Fluorescence Imaging Techniques Research Articles

Early discrimination of mature-and immature-green tomatoes (Solanum lycopersicum L.) using fluorescence imaging method

Detecting mature-green and immature-green tomatoes using non-destructive approaches is a challenge for the fresh produce industry. Hyperspectral fluorescence imaging technique with excitation wavelength at 365 nm and UV–vis CCD camera was used for early non-destructive detection of mature-green and immature-green fruit from 200 randomly harvested green tomatoes. Conventional destructive analysis regarding locule gel development and seed texture were assessed to assign the maturity stage of the fruit. In addition soluble solid content (SSC), pH, total acidity (TA), and color were measured, on the training set and on the prediction set, in this case also after 10 d of storage. Fluorescence intensity at the surface of immature-green fruit was higher in the red region (690 nm) than that of mature-green fruit, suggesting that hyperspectral fluorescence imaging can be an effective classification tool. A univariate classification method was used to distinguish mature-green and immature-green tomatoes based on the grey scale values extracted from fluorescence imaging, with a non-error rate of 96 % in calibration and 100 % in external prediction. Hence, a non-destructive method for the early distinction of mature-green from immature-green tomatoes is available.

A poly-diagnostic study of bipolar high-power magnetron sputtering: role of electrical parameters

Ion acceleration in a bipolar high-power impulse magnetron sputtering discharge working with Ar gas and using a Ti cathode has been investigated. The multiple discharge diagnostic approaches have been implemented, including the target and substrate current probe measurements, mass spectrometry and laser-induced fluorescence. In particular, the current waveforms were analysed in detail, showing the particularities of the current evolution during the negative (plasma) and positive voltage pulses. It is shown that the substrate current during the positive voltage pulse reveals two peaks, corresponding, as suggested, to two groups of the positive ions repulsed from the cathode by the applied positive potential. These observations correlate well with the mass spectrometry measurements as well as with the two-dimensional ion density mapping performed for several discharge conditions by laser-induced fluorescence imaging technique.

Can necrotic bone be objectively identified in chronic fracture related infections? — First clinical experience with an intraoperative fluorescence imaging technique

IntroductionAdequate debridement of necrotic bone is of paramount importance for eradication of infection in chronic osteomyelitis. Currently, no tools are available to detect the exact amount of necrotic bone in order to optimize surgical resection. The aim of the present study was to evaluate the feasibility of an intraoperative illumination method (VELscope®) and the correlation between intraoperative and pathohistological findings in surgically treated chronic fracture related infection patients. MethodsTen consecutive patients with chronic fracture related infections of the lower extremity were included into this prospectively performed case series. All patients had to be treated surgically for fracture related infections requiring bony debridement. An intraoperative illumination method (VELscope®) was used to intraoperatively differentiate between viable and necrotic bone. Tissue samples from the identified viable and necrotic bone areas were histopathologically examined and compared to intraoperative findings. ResultsIn all included patients, the intraoperative illumination was deemed helpful to differentiate between necrotic and viable bone tissues during bony debridement. The histopathological examination of the samples showed good correlation of the intraoperative illumination findings with histopathological signs of necrosis for areas deemed dead and histopathological signs of intact bone for areas deemed vital during illumination. ConclusionThe fluorescence-assisted, intraoperative detection of necrotic and viable bone using the VELscope® is an easy-to-use procedure that can help surgeons to optimize intraoperative bone resection in chronic fracture related infections by unmasking viable from necrotic bone tissue. This may help to improve resection techniques and eventually treatment outcome in patients in the future.

Labeled-protein corona-coated Bi2S3 nanorods targeted to lysosomes for bioimaging and efficient photothermal cancer therapy

One of the main diseases contributing to human death are malignant tumors. Phototherapy is a promising approach for cancer therapy, and functional nanoparticles with targeting ligands are commonly used to improve the therapeutic efficiency. However, recent studies have shown that nanoparticles in contact with a biological fluid can rapidly form a “protein corona” on their surface, which will remarkably decrease the targeting ability. Here, we describe the preparation of hybrid nanomaterials with Bi2S3 nanorods as the core, and fluorescein-isothiocyanate and folic acid-modified human serum albumin (HSA-FITC-FA) as the shell. By using fluorescent binding label (FITC) and imaging techniques, we discovered the image of the cell lysosomes, indicating that the photothermal therapy agent was predominantly targeted to and accumulated in lysosomes. Combined with photothermal therapy agent (Bi2S3 nanorods) and targeting ligand (FA), the obtained product shows enhanced photothermal therapy under near-infrared region laser irradiation. Additionally, SDS-PAGE shows that the modified HSA shell could remarkably reduce the reabsorption of protein corona from blood serum, minimized the adverse effect of protein corona on targetability. Taken together, the results indicate that our strategy has the potential for preparing efficient photothermal nanomaterials with image-guided subcellular organelle-targeting cancer cell ablation ability.

