Fluorescence Imaging Techniques Research Articles

Imaging of peroxynitrite in mitochondria by a near-infrared fluorescent probe with a large Stokes shift

As a result of large imaging depth, low background biofluorescence, facile operation, and high tempo-spatial resolution, the small-molecule near-infrared (NIR) fluorescence imaging technique has been one of the most popular tools for in situ monitoring of cellular oxidative stress (OS) processes. Nevertheless, the recent near infrared probes have low signal-to-noise ratio due to the small stokes shift. To this end, this work quinone-cyanine-7 (QCy7, λmaxabs = 582 nm, ε = 9329 M−1 cm−1 and λmaxem = 719 nm, Ф = 0.12) with large Stokes shift (137 nm) was used as a fluorophore, and 1-methylindoline-2,3-dione was used as the “Turn-ON” cyanine NIR Peroxynitrite (ONOO−) fluorescent probe HJ-ONOO-P3 (with 41-fold fluorescence enhancement with ONOO− concentration in the range of 0–35 μM). The indole quaternary ammonium heterocycle with a positive charge itself could be used as the targeted group for mitochondria. This probe exhibited a rapid response (5 min complete response) and high selectivity and sensitivity to ONOO− (detection limit as low as 25.4 nM). Meanwhile, this probe could achieve the task of in-situ imaging of OS during cellular oxygen-glucose deprivation/reperfusion (OGD/R) and middle cerebral artery occlusion (MCAO) in mice. The results demonstrated that the mitochondria-targeted NIR probe with a large Stokes shift possess great potential in studies of stroke and the induced cellular OS.

Expert consensus on indocyanine green fluorescence imaging for thoracoscopic lung resection (The Version 2022)

The use of the white-light thoracoscopy is hampered by the low contrast between oncologic margins and surrounding normal parenchyma. As a result, many patients with in situ or micro-infiltrating adenocarcinoma have to undergo lobectomy due to a lack of tactile and visual feedback in the resection of solitary pulmonary nodules. Near-infrared (NIR) guided indocyanine green (ICG) fluorescence imaging technique has been widely investigated due to its unique capability in addressing the current challenges; however, there is no special consensus on the evidence and recommendations for its preoperative and intraoperative applications. This manuscript will describe the development process of a consensus on ICG fluorescence-guided thoracoscopic resection of pulmonary lesions and make recommendations that can be applied in a greater number of centers. Specifically, an expert panel of thoracic surgeons and radiographers was formed. Based on the quality of evidence and strength of recommendations, the consensus was developed in conjunction with the Chinese Guidelines on Video-assisted Thoracoscopy, and the National Comprehensive Cancer Network (NCCN) guidelines on the management of pulmonary lesions. Each of the statements was discussed and agreed upon with a unanimous consensus amongst the panel. A total of 6 consensus statements were developed. Fluorescence-guided thoracoscopy has unique advantages in the visualization of pulmonary nodules, and recognition and resection of the anterior plane of the pulmonary segment. The expert panel agrees that fluorescence-guided thoracoscopic surgery has the potential to become a routine operation for the treatment of pulmonary lesions.

Assessing the development status of intraoperative fluorescence imaging for anatomy visualisation, using the IDEAL framework

ObjectivesIntraoperative fluorescence imaging is currently used in a variety of surgical fields for four main purposes: visualising anatomy, assessing tissue perfusion, identifying/localising cancer and mapping lymphatic systems. To establish evidence-based...

Classification of Tea Leaves Based on Fluorescence Imaging and Convolutional Neural Networks.

The development of the smartphone and computer vision technique provides customers with a convenient approach to identify tea species, as well as qualities. However, the prediction model may not behave robustly due to changes in illumination conditions. Fluorescence imaging can induce the fluorescence signal from typical components, and thus may improve the prediction accuracy. In this paper, a tea classification method based on fluorescence imaging and convolutional neural networks (CNN) is proposed. Ultra-violet (UV) LEDs with a central wavelength of 370 nm were utilized to induce the fluorescence of tea samples so that the fluorescence images could be captured. Five kinds of tea were included and pre-processed. Two CNN-based classification models, e.g., the VGG16 and ResNet-34, were utilized for model training. Images captured under the conventional fluorescent lamp were also tested for comparison. The results show that the accuracy of the classification model based on fluorescence images is better than those based on the white-light illumination images, and the performance of the VGG16 model is better than the ResNet-34 model in our case. The classification accuracy of fluorescence images reached 97.5%, which proves that the LED-induced fluorescence imaging technique is promising to use in our daily life.

