small fluorescence probes will greatly enhance selectivity and sensitivity due to avoiding the background fluorescence. Small-molecule probes are practically useful for bioimaging and sensing but always suffer from weak CPL properties. To optimize chiral perturbation, the study of structure-CPL correlation is an urgent necessity. Here, we investigated the influence of 3, 3′-substituent of BINOL on optical properties and found that iodization could significantly enhance the fluorescence quantum yield (ΦF) and dissymmetry factor (|glum|). Based on this finding, we presented a strategy to design dual-readout probes emiting both robust total fluorescence and CPL upon activation. As an application example, we first designed a pair of thiols probes, R/S-P1, with the fluorescence readout showing good linear response to cysteine, and CPL signal displaying significant enhancement with |glum| from undetectable value to 0.7 × 10−3.

AIEgens for Bacterial Imaging and Ablation

Accurate and sensitive diagnosis of pathogenic bacterial infection is a fundamental first step for correct bacteria management, helping to avoid the development of drug-resistant bacteria caused by the inappropriate use and overuse of antibiotics. Fluorescence probes as a promising visual tool can help identify pathogens rapidly and reliably. However, rigidly structured traditional fluorescence probes generally suffer from the drawback of aggregation-caused quenching (ACQ) effect, which greatly undermines their advantages with respect to sensitivity. Luminogens with aggregation-induced emission properties, namely AIEgens, can overcome the ACQ effect and certain AIEgen-based materials are capable of generating reactive oxygen species (ROS) in the aggregate states. Hence, they have become powerful tools for imaging and killing bacteria. This review summarizes the recent advances in AIEgens for the diagnosis and treatment of pathogen infections. Special attention has been paid to the molecular design, the application in bacterial imaging and ablation in vitro and in vivo, and the biocompatibility of AIEgens. Finally, the challenges and prospects are discussed in terms of using AIEgens to advance precision therapies for pathogen infections.

Stimulus-Responsive Room Temperature Phosphorescence Materials: Internal Mechanism, Design Strategy, and Potential Application

ConspectusRoom temperature phosphorescence (RTP) materials, which could respond to external stimuli, such as force, heat, light, electric filed, etc., have drawn increasing attention for their broad application prospects, especially in the fields of anticounterfeiting, sensors, data storage, and so on. In comparison with the traditional fluorescence ones, RTP materials show much longer emission lifetimes, which can be even caught by the naked eye. Thus, the change in emission lifetime under an external stimulus for RTP materials can be also a potential monitoring parameter, in addition to emission color and intensity. In other words, the number of visual monitoring parameters could increase from two to three in RTP materials, which would greatly facilitate their practical applications. Until now, RTP materials have been typically limited to metal-containing inorganic materials, particularly rare-earth phosphors. Their emissions are governed by the slow liberation of trapped charge carriers from isolated traps of impurities, defects, or ions through thermal stimulation with low luminescence efficiency. However, these materials suffer from some intrinsic disadvantages, including high cost, potential toxicity, and instability in aqueous environments. In order to solve these problems, the purely organic RTP materials should be a good choice. However, these kinds of materials are really scarce now, especially for the ones with stimulus response characteristic.To develop purely organic RTP materials with a stimulus response effect, we and other scientists have tried a lot. Luckily, some progresses have been made. In this Account, we present our recent progress on the stimulus-responsive room temperature phosphorescence of organic materials, mainly focusing on the internal mechanism and potential applications. First, the fundamental knowledge is described to illustrate the importance and main principles of the stimulus-responsive RTP effect. Then, some typical stimulus-responsive RTP materials based on different internal mechanisms are discussed. Mainly, two kinds of stimulus-responsive RTP materials were introduced, namely, single-component and multicomponent ones. Correspondingly, their dynamic change of the RTP property under external stimulus occurred based on the distinct internal mechanisms. For single-component materials, the changes in molecular structure, packing, or conformation, have played a significant role in their corresponding stimulus-responsive RTP effect. As for multicomponent materials, the changed oxygen concentration in matrix and intermolecular distance between different components were found more during the stimulus-responsive RTP process. Accordingly, different potential applications were explored based on the different stimulus-responsive RTP processes. With the classification of stimulus-responsive RTP materials based on different internal mechanisms, the corresponding design strategy could be well proposed, thus guiding the further development of this research field.

