Confocal Laser Scanning Fluorescence Research Articles

Biodegradable Nanocomposite of ZnS(Mn) Quantum Dots Immobilized Graphene Oxide for Bioimaging Applications.

Developing a biocompatible and biodegradable graphene-based fluorescent nanoprobe with the ability to visualize live cells could be interesting for intracellular imaging and monitoring the efficiency of chemotherapy. Herein, we report a biodegradable and biocompatible hybrid fluorescent graphene oxide (GO)-ZnS(Mn) composite synthesized via in situ growth of ZnS(Mn) quantum dots (QDs) on the surface of GO in the aqueous medium. The prepared 'GO-ZnS(Mn)' composite was characterized by X-ray diffraction (XRD), X-ray photoelectron spectroscopy (XPS), thermogravimetric analysis (TGA) and high-resolution transmission electron microscopy (HR-TEM) along with selected area electron diffraction (SAED). Further, the fluorescence properties of the GO-ZnS(Mn) composite were studied using fluorescence emission spectroscopy. The composite material exhibited a strong and broad visible light fluorescence from 500 to 600 nm by excitation with 365 nm (UV) light. The cytotoxic experiments of folic acid (FA) conjugated GO-ZnS(Mn) using MTT [(3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide)] assay revealed that the composite had excellent biocompatibility even at higher concentrations up to 200 µg/mL in HeLa cell lines. Next, the bioimaging experiments carried out using confocal fluorescence laser scanning microscopy (CLSM) revealed that GO-ZnS(Mn) composite was taken up by the HeLa cells effectively within 12 h of incubation via receptor (folate) mediated endocytosis with strong fluorescence throughout the cell surface. Finally, the biodegradability of GO-ZnS(Mn) composite was studied by treating it with human myeloperoxidase enzyme (hMPO) isolated from the primary immune cells, neutrophils, which is important to understand the in vivo fate of GO-Zns(Mn). The HR-TEM and Raman analyses confirmed the biodegradation of GO-ZnS(Mn) within 15 h of hMPO treatment. Thus, the biodegradable GO-ZnS (Mn) composite could be helpful for chemotherapy and bioimaging applications.

Fabrication of hydroxyapatite porous body with connective pores using the self-networking property of rice starch powder by heat treatment

• Porous hydroxyapatite ceramics was fabricated. • Gelatinization-retrogradation phenomena of starch was used as pore-networking agent. • Randomly-developed open pores were introduced into the hydroxyapatite matrix. • Pore structure was characterized by confocal scanning laser fluorescence microscopy. Hydroxyapatite porous ceramics were fabricated utilizing the property called gelatinization-retrogradation in which starch particles used as a pore-forming agent form a self-network structure by heat treatment. The pore structures in the hydroxyapatite samples were observed by combining the immersion transmission method and a confocal scanning laser fluorescence microscope. The three-dimensional image created by stacking the cross-sectional observation images showed that randomly-developed open pores were introduced into the matrix.

Wavefront sensor-less adaptive optics using deep reinforcement learning.

Image degradation due to wavefront aberrations can be corrected with adaptive optics (AO). In a typical AO configuration, the aberrations are measured directly using a Shack-Hartmann wavefront sensor and corrected with a deformable mirror in order to attain diffraction limited performance for the main imaging system. Wavefront sensor-less adaptive optics (SAO) uses the image information directly to determine the aberrations and provide guidance for shaping the deformable mirror, often iteratively. In this report, we present a Deep Reinforcement Learning (DRL) approach for SAO correction using a custom-built fluorescence confocal scanning laser microscope. The experimental results demonstrate the improved performance of the DRL approach relative to a Zernike Mode Hill Climbing algorithm for SAO.

Fabrication of Microstructured Surface Topologies for the Promotion of Marine Bacteria Biofilm.