One-step hydrothermal synthesis of Cit-NaYbF4:Er3+ nanocrystals with enhanced red upconversion emission for in vivo fluorescence molecular tomography

Fluorescence imaging techniques represent essential tools in in vitro, preclinical, and clinical studies. In this study, an improved one-step hydrothermal method to synthesize citric acid (CA) modified α-NaYbF4:2%Er3+ nanocrystals was proposed. The introduction of various doping ions into NaYbF4:2%Er3+ and the different valence states of the same ions affect both the crystal size and upconversion luminescence. Therefore, we investigated the upconversion luminescence enhancement of NaYbF4:2%Er3+ by ion doping and find that the upconversion luminescence intensity of the upconversion nanoparticles (UCNPs) co-doped with 5 mol% Fe2+ ions shows the greatest enhancement, especially for red emission at 654 nm. Furthermore, HeLa cells incubated with UCNPs allow for imaging with strong red upconversion emission detection. Confocal laser scanning microscope (CLSM) fluorescent images of HeLa cells indicate that NaYbF4:2%Er/5%Fe2+ leads to a clear outline and improves visualization of the cell morphology. In addition, the CA coated NaYbF4:2%Er3+/5%Fe2+ nanoparticles and NaYbF4:2%Er3+/5%Fe2+ show low cytotoxicity in HeLa cells. Organ imaging reveals the efficiency of these UCNPs to analyze the lungs, liver, and spleen. Together, these results indicate that the Cit-NaYbF4:2%Er3+/5%Fe2+ UCNPs are efficient nanoprobes for fluorescence molecular tomography.

Single-molecule fluorescence imaging techniques reveal molecular mechanisms underlying deoxyribonucleic acid damage repair.

Advances in single-molecule techniques have uncovered numerous biological secrets that cannot be disclosed by traditional methods. Among a variety of single-molecule methods, single-molecule fluorescence imaging techniques enable real-time visualization of biomolecular interactions and have allowed the accumulation of convincing evidence. These techniques have been broadly utilized for studying DNA metabolic events such as replication, transcription, and DNA repair, which are fundamental biological reactions. In particular, DNA repair has received much attention because it maintains genomic integrity and is associated with diverse human diseases. In this review, we introduce representative single-molecule fluorescence imaging techniques and survey how each technique has been employed for investigating the detailed mechanisms underlying DNA repair pathways. In addition, we briefly show how live-cell imaging at the single-molecule level contributes to understanding DNA repair processes inside cells.

Single-Molecule Imaging of Reactive Oxygen Species on a Semiconductor Nano-Heterostructure for Understanding Photocatalytic Heterogeneity in Aqueous.

We constructed a single-molecule fluorescence imaging technique to monitor the spatiotemporal distribution of the hydroxyl radical (•OH) on TiO2-attached multiwalled carbon nanotubes (TiO2-MWCNTs) in aqueous. We found the heterogeneous distribution of •OH is closely related to the composition and heterostructure of the catalysts. The dynamic •OH production rate was evaluated by counting the single-molecule fluorescent bursts. We further confirmed the production of •OH on TiO2-MWCNTs mainly occurred via electron reduction during the aqueous photocatalytic process. Our study reveals the mechanism of reactive oxygen species involved photocatalytic reaction and guides the design of advanced semiconductor photocatalysts.

