Applications of confocal microscopy in industrial solid materials: some examples and a first evaluation

We report here the first results of the application of confocal scanning laser microscopy (CSLM) for the study of the microstructure of solid industrial materials. Glass-fibre-reinforced composites, heterogeneous and conductive polymers, homogeneous as well as heterogeneous catalyst (precursor) specimens and soils were examined. We conclude that both the fluorescence and reflection modes of CSLM can yield valuable information. In particular, the optical sectioning capability of CSLM appears to be of great value as it enables one to access the 3-D organization of the specimen without the need for a difficult and time-consuming specimen preparation procedure. However, local obscuration may be an important factor in confocal image formation, limiting the penetration capabilities of the technique for industrial materials.     

Improved chondrocyte morphology and glycogen retention in the secondary center of ossification following osmium-potassium ferrocyanide fixation

The use of osmium-potassium ferrocyanide as the secondary fixative greatly improved chondrocyte preservation and stabilized the cartilage matrix proteoglycan. The proteoglycan was similar in appearance to that seen following fixation in the presence of cationic dyes. Extensive glycogen preservation was noted in these cells, occupying the area prior to and during the formation of the secondary center of ossification. The volume and organization of the glycogen within the cell cytoplasm were greater than that following buffered osmium fixation, and the cellular vacuoles within were greatly reduced. The cells forming the secondary center prior to the onset of mineralization were of greatest interest, because other studies compared them with the primary growth plate and described them as showing signs of hypertrophy as early as 5 days postnatally, as is found in the primary growth plate. Our observations indicate that glycogen is present in these cells, and cellular enlargement was not present. The cells do not resemble the hypertrophic chondrocytes of the primary growth plate, as far as cytoplasmic content is concerned, and we suggest that they may contribute to the development of the secondary center in a different fashion.     

Radon track imaging in CR-39 plastic detectors using confocal scanning laser microscopy

Confocal scanning laser microscopy (CSLM) has been used to provide the first images of radon track populations in two external CR-39 plastic detectors. Measurements of variables including track area distribution and estimates of the angle of track inclination (dip) derived from surface CSLM sections are presented. CSLM depth slices, combined with three-dimensional (3D) visualization techniques, provide a new, non-destructive way of examining the 2D and 3D geometry of the etched tracks within solid-state nuclear track detectors that may prove useful in complementing existing optical microscopy methods.     

In vivo determination of fibril orientation in plant cell walls with polarization CSLM

Congo Red fluorescence is used to detect cellulose in the wall of plant cells. The orientation of the cellulose fibrils is determined by using polarized light for excitation. The absorption characteristics of Congo Red make this approach a method of choice for applications with any standard confocal scanning laser microscope (CSLM). The semiquantitative character of CSLM observations combined with the non-toxicity of the stain allow a very fast and reliable assessment of cellulose orientation in the wall of living plant cells.     

Dynamic confocal scanning laser microscopy methods for studying milk protein gelation and cheese melting

Dynamic confocal scanning laser microscopy (CSLM) methods were developed to enable observation of milk protein gelation and cheese melting. Protein aggregation and the formation of gel networks in renneted full-fat and low-fat milks and glucono-δ-lactone (GDL)-acidified skim milks were observed by CSLM and observations correlated with increases in shear modulus (G′) and dynamic viscosity (η*) as determined by dynamic amplitude oscillatory rheology. Confocal scanning laser microscopy observation of low-fat and full-fat cheeses showed changes in fat distribution and an increase in staining intensity during cheese melting.     

Fluorescence photobleaching recovery in the confocal scanning light microscope

Measurement of mobilities of species in liquid systems is of great importance for understanding a number of dynamic phenomena. A well known method for measuring mobilities driven by diffusion is fluorescence photobleaching recovery (FPR), also known as fluorescence recovery after photobleaching (FRAP). New FPR recovery equations for three-dimensional (3-D) apertured scanning using a Gaussian approximation for the axial beam profile have been successfully developed and found to provide a solid basis for extraction of the lateral diffusion coefficient from confocal scanning light microscopy (CSLM)-FPR experimental data. The 2-D diffusion coefficients of fluorescent species can be successfully measured by FPR in the CSLM, which has the great advantage that bleaching can be targeted at a well-defined volume element in bulk samples. Two-dimensional diffusion coefficients of 45-nm latex spheres, of FITC molecules and of a 2·45-nm protein-FITC complex in water-glycerol mixtures, measured by FPR in the CSLM, are in close agreement with those calculated from the size of the diffusing species and viscosity of the medium. Diffusion coefficients as high as 2 times 10^?6 cm²/s can be measured.     