Several marine bacteria of the Roseobacter group can inhibit other microorganisms and are especially antagonistic when growing in biofilms. This aptitude to naturally compete with other bacteria can reduce the need for antibiotics in large-scale aquaculture units, provided that their culture can be promoted and controlled. Micropatterned surfaces may facilitate and promote the biofilm formation of species from the Roseobacter group, due to the increased contact between the cells and the surface material. Our research goal is to fabricate biofilm-optimal micropatterned surfaces and investigate the relevant length scales for surface topographies that can promote the growth and biofilm formation of the Roseobacter group of bacteria. In a preliminary study, silicon surfaces comprising arrays of pillars and pits with different periodicities, diameters, and depths were produced by UV lithography and deep reactive ion etching (DRIE) on polished silicon wafers. The resulting surface microscale topologies were characterized via optical profilometry and scanning electron microscopy (SEM). Screening of the bacterial biofilm on the patterned surfaces was performed using green fluorescent staining (SYBR green I) and confocal laser scanning microscopy (CLSM). Our results indicate that there is a correlation between the surface morphology and the spatial organization of the bacterial biofilm.

Effects on Lignin Redistribution in Eucalyptus globulus Fibres Pre-Treated by Steam Explosion: A Microscale Study to Cellulose Accessibility.

The objective of this study was to investigate structural changes and lignin redistribution in Eucalyptus globulus pre-treated by steam explosion under different degrees of severity (S0), in order to evaluate their effect on cellulose accessibility by enzymatic hydrolysis. Approximately 87.7% to 98.5% of original glucans were retained in the pre-treated material. Glucose yields after the enzymatic hydrolysis of pre-treated material improved from 19.4% to 85.1% when S0 was increased from 8.53 to 10.42. One of the main reasons for the increase in glucose yield was the redistribution of lignin as micro-particles were deposited on the surface and interior of the fibre cell wall. This information was confirmed by laser scanning confocal fluorescence and FT-IR imaging; these microscopic techniques show changes in the physical and chemical characteristics of pre-treated fibres. In addition, the results allowed the construction of an explanatory model for microscale understanding of the enzymatic accessibility mechanism in the pre-treated lignocellulose.

Microscopic analysis of the microbiota of three commercial Phytoseiidae species (Acari: Mesostigmata)

Microbes associated with the external and internal anatomy of three commercially available predatory mite species—Phytoseiulus persimilis, Typhlodromips (= Amblyseius) swirskii, and Neoseiulus (= Amblyseius) cucumeris—were examined using light microscopy, confocal laser scanning microscopy and fluorescence in-situ hybridization (FISH). Four microbe morphotypes were observed on external body regions. These included three microfungi-like organisms (named T1, T2 and T3) and rod-shaped bacteria (T4). Morphotypes showed unique distributions on the external body regions and certain microbes were found only on one host species. Microfungi-like T1 were present in all three species whereas T2 and T3 were present in only P. persimilis and T. swirskii, respectively. T1 and T2 microbes were most abundant on the ventral structures of the idiosoma and legs, most frequently associated with coxae, coxal folds, ventrianal shields and epigynal shields. T3 microbes were most abundant on legs and dorsal idiosoma. T4 microbes were less abundant and were attached to epigynal shields of N. cucumeris and T. swirskii. Significant differences in distribution between batches suggest temporal fluctuations in the microbiota of phytoseiids in mass-reared systems. FISH showed bacteria within the alimentary tract, in Malpighian tubules and anal atria. These may aid absorption of excretory products or maintaining gut physiology. We suggest a mechanism by which microbes may be transmitted to offspring and throughout populations. This study aims to improve our knowledge of this poorly understood area and highlights the necessity of understanding the microbiota of Acari.

Impact of Sub-Retinal Fluid on the Long-Term Incidence of Macular Atrophy in Neovascular Age-related Macular Degeneration under Treat & Extend Anti-Vascular Endothelial Growth Factor Inhibitors