A “hydroxyl radicals standard” for evaluating the efficacy of anti-asthma drugs by using a two-photon NIR optical sensor

Asthma is a common chronic inflammatory disease of the airways and a condition closely associated with oxidative stress. However, the lack of effective biomarkers and non-invasive real-time monitoring tools remains a great challenge for the early diagnosis and therapy of asthma. Hydroxyl radicals represent a key reactive oxygen species in biological systems, and therefore the change of hydroxyl radical level may be observed to be elevated in asthmatic mice. To verify this conjecture, in this paper, we designed the first two-photon (TP) near-infrared (NIR) fluorescent probe to be used with high selectivity for monitoring the fluctuating changes of hydroxyl radicals. More importantly, using this new probe, we have successfully observed the changes of hydroxyl radicals during asthma by visualization means for the first time. Immediately following the imaging experiments on mice, we also found that the therapeutic effect of using the combination of budesonide and terbutaline administration in asthmatic mice was effective than what was achieved by the administration of budesonide or terbutaline alone. These findings suggest that this probe has the potential to be a powerful tool for future asthma diagnosis and assessment of the therapeutic effect of asthma co-administration. Notably, it is expected to establish a “hydroxyl radicals standard” for evaluating the efficacy of anti-asthma drug therapy by using fluorescence imaging techniques.

A highly photostable and versatile two-photon fluorescent probe for the detection of a wide range of intracellular nitric oxide concentrations in macrophages and endothelial cells.

Nitric oxide (NO) is involved in many biological processes affecting the cardiovascular, nervous and immune systems. Intracellular NO can be monitored using fluorescent probes in combination with fluorescence imaging techniques. Most of the currently available NO fluorescent molecular probes are excited via one-photon excitation using UV or Vis light, which results in poor penetration and high photodamage to living tissues. Here, we report a two-photon fluorescent molecular probe, DANPY-NO, able to detect NO in live cells. The probe consists of an o-phenylenediamine linked to a naphthalimide core; and operates via photoinduced electron transfer. DANPY-NO exhibits good sensitivity (LOD of 77.8nM) and high selectivity towards NO, and is stable over a broad range of pHs. The probe targeted acidic organelles within macrophages and endothelial cells, and demonstrated enhanced photostability over a commercially available NO probe. DANPY-NO was used to selectively detect endogenous NO in RAW264.7ϒ NO- macrophages, THP-1 human leukemic cells, primary mouse (bone marrow-derived) macrophages and endothelial cells. The probe was also able to detect exogenous NO in endothelial cells and distinguish between increasing concentrations of NO. The NO detection was evidenced using confocal laser scanning and two-photon microscopies, and flow cytometry. Further evidence was obtained by recording the changes in the intracellular fluorescence emission spectrum of the probe. Importantly, the probe displayed negligible toxicity to the analysed biological samples. The excellent sensitivity, selectivity, stability and versatility of DANPY-NO confirm its potential for in vitro and in vivo imaging of NO.

Chlorophyll Fluorescence Imaging (CFI) and Laser-Induced Breakdown Spectroscopy (LIBS) Applied to Investigate Tomato Plants Infected by the Root Knot Nematode (RKN) Meloidogyne incognita and Tobacco Plants Infected by Cymbidium Ringspot Virus

Recently, studies on climate change have highlighted the central role of photosynthetic mechanisms in the defense response of plants to abiotic and biotic stresses. Photo-sensing and photo-activation are innovative technologies applied for the early detection of plant pathogens in order to prevent the dramatic impact they may have on plants. Chlorophyll Fluorescence Imaging (CFI) and Laser-Induced Breakdown Spectroscopy (LIBS) analytical techniques can be used to evaluate the amount of chlorophyll in plants, which can be altered in the case of biotic and abiotic stresses. In this work, both techniques were applied to two pathogenic model systems, i.e., roots of susceptible tomato plants infected by Meloidogyne incognita and Nicotiana benthamiana plants infected by cymbidium ringspot virus. Experimental evidence is provided and discussed showing that specific application protocols of both methods can be used successfully for the early detection of symptoms of the pathogen attacks of Meloidogyne incognita on tomato roots and of cymbidium ringspot virus infected plants. In particular, a decrease in chlorophyll content was measured by fluorescence imaging, and an increase in Mg++ content was determined by LIBS in both the leaves and stems of infected tomato plants and the leaves of infected plants, with respect to control (non-infected) plants. Thus, the two techniques used have been shown to be able to discriminate satisfactorily between control and infected plants and to provide some insight on the underlying mechanisms of plant defenses again nematodes and viruses.

A ciliary synapse for “short-circuit” neuromodulation

Nearly all neurons contain a primary cilium, but little is known about how this compartment contributes to neuromodulatory signaling. In a new study, Sheu etal. use cutting-edge electron microscopy and fluorescence imaging techniques to reveal a new type of synapse that enables chemical transmission between serotonergic axons and the primary cilia of hippocampal neurons.