Effects of desiccation on <i>Euterpe edulis</i> Martius seeds

Information on desiccation sensitivity of Euterpe edulis seeds under two drying rates is presented. The sensitivity was studied during the course of germination and normal germination. The water content was evaluated for both seeds and embryos. Results showed the following: (a) For both drying treatments and for both germination and normal germination, desiccation sensitivity values were higher for measurements based on the water content of the embryo than for those of the seed. (b) For both drying treatments, desiccation sensitivity were higher for normal germination than for germination based on both the embryo and seed water contents. (c) Under the slow drying treatment and for measurements based on the seed water content, critical water content was visible for normal germination but not for germination; (d) Critical water contents for germination and normal germination were more clearly established in the fast drying treatment than they were in the slow drying method based on both the embryo and seed water contents. Critical water contents were not associated with changes in electrolyte leakage, which suggests that conductivity is not a good indicator of physiological seed quality. From the beginning of both drying treatments, changes in nuclei and vacuoles were observed, but, when seed water content was reduced to below critical values, the cells became severely plasmolyzed, the vacuoles highly distorted, and the nuclei formed an almost homogeneous mass with the chromatin and the nucleoplasm, which suggests irreversible DNA damages.     

Fluorescence in the tandem scanning microscope

Our studies have shown that the fluorescence mode can be used to good effect in both tandem scanning microscopes (TSM: direct view confocal microscopes) as well as confocal scanning laser microscopes (CSLM). Applications are presented which show that the two great advantages of TSM are real-time viewing and real colour, which allow faster use and interpretation. CSLM are complementary, not competitive, being currently more sophisticated for low-level fluorescence work. This is equally possible with available TSM, but requires further development using CCD cameras and image-processing systems.     

Prospects for tooth regeneration in the 21st century: a perspective

The prospects for tooth regeneration in the 21st century are compelling. Using the foundations of experimental embryology, developmental and molecular biology, the principles of biomimetics (the mimicking of biological processes), tooth regeneration is becoming a realistic possibility within the next few decades. The cellular, molecular, and developmental "rules" for tooth morphogenesis are rapidly being discovered. The knowledge gained from adult stem cell biology, especially associated with dentin, cartilage, and bone tissue regeneration, provides additional opportunities for eventual tooth organogenesis. The centuries of tooth development using xenotransplantation, allotransplantation, and autotransplantation have resulted in many important insights that can enhance tooth regeneration. In considering the future, several lines of evidence need to be considered: (1) enamel organ epithelia and dental papilla mesenchyme tissues contain stem cells during postnatal stages of life; (2) late cap stage and bell stage tooth organs contain stem cells; (3) odontogenic adult stem cells respond to mechanical as well as chemical "signals"; (4) presumably adult bone marrow as well as dental pulp tissues contain "odontogenic" stem cells; and (5) epithelial-mesenchymal interactions are pre-requisite for tooth regeneration. The authors express "guarded enthusiasm," yet there should be little doubt that adult stem cell-mediated tooth regeneration will be realized in the not too distant future. The prospects for tooth regeneration could be realized in the next few decades and could be rapidly utilized to improve the quality of human life in many nations around the world.     

Fine structural analysis of the epithelial cells in the hepatopancreas of Palaemonetes argentinus (Crustacea, Decapoda, Caridea) in intermoult.

