Confocal Scanning Laser Tomography Research Articles

1640. Toward New Anti-Biofilm Therapies: High Mobility Group Box 1 (HMGB1) Protein and Its Structural Variants Can Be Used to Disrupt Bacterial Biofilms (BBs)

BackgroundBB-related infections are a major public health problem, as they are notoriously refractory to current treatments. One of the defining characteristics of BBs is the extracellular polymeric substance (EPS). Extracellular DNA and the bacterial DNABII family of proteins are key components of EPS and are crucial for BBs structural integrity. It is known that targeting DNABII proteins disrupts BBs. We hypothesized that HMGB1, a DNA-binding eukaryotic protein, could affect BBs as it binds to the same DNA structures as the DNABII proteins. HMGB1 is comprised of 3 domains, A Box, B Box, and C tail, all of which have different functions. We aimed to determine in vitro the effects of HMGB1 and its individual domains against BBs.Methods Klebsiella pneumoniae (KP), a common cause of nosocomial infections, was used for all BBs disruption assays. Human recombinant full-length HMGB1 (rHMGB1; 1–215), a C45S mutation variant (mHMGB1) and the HMGB1 domains A Box (1–89), B Box (90–176), AB Boxes (1–176), B-linker Box (80–179), and B-linker Box C106S were expressed (in E. coli) and purified to >95%. To evaluate the effect of rHMGB1 and the various domains on established BBs, each protein species (200 nM) was added to preformed BBs at 24 hours. At 40 hours the BBs were washed, stained with LIVE/DEAD®, visualized via confocal laser scanning microscopy and images were analyzed by COMSTAT to calculate average thickness and biomass.ResultsExogenous rHMGB1 and its individual domains, with the exception of A Box caused a significant reduction (P < 0.05) in average thickness (AT) and biomass (BM) of KP biofilms when compared with untreated KP biofilms (% reduction mean ± SE in AT: 44% ± 0.33, 75% ± 0.04, 63% ± 0.1, 77% ± 0.03, 64% ± 0.08, 54% ± 0.15 and in BM: 61% ± 0.01, 80% ± 0.01, 68% ± 0.02, 67% ± 0.01, 73% ± 0.02, 56% ± 0.02 induced by rHMGB1, mHMGB1, B-Box, B-linker Box, AB Boxes, and B-linker Box C106S, respectively).ConclusionFull-length recombinant HMGB1 was able to significantly disrupt established KP biofilms as were all truncated HMGB1 forms containing the B Box domain and could potentially be used as a therapeutic treatment for BB-related infections.Disclosures J. Buzzo, ProclaRx: Collaborator, Research support. S. Goodman, ProclaRx: Collaborator and Scientific Advisor, Research support.

Neurochemical fingerprinting of amygdalostriatal and intra-amygdaloid projections: a tracing–immunofluorescence study in the rat

Amygdalostriatal and intra-amygdaloid fiber connectivity was studied in rats via injections of one of the tracers Phaseolus vulgaris leucoagglutinin (PHA-L) or biotinylated dextran amine (BDA) into various amygdaloid nuclei. To determine the neurotransmitter identity of labeled fibers we combined tracer detection with immunofluorescence staining, using antibodies against vesicular transporters (VTs) associated with glutamatergic (VGluT1, VGluT2) or GABAergic (VGAT) neurotransmission. High-magnification confocal laser scanning images were screened for overlap: occurrence inside tracer labeled fibers or axon terminals of immunofluorescence signal associated with one of the VTs.Labeled amygdalostriatal fibers were seen when tracer had been injected into the magnocellular and parvicellular portions of the basal amygdaloid nucleus and the lateral amygdaloid nucleus (nuclei belonging to 'cortical type' amygdaloid nuclei). Intra-amygdaloidal projection fibers were mostly found after tracer injections in the central and medial amygdaloid nuclei ('striatal type' amygdaloid nuclei).Terminals of tracer-labeled amygdalostriatal fibers contained immunofluorescence signal associated mostly with VGluT1 and to a lesser degree with VGluT2 or VGAT. Intra-amygdaloid labeled fibers showed colocalization mostly of VGluT1, followed by VGAT. VGluT2 co-occurred in a minority of intra-amygdaloid tracer-containing fiber terminals.We conclude from our observations that both amygdalostriatal and intra-amygdaloid projections, arising from, respectively, 'cortical type' and 'striatal type' amygdaloid nuclei contain strong glutamatergic and modest GABAergic components. The glutamatergic fibers express either VGluT1 or VGluT2. The absence in large numbers of tracer labeled fibers of expression of one of the selected VTs leads us to suspect that amygdalostriatal projection fibers may contain hitherto neglected neurotransmitters in these connections, e.g., aspartate.