Sub-retinal fluid (SRF) has been discussed as a protective factor against macular atrophy in eyes with neovascular age-related macular degeneration (nAMD).To gauge the impact of SRF on macular atrophy, a database of 310 nAMD eyes was screened for eyes manifesting an SRF-only phenotype under treat & extend anti-VEGF treatment, defined as nAMD expressing CNV exudation beyond the three monthly anti-VEGF loading doses by SRF only without any signs of exudative intra-retinal fluid (IRF) for ≥3 years. Incidence of macular atrophy and treatment responses were evaluated on multimodal imaging, including optical coherence tomography (OCT), blue autofluorescence (BAF) and near-infrared (NIR) confocal scanning laser ophthalmoscopy and fluorescence and indocyanine green angiography (FAG/ICGA). In total, 27 eyes (8.7%) of 26 patients with a mean follow-up of 4.2 ± 0.9 (3–5) years met the inclusion criteria. Mean age was 72 ± 6 (range: 61–86) years. The SRF only phenotype was seen from baseline in 14 eyes (52%), and in 13 eyes (48%) after a mean 1.0 ± 1.3 (1–3) injections. In years 1 to 5, mean 7.5, 5.9, 6.1, 6.1 and 7.0 anti-VEGF injections were given (p = 0.33). Cumulative macular atrophy incidence was 11.5% at year 1, 15.4% throughout years 2 to 4, and 22.4% at year 5. In conclusion, eyes manifesting activity by SRF only in treat & extend anti-VEGF regimen for nAMD seem to exhibit rather low rates of macular atrophy during long-term follow-up. SRF might be an indicator of a more benign form of nAMD.

Changes in microcirculation and structural components of the skin under photodynamic effects

Introduction . Distinctive features of microcirculation and their connection with structural disorders in the skin during photodynamic exposure using various photosensitizers (PS) are not well understood. Objective . Comparative analysis of the relationship between morphological abnormalities and changes in blood flow in the skin during photoactivation of PS, taking into consideration the influence on mast cells (MC), structural elements of the dermis and underlying muscle tissue. Materials and methods . In this work were used male Wistar rats (n=46). PS were administered intravenously: Radachlorin (RDC) (5 mg/kg) 3 hours, Coproporphyrin III (CPP) (10 mg/kg) 24 hours before irradiation. To control the accumulation of PS in the skin, methods of fluorescence videodermatoscopy and confocal scanning laser microscopy (CSLM) were used. Laser irradiation (662 and 635 nm) was performed with an energy density of 50 Jcm –2 , the irradiated surface area was 0,78 cm 2 . Laser Doppler flowmetry was used to assess microcirculation in the skin. Results . According to videodermatoscopy, fluorescence maximum values in the R channel were recorded 6-8 hours after administration, and amounted to 4901 [4190; 6732] a. u. for RDC and 2997 [2678; 3351] a. u. for CPP. Using the CSLM method, it was shown that CPP is poorly visualized in the walls of blood vessels. In experiments with RDC, the structures of areolar tissue of the hypodermis are determined. An hour after laser irradiation, in experiments with RDC, as well as with CPP, a significant decrease in blood flow was noted compared with the control to 3,61±0,7 and 4,27±1,4 PU, respectively. Structural changes when using both PS showed endothelial swelling, vasodilation and congestion, hemorrhages in the dermis, and underlying muscle tissue. The amount of MC was reduced. Conclusions . Laser irradiation with the parameters used in the work leads to a significant decrease in blood flow in the skin in experimental animals as a result of photoactivation of PS. CPP requires a higher radiation energy density. Morphological changes were revealed not only at the level of the epidermis, dermis and hypodermis, but also the underlying striated muscle tissue. In the early stages after exposure, massive degranulation of MC is noted with a decrease in their number.

Unity in diversity: a survey of muscular systems of ctenostome Gymnolaemata (Lophotrochozoa, Bryozoa)

