Correlative light and electron microscopy using immunolabeled sections.

In correlative microscopy, light microscopy provides the overview and orientation of the complex cells and tissue, while electron microscopy offers the detailed localization and correlation of subcellular structures. In this chapter we offer detailed high-quality electron microscopical preparation methods for optimum preservation of the cellular ultrastructure. From such preparations serial thin sections are collected and used for comparative histochemical, immunofluorescence, and immunogold staining.In light microscopy histological stains identify the orientation of the sample and immunofluorescence labeling facilitates to find the region of interest, namely, the labeled cells expressing the macromolecule under investigation. Sections, labeled with immunogold are analyzed by electron microscopy in order to identify the label within the cellular architecture at high resolution.

Correlative light and electron microscopy in parasite research.

The interaction of a parasite and a host cell is a complex process, which involves several steps: (1) attachment to the plasma membrane, (2) entry inside the host cell, and (3) hijacking of the metabolism of the host. In biochemical experiments, only an event averaged over the whole cell population can be analyzed. The power of microscopy, however, is to investigate individual events in individual cells. Therefore, parasitologists frequently perform experiments with fluorescence microscopy using different dyes to label structures of the parasite or the host cell. Though the resolution of light microscopy has greatly improved, it is not sufficient to reveal interactions at the ultrastructural level. Furthermore, only specifically labeled structures can be seen and related to each other. Here, we want to demonstrate the additional value of electron microscopy in this area of research. Investigation of the different steps of parasite-host cell interaction by electron microscopy, however, is often hampered by the fact that there are only a few cells infected, and therefore it is difficult to find enough cells to study. A solution is to profit from low magnification, hence large overview, and specific location of the players by fluorescence labels in a light microscope with the high power resolution and structural information provided by an electron microscope, in short by correlative light and electron microscopy.

A protocol for preparing GFP-labeled neurons previously imaged in vivo and in slice preparations for light and electron microscopic analysis.

In vivo imaging of green fluorescent protein (GFP)-labeled neurons in the intact brain is being used increasingly to study neuronal plasticity. However, interpreting the observed changes as modifications in neuronal connectivity needs information about synapses. We show here that axons and dendrites of GFP-labeled neurons imaged previously in the live mouse or in slice preparations using 2-photon laser microscopy can be analyzed using light and electron microscopy, allowing morphological reconstruction of the synapses both on the imaged neurons, as well as those in the surrounding neuropil. We describe how, over a 2-day period, the imaged tissue is fixed, sliced and immuno-labeled to localize the neurons of interest. Once embedded in epoxy resin, the entire neuron can then be drawn in three dimensions (3D) for detailed morphological analysis using light microscopy. Specific dendrites and axons can be further serially thin sectioned, imaged in the electron microscope (EM) and then the ultrastructure analyzed on the serial images.

Hyperglycemia-induced abnormalities in rat and human corneas: the potential of second harmonic generation microscopy.

BACKGROUND: Second Harmonic Generation (SHG) microscopy recently appeared as an efficient optical imaging technique to probe unstained collagen-rich tissues like cornea. Moreover, corneal remodeling occurs in many diseases and precise characterization requires overcoming the limitations of conventional techniques. In this work, we focus on diabetes, which affects hundreds of million people worldwide and most often leads to diabetic retinopathy, with no early diagnostic tool. This study then aims to establish the potential of SHG microscopy for in situ detection and characterization of hyperglycemia-induced abnormalities in the Descemet's membrane, in the posterior cornea. METHODOLOGY/PRINCIPAL FINDINGS: We studied corneas from age-matched control and Goto-Kakizaki rats, a spontaneous model of type 2 diabetes, and corneas from human donors with type 2 diabetes and without any diabetes. SHG imaging was compared to confocal microscopy, to histology characterization using conventional staining and transmitted light microscopy and to transmission electron microscopy. SHG imaging revealed collagen deposits in the Descemet's membrane of unstained corneas in a unique way compared to these gold standard techniques in ophthalmology. It provided background-free images of the three-dimensional interwoven distribution of the collagen deposits, with improved contrast compared to confocal microscopy. It also provided structural capability in intact corneas because of its high specificity to fibrillar collagen, with substantially larger field of view than transmission electron microscopy. Moreover, in vivo SHG imaging was demonstrated in Goto-Kakizaki rats. CONCLUSIONS/SIGNIFICANCE: Our study shows unambiguously the high potential of SHG microscopy for three-dimensional characterization of structural abnormalities in unstained corneas. Furthermore, our demonstration of in vivo SHG imaging opens the way to long-term dynamical studies. This method should be easily generalized to other structural remodeling of the cornea and SHG microscopy should prove to be invaluable for in vivo corneal pathological studies.