Optical coherence tomography and magnetic resonance imaging visual pathway evaluation in Wolfram syndrome.

The aim of this study was to assess parameters of retinal morphology by using high-definition optical coherence tomography (OCT) in patients with Wolfram syndrome (WFS) and their relation to optic tract atrophy in magnetic resonance imaging (MRI). High-definition OCT and MRI parameters were evaluated in 12 patients with WFS (three males, nine females; median age at examination 12y 8mo, range 10y 2mo-15y 11mo) and referred to 30 individuals with type 1 diabetes (T1D) (12 males, 18 females; median age at examination 20y 5mo, range 16y 8mo-21y 4mo) and 33 typically developing comparison participants (10 males, 23 females; median age at examination 20y 7mo, range 13y-22y 4mo). Total thickness and quadrant thickness of the retinal nerve fibre layer (RNFL), macular full-thickness parameters and macular ganglion cell layer/inner plexiform layer, intraorbital and intracranial thickness of the optical nerve, as well as the optic chiasm and visual tracts were significantly reduced in patients with WFS compared with those having T1D and the typically developing comparison participants. Optic chiasm thickness correlated negatively in patients with WFS with both age (r=-0.79; p=0.002) and duration of diabetes (r=-0.62; p=0.032). Thickness of the intraorbital parts of the optic nerves in patients with WFS correlated positively with thickness of the superior RNFL (r=0.73; p=0.006). High-definition OCT in combination with MRI could become an important tool for evaluating the effectiveness of therapeutic trials in patients with WFS. Provides evidence of significant reduction of retinal parameters and optic nerves in patients with Wolfram syndrome (WFS). Shows correlations between magnetic resonance imaging parameters and retinal morphology parameters in patients with WFS.

A Common Glaucoma-risk Variant of SIX6 Alters Retinal Nerve Fiber Layer and Optic Disc Measures in a European Population: The EPIC-Norfolk Eye Study.

A common missense variant in the SIX6 gene (rs33912345) is strongly associated with primary open-angle glaucoma (POAG). We aimed to examine the association of rs33912345 with optic disc and retinal nerve fiber layer (RNFL) measures in a European population. We examined participants of the population-based EPIC-Norfolk Eye Study. Participants underwent confocal laser scanning tomography (Heidelberg Retina Tomograph II, HRT) to estimate optic disc rim area and vertical cup-disc ratio (VCDR). Scanning laser polarimetry (GDxVCC) was used to estimate average RNFL thickness. The mean of right and left eye values was considered for each participant. Genotyping was performed using the Affymetrix UK Biobank Axiom Array. Multivariable linear regression with the optic nerve head parameter as outcome variable and dosage of rs33912345 genotype as primary explanatory variable was used, adjusted for age, sex, disc area, axial length, and intraocular pressure. We further repeated analyses stratified into age tertiles. In total, 5433 participants with HRT data and 3699 participants with GDxVCC data were included. Each "C" allele of rs33912345 was associated with a smaller rim area (-0.030 mm [95% CI -0.040, -0.020]; P=5.4×10), a larger VCDR (0.025 [95% CI 0.017, 0.033]; P=3.3×10) and a thinner RNFL (-0.39 μm [95% CI -0.62, -0.15]; P=0.001). The RNFL association was strongest in the oldest age tertile, whereas rim area and VCDR associations were strongest in the youngest and oldest age tertiles. The protein-coding SIX6 variant rs33912345, previously associated with POAG, has a functional effect on glaucoma-associated optic nerve head traits in Europeans.

