Two-photon Confocal Microscopy Research Articles

Neural Tracing Protein-Functionalized Nanoparticles Capable of Fast Retrograde Axonal Transport in Live Neurons.

Neural tracing proteins like horseradish peroxidase-conjugated wheat germ agglutinin (WGA-HRP) can target the central nervous system (CNS) through anatomic retrograde transport without crossing the blood-brain barrier (BBB). Conjugating WGA-HRP to nanoparticles may enable the creation of BBB-bypassing nanomedicine. Microfluidics and two-photon confocal microscopy is applied to screen nanocarriers for transport efficacy and gain mechanistic insights into their interactions with neurons. Protein modification of gold nanoparticles alters their cellular uptake at the axonal terminal and activates fast retrograde transport. Trajectory analysis of individual endosomes carrying the nanoparticles reveals a run-and-pause pattern along the axon with endosomes carrying WGA-HRP-conjugated gold nanoparticles exhibiting longer run duration and faster instantaneous velocity than those carrying nonconjugated nanoparticles. The results offer a mechanistic explanation of the different axonal transport dynamics as well as a cell-based functional assay of neuron-targeted nanoparticles with the goal of developing BBB-bypassing nanomedicine for the treatment of nervous system disorders.

Advancing Glucose Sensing Through Auto-Fluorescent Polymer Brushes: From Surface Design to Nano-Arrays.

Designing smart (bio)interfaces with the capability to sense and react to changes in local environments offers intriguing possibilities for new surface-based sensing devices and technologies. Polymer brushes make ideal materials to design such adaptive and responsive interfaces given their large variety of functional and structural possibilities as well as their outstanding abilities to respond to physical, chemical, and biological stimuli. Herein, a practical sensory interface for glucose detection based on auto-fluorescent polymer brushes decorated with phenylboronic acid (PBA) receptors is presented. The glucose-responsive luminescent surfaces, which are capable of translating conformational transitions triggered by pH variations and binding events into fluorescent readouts without the need for fluorescent dyes, are grown from both nanopatterned and non-patterned substrates. Two-photon laser scanning confocal microscopy and atomic force microscopy (AFM) analyses reveal the relationship between the brush conformation and glucose concentration and confirm that the phenylboronic acid functionalized brushes can bind glucose over a range of physiologically relevant concentrations in a reversible manner. The combination of auto-fluorescent polymer brushes with synthetic receptors presents a promising avenue for designing innovative and robust sensing systems, which are essential for various biomedical applications, among other uses.

3D Morphology of Different Crystal Forms in β-Nucleated and Fiber-Sheared Polypropylene: α-Teardrops, α-Teeth, and β-Fans.

Polymorphism of semicrystalline polymers has significant influence on their physical properties, with each form having its advantages and disadvantages. However, real-life polymer processing often results in different coexisting crystal polymorphs, and it remains a challenge to determine their shape, spatial distribution, and volume fraction. Here, i-polypropylene (i-PP) sheets containing both α- and β-forms were prepared either by adding β-nucleating agent or by fiber pulling-induced crystallization. By adding a compatible dye that is partially rejected from the growing crystalline aggregates (spherulites and cylindrites), we visualize the shape of these objects in 3D using two-photon fluorescence confocal microscopy. To distinguish between crystal forms, we take advantage of the difference in dye-retaining ability of the α- and β-aggregates. Even in 2D, fluorescence microscopy (FM) distinguishes the two crystal forms better than polarized microscopy. In 3D imaging, the volume fraction and spatial distribution of α- and β-forms in different morphological types could be determined quantitatively. Morphologies described as α-teeth, β-fans, and α-teardrops were visualized for the first time in 3D. Furthermore, internal and surface microcracks were seen to be associated predominantly with the β-form and around the fiber. Spatial distribution of α- and β-forms was also determined by scanning with a synchrotron X-ray beam. Good agreement was obtained with 3D microscopy, but XRD could not match the detail obtainable by the tomography. The work demonstrates the ability of the 3D imaging method to distinguish different crystal forms and their specific morphologies.

Background Rejection in Two-Photon Fluorescence Image Scanning Microscopy

We discuss the properties of signal strength and integrated intensity in two-photon excitation confocal microscopy and image scanning microscopy. The resolution, optical sectioning and background rejection are all improved over nonconfocal two-photon microscopy. Replacing the pinhole of confocal two-photon microscopy with a detector array increases the peak intensity of the point spread function. The outer pixels of a detector array give signals from defocused regions, and thus the processing of these, such as through subtraction, can further improve optical sectioning and background rejection.