BackgroundMyoanatomical studies of adult bryozoans employing fluorescent staining and confocal laser scanning microscopy (CLSM) have been chiefly conducted on freshwater bryozoans. The diversity of muscular systems in the marine bryozoans is currently not well known with only two species being studied in more detail. The aim of this study is to unravel the diversity of muscle systems of 15 ctenostome bryozoans by phalloidin-coupled fluorescence stainings combined with CLSM.ResultsIn general, the myoanatomy of the selected ctenostomes shows significant similarities and consists of 1) muscles associated with the body wall, 2) apertural muscles, 3) lophophoral muscles, 4) tentacle sheath muscles, 5) digestive tract muscles and 6) the prominent retractor muscles. Differences are present in the arrangement of the apertural muscles from generally three muscles sets of four bundles, which in some species can be partially reduced or modified into a bilateral arrangement. The cardiac region of the digestive tract shows a distinct sphincter in four of the six studied clades. In some cases the cardiac region forms a prominent proventriculus or gizzard. Tentacle sheath muscles in victorelloideans and walkerioideans are arranged diagonally and differ from the simple longitudinal muscle arrangements common to all other taxa. Lophophoral base muscles consist of four sets that vary in the size of the sets and in the shape of the inner lophophoral ring, which either forms a complete ring or separate, intertentacular muscle bundles. The stolon-forming walkeridiodean ctenostomes show prominent transverse muscles in their stolons. These are always present in the shorter side stolons, but their occurrence in the main stolon seems to depend on the colony form, being present in creeping but absent in erect colony forms.ConclusionsThis study represents the first broad survey of muscular systems in adult ctenostome bryozoans and shows a certain degree of conservation in a series of diverse colony forms belonging to five major clades. However, several myoanatomical features such as the cardiac sphincter, basal (possibly transitory) cystid muscles, tentacle sheath muscles or apertural muscle arrangement vary across taxa and thus show a high potential for the assessment of character evolution within ctenostomes. As such, this study represents an essential contribution towards determining and reconstructing the character states of the bryozoan ground pattern once a reliable phylogenetic tree of the whole phylum becomes available.

3D Fluorescence‐Based Security Features by 3D Laser Lithography

Abstract3D optical security features manufactured by multistep 3D optical laser lithography are presented. These microstructures are composed of a nonfluorescent 3D cross‐grid scaffold and fluorescent markers, realized by an acrylate‐based resist containing CdSSe‐based core–shell semiconductor quantum dots, arranged onto this scaffold at will. The readout of these 3D microstructures can be (exclusively) achieved by optical sectioning methods, for example, 3D confocal fluorescence laser scanning microscopy. As examples, structures with five different layers of markers and one emission color and two different colors, respectively, are shown. This class of deterministic 3D microstructures can be embedded into thin polymer films, can be individualized, and allows for plenty of future variations and extensions. It therefore represents an interesting avenue beyond established 2D holographic or 2D fluorescent optical security features.

Gb3 Glycosphingolipids with Fluorescent Oligoene Fatty Acids: Synthesis and Phase Behavior in Model Membranes.

Glycosphingolipids are involved in a number of physiological and pathophysiological processes, and they serve as receptors for a variety of bacterial toxins and viruses. To investigate their function in lipid membranes, fluorescently labeled glycosphingolipids are highly desirable. Herein, a synthetic route to access Gb3 glycosphingolipids with fluorescently labeled fatty acids, consisting of pentaene and hexaene moieties either at the terminus or in the middle of the acyl chain, has been developed. The fluorescent properties of the Gb3 derivatives were investigated in small unilamellar vesicles composed of a raft-like mixture. Phase-separated giant unilamellar vesicles (GUVs) allowed the quantification of the apparent partitioning coefficients of the Gb3 compounds by means of confocal fluorescence laser scanning microscopy. The determined partition coefficients demonstrate that the Gb3 derivatives are preferentially localized in the liquid-disordered (ld ) phase. To analyze whether the compounds behave like their physiological counterparts, Cy3-labeled (Cy: cyanine) Shiga toxin B subunits (STxB) were specifically bound to Gb3 -doped GUVs. However, the protein was favorably localized in the ld phase, in contrast to results reported for STxB bound to naturally occurring Gb3 , which is discussed in terms of the packing density of the lipids in the liquid-ordered (lo ) phase.

Experimental verification of subthreshold laser therapy using conventional pattern scan laser.