A Novel Fluorescence-Labeled Curcumin Conjugate: Synthesis, Evaluation and Imaging on Human Cell Lines

Curcumin, as the main ingredient of the curcuma spice, has increasingly become the target of scientific research. The turmeric root where the spice is obtained from has been widely used in the traditional medicine. Moreover, scientific studies have found that curcumin has anti-inflammatory, anti-cancer, anti-angiogenic effects as well as antibacterial properties. Recently, curcumin has gathered interest as a potential therapeutic agent in the research on Alzheimer's disease. A consistent problem in the investigative and therapeutic applications of curcumin is its poor solubility in aqueous solutions. In the present study, we synthesized a conjugate of curcumin, the amino acid lysine and the fluorescent dye fluorescein. This conjugate was soluble in cell culture medium and facilitated the examination of curcumin with fluorescence imaging methods. We studied the cell growth impact of unmodified curcumin on seven different human cell lines and then analyzed the uptake and cellular localization of our curcumin conjugate with confocal laser scanning imaging and flow cytometry on the seven cell lines. We found that unbound curcumin inhibited cell growth in vitro and was not taken up into the cells. The curcumin conjugate was internalized into the cell cytoplasm in a dot-like pattern and cellular uptake correlated with the cell membrane damage which was measured using propidium iodide. The CAL-72 osteosarcoma cell exhibited 3-4fold increased conjugate uptake and a strong uniform fluorescein staining in addition to the dot-like pattern observed in all cell lines. In conclusion, we successfully synthesized a novel water-soluble fluorescent curcumin conjugate which showed a strong preference for CAL-72 osteosarcoma cells in vitro.

Air-breathing bio-cathodes based on electro-active biochar from pyrolysis of Giant Cane stalks

An innovative low-tech solution to fabricate electro-active biochar (e-biochar) electrodes for bio-electrochemical systems (BES) is proposed. Ligno-cellulosic stalks of Giant Cane (Arundo Donax L.) were subjected to pyrolysis treatment at 900 °C for 1 h. The material kept its original hollow cylindrical shape, rigid morphology and porous texture, as confirmed by 3DX-ray micro-computed tomography. These characteristics are suitable for its use at the air-water interface in BES, as air-breathing bio-cathodes. BET (Brunauer-Emmett-Teller) specific surface area was equal to 114 ± 4 m2 g−1, with more than 95% of pores in the microporosity range (pore diameter < 1 nm). Surface electrocatalytic activity was sufficient to sustain oxygen reduction reaction at pH 7, in terms of both onset potential (−0.02 V vs Ag/AgCl) and reduction limiting current density (1 A m−2). Electrical resistivity measurements confirmed sufficient conductivity (8.9 × 10−3 ± 1 × 10−4 Ω m) of the material and Raman spectroscopy allowed to estimate a graphitization degree in relation to the ID/IG, equal to 2.26. In parallel, the e-biochar were tested as air-exposed bio-cathodes in BES, coupled to carbon cloth bio-anodes. After inoculation with wastewater from swine-farming, current densities were generated in the range of 100–150 mA m−2, along more than 2 months of operation, under sodium acetate feeding. Confocal laser scanning imaging revealed consistent biofilm formation on the water-side surface of the cathodes, while a nearly-complete absence of it at the air-side.These e-biochar electrodes might open innovative perspectives to scale-up BES for different applications. Here, consistent salts depositions on the material after 70 days of exposure to the wastewater, suggest that e-biochar biocathodes might serve to recycle nutrients to agricultural soils, through minerals-enriched biochar.

Dotted Core-Shell Nanoparticles for T1 -Weighted MRI of Tumors.