A near-infrared excitation/emission fluorescent probe for imaging of endogenous cysteine in living cells and zebrafish.

The fluorescence imaging technique provides an essential tool for studying biological systems. However, due to the interference of autofluorescence of biological tissues, the application of short-wavelength fluorescent probes in biological imaging was limited. The near-infrared (NIR) excitation/emission fluorescent probe possesses unique advantages in optical imaging in vivo, including less light scattering, minimal photo-damage to biological samples, deep tissue penetration, and weak autofluorescence interference from complicated biological systems. In this work, a convenient fluorophore (E)-2-[2-(6-hydroxy-2,3-dihydro-1H-xanthen-4-yl)vinyl]-3- methylbenzo[d]thiazol-3-ium iodide (DXM-OH) with NIR excitation and emission was rationally designed and developed. What's more, DXM-OH was applied to construct an "OFF-ON" fluorescent probe (E)-2-{2-[6-(acryloyloxy)-2,3-dihydro-1H- xanthen-4-yl]vinyl}-3-methylbenzo[d]thiazol-3-ium iodide (DXM) for sensitive and selective detection of cysteine (Cys). The experimental results showed that DXM had the advantages of good cell permeability, low toxicity, and excellent optical properties (NIR excitation/emission) and it was successfully applied to image Cys of living cells and zebrafish.

Probing Spin Correlations in a Bose-Einstein Condensate Near the Single-Atom Level.

Using parametric conversion induced by a Shapiro-type resonance, we produce and characterize a two-mode squeezed vacuum state in a sodium spin 1 Bose-Einstein condensate. Spin-changing collisions generate correlated pairs of atoms in the m=±1 Zeeman states out of a condensate with initially all atoms in m=0. A novel fluorescence imaging technique with sensitivity ΔN∼1.6 atom enables us to demonstrate the role of quantum fluctuations in the initial dynamics and to characterize the full distribution of the final state. Assuming that all atoms share the same spatial wave function, we infer a squeezing parameter of 15.3dB.

Manganese–Fluorouracil Metallodrug Nanotheranostic for MRI-Correlated Drug Release and Enhanced Chemoradiotherapy

For cancer therapy, drug delivery systems are often limited by insufficient drug loading capacity, which usually results in systemic toxicity and heavy metabolic burden to excrete the carriers. Her...

A multiscale study on photophysical properties of a novel fluorescent probe for imaging amyloid‐β in Alzheimer's disease

AbstractDetection of amyloid‐β deposition in the brain region is of significant importance for early diagnosis of Alzheimer's disease (AD). In the last few decades, the fluorescent imaging technique has been considered an effective tool for detecting amyloid‐β plaques due to its safety, sensitivity, and operability. Thus, numerous fluorescent probes for amyloid‐β have been developed. The design of a probe with high selectivity and improved sensing performance requires knowledge about the potential binding sites for the probe in amyloid‐β and local microstructure of the probe in different sites. In this study, amyloid‐β‐specific photophysical properties of a novel fluorescent probe (cis‐PAD‐1) are theoretically investigated by using multiscale simulations, including molecular docking and quantum mechanics/molecular mechanics calculations. Binding profile of cis‐PAD‐1 in amyloid‐β has been simulated, and binding affinity of the probe in various sites is calculated. An excited‐state property study on cis‐PAD‐1 illustrates that the probe shows remarkable fluorescence enhancement in amyloid‐β due to the influence of the microenvironment, which is consistent with the experimental observation. Most importantly, two‐photon absorption cross section of the probe is greatly increased in the near‐infrared region when targeting with amyloid‐β owing to the enhanced transition dipole moment. Therefore, one can propose the usage of cis‐PAD‐1 as an excellent candidate in two‐photon fluorescent imaging for amyloid‐β. The detailed investigations provide information on the development and design strategy of a new fluorescent probe for amyloid‐β imaging in AD.

Visualizing the Interfacial Charge Transfer between Photoactive Microcystis aeruginosa and Hydrogenated TiO2.

