Abstract: It is widely accepted that glycine transporters of the GLYT1 type are situated on astrocytes whereas GLYT2 are present on glycinergic neuronal terminals where they mediate glycine uptake. We here used purified preparations of mouse spinal cord nerve terminals (synaptosomes) and of astrocyte-derived subcellular particles (gliosomes) to characterize functionally and morphologically the glial versus neuronal distribution of GLYT1 and GLYT2. Both gliosomes and synaptosomes accumulated [(3)H]GABA through GAT1 transporters and, when exposed to glycine in superfusion conditions, they released the radioactive amino acid not in a receptor-dependent manner, but as a consequence of glycine penetration through selective transporters. The glycine-evoked release of [(3)H]GABA was exocytotic from synaptosomes but GAT1 carrier-mediated from gliosomes. Based on the sensitivity of the glycine effects to selective GLYT1 and GLYT2 blockers, the two transporters contributed equally to evoke [(3)H]GABA release from GABAergic synaptosomes; even more surprising, the 'neuronal' GLYT2 contributed more efficiently than the 'glial' GLYT1 to mediate the glycine effect in [(3)H]GABA releasing gliosomes. These functional results were largely confirmed by confocal microscopy analysis showing co-expression of GAT1 and GLYT2 in GFAP-positive gliosomes and of GAT1 and GLYT1 in MAP2-positive synaptosomes. To conclude, functional GLYT1 are present on neuronal axon terminals and functional GLYT2 are expressed on astrocytes, indicating not complete selectivity of glycine transporters in their glial versus neuronal localization in the spinal cord.

Abstract:

Abstract: The mayor pathologic hallmarks of Alzheimer's disease (AD) are senile plaque and neurofibrillary tangles. Senile plaque are primarily made up of deposits of amyloid-beta protein, a proteolytic product derived from the amyloid precursor protein (APP). APP is a transmembrane protein detected into the endoplasmic reticulum, in the Golgi apparatus, at the cell surface, recycled by endocytosis to endosomes, whose physiological function is unclear. Presenilins (PS), are a component of gamma-secretase complex that cleave alpha-CTFs (carboxy-terminal fragment), or beta-CTFs, leaving 40 or 42 amino acids amyloid-beta peptides and 58 or 56 amino acids intracellular domains (AICD). Where the amyloid-beta peptides is generated is not clear. The study of APP-PS interaction in specific cell compartments provides a good opportunity to light upon the molecular mechanisms regulating the activity of the "gamma-secretase complex," and where beta-amyloid is generated. In our study we used a biophysical assay of protein proximity: fluorescence resonance energy transfer (FRET), that can provide information about molecular interactions when two proteins are in the close proximity (<10 nm), to examine the subcellular localization and interaction between APP and PS1 in human neuroglioma cells (H4). Confocal microscopic analysis reveals extensive colocalization in different cells' compartment, and centrosomal or microtubule organizing center (MTOC) localization of APP and PS1, but not necessarily a close molecular interaction. We used FRET to determine if APP and PS1 interact at the cell centrosome. FRET data suggest a close interaction between APP and PS1 in subcellular compartments and at the centrosome of H4 cells. Using this approach we show that APP and PS1 are closely associated in the centrosomes of the H4 cell.