Gd-based T 1 -weighted contrast agents have dominated the magnetic resonance imaging (MRI) contrast agent market for decades. Nevertheless, they are reported to be nephrotoxic and the U.S. Food and Drug Administration has issued a general warning concerning their use. In order to reduce the risk of nephrotoxicity, the MRI performance of the Gd-based T 1 -weighted contrast agents needs to be improved to allow a much lower dosage. In this study, novel dotted core-shell nanoparticles (FeGd-HN3-RGD2) with superhigh r 1 value (70.0 mM-1 s-1 ) and very low r 2 /r 1 ratio (1.98) are developed for high-contrast T 1 -weighted MRI of tumors. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and histological analyses show good biocompatibility of FeGd-HN3-RGD2. Laser scanning confocal microscopy images and flow cytometry demonstrate active targeting to integrin αv β3 positive tumors. MRI of tumors shows high tumor ΔSNR for FeGd-HN3-RGD2 (477 ± 44%), which is about 6-7-fold higher than that of Magnevist (75 ± 11%). MRI and inductively coupled plasma results further confirm that the accumulation of FeGd-HN3-RGD2 in tumors is higher than liver and spleen due to the RGD2 targeting and small hydrodynamic particle size (8.5 nm), and FeGd-HN3-RGD2 is readily cleared from the body by renal excretion.

Easy Employment and Crosstalk-Free Detection of Seven Fluorophores in a Widefield Fluorescence Microscope.

Immunofluorescence staining has become an essential tool in pathology and biomedical sciences to identify rare cells, cell–cell interactions, and submicroscopic cellular components. Many experimental settings, however, suffer from the fact that traditional widefield fluorescence microscopy is usually restricted to imaging three or four fluorophores only. Due to a lack of morphological information and a high detection limit, even flow cytometry—which is capable of staining 20 or more fluorophores at the same time—is limited in its applicability, especially in areas such as rare cell detection. Other advanced imaging approaches, such as confocal laser scanning microscopy and imaging flow cytometry, may be addressing these shortcomings, but in turn require sophisticated downstream data processing and high capital outlay. Here, we describe a new method and filter set-up to routinely employ up to seven fluorophores on a traditional widefield fluorescence microscope equipped with a standard high-pressure mercury light source. Quantification of crosstalk between channels and actual seven-color imaging of cancer cells spiked into leukocytes demonstrate that there is no need for digital compensation correction algorithms. Our set-up thus permits a detailed analysis of rare cell populations, co-localization of antigens, and cell morphology in a standard research or routine laboratory setting.

Synthesis of tacrolimus nanocrystals and artificial tears surface modification

Objective To synthesize a dual-function nanodrug with immunosuppression of tacrolimus (FK506) and lubrication of artificial tear carboxymethylcellulose sodium (CMC), and provide a basis for the clinical treatment of dry eye syndrome. Methods The FK506 nanocrystals were prepared by ultrasonic method, and polyallylamine hydrochloride (PAH) and CMC were deposited alternatively on the surface of FK506 nanocrystals using layer-by-layer (LbL) self-assembly technology to prepare the bifunctional nanodrug. The morphology, particle size, surface charge, and composition of the nanodrug were analyzed. Results The particle size of FK506 nanocrystals was uniform, and the FK506-(PAH/CMC)3 was approximately spherical with uneven surface. Zeta potential detection results showed that the charge changed alternatively with the increasing of layer number. The results of laser scanning confocal imaging and infrared spectroscopy showed that PAH and CMC were successfully modified on the surface of FK506 nanocrystals. Conclusion The prepared FK506-(PAH/CMC)3 nanodrug is expected to provide a scientific basis for the combined treatment of dry eye. Key words: Tacrolimus; Nanocrystals; Artificial tear; Dry eyes

Evaluation of two‐dimensional Bruch's membrane opening minimum rim area for glaucoma diagnostics in a large patient cohort