Light-induced degradation of phyA is promoted by transfer of the photoreceptor into the nucleus.

Higher plants possess multiple members of the phytochrome family of red, far-red light sensors to modulate plant growth and development according to competition from neighbors. The phytochrome family is composed of the light-labile phyA and several light-stable members (phyB-phyE in Arabidopsis). phyA accumulates to high levels in etiolated seedlings and is essential for young seedling establishment under a dense canopy. In photosynthetically active seedlings high levels of phyA counteract the shade avoidance response. phyA levels are maintained low in light-grown plants by a combination of light-dependent repression of PHYA transcription and light-induced proteasome-mediated degradation of the activated photoreceptor. Light-activated phyA is transported from the cytoplasm where it resides in darkness to the nucleus where it is needed for most phytochrome-induced responses. Here we show that phyA is degraded by a proteasome-dependent mechanism both in the cytoplasm and the nucleus. However, phyA degradation is significantly slower in the cytoplasm than in the nucleus. In the nucleus phyA is degraded in a proteasome-dependent mechanism even in its inactive Pr (red light absorbing) form, preventing the accumulation of high levels of nuclear phyA in darkness. Thus, light-induced degradation of phyA is in part controlled by a light-regulated import into the nucleus where the turnover is faster. Although most phyA responses require nuclear phyA it might be useful to maintain phyA in the cytoplasm in its inactive form to allow accumulation of high levels of the light sensor in etiolated seedlings.

Correlative microscopy.

In recent years correlative microscopy, combining the power and advantages of different imaging system, e.g., light, electrons, X-ray, NMR, etc., has become an important tool for biomedical research. Among all the possible combinations of techniques, light and electron microscopy, have made an especially big step forward and are being implemented in more and more research labs. Electron microscopy profits from the high spatial resolution, the direct recognition of the cellular ultrastructure and identification of the organelles. It, however, has two severe limitations: the restricted field of view and the fact that no live imaging can be done. On the other hand light microscopy has the advantage of live imaging, following a fluorescently tagged molecule in real time and at lower magnifications the large field of view facilitates the identification and location of sparse individual cells in a large context, e.g., tissue. The combination of these two imaging techniques appears to be a valuable approach to dissect biological events at a submicrometer level. Light microscopy can be used to follow a labelled protein of interest, or a visible organelle such as mitochondria, in time, then the sample is fixed and the exactly same region is investigated by electron microscopy. The time resolution is dependent on the speed of penetration and fixation when chemical fixatives are used and on the reaction time of the operator for cryo-fixation. Light microscopy can also be used to identify cells of interest, e.g., a special cell type in tissue or cells that have been modified by either transfections or RNAi, in a large population of non-modified cells. A further application is to find fluorescence labels in cells on a large section to reduce searching time in the electron microscope. Multiple fluorescence labelling of a series of sections can be correlated with the ultrastructure of the individual sections to get 3D information of the distribution of the marked proteins: array tomography. More and more efforts are put in either converting a fluorescence label into an electron dense product or preserving the fluorescence throughout preparation for the electron microscopy. Here, we will review successful protocols and where possible try to extract common features to better understand the importance of the individual steps in the preparation. Further the new instruments and software, intended to ease correlative light and electron microscopy, are discussed. Last but not least we will detail the approach we have chosen for correlative microscopy.