Natural Solar Irradiation Produces Fluorescent and Biodegradable Nanoplastics.

Nanoplastics (NPs) have raised global concern owing to their potential health effects. Herein, after simulated and natural solar irradiation, polyethylene, polypropylene, polystyrene, and poly(vinyl chloride) nanoplastics (PVC NPs) were observed to exhibit enhanced fluorescence, particularly PVC NPs. Furthermore, the role of photoaged NPs as a potential fluorescence indicator was evaluated by exposing a model aquatic organism Daphnia magna to these NPs. Our results revealed that photoaged NPs exhibited strong fluorescence owing to the generation of conjugated π bonds, which can achieve π-π* electron transition with low energy consumption. Photogenerated fluorescence also enabled the photoaged NPs to act as efficient fluorescent tracers, which can help track NP migration in various organisms. The results of two-photon laser confocal scanning microscopy revealed that the photoaged NPs could translocate across biological barriers and accumulate in extraintestinal tissues in addition to being ingested and excreted. Moreover, compared with pristine NPs, the photoaged NPs underwent biodegradation more easily, probably because of increased hydrophilicity due to photogenerated oxygen-containing moieties. Therefore, in addition to producing fluorescent NPs without the attachment of external fluorescent dyes, the natural photoaging process can promote the migration and degradation of photoaged NPs in food chains.

Morphology of Shear-Induced Polymer Cylindrites Revealed by 3D Optical Imaging.

Two-photon confocal laser microscopy was used to obtain three-dimensional (3D) images of the morphology of poly(lactic acid) after shear-induced crystallization. The necessary fluorescence contrast was achieved by doping the polymer with Nile Red. The dye gets partially rejected from the growing crystalline aggregates during their formation, thus creating a renderable high-low fluorescence boundary outlining the shape of the aggregates. Parallel-plate melt-shearing and pulling a glass fiber through the melt were used as the two methods to achieve shear-induced crystallization. This study focuses on the shape of the resulting cylindrites, i.e., large-diameter shish-kebabs. The first 3D images of polymer cylindrites show that, if far from boundaries, they are circular cylinders, highly regular after fiber pull, but less so after parallel-plate shear. In the latter case, the cylindrite reveals the trajectory of the foreign particle that had nucleated its growth. Interestingly, lateral growth of the cylindrites was found to accelerate toward the sample surface when approaching it, giving the cylindrite an elliptical cross section. Furthermore and surprisingly, in the case of fiber pull, a row of spherulites is nucleated at the polymer-substrate interface nearest to the fiber, aligned along the fiber axis and appearing ahead of the rest of the space-filling spherulites. Both the phenomena, elliptical cylindrites and row of spherulites, are attributed to negative pressure buildup peaking at the cylindrite growth front and at the nearby film surface caused by crystallization-induced volume contraction. The pressure and flow distribution in the system is confirmed by numerical simulation. The results illustrate the value of 3D imaging of crystalline morphology in polymer science and polymer processing industry.

Dual transcriptome of Streptococcus mutans and Candida albicans interplay in biofilms

ABSTRACT Objective To assess the interactions between Streptococcus mutans and Candida albicans during cariogenic biofilm formation. Methods The S. mutans and C. albicans duo-species biofilms were formed in 1% sucrose to mimic the high caries risk challenges. The biofilm structure was assessed using two-photon laser confocal microscopy. The transcriptome of 48h-biofilms was assessed by RNA-Seq. The expression of S. mutans and C. albicans virulence genes was examined via real-time reverse transcription-polymerase chain reaction. Results The morphogenesis of C. albicans-S. mutans duo-species biofilms was significantly altered when comparing to S. mutans or C. albicans single-species biofilm. Duo-species biofilms exhibited unique expression profile with a large number of differentially expressed genes (DEGs), including a higher expression of S. mutans atpD (acid-adaptive), C. albicans CHT2 (fungal cell wall chitin remodeling), and C. albicans SOD3 (cytotoxic oxygen radical destroying) (p < 0.05). KEGG pathway analyses further revealed that the majority of the up-regulated DEGs are related to microbial metabolism. Furthermore, the expressions of S. mutans and C. albicans key virulence genes (gtfB, gtfC, gtfD, ECE1, HWP1, ERG4, CHT2) were associated with sugar availability-related and time-related dynamics. Conclusion Cross-kingdom interactions impact S. mutans-C. albicans biofilm formations and dynamic expressions of virulence genes.