To characterize the two-dimensional parameter Bruch's membrane opening minimum rim area (BMO-MRA) in spectral domain optical coherence tomography (SD-OCT) of the optic nerve head (ONH) compared to minimum rim width (BMO-MRW) and retinal nerve fibre layer (RNFL) thickness in a large patient cohort. Case-control, cross-sectional study of 705 eyes of 445 participants. A total of 449 eyes with glaucoma, 67 eyes with ocular hypertension and 189 healthy controls, underwent SD-OCT and confocal laser scanning tomography (CSLT), visual field testing and clinical examination. Morphometric ONH parameters, visual field function and diagnostic power were compared. Main outcome measures were SD-OCT-derived BMO-MRA, BMO-MRW, RNFL thickness and CSLT-derived rim area (DM-RA). Mean ONH area was 2.11±0.57mm2 ; mean BMO area was 1.89±0.45mm2 . Correlation of mean deviation in visual field to morphometric parameters was ρ=0.70, (p<0.001) for RNFL thickness, ρ=0.68 (p<0.001) for BMO-MRA, ρ=0.66 (p<0.001) for BMO-MRW. These correlations were not significantly different (p>0.05), while DM-RA correlated significantly worse (ρ=0.55; p<0.001). In receiver operating characteristics, the calculated area under the curve (AUC) and sensitivity at 90% specificity to differentiate glaucoma were 0.87% and 70.1% for BMO-MRA, 0.86% and 68.1% for RNFL thickness, 0.84% and 66.0% for BMO-MRW, 0.82% and 51.3% for DM-RA. In a heterogenous clinical cohort of glaucoma patients, all analysed SD-OCT parameters excel DM-RA of CSLT. The two-dimensional parameter BMO-MRA shows comparable levels of diagnostic power to detect glaucoma compared to established parameters BMO-MRW and RNFL thickness. Given higher comparability between ONH sizes, BMO-MRA might become an additional standard tool in SD-OCT imaging for glaucoma.

Structural and functional analyses of the optic nerve and lateral geniculate nucleus in glaucoma

PurposeTo analyze the correlation between structural characteristics of intraorbital optic nerve (ION) and lateral geniculate nucleus (LGN) measured by 3-Tesla magnetic resonance imaging (3T MRI), and the severity of glaucomatous damage.MethodsIn this cross-sectional study, 41 glaucoma patients and 12 age- and sex-matched controls underwent standard automated perimetry (SAP) and frequency doubling technology (FDT) as functional evaluation; optic disc stereophotograph, spectral-domain optical coherence tomography (OCT) and confocal scanning laser tomography as ocular structural evaluation; and 3T MRI. Structure-structure and structure-function correlation were performed using bootstrap resampling method for clustered data.ResultsThe ION mean diameter and cross-sectional area were different between glaucoma and control groups at 5mm and 10mm (all, p≤0.011) from the globe, but not at 15mm (both, p≥0.067). LGN height was significantly lower in glaucoma group (p = 0.005). OCT rim area and functional parameters (SAP and FDT) correlated significantly with all ION segments, showing stronger correlations at 10 and 15 mm. ION parameters at 10 and 15 mm presented mild-to-moderate correlation with OCT peripapillary nerve fiber layer thickness, and ION at 15mm had mild association with the neuroretinal rim area on stereophotographs. Although LGN height was significantly smaller in glaucoma group (p = 0.005), LGN parameters were not associated with any ocular structural or functional parameter.ConclusionAssessment of central and peripheral nervous systems using 3T MRI confirmed that glaucoma patients had smaller ION dimensions and LGN height compared to the control group. In general, ION dimensions presented mild to moderate correlations with functional and ocular structural parameters. Although ION had significant correlations at any distance from the eye, the ION distal locations correlated better with OCT results and functional parameters. However, LGN parameters were not associated with functional or ocular structural parameters.

Short-Term Effects of Overnight Orthokeratology on Corneal Sub-basal Nerve Plexus Morphology and Corneal Sensitivity.