Distribution of the monocarboxylate transporter MCT2 in human cerebral cortex: an immunohistochemical study

The monocarboxylate transporter MCT2 belongs to a large family of membrane proteins involved in the transport of lactate, pyruvate and ketone bodies. Although its expression in rodent brain has been well documented, the presence of MCT2 in the human brain has been questioned on the basis of low mRNA abundance. In this study, the distribution of the monocarboxylate transporter MCT2 has been investigated in the cortex of normal adult human brain using an immunohistochemical approach. Widespread neuropil staining in all cortical layers was observed by light microscopy. Such a distribution was very similar in three different cortical areas investigated. At the cellular level, the expression of MCT2 could be observed in a large number of neurons, in fibers both in grey and white matter, as well as in some astrocytes, mostly localized in layer I and in the white matter. Double staining experiments combined with confocal microscopy confirmed the neuronal expression but also suggested a preferential postsynaptic localization of synaptic MCT2 expression. A few astrocytes in the grey matter appeared to exhibit MCT2 labelling but at low levels. Electron microscopy revealed strong MCT2 expression at asymmetric synapses in the postsynaptic density and also within the spine head but not in the presynaptic terminal. These data not only demonstrate neuronal MCT2 expression in human, but since a portion of it exhibits a distinct synaptic localization, it further supports a putative role for MCT2 in adjustment of energy supply to levels of activity.

Interstitial cells of Cajal are normally distributed in both ganglionated and aganglionic bowel in Hirschsprung's disease

Résumé Introduction : La chirurgie de la maladie de Hirschsprung est fréquemment compliquée d'une atteinte post-opératoire de la motilité intestinale. Des anomalies du système nerveux entérique (SNE) telles que la dysplasie neuronale intestinale de type B, l'hypoganglionose ou l'aganglionose, présents dans le segment abaissé, peuvent être la cause de certaines de ces complications mais aucune information n'est disponible quant au rôle des cellules interstitielles de Cajal (CIC) sur la motilité intestinale dans la phase post-opératoire. Ces cellules sont considérées avoir un rôle de pacemaker dans le tractus gastro-intestinal. L'objectif de cette étude était de décrire la distribution des CIC dans le segment proximal du côlon réséqué lors de cures chirurgicales de maladie de Hirschsprung et de confronter ces observations à l'évolution clinique post-opératoire. Matériel et Méthodes : L'incidence des complications post-opératoires a été déterminée par une revue rétrospective des dossiers de 48 patients opérés pour maladie de Hirschspung entre 1977 et 1999 et par l'étude histologique et immuno-histochimique des pièces réséquées chez ces patients. Nous avons comparé la distribution des CIC dans le segment proximal du côlon avec celle du côlon sain de 16 enfants contrôles par microscopie optique. L'immunohistochimie au c-Kit a été utilisée pour marquer spécifiquement les CIC sur échantillons paraffinés. Ces résultats ont ensuite été corrélés avec l'étude du SNE de ces mêmes segments, déterminée par immunohistochimie au CD56 et au protein gene product 9.5. Résultats Les complications post-opératoires suivantes furent identifiées : constipation 46%, constipation avec incontinence 15%, entérocolite 8%, décès 4% (probablement sur entérocolite). La distribution des CIC dans les segments proximaux réséqués chez les enfants avec maladie de Hirschsprung était identique à celle observée dans les segments de côlon sain, et ce indépendamment de la distribution normale ou anormale du SNE. Chez les enfants opérés pour maladie de Hirschsprung les segments réséqués présentaient les anomalies d'innervation suivantes : aganglionose 10.4%, hypoganglionose 12.5%, dysplasie neuronale intestinale de type B 6.3%, autres dysganglionoses 14.6%. Aucune relation entre ces anomalies d'innervation et les complications post-opératoires n'a été mise en évidence. Conclusion : La distribution des CIC est normale chez les patient opérés pour maladie de Hirschsprung, et ne contribue donc pas aux atteintes post-opératoires de la motilité intestinale. Cela signifie aussi que le réseau de CIC se développe noinialement dans le côlon humain, même en présence d'une innervation colique anormale ou absente. Abstract: Surgery for Hirschsprung's disease is often complicated by post-operative bowel motility disorders. The impact of intestinal neural histology on the surgical outcome has been previously studied, but no information is available concerning the influence of the distribution of interstitial cells of Cajal (ICC) on these complications. These cells are considered to be pacemakers in the gastrointestinal tract. The aim of this study was to assess the distribution of ICC in the proximal segment of resected bowel in Hirschsprung's disease and confront these results with the clinical outcome. Using immunohistochemistry for light microscopy, we compared the pattern of distribution of ICC in the proximal segment of resected bowel in Hirschsprung's disease with that in normal colon. We correlated these results with the corresponding neural intestinal histology determined by CD56 and the protein gene product 9.5 immunohistochemistry. The distribution of ICC in the proximal segment of resected bowel is identical to that of normal colon, regardless of normal or abnormal colon innervation. ICC distribution does not seem to contribute to post-operative bowel motility disorders in patients operated for Hirschsprung's disease.