The aim of this study is to describe the ultrastructure of the hepatopancreas of P. argentinus in intermoult. P. argentinus hepatopancreas was studied using standard TEM techniques. Each tubule consists of four cellular types: E (embryonic), F (fibrillar), R (resorptive) and B (blister like). E-cells have embryonic features and some of them were found in mitosis. F, R and B cells possess an apical brush border. F-cells have a central or basal nucleus, a conspicuous RER, and dilated Golgi cisternae. R cells show a polar organization of organelles in three areas: apical, with numerous mitochondria and sER tubules, a central area with the nucleus and RER, and a basal area containing a sER-like tubule system and mitochondria. B-cells were observed at different stages of their life cycle. In an early differentiation stage they comprise an apical endocytotic complex and Golgi vesicles. The fusion of endocytotic and Golgi vesicles originates subapical vacuoles. During maturation, a big central vacuole is formed by coalescence of subapical vacuoles. The central vacuole is eliminated by holocrine secretion. The ultrastructure suggests that F-cells synthesize proteins, R-cells storage nutrients and B-cells have a secretory or excretory function, and confirms the independent origin of F, B and R cells from the embryonic cells.     

Ultrastructural investigation of neurons identified and localized using the confocal scanning laser microscope

Studies of labeled neurons at the light-microscopic level often pinpoint a substructure of particular interest, i.e., a synapse or a spine. An ultrastructural investigation would explain a lot about how these structures arose, how they function, and how they are regulated. Finding a small region in a large block can require constant checking during sectioning, until past the structure. In our pursuit of the synaptic structure of varicosities on the axons of neurons identified physiologically and morphologically at the light level, we have combined confocal scanning laser microscopy (CSLM) with conventional and high-voltage electron microscopy (EM). CSLM images were collected in the reflection mode to view neurons filled with horseradish peroxidase and stained with nickel-intensified diaminobenzidine, which is compatible with EM. The CSLM optical sections provided a record of what one should expect to see at regular intervals throughout the depth of the tissue block. We have shown that the CSLM greatly simplified the task of localizing small structures in brain tissue prepared for EM.     

Three-dimensional image restoration and super-resolution in fluorescence confocal microscopy

Confocal scanning laser microscopy (CSLM) provides optical sectioning of a fluorescent sample and improved resolution with respect to conventional optical microscopy. As a result, three-dimensional (3-D) imaging of biological objects becomes possible. A difficulty is that the lateral resolution is better than the axial resolution and, thus, the microscope provides orientation-dependent images. However, a theoretical investigation of the process of image formation in CSLM shows that it must be possible to improve the resolution obtained in practice. We present two methods for achieving such a result in the case of 3-D fluorescent objects. The first method applies to conventional CSLM, where the image is detected only on the optical axis for any scanning position. Since the resulting 3-D image is the convolution of the object with the impulse-response function of the instrument, the problem of image restoration is a deconvolution problem and is affected by numerical instability. A short introduction to the linear methods developed for obtaining stable solutions of these problems (the so-called regularization theory of ill-posed problems) is given and an application to a real image is discussed. The second method applies to a new version of CSLM proposed in recent years. In such a case the full image must be measured by a suitable array of detectors. For each scanning position the data are not single numbers but vectors. Then, in order to recover the object, one must solve a Fredholm integral equation of the first kind. A method for the solution of this equation is presented and the possibility of achieving super-resolution is demonstrated. More precisely, we show that it is possible to improve by about a factor of 2 the resolution of conventional CSLM both in the lateral and axial directions.     

Measurements of fibre direction in reinforced polymer composites

High-quality data on the three-dimensional (3-D) spatial distributions of glass and carbon fibres in fibre-reinforced polymer composites are important for both process control and the modelling of the mechanical and thermal properties of these composites. The advent of economical, high-speed, image analyser systems has enabled numerous research groups to measure directional distributions of fibre samples. Specimens are microtomed and polished and, using optical reflection microscopy, thousands of elliptical fibre images may be analysed within a short period of time. From the eccentricity of the fibre images, estimation of the angles (θ, φ) of each fibre relative to the vertical axis and within the measurement plane is deduced. However, this measurement is subject to considerable error. The confocal scanning laser microscope (CSLM), operating in fluorescence mode or reflection mode, is capable of improving the angular resolution (δθ, δφ) for all fibre directions. The ability of the CSLM to optically section glass and carbon fibre-reinforced polymer composites down to depths of 20 or 30 μm allows the user to determine accurate fibre directions from the apparent movement of fibre profiles. The CSLM has the potential for standardizing measurements of 3-D fibre directions in polymer composites and providing the quality directional data which are required for the theoretical modelling of composite processing and composite strength.     

