Confocal Image 3D Surface Measurement with Optical Fiber Plate

A whole-field 3D surface measurement system for semiconductor wafer inspection is described.The system consists of an optical fiber plate,which can split the light beam into N^2 subbeams to realize the whole-field inspection.A special prism is used to separate the illumination light and signal light.This setup is characterized by high precision,high speed and simple structure.

3D Confocal Raman Imaging of Oil-Rich Emulsion from Enzyme-Assisted Aqueous Extraction of Extruded Soybean Powder

The understanding of the structure morphology of oil-rich emulsion from enzyme-assisted extraction processing(EAEP)was a critical step to break the oil-rich emulsion structure in order to recover oil.Albeit EAEP method has been applied as an alternative way to conventional solvent extraction method,the structure morphology of oil-rich emulsion was still unclear.The current study aimed to investigate the structure morphology of oil-rich emulsion from EAEP using 3 D confocal Raman imaging technique.With increasing the enzymatic hydrolysis duration from 1 to 3 h,the stability of oil-rich emulsion was decreased as visualized in the 3 D confocal Raman images that the protein and oil were mixed together.The subsequent Raman spectrum analysis further revealed that the decreased stability of oil-rich emulsion was due to the protein aggregations via SS bonds or protein-lipid interactions.The conformational transfer in protein indicated the formation of a compact structure.

Optical investigations reveal the effects of 2-aminoethyldiphenyl borate on STIM1 puncta formation

2-A minoethyldiphenyl borate(2-APB)is the most commonly used pharmacological agent in the study of calcium release-activated channels(CRACa);however,its inhibitory mechanism to CRACs remains unclear.To address this issue,we systematically employed confocal imaging,dual-wavelength excitation photometry and FRET to examine the effects of 2-APB on the dynamic activities and function of STIM1 and Orail,two key components of CRACs.Imaging results support that there are two signaling pathways(Orail-independent and Orail-dependent)for the formation of STM1 puncta.2 APB could dose dependently block Orail-independent but not Oril-dependent STIM1 puncta formation,despite its obvious inhibition effect on store-opented Ca^(2+)entry(SOCE).In addition,we found that although 2-APB could not visibly alter near plasma membrane CAD-eYFP localization,it could completely block CAD-YFP-induced constitutive Ca^(2+)entry and promnote the interaction between Orail and CAD by FRET mea-surements.Therefore,we proposed that inhibitory action of 2-APB on SOCE might attribute to its direct inhbitory effects on Orail channel itself,but not the interference on puncta formation between STIM1 and Orail.

Highly specific characterization and discrimination of monosodium urate crystals in gouty arthritis based on aggregation-induced emission luminogens

Existing technologies used to detect monosodium urate(MSU)crystals for gout diagnosis are not ideal due to their low sensitivity and complexity of operation.The purpose of this study was to explore whether aggregation-induced emission luminogens(AIEgens)can be used for highly specific imaging of MSU crystals to assist in the diagnosis of gout.First,we developed a series of luminogens(i.e.,tetraphenyl ethylene(TPE)-NH_(2),TPE-2NH_(2),TPE-4NH_(2),TPE-COOH,TPE-2COOH,TPE-4COOH,and TPE-Ketoalkyne),each of which was then evenly mixed with MSU crystals.Next,optimal fluorescence imaging of each of the luminogens was characterized by a confocal laser scanning microscope(CLSM).This approach was used for imaging standard samples of MSU,hydroxyapatite(HAP)crystals,and mixed samples with 1:1 mass ratio of MSU/HAP.We also imaged samples from mouse models of acute gouty arthritis,HAP deposition disease,and comorbidities of interest.Subsequently,CLSM imaging results were compared with those of compensated polarized light microscopy,and we assessed the biosafety of TPE-Ketoalkyne in the RAW264.7 cell line.Finally,CLSM time series and three-dimensional imaging were performed on MSU crystal samples from human gouty synovial fluid and tophi.As a promising candidate for MSU crystal labeling,TPE-Ketoalkyne was found to detect MSU crystals accurately and rapidly in standard samples,animal samples,and human samples,and could precisely distinguish gout from HAP deposition disease.This work demonstrates that TPE-Ketoalkyne is suitable for highly specific and timely imaging of MSU crystals in gouty arthritis and may facilitate future research on MSU crystal-related diseases.

Dark-field line confocal imaging with point confocality and extended line field for bulk defects detection

Fabrication of high-quality optics puts a strong demand on high-throughput detection of macroscopic bulk defects in optical components.A dark-field line confocal imaging method is proposed with two distinct advantages:(ⅰ)a point-to-line confocal scheme formed by a columnar elliptical mirror and an optical fiber bundle breaks through the constraint on light collection angle and field of view in the traditional line confocal microscopy using an objective,allowing for an extended confocal line field of more than 100 mm while maintaining a light collection angle of 27°;(ⅱ)the bulk defects are independently illuminated as a function of time to eliminate the cross talk in the direction of the confocal slit,thus preserving point confocality and showing the optical section thicknesses to be 162μm in the axial direction,and 19 and 22μm in the orthogonal transverse directions.The experimental results verify that the method has a minimum detectable bulk defect of less than 5μm and an imaging efficiency of 400 mm2/s.The method shows great potential in high-throughput and highsensitivity bulk defects detection.