PurposeLeading-edge therapeutic laser technologies are not available at every medical facility; therefore, alternative approaches incorporating novel advances in digital and laser technology into more readily available conventional methods have generated significant research interest. Using a rabbit model, this study investigated whether the algorithm used in the Endpoint Management (EM) software system of the latest devices could enable subthreshold laser treatment in conventional retinal tissue laser therapy systems.Materials and methodsTwo types of devices were used, the PASCAL Streamline 577 and the MC 500-Vixi™, and the laser method was classified into three categories: EM; single-shot using PASCAL with arbitrary energy settings (PSS-SDM); and MC500-VixiTM (VX-SDM), which were performed in eight eyes from four Dutch-Belted rabbits. In EM, 100 mW (100%) was set as a landmark, and the laser energy parameters were gradually decreased to 80%, 60%, 50%, 40%, 30%, 20%, and 10%, using a 2 × 3 square pattern. In PSS-SDM and VX-SDM, as control, the laser energy was gradually decreased to 100, 80, 60, 50, 40, 30, 20, and 10 mW. The laser settings were fixed at 200 μm, 20 ms, and a wavelength of 577 μm. To identify and compare the extent of tissue damage at each spot size, optical coherence tomography (OCT) and histological findings were used to construct a three-dimensional histopathology image using a confocal laser scanning fluorescence microscope.ResultsThe spot size at 50% setting on EM was 7183 μm2; PSS-SDM required 50 mW (5503 μm2) to 60 mW (10279 μm2) and VX-SDM required 50 mW (7423 μm2) to create the approximate spot size. Furthermore, at 50 mW of PSS-SDM and VX-SDM, the extent of tissue damage in all three methods was generally in accord with the outer nuclear layer by OCT and inner nuclear layer by histopathological imaging.ConclusionThese findings suggest that it may be possible to perform subthreshold laser therapy using approximations from the EM algorithm.

Application of fluorescently labelled lectins for the study of polysaccharides in biofilms with a focus on biofouling of nanofiltration membranes

The biofilm state is the dominant microbial lifestyle in nature. A biofilm can be defined as cells organised as microcolonies embedded in an organic polymer matrix of microbial origin living at an interface between two different liquids, air and liquid, or solid and liquid. The biofilm matrix is made of extracellular polymeric substances, polysaccharides being considered as the major structural components of the matrix. Fluorescently labelled lectins have been widely used to stain microbial extracellular glycoconjugates in natural and artificial environments, and to study specific bacterial species or highly complex environments. Biofilm development at the membrane surface conducting to biofouling is one of the major problems encountered during drinking water production by filtration. Biofouling affects the durability and effectiveness of filtration membranes. Biofouling can be reduced by pretreatments in order to control two key parameters of water, the bioavailable organic matter concentration and the concentration of live bacteria. Nanofiltration (NF) is a high technology process particularly suited to the treatment of surface waters to produce drinking water that is highly sensitive to biofouling. The development of strategies for fouling prevention and control requires characterizing the fouling material composition and organisation before and after NF membrane cleaning. The aim of this review is to present basics of biofilm analyses after staining with fluorescently labelled lectins and to focus on the use of fluorescent lectins and confocal laser scanning microscopy to analyse NF membrane biofouling.

Differential Sensitivity of Madin-darby Canine Kidney (MDCK) Cells to Epinephrine.

Catecholamines regulate a variety of cellular functions in the mammalian kidney. The present study was aimed to investigate the differential sensitivity of Madin-Darby Kidney Cells (MDCK cells) to epinephrine in a dose-dependent manner. The loss of adhesion and altered cell shape were observed in MDCK cells. The presence of apoptosis and necrosis were studied by the fluorescence microscope and Confocal Laser Scanning Microscope (CLSM). Scanning Electron Microscope (SEM) analysis showed several surface microvilli, and cells were rounded having ruffled and crenated surface. Agarose gel electrophoresis study showed the presence of smearing, which further confirms the occurrence of necrosis. The fluorescence staining study showed the increased reactive oxygen species (ROS) level. Up-regulation of p53, bax, and caspase 3 mRNA expressions was evidenced by quantitative PCR (qPCR). Caspase 3 activity was also increased in epinephrine treated cells. Our experimental results do not imply that the epinephrine should not be used in the clinical treatments. However, our results add a research note of caution on the possible cytotoxic effect of maximal doses of epinephrine over a prolonged time.

Substrate Elastic Modulus Regulates the Morphology, Focal Adhesions, and α-Smooth Muscle Actin Expression of Retinal Müller Cells.