Long-term monitoring of live cell proliferation in presence of PVP-Hypericin: a new strategy using ms pulses of LED and the fluorescent dye CFSE

During fluorescent live cell imaging it is critical to keep excitation light dose as low as possible, especially in the presence of photosensitizer drugs, which generate free radicals upon photobleaching. During fluorescent imaging, stress by excitation and free radicals induces serious cell damages that may arrest the cell cycle. This limits the usefulness of the technique for drug discovery, when prolonged live cell imaging is necessary. This paper presents a strategy to provide gentle experimental conditions for dynamic monitoring of the proliferation of human lung epithelial carcinoma cells (A549) in the presence of the photosensitizer Polyvinylpyrrolidone-Hypericin. The distinctive strategy of this paper is based on the stringent environmental control and optimizing the excitation light dose by (i) using a low-power pulsed blue light-emitting diode with short pulse duration of 1.29 ms and (ii) adding a nontoxic fluorescent dye called carboxyfluorescein-diacetate-succinimidyl-ester (CFSE) to improve the fluorescence signals. To demonstrate the usefulness of the strategy, fluorescence signals and proliferation of dual-marked cells, during 5-h fluorescence imaging under pulsed excitation, were compared with those kept under continuous excitation and nonmarked reference cells. The results demonstrated 3% cell division and 2% apoptosis due to pulsed excitation compared to no division and 85% apoptosis under the continuous irradiation. Therefore, our strategy allows live cell imaging to be performed over longer time scales than with conventional continuous excitation.     

Inhibitory effects of Broccolini leaf flavonoids on human cancer cells

Broccolini (Brassica oleracea Italica × Alboglabra) is a hybrid between broccoli and Gai Lan, also known as Chinese broccoli and Chinese kale. The aim of this study was to assess the antitumor activity of Broccolini leaf flavonoids (BLF). Cell growth inhibition was evaluated using a standard colorimetric MTT assay, cellular morphology was observed using phase contrast microscopy and flow cytometry was introduced to further investigate cells apoptosis effect. The results showed that BLF possess a dose-dependent antiproliferative effects on four human cancer cell lines (SW480, HepG2, Hela, and A549) and apoptosis induction activity on SW480 cell line. Thus, the hybrid species Broccolini could be considered as a functional vegetable with potential in assisting for the treatment of four human cancers examined here.     

Detection of ROS and translocation of ERP-57 in apoptotic induced human neuroblastoma (SH-SY5Y) cells

Several toxic compounds are known to induce apoptosis in mammalian cell lines. The human neuroblastoma cells (SH-SY5Y) were exposed to the phosphatase inhibiting toxin okadaic acid (OA) or hydrogen peroxide (H2O2) to induce apoptosis as well as generate reactive oxygen species (ROS). Mitoxantrone (MXT) was used as a positive control for apoptosis. The SH-SY5Y cells were transfected with eukaryotic expression plasmid pHyPer-dMito encoding mitochondrial-targeted fluorescent or pHyPer-dCito encoding cytoplasmic-targeted fluorescent sensor for hydrogen peroxide (HyPer). The ERp57, also called GRP58 (Glucose-regulated protein 58), is a stress protein induced in conditions like glucose starvation and viral infection. Recently ERp57 was shown to translocate from the endoplasmatic reticulum to the cell surface in anthracycline-induced apoptotic cells. ERp57 co-translocation together with calreticulin has been suggested to be crucial for recognizing tumor cells to induce immunogenic cell death. ERp57 translocation after exposure to okadaic acid was studied using immunofluorescence and confocal microscopy. These studies indicated that okadaic acid has induced the translocation of ERp57 to the cellular membrane.     

Endocytic pathways in the olfactory and vomeronasal epithelia of the mouse: ultrastructure and uptake of tracers.