Bio-inspired nickel nanoparticles of pyrimidine-Schiff base: In vitro anticancer, BSA and DNA interactions, molecular docking and antioxidant studies

In this work, interactions of pyrimidine derivative Schiff base ligand (DMPMM) were studied and its stabilized powder nickel nanoparticles (DMPMM-NiNPs) were synthesized and various biological studies were evaluated. DNA binding studies of CT-DNA with prepared compounds in Tris-HCl/NaCl buffer were carried out by traditional UV-Visible and fluorescence spectroscopic methods, viscosity measurements and cyclic voltammetry. Results showed that the small scale of DMPMM had less activity to interact with biological systems and when it assembled on nickel nanoparticles surface the activity increased. Thermal denaturation and sonochemical denaturation studies of DNA with the presence and the absence of our compounds also were done by UV-Visible spectroscopic method and its results indicated that the synthesized compounds increased the denaturation temperature. BSA binding studies of synthesized compounds were done by UV-Visible and fluorescence spectroscopy. Molecular docking of prepared ligand and its nanoparticles with biomolecules (DNA and BSA) were studied. Antimicrobial studies of the DMPMM and DMPMM-NiNPs were carried out by Agar-Agar well diffusion method. Anticancer studies results evidenced that the synthesized DMPMM-NiNPs had good selectivity to control the growth of cancer cells without damaging the normal cells. Various antioxidant scavenging studies results have shown that DMPMM and DMPMM-NiNPs have significant antioxidant activity. Highlights Stable and solid nickel nanoparticles were prepared. The size of the prepared nickel nanoparticles was nearly 3 to 8 nm. Organic ligand capped nickel nanoparticles interacted with DNA and BSA. Ni nanoparticles increased the denaturation temperature of DNA. It was found to have good anticancer activity with fewer side effects than cisplatin. Communicated by Ramaswamy H. Sarma

Imaging and Quantification of mRNA Molecules at Single-Cell Resolution in the Human Fungal Pathogen Candida albicans.

ABSTRACTThe study of gene expression in fungi has typically relied on measuring transcripts in populations of cells. A major disadvantage of this approach is that the transcripts’ spatial distribution and stochastic variation among individual cells within a clonal population is lost. Traditional fluorescence in situ hybridization techniques have been of limited use in fungi due to poor specificity and high background signal. Here, we report that in situ hybridization chain reaction (HCR), a method that employs split-initiator probes to trigger signal amplification upon mRNA-probe hybridization, is ideally suited for the imaging and quantification of low-abundance transcripts at single-cell resolution in the fungus Candida albicans. We show that HCR allows the absolute quantification of transcripts within a cell by microscopy as well as their relative quantification by flow cytometry. mRNA imaging also revealed the subcellular localization of specific transcripts. Furthermore, we establish that HCR is amenable to multiplexing by visualizing different transcripts in the same cell. Finally, we combine HCR with immunostaining to image specific mRNAs and proteins simultaneously within a single C. albicans cell. The fungus is a major pathogen in humans where it can colonize and invade mucosal surfaces and most internal organs. The technical development that we introduce, therefore, paves the way to study the patterns of expression of pathogenesis-associated C. albicans genes in infected organs at single-cell resolution.IMPORTANCE Tools to visualize and quantify transcripts at single-cell resolution have enabled the dissection of spatiotemporal patterns of gene expression in animal cells and tissues. Yet the accurate quantification of transcripts at single-cell resolution remains challenging for the much smaller microbial cells. Widespread phenomena such as stochastic variation in transcript levels among cells—even within a clonal population—seem to play important roles in the biology of many microorganisms. Investigating this process requires microbial cell-optimized procedures to image and measure mRNAs at single-molecule resolution. In this report, we adapt and expand in situ hybridization chain reaction (HCR) combined with split-initiator probes to visualize transcripts in the human-pathogenic fungus Candida albicans at high resolution.