The stiffness of the extracellular matrix has been shown to regulate cell adhesion, migration, and transdifferentiation in fibrotic processes. Retinal Müller cells have been shown to be mechanosensitive; they are involved in fibrotic vitreoretinal diseases. Since fibrosis increases the rigidity of the extracellular matrix, our aim was to develop an in vitro model for studying Müller cell morphology and differentiation state in relation to matrix stiffness. A spontaneously immortalized human Müller cell line (MIO-M1) was cultured on type I collagen-coated polyacrylamide gels with Young's moduli ranging from 2 to 92 kPa. Cell surface area, focal adhesion, and the expression and morphology of α-smooth muscle actin induced by transforming growth factor β (TGF-β [10 ng/mL for 48 hours]) were analyzed by immunocytology. The images were documented by using fluorescence microscopy and confocal scanning laser microscopy. MIO-M1 cells cultured on stiff substrates exhibited a significant increase in cell surface area, stress fiber, and mature focal adhesion formation. Furthermore, Müller cells treated with TGF-β1 and TGF-β2 and cultured on stiff substrates showed an increased incorporation of α-smooth muscle actin into stress fibers when compared to those grown on soft surfaces. Compliance of the surrounding matrix seems to influence the morphology and contraction of retinal Müller cells in fibrotic conditions. Development of an in vitro model simulating both the normally compliant retinal tissue and the rigid retinal fibrotic tissue helps fill the gap between the results of petri-dish cell culture with rigid surfaces and in vivo findings.

In Vitro Spoilation of Silicone-Hydrogel Soft Contact Lenses in a Model-Blink Cell

We developed an in vitro model-blink cell that reproduces the mechanism of in vivo fouling of soft contact lenses. In the model-blink cell, model tear lipid directly contacts the lens surface after forced aqueous rupture, mirroring the pre-lens tear-film breakup during interblink. Soft contact lenses are attached to a Teflon holder and immersed in artificial tear solution with protein, salts, and mucins. Artificial tear-lipid solution is spread over the air/tear interface as a duplex lipid layer. The aqueous tear film is periodically ruptured and reformed by withdrawing and reinjecting tear solution into the cell, mimicking the blink-rupture process. Fouled deposits appear on the lenses after cycling, and their compositions and spatial distributions are subsequently analyzed by optical microscopy, laser ablation electrospray ionization mass spectrometry, and two-photon fluorescence confocal scanning laser microscopy. Discrete deposit (white) spots with an average size of 20 to 300 μm are observed on the studied lenses, confirming what is seen in vivo and validating the in vitro model-blink cell. Targeted lipids (cholesterol) and proteins (albumin from bovine serum) are identified in the discrete surface deposits. Both lipid and protein occur simultaneously in the surface deposits and overlap with the white spots observed by optical microscopy. Additionally, lipid and protein penetrate into the bulk of tested silicone-hydrogel lenses, likely attributed to the bicontinuous microstructure of oleophilic silicone and hydrophilic polymer phases of the lens. In vitro spoilation of soft contact lenses is successfully achieved by the model-blink cell confirming the tear rupture/deposition mechanism of lens fouling. The model-blink cell provides a reliable laboratory tool for screening new antifouling lens materials, surface coatings, and care solutions.

Fluorescence and Label Free Impedimetric DNA Detection on SnO2 Nanopillars

AbstractSnO2 is a n‐type semiconductive oxide with attractive characteristics mainly based on easy elaboration and functionalization routes in addition to chemical robustness. We have fabricated SnO2 nanopillars based DNA sensor to perform the label free (without any redox compound) impedimetric DNA detection. The non faradaic electrochemical impedance spectroscopic (EIS) behavior and more particularly the evolution of the polarization resistance the SnO2 nanopillars has been thoroughly investigated upon the different steps of their functionalization process up to DNA hybridization. Similarly to our previous study on planar 2D SnO2 surfaces, the DNA hybridization induces a systematic increase of the polarization resistance, the magnitude of which decreases with the target DNA concentration. This DNA concentration dependence matches the one obtained from epifluorescence intensity measurements. A common value of DNA detection limit, i.e. 2 nM, is found from both measurement techniques. Interestingly the 3D view intensity obtained by confocal scanning laser fluorescence microscopy confirms that the DNA molecules are mainly grafted along the SnO2 nanopillars. Finally both impedance and fluorescence measurements obtained in the case of 1‐ and 2‐base mismatch hybridizations demonstrate the selectivity of the SnO2 nanopillars based DNA sensor. These preliminary results open the way to further investigations on the influence of both the shape ratio and electrical properties of the SnO2 nanopillars on the impedance variations related to DNA hybridization, notably in view of improving the sensor performances.