Optical Visualization of Red-GQDs' Organelles Distribution and Localization in Living Cells.

Recently, there has been a rapidly expanding interest in a new nanomaterial, graphene quantum dots (GQDs), owing to its profound potential in various advanced applications. At present, the study of GQDs mainly focuses on the new synthesis methods and surface modification. However, revealing the intracellular distribution of GQDs is currently not available, limiting in-depth understanding of its biological regulatory mechanism. To fill up this gap, the visualization study of red fluorescent graphene quantum dots (Red-GQDs) is helpful to clarify their subcellular distribution and metabolism in living cells system. Here, in this study, two-photon laser confocal microscopy was used to deeply analyze the uptake and subcellular distribution of Red-GQDs by HeLa cells at different concentrations and times through visual observation and discussed the effect of Red-GQDs on the metabolic of HeLa cells. The results indicated that Red-GQDs could be well-absorbed by HeLa cells and further revealed the differential distribution of Red-GQDs in different organelles (lysosomes and mitochondria) in a time-dependent manner. In addition, we confirmed that Red-GQDs significantly affect cell biological functions. Low concentrations of Red-GQDs are related to the autophagy pathway of cells, and high concentrations of Red-GQDs can induce ferroptosis in cells and promote the secretion of cellular exosomes. In the present study, the distribution and metabolic pathways of Red-GQDs in the subcellular structure of cells were characterized in detail through visual analysis, which can bring positive reference for the application of Red-GQDs in the future.

Two-photon ratiometric carbon dot-based probe for real-time intracellular pH monitoring in 3D environment

• Design and development of novel pH-sensitive two-photon ratiometric carbon dot-based probe was presented. • Probe possessed remarkable thermal stability, photostability, two-photon NIR excitation capability, and biocompatibility. • Probe demonstrated superb reversibility and ratiometric response to pH changes (4–10), upon single wavelength excitation. • Precise real-time monitoring and quantification of the intracellular pH values in 2D and 3D environments was performed. Designing a two-photon ratiometric pH-sensitive nanoprobe for real time monitoring of intracellular pH in biological environment is of great importance for better understanding the pathogenesis of diseases and the design of intracellular drug delivery-based system. However, the development of such probe remains a challenge. Here, we report for the first time the design and development of the two-photon ratiometric carbon dot-based nanoprobe and demonstrate its capability to monitor and quantify the intracellular cytoplasmic pH value, in real time. The nanoprobe comprised of a fluorescent carbon dot functionalized with a pH-responsive fluorescein dye (FACD). With increasing pH, FACD exhibits a clear ratiometric change in the emission intensity ratio, with sensitivity across a wide pH range in both extracellular and intracellular compartments. FACD is non-toxic to adipose stem cells in cell imaging experimental conditions and exhibits remarkable thermal stability, photostability, and two-photon near-infrared excitation capability. Using real-time dual-channel two-photon confocal microscopy we demonstrate the great potential of FACD as an efficient nanoprobe with high accuracy for the intracellular sensing of pH in living adipose stem cells seeded on either cell-culture dishes or on a 3D printed bioactive scaffold.

Harnessing Electron Spin Hyperpolarization in Chromophore-Radical Spin Probes for Subcellular Resolution in Electron Paramagnetic Resonance Imaging: Concept and Feasibility.

Obtaining a subcellular resolution for biological samples doped with stable radicals at room temperature (RT) is a long-sought goal in electron paramagnetic resonance imaging (EPRI). The spatial resolution in current EPRI methods is constrained either because of low electron spin polarization at RT or the experimental limitations associated with the field gradients and the radical linewidth. Inspired by the recent demonstration of a large electron spin hyperpolarization in chromophore-nitroxyl spin probe molecules, the present work proposes a novel optically hyperpolarized EPR imaging (OH-EPRI) method, which combines the optical method of two-photon confocal microscopy for hyperpolarization generation and the rapid scan (RS) EPR method for signal detection. An important aspect of OH-EPRI is that it is not limited by the abovementioned restrictions of conventional EPRI since the large hyperpolarization in the spin probes overcomes the poor thermal spin polarization at RT, and the use of two-photon optical excitation of the chromophore naturally generates the required spatial resolution, without the need for any magnetic field gradient. Simulations based on time-dependent Bloch equations, which took into account both the RS field modulation and the hyperpolarization generation by optical means, were performed to examine the feasibility of OH-EPRI. The simulation results revealed that a spatial resolution of up to 2 fL can be achieved in OH-EPRI at RT under in vitro conditions. Notably, the majority of the requirements for an OH-EPRI experiment can be fulfilled by the currently available technologies, thereby paving the way for its easy implementation. Thus, the proposed method could potentially bridge the sensitivity gap between the optical and magnetic imaging techniques.