The effect of standard and high-fluence corneal cross-linking (CXL) on cornea and limbus.

PURPOSE: When treating peripheral ectatic disease-like pellucid marginal degeneration (PMD), corneal cross-linking with UV-A and riboflavin (CXL) must be applied eccentrically to the periphery of the lower cornea, partly irradiating the corneal limbus. Here, we investigated the effect of standard and double-standard fluence corneal cross-linking with riboflavin and UV-A (CXL) on cornea and corneal limbus in the rabbit eye in vivo. METHODS: Epithelium-off CXL was performed in male New Zealand White rabbits with two irradiation diameters (7 mm central cornea, 13 mm cornea and limbus), using standard fluence (5.4 J/cm(2)) and double-standard fluence (10.8 J/cm(2)) settings. Controls were subjected to epithelial removal and riboflavin instillation, but were not irradiated with UV-A. Following CXL, animals were examined daily until complete closure of the epithelium, and at 7, 14, 21, and 28 days. Animals were killed and a corneoscleral button was excised and processed for light microscopy and immunohistochemistry. RESULTS: For both irradiation diameters and fluences tested, no signs of endothelial damage or limbal vessel thrombosis were observed, and time to re-epithelialization was similar to untreated controls. Histological and immunohistochemical analysis revealed no differences in the p63 putative stem cell marker expression pattern. CONCLUSIONS: Even when using fluence twice as high as the one used in current clinical CXL settings, circumferential UV-A irradiation of the corneal limbus does not alter the regenerative capacity of the limbal epithelial cells, and the expression pattern of the putative stem cell marker p63 remains unchanged. This suggests that eccentric CXL may be performed safely in PMD.

Effectiveness of intermittent preventive treatment with sulfadoxine-pyrimethamine during pregnancy on placental malaria, maternal anaemia and birthweight in areas with high and low malaria transmission intensity in Tanzania.