Improving contrast and sectioning power in confocal imaging by third harmonic generation in SiOx nanocrystallites

We present a new optical microscope in which the light transmitted by a sample-scanned transmission confocal microscope is frequency-tripled by SiOx nanocrystallites in lieu of being transmitted by a confocal pinhole. This imaging technique offers an increased contrast and a high scattered light rejection. It is demonstrated that the contrast close to the Sparrow resolution limit is enhanced and the sectioning power are increased with respect to the linear confocal detection mode. An experimental implementation is presented and compared with the conventional linear confocal mode.

Integrated identification of growth pattern and taxon of bacterium in gut microbiota via confocal fluorescence imaging‐oriented single‐cell sequencing

Despite the fast progress in our understanding of the complex functions of gut microbiota,it is still challenging to directly investigate the in vivo microbial activities and processes on an individual cell basis.To gain knowledge of the indigenous growth/division patterns of the diverse mouse gut bacteria with a relatively high throughput,here,we propose an integrative strategy,which combines the use of fluorescent probe labeling,confocal imaging with single‐cell sorting,and sequencing.Mouse gut bacteria sequentially labeled by two fluorescent D‐amino acid probes in vivo were first imaged by confocal microscopy to visualize their growth patterns,which can be unveiled by the distribution of the two fluorescence signals on each bacterium.Bacterial cells of interest on the imaging slide were then sorted using a laser ejection equipment,and the collected cells were then sequenced individually to identify their taxa.Our strategy allows integrated acquirement of the growth pattern knowledge of a variety of gut bacteria and their genomic information on a single‐cell basis,which should also have great potential in studying many other complex bacterial systems.

The First Olympus Confocal Micro Imaging Competition China Award Ceremony

On January 20, 2010, the award ceremony for the First Olympus Confocal Micro Imaging Competition China was held in Beijing. After rounds of judging and competition, 16 photos finally won the prize.The First Olympus Confocal MicroImaging Competition China Award Ceremony was organized by Sciencenet.

Cellular Tolerance, Accumulation and Distribution of Cadmium in Leaves of Hyperaccumulator Picris divaricata

Knowledge of cellular metal homeostasis will provide a better understanding of the mechanisms involved in metal tolerance and hyperaccumulation in metal-hyperaccumulating plants. Energy dispersive X-ray spectrometry （EDS） was used to determine the localization of cadmium （Cd） in leaves of the Zn/Cd hyperaccumulator Picris divaricata which had a shoot Cd concentration of 565 mg kg-1 after 2 weeks of growth in solution culture supplying 10μ tmol L^-1 CdCl2. The results indicated that Cd was distributed mainly in the trichomes, upper and lower epidermis and bundle sheath cells, with a relatively low level of Cd in mesophyll cells. Mesophyll protoplasts isolated from leaves remained viable after 24 h exposure to CdCl2 at a concentration up to 1 mmol L^-1, indicating their high tolerance to Cd. The intracellular Cd was visualized by staining with Leadmium Green dye, a cellular permeable Cd fluorescence probe. The results showed that the majority of protoplasts （〉 82%） did not accumulate Cd, with only a minority （〈 18%） showing Cd accumulation. In the Cd-accumulating protoplasts, Cd accumulation was depressed by the addition of Fe^2＋, Mn^2＋ and the metabolic inhibitor carbonyl cyanide m-chlorophenylhydrazone （CCCP）, but not by Ca^2＋ or Zn^2＋. Furthermore, the entire process of Cd uptake from external solution into the cytoplasm and subsequent sequestration into vacuoles was successfully recorded by confocal images. These results suggested that reduced cellular Cd accumulation and efficient Cd vacuolar sequestration in mesophyll cells might be responsible for cellular Cd tolerance and distribution in the leaves of P. divaricata.

Fluorescent Superlattices of Gold Nanoparticles:A New Class of Functional Materials

Fluorescent three-dimensional(3-D)superlattices of dansyl glutathione protected gold nanoparticles,with potential applications in molecular detection,have been synthesized at an air/water interface by controlling the pH of the nanoparticle suspension.The number of fluorophores per nanoparticle was calculated to be~127.Morphologies of the superlattice crystals were examined using scanning electron microscopy(SEM).Most of the crystals observed were triangular in shape.High-resolution transmission electron microscopy(HRTEM)and small angle X-ray scattering(SAXS)were used to study the packing of nanoparticles in these crystals.Both these studies showed that the nanoparticles were arranged in a face-centered cubic(fcc)pattern with a particle-particle distance(center-center)of~10.5 nm.Evolution of the crystal morphologies with time was also examined.The fluorescence properties of these triangles were studied using confocal fluorescence imaging and confocal Raman mapping,which were in good agreement with the morphologies observed by SEM.The superlattice exhibits near-infrared(NIR)absorption in the range 1100-2500 nm.Easy synthesis of such functional nanoparticle-based solids makes it possible to use them in novel applications.We utilized the fluorescence of dansyl glutathione gold superlattice crystals for the selective detection of bovine serum albumin(BSA),the major protein constituent of blood plasma,based on the selective binding of the naphthalene ring of the dansyl moiety with site I of BSA.