Glutathione-capped gold nanoclusters as near-infrared-emitting efficient contrast agents for confocal fluorescence imaging of tissue-mimicking phantoms.

An innovative research has been conducted focused on demonstrating the ability of novel dual-emissive glutathione-stabilized gold nanoclusters (GSH-AuNCs) to perform bright near-infrared (NIR)-emitting contrast agents inside tissue-mimicking agarose-phantoms via two complementary confocal fluorescence imaging techniques. First, using a new and fast microwave-assisted approach, we synthesized photostable dual-emitting GSH-AuNCs with an average size of 3.2 ± 0.4 nm and NIR emission quantum yield of 9.9%. Steady-state fluorescence measurements coupled with fluorescence lifetime imaging microscopy (FLIM) assays performed on lyophilized GSH-AuNCs revealed that the obtained GSH-AuNCs exhibit PL emissions at 610nm (red PL) and, respectively, 800nm (NIR PL) in both solution and powder solid-state. Time-resolved fluorescence measurements showed that the two PL components are characterized by average lifetimes of 407ns (red PL) and 1821ns (NIR PL), respectively. Additionally, due to a partial overlap between the red PL and the absorption of the NIR PL, an energy transfer between the two coexisting emissive centers was discovered and confirmed via steady-state and time-resolved fluorescence measurements. Furthermore, the FLIM analysis performed on powder GSH-AuNCs under 640nm, an excitation more suitable for bioimaging applications, revealed a homogeneous and photostable NIR PL signal from GSH-AuNCs. Finally, the ability of GSH-AuNCs to operate as reliable NIR-emitting contrast agents inside tissue-mimicking agarose-phantoms was demonstrated here for the first time via complementary FLIM and re-scan confocal fluorescence imaging techniques. In consequence, GSH-AuNCs show great promise for future in vivo imaging applications via confocal fluorescence microscopy.

Bioinspired Polysaccharide Derivative with Efficient and Stable Lubrication for Silicon-Based Devices.

It is becoming increasingly important to synthesize efficient biomacromolecule lubricants suitable for medical devices. Even though the development of biomimetic lubricants has made great progress, the current system suitable for hydrophobic silicone-based medical devices is highly limited. In this work, we synthesize one kind of novel polysaccharide-derived macromolecule lubricant of chitosan (CS) grafted polyethylene glycol (PEG) chains and catechol groups (CT) (CS-g-PEG-g-CT). CS-g-PEG-g-CT shows good adsorption ability by applying quantitative analysis of quartz crystal microbalance (QCM), attenuated total reflectance-Fourier transform infrared spectroscopy (ATR-FTIR), and confocal fluorescence imaging technique, as well as the typical shear-thinning feature. CS-g-PEG-g-CT exhibits low and stable coefficients of friction (COFs) (0.01-0.02) on polydimethylsiloxane (PDMS) surfaces at a wide range of mass concentrations in diverse media including pure water, physiological saline, and PBS buffer solution and is even tolerant to various normal loads and sliding frequencies for complex pressurizing or shearing environments. Subsequently, systematic surface characterizations are used to verify the dynamic attachment ability of the CS-g-PEG-g-CT lubricant on the loading/shearing process. The lubrication mechanism of CS-g-PEG-g-CT can be attributed to the synergy of strong adsorption from catechol groups to form a uniform assembly layer, excellent hydration effect from PEG chains, and typical shear-thinning feature to dissipate viscous resistance. Surprisingly, CS-g-PEG-g-CT exhibits efficient lubricity on silicone-based commercial contact lenses and catheters. The current macromolecule lubricant demonstrates great real application potential in the fields of medical devices and disease treatments.

Pancreatic fluorescence using continuous indocyanine greeninfusion.

Pancreatic fluorescence using continuous indocyanine greeninfusion.

A Multiscale Molecular Dynamic Analysis Reveals the Effect of Sialylation on EGFR Clustering in a CRISPR/Cas9-Derived Model.