To assess the effects of a short period of orthokeratology (OK) on corneal sub-basal nerve plexus (SBNP) morphology and corneal sensitivity. Measurements were made in 56 right eyes of 56 subjects with low-to-moderate myopia who wore 2 OK lens designs (Group CRT: HDS 100 Paragon CRT, n=35; Group SF: Seefree; n=21) for a period of 1 month and in 15 right eyes of noncontact lens wearers as controls. The variables determined in each participant were corneal sensitivity using a Cochet-Bonnet esthesiometer and 12 SBNP variables determined on laser scanning confocal microscopy images using 3 different software packages. Correlation between SBNP architecture and corneal sensitivity was also examined. Few changes were observed over the 1-month period in the variables examined in the OK treatment and control groups. However, significant reductions were detected over time in the number of nerves in the central cornea in the groups CRT (P=0.029) and SF (P=0.043) and in central corneal sensitivity in CRT (P=0.047) along with significant increases in central and midperipheral corneal Langerhans cell counts in SF (P=0.001 and 0.048, respectively). This study provides useful data to better understand the anatomical changes induced by OK in corneal SBNP. The different response observed to the 2 OK lens designs requires further investigation.

Bruch's membrane opening-based optical coherence tomography of the optic nerve head: a useful diagnostic tool to detect glaucoma in macrodiscs.

PurposeTo compare Bruch's membrane opening (BMO)-based spectral domain optical coherence tomography (SD-OCT) and margin based confocal scanning laser tomography (CSLT) of the optic nerve head (ONH) to visual field function in large optic discs (macrodiscs) and to assess performance for glaucoma detection.MethodsIn a case-control, cross-sectional study, 125 eyes of 125 patients with disc size >2.45 mm2, thereof 44 glaucoma and 11 ocular hypertension (OHT) patients and 70 healthy controls underwent SD-OCT and CSLT examination, visual field testing and clinical evaluation. Mean outcome measures BMO-based minimum rim width (BMO-MRW), retinal nerve fiber layer thickness (RNFLT) in SD-OCT, and rim area measured in CSLT were compared and correlated to visual field function.ResultsAll participants had a mean disc area of 2.91±0.38 mm2 in CSLT and a BMO area of 2.45±0.39 mm2 (r=0.76;P<0.001). In glaucoma patients, visual field mean deviation was -10.0±6.1 dB. Global BMO-MRW correlated better to visual field function (Spearman's Rho (ρ)=0.71; P<0.001) than RNFLT (ρ=0.52;P<0.001) and CSLT rim area (ρ=0.63; P<0.001). BMO-MRW was significantly decreased with higher visual field loss (P<0.001). In ROC analysis, diagnostic power to differentiate glaucoma patients and healthy controls was highest for BMO-MRW (Area under curve, AUC=0.96; sensitivity at 95% specificity=82%). Rim area in CSLT (AUC=0.91; sensitivity=61.0%; P=0.04) and RNFLT (AUC=0.89; sensitivity=61%; P=0.01) were significantly less powerful.ConclusionsIn macrodiscs, BMO-MRW has the best diagnostic power to discriminate glaucoma patients from normal controls compared to RNFLT and rim area in CSLT. Additionally, BMO-MRW seems to reflect the structure-function relationship better than the other two parameters.

A new approach to coat PA12 powders with laser-generated nanoparticles for selective laser sintering

The modification of selective laser sintering (SLS) powder materials by nanoadditives offers the possibility to adapt the powder properties to the laser sintering process or the resulting part properties. To avoid agglomeration of the nanofiller, a new approach in which surfactant-free laser-generated colloidal nanoparticles are adsorbed onto the polymer surface directly in an aqueous solution is demonstrated. Based on this novel approach, polyamide 12 (PA12) powders are decorated with metal and oxide nanoparticles and processed via SLS. Electron microscopy and confocal laser scanning imaging are utilized to analyze the dispersion of the filler.