Bimodal in vivo imaging provides early assessment of stem-cell-based photoreceptor engraftment.

Subretinal transplantation of stem-cell-derived photoreceptor precursor cells (PPCs) is a promising and innovative approach to treating a range of blinding diseases. However, common barriers to efficient preclinical transplantation comes in the form of suboptimal graft architecture, limited graft survival, and immune-rejection, each of which cannot be assessed using conventional in vivo imaging (i.e., rodent ophthalmoscopy). With the majority of PPCs reported to die within the first few weeks after transplantation, understanding the mechanisms of graft failure, and ultimately devising preventative methods, currently relies on lengthy end point histology. To address these limitations, we hypothesized that combining two imaging modalities, optical coherence tomography (OCT) and fluorescence confocal scanning laser ophthalmoscopy (fcSLO), could provide a more rapid and comprehensive view of PPC engraftment. Human ESC-derived PPCs were transplanted into 15 retinal dystrophic rats that underwent bimodal imaging at 0, 8, and 15 days posttransplant. Bimodal imaging provided serial detection of graft: placement, architecture, and survival; each undetectable under ophthalmoscopy. Bimodal imaging determined graft placement to be either: subretinal (n=7), choroidal (n=4), or vitreal (n=4) indicating neural retinal perforation. Graft architecture was highly variable at the time of transplantation, with notable redistribution over time, while complete, or near complete, graft loss was observed in the majority of recipients after day 8. Of particular importance was detection of vitreal aggregates overlying the graft-possibly an indicator of host-site inflammation and rejection. Early real-time feedback of engraftment has the potential to greatly increase efficiency of preclinical trials in cell-based retinal therapeutics.

B-Spline potential function for maximum a-posteriori image reconstruction in fluorescence microscopy

An iterative image reconstruction technique employing B-Spline potential function in a Bayesian framework is proposed for fluorescence microscopy images. B-splines are piecewise polynomials with smooth transition, compact support and are the shortest polynomial splines. Incorporation of the B-spline potential function in the maximum-a-posteriori reconstruction technique resulted in improved contrast, enhanced resolution and substantial background reduction. The proposed technique is validated on simulated data as well as on the images acquired from fluorescence microscopes (widefield, confocal laser scanning fluorescence and super-resolution 4Pi microscopy). A comparative study of the proposed technique with the state-of-art maximum likelihood (ML) and maximum-a-posteriori (MAP) with quadratic potential function shows its superiority over the others. B-Spline MAP technique can find applications in several imaging modalities of fluorescence microscopy like selective plane illumination microscopy, localization microscopy and STED.

Severe, early axonal degeneration following experimental anterior ischemic optic neuropathy.

Anterior ischemic optic neuropathy (AION) is the most common acute optic neuropathy in adults older than 50 and leads to axonal degeneration, thinning of the retinal nerve fiber layer and loss of the retinal ganglion cells (RGCs). We used experimental AION model to study early axonal changes following ischemia. We induced optic nerve head ischemia in adult mice using photochemical thrombosis and analyzed retinal changes within 1 week. We used confocal scanning laser ophthalmoscopy (cSLO) and fluorescence microscopy of retinal whole mount preparations to analyze axonal degeneration in Thy1-YFP-H mice and those injected with annexin-V-A488 intravitreally. Three days after AION, morphometric analyses in Thy1-YFP-H mice revealed evidence of early axonal changes, including swollen or branched axonal stumps. There was also a beads-on-a-string appearance of YFP expression. The axonal enlargements occurred at an interval of 17 ± 1 μm or 6 ± 0 enlargements/100 μm. At day 7 after AION, the degenerating intraretinal RGC axons exhibited intense annexin-V-A488 staining (P = 0.002). The annexin-V staining pattern was fragmented, with intersegment interval of 20.1 ± 1.4 μm or 5.8 ± 0.4 annexin-V-A488(+) fragments/100 μm, which were similar to that of degenerating Thy1-YFP(+) axons. Following a photochemical thrombosis model of AION, RGC axons displayed severe degenerative changes within 1 week, suggesting that after ischemia, RGC axons may degenerate in a temporally and spatially distinct fashion from that of the soma. Our findings also further established annexin-V as a useful marker of retinal degeneration because it strongly labeled dying RGC axons.