Abstract: The aim of this study has been the biochemical demonstration of the presence of gamma-aminobutyric acid (GABA) in the Mediterranean sea fan Eunicella cavolini by means of high-performance liquid chromatography, and the description of the distribution pattern of GABA and its related molecules, glutamic acid decarboxylase (GAD), vesicular GABA transporter (VGAT) and one of the GABA receptors (GABA(B) R) by immunohistochemical methods. The interrelationships of GABA, GAD and GABA receptor immunoreactivity have been established by using double-immunohistochemical methods and confocal microscopy. The immunodetection of monoclonal and/or polyclonal antibodies has revealed GABA immunoreactivity throughout the polyp tissue, both in neuronal and non-neuronal elements. GAD immunoreactivity has been mostly localized in the neuronal compartment, contacting epithelial and muscular elements. GABA(B) R immunoreactivity appears particularly intense in the nematocytes and in the oocyte envelope; its presence in GAD-immunoreactive neurons in the tentacles suggests an autocrine type of regulation. Western blot analysis has confirmed that a GABA(B) R, with a molecular weight of 142 kDa, similar to that of rat brain, is present in E. cavolini polyp tissue. The identification of the sites of the synthesis, vesicular transport, storage and reception of GABA strongly suggests the presence of an almost complete set of GABA-related molecules for the functioning of the GABAergic system in this simple nervous system. The distribution of these different immunoreactivities has allowed us to hypothesize GABA involvement in nematocyst discharge, in body wall and enteric muscular contraction, in neuronal integration and in male gametocyte differentiation.

Abstract: In the last decade, fluorescence resonance energy transfer (FRET) has become a useful technique for studying intermolecular interactions applied to the analysis of biological systems. Although FRET measurements may be very helpful in the comprehension of different cellular processes, it can be difficult to obtain quantitative results, hence the necessity of studying FRET on controllable systems. Here, a fuzzy nanostructured system called a nanocapsule is presented as a nanometric-device allowing distance modulation, thus preserving photophysical properties of fluorescent dyes and exhibiting good potential features for improving quantitative FRET analysis. We evaluated the behavior of such a sample using four FRET methods (three of them based on steady-state fluorescence and one using lifetime measurements). Within some limitations that can be overcome, these nanodevices have the potential to serve as a benchmark system for characterizing new FRET couples and to develop quantitative approaches for FRET analysis.

Abstract: PURPOSE: Calcium ions play a pivotal role in phototransduction. In this study, the presence and functional role of the adenosine diphosphoribosyl (ADPR)-cyclase-cyclic ADP-ribose (cADPR) system in bovine retinal rod outer segments (ROS) was investigated. METHODS: A Ca(2+) release from osmotically intact ROS discs elicited by cADPR was studied in the presence of the Ca(2+) tracer fluo-3. Endogenous cyclic guanosine diphosphate ribose (cGDPR) formation in discs was investigated by spectrophotometric detection of its synthesis from nicotinamide guanine dinucleotide (NGD(+)). ADPR-cyclase was also investigated at a structural level on mildly denaturing SDS-PAGE by production of cyclic inosine diphosphate ribose from nicotinamide hypoxantine dinucleotide (NHD(+)). Western immunoblot analysis with a specific antibody was conducted to verify the presence of ryanodine-sensitive Ca(2+) channels (RyRs) in ROS discs. RESULTS: cADPR-dependent Ca(2+) release was a linear function of extravesicular free Ca(2+) concentration, between 200 and 900 nM Ca(2+). When free Ca(2+) was 203 +/- 10 nM the mean Ca(2+) release was 23 +/- 3 pmol/mL per milligram protein. The average rate of cGDPR production was 13 +/- 2 nmol cGDPR/min per milligram protein, by a putative enzyme with an apparent molecular mass of 53 +/- 1 kDa. ROS ADPR-cyclase was localized in the membranous fraction. No nicotinamide adenine dinucleotide glycohydrolase (NADase) activity was detected. The presence of RyR channels in pure disc preparations was confirmed by confocal laser scanning microscopy. CONCLUSIONS: A cADPR metabolism may be present in retinal ROS discs, which may be Ca(2+) stores operated by cADPR. A model is proposed for the physiological role of cADPR-mediated Ca(2+) release in bovine ROS.