Synthesis of Biotin-Modified Galactosylated Chitosan Nanoparticles and Their Characteristics in Vitro and in Vivo

Background/Aims: Our previous study found that a nanoparticle drug delivery system that operates as a drug carrier and controlled release system not only improves the efficacy of the drugs but also reduces their side effects. However, this system could not efficiently target hepatoma cells. The aim of this study was to synthesize biotin-modified galactosylated chitosan nanoparticles (Bio-GC) and evaluate their characteristics in vitro and in vivo. Methods: Bio-GC nanomaterials were synthesized, and confirmed by fourier transform infrared spectroscopy (FT-IR) and hydrogen-¹ nuclear magnetic resonance (¹H-NMR). The liver position and cancer target property of Bio-GC nanoparticles in vitro and in vivo was tested by confocal laser and small animal imaging system. The characteristics of Bio-GC/5-fluorouracil (5-FU) nanoparticles in vitro and in vivo were explored by cell proliferation, migration and cytotoxicity test, or by animal experiment. Results: Bio-GC nanoparticles were synthesized with biodegradable chitosan as the nanomaterial skeleton with biotin and galactose grafts. Bio-GC was confirmed by FT-IR and ¹H-NMR. Bio-GC/5-FU nanoparticles were synthesized according to the optimal mass ratio for Bio-GC/5-FU (1: 4) and had a mean particle size of 81.1 nm, zeta potential of +39.2 mV, and drug loading capacity of 8.98%. Bio-GC/5-FU nanoparticles had sustained release properties (rapid, steady, and slow release phases). Bio-GC nanoparticles targeted liver and liver cancer cell in vitro and in vivo, and this was confirmed by confocal laser scanning and small animal imaging system. Compared with GC/5-FU nanoparticles, Bio-GC/5-FU nanoparticles showed more specific cytotoxic activity in a dose- and time-dependent manner and a more obvious inhibitory effect on the migration of liver cancer cells. In addition, Bio-GC/5-FU nanoparticles significantly prolonged the survival time of mice in orthotopic liver cancer transplantation model compared with other 5-FU nanoparticles or 5-FU alone. Bio-GC (0.64%) nanomaterial had no obvious cytotoxic effects on cells; thus, the concentration of Bio-GC/5-FU nanoparticles used was only 0.04% and showed no toxic effects on the cells. Conclusion: Bio-GC is a liver- and cancer-targeting nanomaterial. Bio-GC/5-FU nanoparticles as drug carriers have stronger inhibitory effects on the proliferation and migration of liver cancer cells compared with 5-FU in vitro and in vivo.

Laser Scanning Confocal Image Alignment Assists Sub-micron Accuracy Micro-assembly

The image resolution of the ordinary optical microscope in the assembly system can’t break through the optical diffraction limit, and the accuracy of alignment detection can’t reach the sub-micron level. So we build the micro assembly system based on the principle of confocal image alignment to solve the above problems. In this paper, the basic principle and system of confocal image alignment assembly are studied, and the levelling calibration method of the platform is also studied. After the calibration, the alignment resolution of the micro assembly system based on the principle of confocal image alignment can reach 0.5μm.The confocal image alignment principle is verified by the assembly application experiment of inserting a 125-μm-diameter optical fiber into a 127-μm-diameterglass micro tubule. The confocal image alignment principle and the calibration method can greatly improve the accuracy and efficiency of the micro assembly.

High-resolution Confocal Imaging of the Blood-brain Barrier: Imaging, 3D Reconstruction, and Quantification of Transcytosis

The blood-brain barrier (BBB) is a dynamic multicellular interface that regulates the transport of molecules between the circulation and the brain. Transcytosis across the BBB regulates the delivery of hormones, metabolites, and therapeutic antibodies to the brain parenchyma. Here, we present a protocol that combines immunofluorescence of free-floating sections with laser scanning confocal microscopy and image analysis to visualize subcellular organelles within endothelial cells at the BBB. Combining this data-set with 3D image analysis software allows for the semi-automated segmentation and quantification of capillary volume and surface area, as well as the number and intensity of intracellular organelles at the BBB. The detection of mouse endogenous immunoglobulin (IgG) within intracellular vesicles and their quantification at the BBB is used to illustrate the method. This protocol can potentially be applied to the investigation of the mechanisms controlling BBB transcytosis of different molecules in vivo.