OBJECTIVE: To assess the effectiveness of IPTp in two areas with different malaria transmission intensities. METHODS: Prospective observational study recruiting pregnant women in two health facilities in areas with high and low malaria transmission intensities. A structured questionnaire was used for interview. Maternal clinic cards and medical logs were assessed to determine drug intake. Placental parasitaemia was screened using both light microscopy and real-time quantitative PCR. RESULTS: Of 350 pregnant women were recruited and screened for placental parasitaemia, 175 from each area. Prevalence of placental parasitaemia was 16.6% (CI 11.4-22.9) in the high transmission area and 2.3% (CI 0.6-5.7) in the low transmission area. Being primigravida and residing in a high transmission area were significant risk factors for placental malaria (OR 2.4; CI 1.1-5.0; P = 0.025) and (OR 9.4; CI 3.2-27.7; P < 0.001), respectively. IPTp was associated with a lower risk of placental malaria (OR 0.3; CI 0.1-1.0; P = 0.044); the effect was more pronounced in the high transmission area (OR 0.2; CI 0.06-0.7; P = 0.015) than in the low transmission area (OR 0.4; CI 0.04-4.5; P = 0.478). IPTp use was not associated with reduced risk of maternal anaemia or low birthweight, regardless of transmission intensity. The number needed to treat (NNT) was four (CI 2-6) women in the high transmission area and 33 (20-50) in the low transmission area to prevent one case of placental malaria. CONCLUSION: IPTp may have an effect on lowering the risk of placental malaria in areas of high transmission, but this effect did not translate into a benefit on risks of maternal anaemia or low birthweight. The NNT needs to be considered, and weighted against that of other protective measures, eventually targeting areas which are above a certain threshold of malaria transmission to maximise the benefit.

Nuclear accumulation of the phytochrome A photoreceptor requires FHY1.

The phytochrome family of red/far-red (R/FR)-responsive photoreceptors plays a key role throughout the life cycle of plants . Arabidopsis has five phytochromes, phyA-phyE, among which phyA and phyB play the most predominant functions . Light-regulated nuclear accumulation of the phytochromes is an important regulatory step of this pathway, but to this date no factor specifically required for this event has been identified . Among all phyA signaling mutants, fhy1 and fhy3 (far-red elongated hypocotyl 1 and 3) have the most severe hyposensitive phenotype, indicating that they play particularly important roles . FHY1 is a small plant-specific protein of unknown function localized both in the nucleus and the cytoplasm . Here we show that FHY1 is specifically required for the light-regulated nuclear accumulation of phyA but not phyB. Moreover, phyA accumulation is only slightly affected in fhy3, indicating that the diminished nuclear accumulation of phyA observed in fhy1 seedlings is not simply a general consequence of reduced phyA signaling. By in vitro pull-down and yeast two-hybrid analyses, we demonstrate that FHY1 physically interacts with phyA, preferentially in its active Pfr form. Furthermore, FHY1 and phyA colocalize in planta. We therefore identify the first component required for light-regulated phytochrome nuclear accumulation.

Peroxisome-proliferator-activated receptor (PPAR)-gamma activation stimulates keratinocyte differentiation.

Previous studies demonstrated that peroxisome-proliferator-activated receptor (PPAR)-alpha or PPAR-delta activation stimulates keratinocyte differentiation, is anti-inflammatory, and improves barrier homeostasis. Here we demonstrate that treatment of cultured human keratinocytes with ciglitazone, a PPAR-gamma activator, increases involucrin and transglutaminase 1 mRNA levels. Moreover, topical treatment of hairless mice with ciglitazone or troglitazone increases loricrin, involucrin, and filaggrin expression without altering epidermal morphology. These results indicate that PPAR-gamma activation stimulates keratinocyte differentiation. Additionally, PPAR-gamma activators accelerated barrier recovery following acute disruption by either tape stripping or acetone treatment, indicating an improvement in permeability barrier homeostasis. Treatment with PPAR-gamma activators also reduced the cutaneous inflammatory response that is induced by phorbol 12-myristate-13-acetate, a model of irritant contact dermatitis and oxazolone, a model of allergic contact dermatitis. To determine whether the effects of PPAR-gamma activators are mediated by PPAR-gamma, we next examined animals deficient in PPAR-gamma. Mice with a deficiency of PPAR-gamma specifically localized to the epidermis did not display any cutaneous abnormalites on inspection, but on light microscopy there was a modest increase in epidermal thickness associated with an increase in proliferating cell nuclear antigen (PCNA) staining. Key functions of the skin including permeability barrier homeostasis, stratum corneum surface pH, and water-holding capacity, and response to inflammatory stimuli were not altered in PPAR-gamma-deficient epidermis. Although PPAR-gamma activators stimulated loricrin and filaggrin expression in wild-type animals, however, in PPAR-gamma-deficient mice no effect was observed indicating that the stimulation of differentiation by PPAR-gamma activators is mediated by PPAR-gamma. In contrast, PPAR-gamma activators inhibited inflammation in both PPAR-gamma-deficient and wild-type mouse skin, indicating that the inhibition of cutaneous inflammation by these PPAR-gamma activators does not require PPAR-gamma in keratinocytes. These observations suggest that thiazolidindiones and perhaps other PPAR-gamma activators maybe useful in the treatment of cutaneous disorders.