Bacterial and viral pathogens can modulate the glycosylation of key host proteins to facilitate pathogenesis by using various glycosidases, particularly sialidases. Epidermal growth factor receptor (EGFR) signaling is activated by ligand-induced receptor dimerization and oligomerization. Ligand binding induces conformational changes in EGFR, leading to clusters and aggregation. However, information on the relevance of EGFR clustering in the pattern of glycosylation during bacterial and viral invasion remains unclear. In this study, (1) we established CRISPR/Cas9-mediated GFP knock-in (EGFP-KI) HeLa cells expressing fluorescently tagged EGFR at close to endogenous levels to study EGF-induced EGFR clustering and molecular dynamics; (2) We studied the effect of sialylation on EGF-induced EGFR clustering and localization in live cells using a high content analysis platform and raster image correlation spectroscopy (RICS) coupled with a number and brightness (N&B) analysis; (3) Our data reveal that the removal of cell surface sialic acids by sialidase treatment significantly decreases EGF receptor clustering with reduced fluorescence intensity, number, and area of EGFR-GFP clusters per cell upon EGF stimulation. Sialylation appears to mediate EGF-induced EGFR clustering as demonstrated by the change of EGFR-GFP clusters in the diffusion coefficient and molecular brightness, providing new insights into the role of sialylation in EGF-induced EGFR activation; and (4) We envision that the combination of CRISPR/Cas9-mediated fluorescent tagging of endogenous proteins and fluorescence imaging techniques can be the method of choice for studying the molecular dynamics and interactions of proteins in live cells.

Design and Development of Benzothiazole-Based Fluorescent Probes for Selective Detection of Aβ Aggregates in Alzheimer's Disease.

The formation and accumulation of amyloid beta (Aβ) peptide are considered the crucial events that are responsible for the progression of Alzheimer's disease (AD). Herein, we have designed and synthesized a series of fluorescent probes by using electron acceptor-donor end groups interacting with a π-conjugating system for the detection of Aβ aggregates. The chemical structure of these probes denoted as RMs, having a conjugated π-system (C═C), showed a maximum emission in PBS (>600 nm), which is the best range for a fluorescent imaging probe. Among all these probes, RM-28 showed an excellent fluorescence property with an emission maximum of >598 nm upon binding to Aβ aggregates. RM-28 also showed high sensitivity (7.5-fold) and high affinities toward Aβ aggregates (Kd = 175.69 ± 4.8 nM; Ka = 0.5 × 107 M-1). It can cross the blood-brain barrier of mice efficiently. The affinity of RM-28 toward Aβ aggregates was observed in 3xTg-AD brain sections of the hippocampus and cortex region using a fluorescent imaging technique, as well as an in vitro fluorescence-based binding assay with Aβ aggregates. Moreover, RM-28 is highly specific to Aβ aggregates and does not bind with intracellular proteins like bovine serum albumin (BSA) and α-synuclein (α-Syn) aggregates. The results indicate that the probe RM-28 emerges as an efficient and veritable highly specific fluorescent probe for the detection of Aβ aggregates in both in vitro and in vivo model systems.

Bright single-photon sources in the telecom band by deterministically coupling single quantum dots to a hybrid circular Bragg resonator

High-performance solid-state quantum sources in the telecom band are of paramount importance for long-distance quantum communications and the quantum Internet by taking advantage of a low-loss optical fiber network. Here, we demonstrate bright telecom-wavelength single-photon sources based on In(Ga)As/GaAs quantum dots (QDs) deterministically coupled to hybrid circular Bragg resonators (h-CBRs) by using a wide-field fluorescence imaging technique. The QD emissions are redshifted toward the telecom O-band by using an ultra-low InAs growth rate and an InGaAs strain reducing layer. Single-photon emissions under both continuous wave (CW) and pulsed operations are demonstrated, showing high brightness with count rates of 1.14 MHz and 0.34 MHz under saturation powers and single-photon purities of g ( 2 ) ( 0 ) = 0.11 ± 0.02 (CW) and g ( 2 ) ( 0 ) = 0.087 ± 0.003 (pulsed) at low excitation powers. A Purcell factor of 4.2 with a collection efficiency of 11.2 % ± 1 % at the first lens is extracted, suggesting efficient coupling between the QD and h-CBR. Our work contributes to the development of highly efficient single-photon sources in the telecom band for fiber-based quantum communication and future distributed quantum networks.