Abstract: We report the analysis of the fluorescence intensity fluctuations of single proteins of a GFP mutant, GFPmut2, embedded in a polyelectrolyte nanocapsule adsorbed on thin conductive layers. Our results, based on single molecule fluorescence spectroscopy, indicate that the fluorescence blinking dynamics of GFP is strongly dependent on the bulk conductivity of the metal layer substrate, on the distance from the conductive surfaces and on the amplitude of the voltage applied to the poly-electrolyte layers. These findings suggest that fluorescence blinking itself might be employed as a reporter signal in nano-bio-technology applications.

Abstract: Layer-by-layer technique is used to adsorb a uniform ultrathin layer of fluorescently labelled polyelectrolytes on a glass cover slip. Due to their thickness, uniformity and fluorescence properties, these ultrathin layers may serve as a simple and applicable standard to directly measure the z-response of different scanning optical microscopes. In this work we use ultrathin layers to measure the z-response of confocal, two-photon excitation and 4Pi laser scanning microscopes. Moreover, due to their uniformity over a wide region, i.e. cover slip surface, it is possible to quantify the z-response of the system over a full field of view area. This property, coupled with a bright fluorescence signal, enables the use of polyelectrolyte layers for representation on sectioned imaging property charts: a very powerful method to characterize image formation properties and capabilities (z-response, off-axis aberration, spherical aberration, etc.) of a three-dimensional scanning system. The sectioned imaging property charts method needs a through-focus dataset taken from such ultrathin layers. Using a comparatively low illumination no significant bleaching occurs during the excitation process, so it is possible to achieve long-term monitoring of the z-response of the system. All the above mentioned properties make such ultrathin layers a suitable candidate for calibration and a powerful tool for real-time evaluation of the optical sectioning capabilities of different three-dimensional scanning systems especially when coupled to sectioned imaging property charts.

Abstract: The amyloid precursor protein (APP) and the presenilins 1 and 2 are genetically linked to the development of familial Alzheimer disease. APP is a single-pass transmembrane protein and precursor of fibrillar and toxic amyloid-beta peptides, which are considered responsible for Alzheimer disease neurodegeneration. Presenilins are multipass membrane proteins, involved in the enzymatic cleavage of APP and other signaling receptors and transducers. The role of APP and presenilins in Alzheimer disease development seems to be related to the formation of amyloid-beta peptides; however, their physiological function, reciprocal interaction, and molecular mechanisms leading to neurodegeneration are unclear. APP and presenilins are also involved in multiple interactions with intracellular proteins, the significance of which is under investigation. Among the different APP-interacting proteins, we focused our interest on the GRB2 adaptor protein, which connects cell surface receptors to intracellular signaling pathways. In this study we provide evidence by co-immunoprecipitation experiments, confocal and electron microscopy, and by fluorescence resonance energy transfer experiments that both APP and presenilin1 interact with GRB2 in vesicular structures at the centrosome of the cell. The final target for these interactions is ERK1,2, which is activated in mitotic centrosomes in a PS1- and APP-dependent manner. These data suggest that both APP and presenilin1 can be part of a common signaling pathway that regulates ERK1,2 and the cell cycle.

Abstract: The morphological and mechanical properties of encapsulated yeast cells (Saccharomyces cerevisiae) have been investigated by atomic force microscopy (AFM). Single living cells have been coated through the alternate deposition of oppositely charged polyelectrolyte (PE) layers. The properties of cells coated by different numbers of PE layers and from PE solutions of different ionic strength have been investigated. AFM imaging indicates an increase in PE coating stability when decreasing the solution ionic strength. The Young's moduli of the different examined systems have been evaluated through a quantitative analysis of force-distance curves by using the Hertz-Sneddon model. The analysis indicates an increase in hybrid system stiffness when lowering the ionic strength of the PE solution. An evaluation of the viability of encapsulated cells was obtained by confocal laser scanning microscopy (CLSM) measurements. CLSM analysis indicates that cells preserve their subcellular structure and duplication capability after encapsulation. By coupling AFM and CLSM data, a correlation between local stiffness and duplication rate was obtained.