Exploring photoactive biotic-abiotic conjugations is of great importance for a variety of applications, but it remains difficult to probe the interfacial transfer of photoinduced charge carriers. In this work, Kelvin probe force microscopy, together with fluorescence imaging technique, were used to visually observe the spatial distribution and interfacial behavior of photocarriers in Microcystis aeruginosa/TiO2 hybrids. Experimental investigations suggested that photosynthetic microalgae cells were prone to trap photoholes from TiO2 photocatalysts. Oxygen vacancy defects in semiconductor exhibited significant impact on the charge migration, as the surface photovoltage of hydrogenated TiO2/microalgae hybrid was much higher than the pristine system. Profiting from the bioenhanced charge separation, biotic-abiotic architecture presented remarkably increased activity for photocatalytic inactivation of microalgae microorganisms. This work not only highlights the visual techniques for understanding the charge transfer around biotic-abiotic interface, but also provides a bioenhanced conjugation for the photocatalytic elimination of microorganisms in water treatment applications.

Intercellular Bioimaging and Biodistribution of Gold Nanoparticle-Loaded Macrophages for Targeted Drug Delivery

In order to effectively apply nanoparticles to clinical use, macrophages have been used as vehicles to deliver genes, drugs or nanomaterials into tumors. In this study, the effectiveness of macrophage as a drug delivery system was validated by biodistribution imaging modalities at intercellular and ex vivo levels. We focused on biodistribution imaging, namely, the characterization of the gold nanoparticle-loaded macrophages using intracellular holotomography and target delivery efficiency analysis using ex vivo fluorescence imaging techniques. In more detail, gold nanoparticles (AuNPs) were prepared with trisodium citrate method and loaded into macrophage cells (RAW 264.7). First, AuNPs loading into macrophages was confirmed using the conventional ultraviolet-visible (UV-VIS) spectroscopy and inductively coupled plasma-mass spectrometry (ICP-MS). Then, the holotomographic imaging was employed to characterize the intracellular biodistribution of the AuNPs-loaded macrophages. The efficacy of target delivery of the well AuNPs uptake macrophages was studied in a mouse model, established via lipopolysaccharide (LPS)-induced inflammation. The fluorescent images and the ex vivo ICP-MS evaluated the delivery efficiency of the AuNPs-loaded macrophages. Results revealed that the holotomographic imaging techniques can be promising modalities to understand intracellular biodistribution and ex vivo fluorescence imaging can be useful to validate the target delivery efficacy of the AuNPs-loaded macrophages.

Identification of Pathways of A498 Human Kidney Carcinoma Cell Death under the Action of Gratiola officinalis L. Extract and Green Tea Flavonoids Using Fluorescence Imaging Techniques

Using fluorescence methods for visualization of human tumor cell apoptosis and necrosis in vitro, the antitumor effects of flavonoid-containing Gratiola officinalis L. and green tea extracts at ultralow concentrations on A498 human kidney carcinoma cells after 24 and 48 h were compared. Fluorescent dyes (propidium iodide and acridine orange) were used in the “living and dead” test, which made it possible to identify the total number of cells that died by necrosis and apoptosis, as well as cells in which apoptosis has started and apoptotic bodies or pycnosis of the nucleus were detected. Gratiola officinalis L. extract had the most pronounced cytostatic activity 48 h after exposure; the number of cells per field of view decreased by a factor of 1.5 compared with the control. Green tea extract at a concentration of 0.0288 mg/mL had the most pronounced cytotoxic activity; its manifestation was growing 48 h after exposure. Gratiola officinalis L. extract exhibited cytotoxic activity at lower concentrations (0.0036 mg/mL).

엽록소 형광이미징 기술을 이용한 키위과일의 생체중 예측

엽록소 형광이미징 기술을 이용한 키위과일의 생체중 예측

Impact of nano-conjugate on Drosophila for early diagnosis of Alzheimer’s disease

Alzheimer’s disease (AD) is an irreversible neurodegenerative disease. Recent identification of AD biomarkers has led to the diagnosis of AD before the onset of dementia. It has been shown that Drosophila melanogaster is a valuable model for studying human neurodegeneration, including AD. According to its properties, curcumin shows promising potential for the diagnosis of AD. In order to improve its use, new formulations, including nanotechnology-based delivery systems, have been applied. The current study aims to diagnose AD by detecting the accumulation of amyloid beta-peptide via carbon-dot–curcumin nanoparticle conjugation in Drosophila. The accumulation of amyloid beta-peptide has been detected via the conjugate using the fluorescence imaging technique. These results suggest that carbon-dot–curcumin nanoparticle conjugation could be used as a diagnostic tool for AD.