Study on the Absorption and Conduction Properties of Vanisulfane in Tobacco

The purposes of this study were to explore the systemic properties of vanisulfane in tobacco plant and to provide a reference for the rational use of vanisulfane in the field. After the tobacco plants were treated by hydroponics and foliar spraying, the contents of vanisulfane in root and stem leaf were detected by high-performance liquid chromatography tandem high-resolution mass spectrometry (UPLC-HRMS), and the position of vanisulfane in root and stem leaf was real-time observed through fluorescence two-photon confocal microscope. UPLC-HRMS results showed that the contents of vanisulfane in root and stem leaf gradually increased with the extension of processing time, and after 12 h treatment, the contents of vanisulfane in root and stem leaf reached the maximum levels of 31.95 and 0.215 mg/kg, respectively. In addition, fluorescence two-photon confocal microscope results showed that vanisulfane could observe in the root and stem leaf. These results showed that vanisulfane had excellent upward and downward of systemic in tobacco plants, which is helpful to guide a reference for the rational use of vanisulfane in the field.

Low-invasive 5D visualization of mitotic progression by two-photon excitation spinning-disk confocal microscopy

Non-linear microscopy, such as multi-photon excitation microscopy, offers spatial localities of excitations, thereby achieving 3D cross-sectional imaging with low phototoxicity even in thick biological specimens. We had developed a multi-point scanning two-photon excitation microscopy system using a spinning-disk confocal scanning unit. However, its severe color cross-talk has precluded multi-color simultaneous imaging. Therefore, in this study, we introduced a mechanical switching system to select either of two NIR laser light pulses and an image-splitting detection system for 3- or 4-color imaging. As a proof of concept, we performed multi-color fluorescent imaging of actively dividing human HeLa cells and tobacco BY-2 cells. We found that the proposed microscopy system enabled time-lapse multi-color 3D imaging of cell divisions while avoiding photodamage. Moreover, the application of a linear unmixing method to the 5D dataset enabled the precise separation of individual intracellular components in multi-color images. We thus demonstrated the versatility of our new microscopy system in capturing the dynamic processes of cellular components that could have multitudes of application.

Effect of annealing atmosphere on the luminescence characteristics of CVD-ZnSe

The influence of the atmosphere of CVD-ZnSe annealing carried out in modes close to the procedure of their doping with iron on the luminescent characteristics of these crystals is investigated. It was found that annealing in the atmosphere of argon and selenium leads to a qualitative change in the impurity-defect composition and to a complex spatial distribution of the luminescent characteristics of CVD-ZnSe, especially in the crystal region at a distance of up to 400 microns from the surface. In the case of annealing in zinc, the luminescent characteristics are more uniform, with the exception of the near-surface zone with a width of about 100 microns, which has a high luminescence intensity in the entire spectral range under study (0.44-0.72 microns). The obtained results are interpreted on the basis of the assumption about the evaporation of zinc during annealing in argon and selenium. Based on the analysis of the spatial distribution of luminescence, assumptions are made about the nature of the observed impurity-defect centers. Keywords: two-photon confocal microscopy, CVD-ZnSe, impurity-defect composition, high temperature annealing.

Halogen-modified carbazole derivatives for lipid droplet-specific bioimaging and two-photon photodynamic therapy

Lipid droplets (LDs) are dynamic multifunctional organelles that participate in the regulation of many metabolic processes, visualization of which is necessary for biological research. In this work, a series of two-photon responsive fluorescent probes (C-H, C-Br, and C-I) based on carbazole units were designed and synthesized. Thereinto, an iodine-modified carbazole derivative C-I exhibited an exciting lipid droplet targeting ability due to its excellent lipophilicity. Meanwhile, benefiting from its larger Stokes shift and two-photon absorption cross-section, C-I was employed for two-photon confocal laser scanning microscopy (CLSM) and stimulated emission depletion (STED) microscopy imaging to observe LDs more accurately. In addition, given the heavy atom effect, C-I can effectively generate reactive oxygen species (ROS) leading to cancer cell apoptosis under near-infrared light irradiation. Notably, we explained the process of cell apoptosis through in vitro simulation experiments. This study provides a promising platform for visualization of lipid droplets.

Micro-distribution of arsenic and polycyclic aromatic hydrocarbons and their interaction in Pteris vittata L.