Imaging of tophaceous gout: computed tomography provides specific images compared with magnetic resonance imaging and ultrasonography.

OBJECTIVE: To determine the usefulness of computed tomography (CT), magnetic resonance imaging (MRI), and Doppler ultrasonography (US) in providing specific images of gouty tophi. METHODS: Four male patients with chronic gout with tophi affecting the knee joints (three cases) or the olecranon processes of the elbows (one case) were assessed. Crystallographic analyses of the synovial fluid or tissue aspirates of the areas of interest were made with polarising light microscopy, alizarin red staining, and x ray diffraction. CT was performed with a GE scanner, MR imaging was obtained with a 1.5 T Magneton (Siemens), and ultrasonography with colour Doppler was carried out by standard technique. RESULTS: Crystallographic analyses showed monosodium urate (MSU) crystals in the specimens of the four patients; hydroxyapatite and calcium pyrophosphate dihydrate (CPPD) crystals were not found. A diffuse soft tissue thickening was seen on plain radiographs but no calcifications or ossifications of the tophi. CT disclosed lesions containing round and oval opacities, with a mean density of about 160 Hounsfield units (HU). With MRI, lesions were of low to intermediate signal intensity on T(1) and T(2) weighting. After contrast injection in two cases, enhancement of the tophus was seen in one. Colour Doppler US showed the tophi to be hypoechogenic with peripheral increase of the blood flow in three cases. CONCLUSION: The MR and colour Doppler US images showed the tophi as masses surrounded by a hypervascular area, which cannot be considered as specific for gout. But on CT images, masses of about 160 HU density were clearly seen, which correspond to MSU crystal deposits.

L'application de la spectroscopie Raman en criminalistique pour l'analyse du colorant des fibres textiles en acrylique, coton et laine