Preparation of amylose-poly(methyl methacrylate) inclusion complex as a smart nanocarrier with switchable surface hydrophilicity

Amylose, as a linear biopolymer, tends to form helical inclusion complexes with suitable guest species. This is of great importance for a variety of applications, especially in the pharmaceutical and food industry. In this study, we propose an approach for the preparation of a novel inclusion complex with switchable surface hydrophilicity. For this purpose, amylose was first conjugated to ethylene diamine hydrophilic residues. Then, the short chains of the hydrophobic poly(methyl methacrylate, PMMA) were grafted onto the cavity of amylose through atom transfer radical polymerization (ATRP). According to CD spectroscopy results, the amylose-PMMA inclusion complexes displayed solvent-directed helical chirality inversion using either DMSO or water as a solvent. Fluorescence imaging, AFM and DLS techniques revealed the solvent-dependent surface hydrophilicity of the amylose-PMMA inclusion complex. Interestingly, its morphological studies displayed a central cavity, which makes it suitable for carrying cargoes in drug delivery applications. Obtaining the amylose-polymer inclusion complexes with tailorable hydrophilicity of both the exterior surface and the interior cavity can be of paramount importance for a wide variety of bio-applications.

Insights Into Spinal Dorsal Horn Circuit Function and Dysfunction Using Optical Approaches.

Somatosensation encompasses a variety of essential modalities including touch, pressure, proprioception, temperature, pain, and itch. These peripheral sensations are crucial for all types of behaviors, ranging from social interaction to danger avoidance. Somatosensory information is transmitted from primary afferent fibers in the periphery into the central nervous system via the dorsal horn of the spinal cord. The dorsal horn functions as an intermediary processing center for this information, comprising a complex network of excitatory and inhibitory interneurons as well as projection neurons that transmit the processed somatosensory information from the spinal cord to the brain. It is now known that there can be dysfunction within this spinal cord circuitry in pathological pain conditions and that these perturbations contribute to the development and maintenance of pathological pain. However, the complex and heterogeneous network of the spinal dorsal horn has hampered efforts to further elucidate its role in somatosensory processing. Emerging optical techniques promise to illuminate the underlying organization and function of the dorsal horn and provide insights into the role of spinal cord sensory processing in shaping the behavioral response to somatosensory input that we ultimately observe. This review article will focus on recent advances in optogenetics and fluorescence imaging techniques in the spinal cord, encompassing findings from both in vivo and in vitro preparations. We will also discuss the current limitations and difficulties of employing these techniques to interrogate the spinal cord and current practices and approaches to overcome these challenges.

PLIF measurements of instantaneous flame structures and curvature of an acoustically excited turbulent premixed flame

In this work, we experimentally measured the instantaneous flame structure and curvature of a methane fueled premixed turbulent acoustically-excited flame in a longitudinal combustor. For this, we implemented simultaneous formaldehyde (CH2O) and hydroxyl (OH) planar laser-induced fluorescence (PLIF) and high-speed schlieren imaging techniques to quantify the dynamic response of this acoustically-excited flame, which was fixed at 260 Hz. The Reynold number of the jet flow was varied from 1216 to 8108. Both imaging techniques revealed that the vortex appeared at the flame top in the presence of acoustic excitations. As the jet velocity increased, the jet flame was squeezed dramatically along the axial direction. The CH2O-PLIF is found to successfully capture the evolution of vortex shedding process. The vortex appears as a sharp angle, when the acoustically-excited flame is squeezed. The vortex size is then increased gradually over an acoustic fluctuation period. To measure the heat release rate from the flame, we used the pixel-by-pixel product of OH×CH2O as a quantitative index. Based on the experimentally measured CH2O, OH and their product characterizing the heat release, it is shown that the more the flame is squeezed by the acoustic waves, the more CH2O is produced and the more unsteady heat is released. Finally, the characteristics of the jet flame curvature were investigated. The variation of the probability density function of the flame curvature revealed that the acoustic excitation with a proper phase could smooth the flame front.