Interactive effects of inorganic arsenic (As) species and polycyclic aromatic hydrocarbons (PAHs) on their uptake, accumulation and translocation in the hyperaccumulator Pteris vittata L. (P. vittata) were studied hydroponically. The presence of PAHs hindered As uptake and acropetal translocation by P. vittata, decreasing As concentrations by 29.8%–54.5% in pinnae, regardless of the initial As speciation. The inhibitive effect of PAHs was 1.6–8.7 times greater for arsenite [As(III)] than for arsenate [As(V)]. Similarly, inorganic As inhibited the uptake of fluorene (FLU) and benzo[a]pyrene (BaP) by P. vittata roots by 0.4%–21.7% and by 33.1%–69.7%, respectively. Interestingly, coexposure to As and PAHs slightly enhanced the translocation of PAHs by P. vittata with their concentrations increased 0.3 to 0.8 times in shoots, except for the As(III)+BaP treatment. The antagonistic interaction between As and PAHs uptake is likely caused by competitive inhibition or oxidative stress injury. By using synchrotron radiation micro X-ray fluorescence imaging, high concentrations of As were found distributed throughout the microstructures far from main vein of the pinnae when coexposed with PAHs, the opposite of what was observed with exposure to As only. PAHs could also significantly inhibit the accumulation and distribution of As in vascular bundles in rachis treated with As(III). The results of two-photon laser scanning confocal microscopy revealed that PAHs were mainly distributed in the vascular cylinder, epidermal cells, vascular bundles, epidermis and vein tissues, and this was independent of As speciation and treatment. This work offers new positive evidence for the interaction between As and PAHs in P. vittata, presents new information on the underlying mechanisms for interactions of As and PAHs affecting their uptake and translocation within P. vittata L., and provides direction for future research on the mechanisms of PAHs uptake by plants.

Spatiotemporal patterning of photoresponsive DNA-based hydrogels to tune local cell responses

Understanding the spatiotemporal effects of surface topographies and modulated stiffness and anisotropic stresses of hydrogels on cell growth remains a biophysical challenge. Here we introduce the photolithographic patterning or two-photon laser scanning confocal microscopy patterning of a series of o-nitrobenzylphosphate ester nucleic acid-based polyacrylamide hydrogel films generating periodically-spaced circular patterned domains surrounded by continuous hydrogel matrices. The patterning processes lead to guided modulated stiffness differences between the patterned domains and the surrounding hydrogel matrices, and to the selective functionalization of sub-regions of the films with nucleic acid anchoring tethers. HeLa cells are deposited on the circularly-shaped domains functionalized with the MUC-1 aptamers. Initiation of the hybridization chain reaction by nucleic acid tethers associated with the continuous hydrogel matrix results in stress-induced ordered orthogonal shape-changes on the patterned domains, leading to ordered shapes of cell aggregates bound to the patterns.

Novel (Phenothiazinyl)Vinyl-Pyridinium Dyes and Their Potential Applications as Cellular Staining Agents.

We report here the synthesis and structural characterization of novel cationic (phenothiazinyl)vinyl-pyridinium (PVP) dyes, together with optical (absorption/emission) properties and their potential applicability as fluorescent labels. Convective heating, ultrasound irradiation and mechanochemical synthesis were considered as alternative synthetic methodologies proficient for overcoming drawbacks such as long reaction time, nonsatisfactory yields or solvent requirements in the synthesis of novel dye (E)-1-(3-chloropropyl)-4-(2-(10-methyl-10H-phenothiazin-3-yl)vinyl)pyridin-1-ium bromide 3d and its N-alkyl-2-methylpyridinium precursor 1c. The trans geometry of the newly synthesized (E)-4-(2-(7-bromo-10-ethyl-10H-phenothiazin-3-yl)vinyl)-1-methylpyridin-1-ium iodide 3b and (E)-1-methyl-4-(2-(10-methyl-10H-phenothiazin-3-yl)vinyl)pyridin-1-ium tetrafluoroborate 3a′ was confirmed by single crystal X-ray diffraction. A negative solvatochromism of the dyes in polar solvents was highlighted by UV-Vis spectroscopy and explanatory insights were supported by molecular modeling which suggested a better stabilization of the lowest unoccupied molecular orbitals (LUMO). The photostability of the dye 3b was investigated by irradiation at 365 nm in different solvents, while the steady-state and time-resolved fluorescence properties of dye 3b and 3a′ in solid state were evaluated under one-photon excitation at 485 nm. The in vitro cytotoxicity of the new PVP dyes on B16-F10 melanoma cells was evaluated by WST-1 assay, while their intracellular localization was assessed by epi-fluorescence conventional microscopy imaging as well as one- and two-photon excited confocal fluorescence lifetime imaging microscopy (FLIM). PVP dyes displayed low cytotoxicity, good internalization inside melanoma cells and intense fluorescence emission inside the B16-F10 murine melanoma cells, making them suitable staining agents for imaging applications.