RESUME La méthode de la spectroscopie Raman est une technique d'analyse chimique basée sur l'exploitation du phénomène de diffusion de la lumière (light scattering). Ce phénomène fut observé pour la première fois en 1928 par Raman et Krishnan. Ces observations permirent à Raman d'obtenir le Prix Nobel en physique en 1930. L'application de la spectroscopie Raman a été entreprise pour l'analyse du colorant de fibres textiles en acrylique, en coton et en laine de couleurs bleue, rouge et noire. Nous avons ainsi pu confirmer que la technique est adaptée pour l'analyse in situ de traces de taille microscopique. De plus, elle peut être qualifiée de rapide, non destructive et ne nécessite aucune préparation particulière des échantillons. Cependant, le phénomène de la fluorescence s'est révélé être l'inconvénient le plus important. Lors de l'analyse des fibres, différentes conditions analytiques ont été testées et il est apparu qu'elles dépendaient surtout du laser choisi. Son potentiel pour la détection et l'identification des colorants imprégnés dans les fibres a été confirmé dans cette étude. Une banque de données spectrale comprenant soixante colorants de référence a été réalisée dans le but d'identifier le colorant principal imprégné dans les fibres collectées. De plus, l'analyse de différents blocs de couleur, caractérisés par des échantillons d'origine inconnue demandés à diverses personnes, a permis de diviser ces derniers en plusieurs groupes et d'évaluer la rareté des configurations des spectres Raman obtenus. La capacité de la technique Raman à différencier ces échantillons a été évaluée et comparée à celle des méthodes conventionnelles pour l'analyse des fibres textiles, à savoir la micro spectrophotométrie UV-Vis (MSP) et la chromatographie sur couche mince (CCM). La technique Raman s'est révélée être moins discriminatoire que la MSP pour tous les blocs de couleurs considérés. C'est pourquoi dans le cadre d'une séquence analytique nous recommandons l'utilisation du Raman après celle de la méthode d'analyse de la couleur, à partir d'un nombre de sources lasers le plus élevé possible. Finalement, la possibilité de disposer d'instruments équipés avec plusieurs longueurs d'onde d'excitation, outre leur pouvoir de réduire la fluorescence, permet l'exploitation d'un plus grand nombre d'échantillons. ABSTRACT Raman spectroscopy allows for the measurement of the inelastic scattering of light due to the vibrational modes of a molecule when irradiated by an intense monochromatic source such as a laser. Such a phenomenon was observed for the first time by Raman and Krishnan in 1928. For this observation, Raman was awarded with the Nobel Prize in Physics in 1930. The application of Raman spectroscopy has been undertaken for the dye analysis of textile fibers. Blue, black and red acrylics, cottons and wools were examined. The Raman technique presents advantages such as non-destructive nature, fast analysis time, and the possibility of performing microscopic in situ analyses. However, the problem of fluorescence was often encountered. Several aspects were investigated according to the best analytical conditions for every type/color fiber combination. The potential of the technique for the detection and identification of dyes was confirmed. A spectral database of 60 reference dyes was built to detect the main dyes used for the coloration of fiber samples. Particular attention was placed on the discriminating power of the technique. Based on the results from the Raman analysis for the different blocs of color submitted to analyses, it was possible to obtain different classes of fibers according to the general shape of spectra. The ability of Raman spectroscopy to differentiate samples was compared to the one of the conventional techniques used for the analysis of textile fibers, like UV-Vis Microspectrophotometry (UV-Vis MSP) and thin layer chromatography (TLC). The Raman technique resulted to be less discriminative than MSP for every bloc of color considered in this study. Thus, it is recommended to use Raman spectroscopy after MSP and light microscopy to be considered for an analytical sequence. It was shown that using several laser wavelengths allowed for the reduction of fluorescence and for the exploitation of a higher number of samples.

Ocular drug delivery targeting the retina and retinal pigment epithelium using polylactide nanoparticles.

PURPOSE: To study the kinetics of polylactide (PLA) nanoparticle (NP) localization within the intraocular tissues and to evaluate their potential to release encapsulated material. METHODS: A single intravitreous injection (5 micro L) of an NP suspension (2.2 mg/mL) encapsulating either Rh-6G (Rh) or Nile red (Nr) was performed. Animals were killed at various times, and the NPs localization within the intraocular tissues was studied by environmental scanning electron microscopy (ESEM), confocal microscopy, light microscopy histology, fluorescence microscopy, and immunohistochemistry. Eyes injected with blank NPs, free Rh, or PBS solution were used as the control. RESULTS: ESEM showed the flow of the NPs from the site of injection into the vitreous cavity and their rapid settling on the internal limiting membrane. Histology demonstrated the anatomic integrity of the injected eyes and showed no toxic effects. A mild inflammatory cell infiltrate was observed in the ciliary body 6 hours after the injection and in the posterior vitreous and retina at 18 to 24 hours. The intensity of inflammation decreased markedly by 48 hours. Confocal and fluorescence microscopy and immunohistochemistry showed that a transretinal movement of the NPs was gradually taking place with a later localization in the RPE cells. Rh encapsulated within the injected NPs diffused and stained the retina and RPE cells. PLA NPs were still present within the RPE cells 4 months after a single intravitreous injection. CONCLUSIONS: Intravitreous injection of PLA NPs appears to result in transretinal movement, with a preferential localization in the RPE cells. Encapsulated Rh diffuses from the NPs and stains the neuroretina and the RPE cells. The findings support the idea that specific targeting of these tissues is feasible. Furthermore, the presence of the NPs within the RPE cells 4 months after a single injection shows that a steady and continuous delivery of drugs can be achieved.