Mammalian olfactory neurons possess a well-developed system of endocytic vesicles, endosomes, and lysosomes in their dendrites and perikarya. Vomeronasal neurons are similar and also contain much perikaryal agranular endoplasmic reticulum (AER). Olfactory supporting cells contain endocytic vesicles and endosomes associated closely with abundant fenestrated AER, and vesicles and numerous large dense vacuoles are present basally. Vomeronasal supporting cells have little AER, and few dense vacuoles occur in their bases. In olfactory neurons, ultrastructural tracers (0.08% horseradish peroxidase, thorium dioxide, ferritin) are endocytosed by olfactory receptor endings and transported to the cell body, where their movement is halted in lysosomes. Higher concentrations (1%) of horseradish peroxidase penetrate olfactory receptor plasma membranes and intercellular junctions. In olfactory supporting cells, endocytosed tracers pass through endosomes to accumulate in dense basal vacuoles. These observations indicate that olfactory sensory membranes are rapidly cycled and that endocytosed materials are trapped within the epithelium. It is proposed that in the olfactory epithelium, endocytosis presents redundant odorants to the enzymes of the supporting cell AER to prevent their accumulation, whereas in the vomeronasal epithelium the receptor cells carry out this activity.     

An extract of <i>Hypericum perforatum</i> induces wound healing through inhibitions of Ca²⁺ mobilizations,mitochondrial oxidative stress and cell death in epithelial cells: Involvement of TRPM2 channels

The wound is induced by several mechanical and metabolic factors. In the etiology of the wound recovery, excessive oxidative stress, calcium ion (Ca²⁺) influx, and apoptosis have important roles. Ca²⁺-permeable TRPM2 channel is activated by oxidative stress. Protective roles of Hypericum perforatum extract (HP) on the mechanical nerve injury-induced apoptosis and oxidative toxicity through regulation of TRPM2 in the experimental animals were recently reported. The potential protective roles in HP treatment were evaluated on the TRPM2-mediated cellular oxidative toxicity in the renal epithelium (MPK) cells. The cells were divided into three groups as control, wound, and wound + HP treatment (75 µM for 72 h). Wound diameters were more importantly decreased in the wound+HP group than in the wound group. In addition, the results of laser confocal microscopy analyses indicated protective roles of HP and TRPM2 antagonists (N-(p-Amylcinnamoyl) anthranilic acid and 2-aminoethyl diphenylborinate) against the wound-induced increase of Ca²⁺ influx and mitochondrial ROS production. The wound-induced increase of early (annexin V-FITC) apoptosis and late (propidium iodide) apoptosis were also decreased in the cells by the HP treatment. In conclusion, HP treatment acted protective effects against wound-mediated oxidative cell toxicity and apoptosis through TRPM2 inhibition. These effects may be attributed to their potent antioxidant effect.     

Ultrastructural characteristics of ovine bone marrow‐derived mesenchymal stromal cells cultured with a silicon stabilized tricalcium phosphate bioceramic

Bioceramics are being used in experimental bone engineering application in association with bone marrow derived mesenchymal stem cells (BM‐MSCs) as a new therapeutic tool, but their effects on the ultrastructure of BM‐MSCs are yet unknown. In this study we report the morphological features of ovine (o)BM‐MSCs cultured with Skelite, a resorbable bioceramic based on silicon stabilized tricalcium phosphate (SiTCP), able to promote the repair of induced bone defect in sheep model. oBM‐MSCs were isolated from the iliac crest, cultured until they reached near‐confluence and incubated with SiTCP. After 48 hr the monolayers were highly damaged and only few cells adhered to the plastic. Thus, SiTCP was removed, and after washing the cells were cultured until they became confluent. Then, they were trypsinizated and processed for transmission electron microscopy (TEM) and RT‐PCR analysis. RT‐PCR displayed that oBM‐MSCs express typical surface marker for MSCs. TEM revealed the presence of electron‐lucent cells and electron‐dense cells, both expressing the CD90 surface antigen. The prominent feature of electron‐lucent cells was the concentration of cytoplasmic organelles around the nucleus as well as large surface blebs containing glycogen or profiles of endoplasmic reticulum. The dark cells had a multilocular appearance by the presence of peripheral vacuoles. Some dark cells contained endocytic vesicles, lysosomes, and glycogen aggregates. oBM‐MSCs showed different types of specialized interconnections. The comparison with ultrastructural features of untreated oBM‐MSCs suggests the light and dark cells are two distinct cell types which were differently affected by SiTCP bioceramic. Skelite cultured ovine BM‐MSCs display electron‐dense and electron‐lucent cells which are differently affected by this bioceramic. This suggests that they could play a different role in bioceramic based therapy.