High-resolution Confocal Imaging of the Blood-brain Barrier: Imaging, 3D Reconstruction, and Quantification of Transcytosis.

The blood-brain barrier (BBB) is a dynamic multicellular interface that regulates the transport of molecules between the circulation and the brain. Transcytosis across the BBB regulates the delivery of hormones, metabolites, and therapeutic antibodies to the brain parenchyma. Here, we present a protocol that combines immunofluorescence of free-floating sections with laser scanning confocal microscopy and image analysis to visualize subcellular organelles within endothelial cells at the BBB. Combining this data-set with 3D image analysis software allows for the semi-automated segmentation and quantification of capillary volume and surface area, as well as the number and intensity of intracellular organelles at the BBB. The detection of mouse endogenous immunoglobulin (IgG) within intracellular vesicles and their quantification at the BBB is used to illustrate the method. This protocol can potentially be applied to the investigation of the mechanisms controlling BBB transcytosis of different molecules in vivo.

Cisplatin and doxorubicin high-loaded nanodrug based on biocompatible thioether- and ethane-bridged hollow mesoporous organosilica nanoparticles

Herein, a mesoporous organosilica nanoparticle (MON) based nanodrug highly loaded with cisplatin (CDDP) and doxorubicin (DOX) (denoted as MONs/CDDP/DOX) has been successfully prepared for the first time. The MONs are characterized with core-contained double hollow shells, thioether and ethane groups separately incorporated frameworks, uniform diameter (420 nm), large surface area (592 m2/g), and ordered pore size (2.5 nm). The safety evaluation of the MONs based on cell viability, haemolytic activity, histological change, and serum biochemical index demonstrates that they have excellent biological compatibility. The efficient uptake of the MONs by human breast cancer MCF-7 cells is further confirmed via confocal laser scanning imaging and flow cytometry. Importantly, the contents of CDDP and DOX in the MONs/CDDP/DOX nanodrug are as high as 120 mg/g and 85 mg/g, respectively. Therefore, the MONs/CDDP/DOX shows a significant improved killing effect against human breast cancer MCF-7 cells compared with sole DOX or CDDP loaded MONs, demonstating the promise of the nanodrug for cancer treatment.

Imaging Amyloid Tissues Stained with Luminescent Conjugated Oligothiophenes by Hyperspectral Confocal Microscopy and Fluorescence Lifetime Imaging

Proteins that deposit as amyloid in tissues throughout the body can be the cause or consequence of a large number of diseases. Among these we find neurodegenerative diseases such as Alzheimer's and Parkinson's disease afflicting primarily the central nervous system, and systemic amyloidosis where serum amyloid A, transthyretin and IgG light chains deposit as amyloid in liver, carpal tunnel, spleen, kidney, heart, and other peripheral tissues. Amyloid has been known and studied for more than a century, often using amyloid specific dyes such as Congo red and Thioflavin T (ThT) or Thioflavin (ThS). In this paper, we present heptamer-formyl thiophene acetic acid (hFTAA) as an example of recently developed complements to these dyes called luminescent conjugated oligothiophenes (LCOs). hFTAA is easy to use and is compatible with co-staining in immunofluorescence or with other cellular markers. Extensive research has proven that hFTAA detects a wider range of disease associated protein aggregates than conventional amyloid dyes. In addition, hFTAA can also be applied for optical assignment of distinct aggregated morphotypes to allow studies of amyloid fibril polymorphism. While the imaging methodology applied is optional, we here demonstrate hyperspectral imaging (HIS), laser scanning confocal microscopy and fluorescence lifetime imaging (FLIM). These examples show some of the imaging techniques where LCOs can be used as tools to gain more detailed knowledge of the formation and structural properties of amyloids. An important limitation to the technique is, as for all conventional optical microscopy techniques, the requirement for microscopic size of aggregates to allow detection. Furthermore, the aggregate should comprise a repetitive β-sheet structure to allow for hFTAA binding. Excessive fixation and/or epitope exposure that modify the aggregate structure or conformation can render poor hFTAA binding and hence pose limitations to accurate imaging.