Ratio-Adjustable Upconversion Luminescence Nanoprobe for Ultrasensitive In Vitro Diagnostics.

The development of precise medicine requires diagnostic probes to simultaneously satisfy an excellent detection limit and a wide linear analysis range because of enormous individual-discrepancy of disease biomarker concentrations, yet it remains challenging. Herein, an upconverison nanoprobe with a luminescence ratio flexibly tailored was designed for ultrasensitive monitoring exhaled nitric oxide to indicate the clinical course of asthma. Two independent emissions from the same nanoprobe can be discretionarily modulated to vary their intensity ratios for adapting different analysis requirements. Delightfully, this novel nanoprobe demonstrated a 100-fold lower detection limit compared with the traditional ratiometric fluorescence manner and a more broad linear detection range from the subpart per billion (ppb) level to hundreds of ppb. This ratio-adjustable fluorescence detection strategy holds great potential for miscellaneous disease diagnosis applications.

Potent inhibitors of toxic alpha-synuclein identified via cellular time-resolved FRET biosensors

We have developed a high-throughput drug discovery platform, measuring fluorescence resonance energy transfer (FRET) with fluorescent alpha-synuclein (αSN) biosensors, to detect spontaneous pre-fibrillar oligomers in living cells. Our two αSN FRET biosensors provide complementary insight into αSN oligomerization and conformation in order to improve the success of drug discovery campaigns for the treatment of Parkinson’s disease. We measure FRET by fluorescence lifetime, rather than traditional fluorescence intensity, providing a structural readout with greater resolution and precision. This facilitates identification of compounds that cause subtle but significant conformational changes in the ensemble of oligomeric states that are easily missed using intensity-based FRET. We screened a 1280-compound small-molecule library and identified 21 compounds that changed the lifetime by >5 SD. Two of these compounds have nanomolar potency in protecting SH-SY5Y cells from αSN-induced death, providing a nearly tenfold improvement over known inhibitors. We tested the efficacy of several compounds in a primary mouse neuron assay of αSN pathology (phosphorylation of mouse αSN pre-formed fibrils) and show rescue of pathology for two of them. These hits were further characterized with biophysical and biochemical assays to explore potential mechanisms of action. In vitro αSN oligomerization, single-molecule FRET, and protein-observed fluorine NMR experiments demonstrate that these compounds modulate αSN oligomers but not monomers. Subsequent aggregation assays further show that these compounds also deter or block αSN fibril assembly.

Genomic cytometry is maturing but has single-cell multiomics hit puberty yet?

Genomic cytometry is maturing but has single-cell multiomics hit puberty yet?

Organelle-specific phase contrast microscopy enables gentle monitoring and analysis of mitochondrial network dynamics.

Mitochondria are delicate organelles that play a key role in cell fate. Current research methods rely on fluorescence labeling that introduces stress due to photobleaching and phototoxicity. Here we propose a new, gentle method to study mitochondrial dynamics, where organelle-specific three-dimensional information is obtained in a label-free manner at high resolution, high specificity, and without detrimental effects associated with staining. A mitochondria cleavage experiment demonstrates that not only do the label-free mitochondria-specific images have the required resolution and precision, but also fairly include all cells and mitochondria in downstream morphological analysis, while fluorescence images omit dim cells and mitochondria. The robustness of the method was tested on samples of different cell lines and on data collected from multiple systems. Thus, we have demonstrated that our method is an attractive alternative to study mitochondrial dynamics, connecting behavior and function in a simpler and more robust way than traditional fluorescence imaging.