Three-Dimensional Virtual Optical Clearing With Cycle-Consistent Generative Adversarial Network

High-throughput deep tissue imaging and chemical tissue clearing protocols have brought out great promotion in biological research. However, due to uneven transparency introduced by tissue anisotropy in imperfectly cleared tissues, fluorescence imaging based on direct chemical tissue clearing still encounters great challenges, such as image blurring, low contrast, artifacts and so on. Here we reported a three-dimensional virtual optical clearing method based on unsupervised cycle-consistent generative adversarial network, termed 3D-VoCycleGAN, to digitally improve image quality and tissue transparency of biological samples. We demonstrated the good image deblurring and denoising capability of our method on imperfectly cleared mouse brain and kidney tissues. With 3D-VoCycleGAN prediction, the signal-to-background ratio (SBR) of images in imperfectly cleared brain tissue areas also showed above 40% improvement. Compared to other deconvolution methods, our method could evidently eliminate the tissue opaqueness and restore the image quality of the larger 3D images deep inside the imperfect cleared biological tissues with higher efficiency. And after virtually cleared, the transparency and clearing depth of mouse kidney tissues were increased by up to 30%. To our knowledge, it is the first interdisciplinary application of the CycleGAN deep learning model in the 3D fluorescence imaging and tissue clearing fields, promoting the development of high-throughput volumetric fluorescence imaging and deep learning techniques.

Ultrabright Föster Resonance Energy Transfer Nanovesicles: The Role of Dye Diffusion.

The development of contrast agents based on fluorescent nanoparticles with high brightness and stability is a key factor to improve the resolution and signal-to-noise ratio of current fluorescence imaging techniques. However, the design of bright fluorescent nanoparticles remains challenging due to fluorescence self-quenching at high concentrations. Developing bright nanoparticles showing FRET emission adds several advantages to the system, including an amplified Stokes shift, the possibility of ratiometric measurements, and of verifying the nanoparticle stability. Herein, we have developed Förster resonance energy transfer (FRET)-based nanovesicles at different dye loadings and investigated them through complementary experimental techniques, including conventional fluorescence spectroscopy and super-resolution microscopy supported by molecular dynamics calculations. We show that the optical properties can be modulated by dye loading at the nanoscopic level due to the dye’s molecular diffusion in fluid-like membranes. This work shows the first proof of a FRET pair dye’s dynamism in liquid-like membranes, resulting in optimized nanoprobes that are 120-fold brighter than QDot 605 and exhibit >80% FRET efficiency with vesicle-to-vesicle variations that are mostly below 10%.

Dynamic Mode Decomposition of Fluorescence Loss in Photobleaching Microscopy Data for Model-Free Analysis of Protein Transport and Aggregation in Living Cells.

The phase separation and aggregation of proteins are hallmarks of many neurodegenerative diseases. These processes can be studied in living cells using fluorescent protein constructs and quantitative live-cell imaging techniques, such as fluorescence recovery after photobleaching (FRAP) or the related fluorescence loss in photobleaching (FLIP). While the acquisition of FLIP images is straightforward on most commercial confocal microscope systems, the analysis and computational modeling of such data is challenging. Here, a novel model-free method is presented, which resolves complex spatiotemporal fluorescence-loss kinetics based on dynamic-mode decomposition (DMD) of FLIP live-cell image sequences. It is shown that the DMD of synthetic and experimental FLIP image series (DMD-FLIP) allows for the unequivocal discrimination of subcellular compartments, such as nuclei, cytoplasm, and protein condensates based on their differing transport and therefore fluorescence loss kinetics. By decomposing fluorescence-loss kinetics into distinct dynamic modes, DMD-FLIP will enable researchers to study protein dynamics at each time scale individually. Furthermore, it is shown that DMD-FLIP is very efficient in denoising confocal time series data. Thus, DMD-FLIP is an easy-to-use method for the model-free detection of barriers to protein diffusion, of phase-separated protein assemblies, and of insoluble protein aggregates. It should, therefore, find wide application in the analysis of protein transport and aggregation, in particular in relation to neurodegenerative diseases and the formation of protein condensates in living cells.