Self-Assembled Type I Collagen-Apatite Fibers with Varying Mineralization Extent and Luminescent Terbium Promote Osteogenic Differentiation of Mesenchymal Stem Cells.

This work explores in depth the simultaneous self-assembly and mineralization of type I collagen by a base-acid neutralization technique to prepare biomimetic collagen-apatite fibrils with varying mineralization extent and doped with luminescent bactericidal Tb3+ ions. Two variants of the method are tested: base-acid titration, a solution of Ca(OH)2 is added dropwise to a stirred solution containing type I collagen dispersed in H3 PO4 ; and direct mixing, the Ca(OH)2 solution is added by fast dripping onto the acidic solution. Only the direct mixing variant yielded an effective control of calcium phosphate polymorphism. Luminescence spectroscopy reveals the long luminescence lifetime and high relative luminescence intensity of the Tb3+ -doped materials, while two-photon confocal fluorescence microscopy shows the characteristic green fluorescence light when using excitation wavelength of 458nm, which is not harmful to bone tissue. Cytotoxicity/viability tests reveal that direct mixing samples show higher cell proliferation than titration samples. Additionally, osteogenic differentiation essays show that all mineralized fibrils promote the osteogenic differentiation, but the effect is more pronounced when using samples prepared by direct mixing, and more notably when using the Tb3+ -doped mineralized fibrils. Based on these findings it is concluded that the new nanocomposite is an ideal candidate for bone regenerative therapy.

Effect of Zinc Atmosphere upon Doping ZnSe : Fe Crystals on the Composition and Distribution of Impurity-Defect Centers

Zinc selenide crystals doped with iron by high-temperature diffusion in zinc and argon atmospheres are studied by the two-photon confocal microscopy. The effect of the atmosphere on the spectra and spatial distribution of two-photon excited luminescence in the range from 425 to 725 nm is studied. It is shown that doping with zinc results in the absence of impurity-defect centers formed in samples doped in an inert argon atmosphere.

The distribution of perivascular-resident cells in blood-labyrinth barrier observed with two-photon fluorescence microscope and Imaris deconvolution

Objective:To determine the cell types composing blood-labyrinth barrier in stira vascularis of cochlear lateral wall, analyze the distribution of these composing cells in blood-labyrinth barrier, and to investigate the relationship between perivascular-resident macrophages （PVMs）, endothelial cells and pericytes in blood-labyrinth barrier. Method:Cochlear lateral wall tissues were harvested from adult GFP-transgenic mice（C57BL/6）. Then the isolated whole stria vascularis tissue was scanned at 0.5 um intervals on the Z axis by two-photon confocal microscope and a 3D-structure of stria vascularis was reconstructed to observe the distribution of capillaries in blood-labyrinth barrier. Cochlear stria vascularis isolated from Balb/c mice was stained by mulit-immunofluorescence and then 3D real time deconvolution of stria vascularis was performed by Imaris software to investigate the distribution of PVMs and pericytes, and their contacting with basement membrane of capillaries was also observed. The ultrastructure of endothelial cells, pericytes and PVMs in blood-labyrinth barrier was observed using transmission electron microscope. Result:The vessels in stria vascularis are parallel to modiolus, distinct from that in spiral ligament which are perpendicular to modiolus. Numerous pericyts in stria vascularis are ensheathed by a vascular basement membrane shared with endothelial cells and closely attaching to the lateral wall of endothelial cells, while PVMs are located outside basement membrane of capillaries. Unlike pericytes that surround one capillary, PVMs branch to connect with more than one capillary. Conclusion:Serial layers on the Z axis scanned by two-photon confocal microscope and a 3D-structure reconstructed by Imaris 3D deconvolution helps to display the micro structure of capillaries in cochlear lateral wall clearly, which could be applied to analyze the 3D structure and function of blood-labyrinth barrier. PVMs in stria vascularis contact with more than one vessel through cytoplasmic processes, suggesting that PVMs may play a more significant role than pericytes in the integrity of blood-labyrinth barrier.