Direct Imaging of Titania Nanotubes Located in Mouse Neural Stem Cell Nuclei

Titania nanotubes(TiO2-NTs)are a potential drug vehicle for use in nanomedicine.To this end,a preliminary study of the interaction of a model cell with TiO2-NTs has been carried out.TiO2-NTs were first conjugated with a fl uorescent label,fl uorescein isothiocyanate(FITC).FITC-conjugated titania nanotubes(FITC-TiO2-NTs)internalized in mouse neural stem cells(NSCs,line C17.2)can be directly imaged by confocal microscopy.The confocal imaging showed that FITC-TiO2-NTs readily entered into the cells.After co-incubation with cells for 24 h,FITC-TiO2-NTs localized around the cell nucleus without crossing the karyotheca.More interestingly,the nanotubes passed through the karyotheca entering the cell nucleus after co-incubation for 48 h.Atomic force microscopy(AFM)and transmission electron microscopy(TEM)were also employed in tracking the nanotubes in the cell.These results will be of benefit in future studies of TiO2-NTs for use as a drug vehicle,particularly for DNA-targeting drugs.

Establishment of Multi-Site Infection Model in Zebrafish Larvae for Studying Staphylococcus aureus Infectious Disease

Zebrafish（Danio rerio） is an ideal model for studying the mechanism of infectious disease and the interaction between host and pathogen.As a teleost,zebrafish has developed a complete immune system which is similar to mammals.Moreover,the easy acquirement of large amounts of transparent embryos makes it a good candidate for gene manipulation and drug screening.In a zebrafish infection model,all of the site,timing,and dose of the bacteria microinjection into the embryo are important factors that determine the bacterial infection of host.Here,we established a multi-site infection model in zebrafish larvae of 36 hours post-fertilization（hpf） by micro-injecting wild-type or GFP-expressing Staphylococcus aereus（5.aureus） with gradient burdens into different embryo sites including the pericardial cavity（PC）,eye,the fourth hindbrain ventricle（4V）,yolk circulation valley（YCV）,caudal vein（CV）,yolk body（YB）,and Duct of Cuvier（DC） to resemble human infectious disease.With the combination of GFP-expressing S.aureus and transgenic zebrafish Tg（corola：eGFP;lyz：Dsred） and Tg（lyz：Dsred） lines whose macrophages or neutrophils are fluorescent labeled,we observed the dynamic process of bacterial infection by in vivo multicolored confocal fluorescence imaging.Analyses of zebrafish embryo survival, bacterial proliferation and myeloid cells phagocytosis show that the site- and dose-dependent differences exist in infection of different bacterial entry routes.This work provides a consideration for the future study of pathogenesis and host resistance through selection of multi-site infection model.More interaction mechanisms between pathogenic bacteria virulence factors and the immune responses of zebrafish could be determined through zebrafish multi-site infection model.

A novel TICT-based near-infrared fluorescent probe for light-up sensing and imaging of human serum albumin in real samples

Human serum albumin(HSA) has emerged as a pivotal biomarker and prognostic indicator for various human diseases. Real-time sensing and visual tracking of HSA in plasma or other biological systems will immensely facilitate the basic researchers and clinicians to better understand HSA-associated biological processes. Herein, a novel near-infrared(NIR) fluorescent probe(7-HTCF) was rationally constructed for light-up sensing and in-situ imaging of HSA in real samples, based on the principle of twisted intramolecular charge transfer(TICT). Under physiological conditions, 7-HTCF could be efficiently trapped by HSA to form a stable complex via binding on a non-drug binding site, while the complex emitted strong fluoresce signals around 670 nm. Further investigations demonstrated that 7-HTCF displayed a great combination of excellent selectivity and good chemical stability, as well as rapid fluorescent response and ultra-high sensitivity for HSA detection. Particularly, the newly developed light-up probe has been successfully utilized for quantitative detection of HSA in diluted plasma samples, while its readouts are hardly affected by the addition of therapeutic agents and herbal medicines. 7-HTCF is also successfully used for in-situ imaging of the reabsorbed HSA in living renal cells, while this dye exhibits good cell permeability and high resolution for in-situ imaging in living cells. Collectively, a novel TICT-based near-infrared fluorescent probe was devised for highly selective and ultra-sensitive sensing of HSA in plasma samples or imaging HSA in living cells, which offered a practical tool for clinical tests and for exploring HSA-associated biological processes.