An inertia-free beam scanning device for single-wavelength 2PE-STED nanoscopy

Two-photon excitation stimulated emission depletion nanoscopy (2PE-STED) is a fluorescence imaging technique ideal for significantly improving the spatial resolution when observing scattering tissue in fixed, in vitro, and in vivo specimens. Both 2PE and STED are beam scanning techniques. The image acquisition is commonly realized by raster scanning, and a pair of galvanometric mirrors are the most used approach. In 2PE fluorescence microscopy, acousto-optical deflectors (AODs) are also used because they allow random-access scanning. However, since the AOD working principle is diffraction of light, these devices are of limited use when it is necessary to deflect multiple beams of different wavelengths and polarization, e.g. in STED nanoscopy. Here, we present the first implementation of single wavelength 2PE-STED that enables a smart beam scanning system based on AODs technology.

Laparoscopic anatomical liver resection guided by real-time indocyanine green fluorescence imaging: experience and lessons learned from the initial series in a single center.

Anatomical liver resection is an established procedure for primary hepatic tumors. Laparoscopic anatomical hepatectomy has been proven to be technically achievable from S1 to S8 in experienced hands. The indocyanine green (ICG) fluorescence imaging technique offers a novel tool of intraoperative visualization in hepatobiliary surgery. This study aims to investigate the feasibility of laparoscopic anatomical liver resection based on segmental staining using real-time ICG fluorescence. From December 2015 to October 2017, 36 patients in our institute underwent lap-ALR using real-time ICG fluorescence mapping of the tumor-bearing portal territory. The procedural and perioperative data were collected and analyzed. In our case series, we successfully performed the fashion of positive staining mostly in segmentectomy or sub-segmentectomy by individually injecting 5-10ml of ICG (0.025mg/ml) into its feeding portal branch guided by intraoperative ultrasound, and the negative staining mainly for sectionectomy, hemihepatectomy and multi-segmentectomy by interrupting the Glissonean pedicle serving the tumor-bearing segments and systemically injecting 1ml of ICG (2.5mg/ml). Our total successful rate of staining is 53%. No conversion to laparotomy, Clavien III-IV complication or 90-day mortality occurred. Valuable technical feedback, experience and lessons are learned from this initial practice. Real-time ICG fluorescence imaging adds much precision to laparoscopic anatomical hepatectomy. The success of segmental staining requires a high proficiency of IOUS and skillful interpretation of preoperative 3D simulation. Decision-making on the fashions of positive and negative staining have been initially recommended. Multi-centered practice and technical modification are necessary to standardize its application.

Blind deconvolution for spike inference from fluorescence recordings

The parallel developments of genetically-encoded calcium indicators and fast fluorescence imaging techniques allows one to simultaneously record neural activity of extended neuronal populations in vivo. To fully harness the potential of functional imaging, one needs to infer the sequence of action potentials from fluorescence traces. Here we build on recently proposed computational approaches to develop a blind sparse deconvolution (BSD) algorithm based on a generative model for inferring spike trains from fluorescence traces. BSD features, (1) automatic (fully unsupervised) estimation of the hyperparameters, such as spike amplitude, noise level and rise and decay time constants, (2) a novel analytical estimate of the sparsity prior, which yields enhanced robustness and computational speed with respect to existing methods, (3) automatic thresholding for binarizing spikes that maximizes the precision-recall performance, (4) super-resolution capabilities increasing the temporal resolution beyond the fluorescence signal acquisition rate. BSD also uniquely provides theoretically-grounded estimates of the expected performance of the spike reconstruction in terms of precision-recall and temporal accuracy for each recording. The performance of the algorithm is established using synthetic data and through the SpikeFinder challenge, a community-based initiative for spike-rate inference benchmarking based on a collection of joint electrophysiological and fluorescence recordings. Our method outperforms classical sparse deconvolution algorithms in terms of robustness, speed and/or accuracy and performs competitively in the SpikeFinder challenge. This algorithm is modular, easy-to-use and made freely available. Its novel features can thus be incorporated in a straightforward way into existing calcium imaging packages.

Azetidine-Containing Heterospirocycles Enhance the Performance of Fluorophores.

Fluorescent dyes are extensively utilized in various fluorescence imaging techniques. However, many existing modification strategies could not balance the performance (such as brightness, photostability, water solubility, and permeability) of fluorophores. Herein we report a general strategy to enhance the performance of donor-acceptor-type fluorophores by introducing azetidine-containing heterospirocycles to the commonly used fluorophore scaffolds. Such a strategy turned out to be a general way to develop high-quality fluorophores.