Abstract: The capability of certain heavy metal ions to induce fluorescence decrease by a quenching mechanism suggested us to design and build a sensor potentially tunable for different ions at different concentrations. We propose a quenching-based sensor exploiting a nanostructured architecture in which fluorescent molecules (the sensing probe) are entrapped to recognize a specific analyte (heavy metal ions) through an optical transduction. The polyelectrolyte nanostructured system, named nanocapsule, improves the fluorophore-ion quenching sensitivity allowing a micromolar detection. Furthermore we couple our sensor with an electrical device in order to refine the sensing procedure: the electric field created allows a metal ions spatial gradient, necessary to detect a specific element on a single sample solution, avoiding a comparative analysis with an intensity reference value. Results obtained will show the advantages and the potentialities of our system as a smart toolbox for metal ions detection.

Abstract: The quantitative analysis of fluorescence perturbation experiments such as fluorescence recovery after photobleaching (FRAP) requires suitable analytical models to be developed. When diffusion in 3D environments is considered, the description of the initial condition produced by the perturbation (i.e., the photobleaching of a selected region in FRAP) represents a crucial aspect. Though it is widely known that bleaching profiles approximations can lead to errors in quantitative FRAP measurements, a detailed analysis of the sources and the effects of these approximations has never been conducted until now. In this study, we measured the experimental 3D bleaching distributions obtained in conventional and two-photon excitation schemes and analyzed the deviations from the ideal cases usually adopted in FRAP experiments. In addition, we considered the non-first-order effects generated by the high energy pulses usually delivered in FRAP experiments. These data have been used for finite-element simulations mimicking FRAP experiments on free diffusing molecules and compared with FRAP model curves based on the ideal bleach distributions. The results show that two-photon excitation more closely fits ideal bleaching patterns even in the event of fluorescence saturation, achieving estimations of diffusion coefficients within 20% accuracy of the correct value.

Abstract: Vertebrate retinal rod outer segment disks house the proteins involved in the phototransduction cascade that converts light into neuronal signal. We develop a technique for the immunofluorescent labeling of osmotically intact isolated rod outer segment disks for confocal laser scanning microscopy imaging. Osmotically intact Ficoll-flotation isolated bovine disks are directly labeled with antibodies in solution. For the first time, osmotically intact single disks can be visualized. Thus, imaging of purified disks, based on advanced optical techniques, may serve as a powerful complement to other methods in studies on phototransduction. In fact, even though much is known about the rod outer segment photoresponse, some unanswered questions remain, particularly about ATP supply, light adaptation, and morphogenesis.

Abstract: In a significant step towards the construction of the semi-synthetic minimal cell, a protein expression system with a minimal set of pure and specific enzymes is required. A novel cell-free transcription and translation system named PURESYSTEM (PS), consisting of a specified set of 36 enzymes and ribosomes, has been entrapped in POPC liposomes for protein synthesis. The PS has been used to transcribe and translate an Enhanced Green Fluorescent Protein (EGFP) gene from plasmid DNA. The synthesis is confirmed by the EGFP fluorescence emitting liposomes on fluorometric analysis and on confocal microscopy analysis. Furthermore the PS encapsulated into POPC liposomes can drive the expression of the plsB and plsC genes encoding for the sn-glycerol-3-phosphate acyltransferase (GPAT) and 1-acyl-sn-glycerol-3-phosphate acyltransferase (LPAAT) involved in the first step of the "salvage pathway" for synthesis of POPC. The expression of GPAT and LPAAT in liposomes would in principle allow the production of the cell boundary from within.

Abstract: The effect of euplotin C -- a lipophilic bioactive metabolite produced by the ciliate Euplotes crassus -- on the kinetics of both phagocytosis of latex particles and fluid-phase uptake of dextran, was studied in the single-cell ciliate Paramecium primaurelia. The inhibition of food vacuole formation was concentration- and time-dependent (p<0.001), even if euplotin C did not completely block the phagocytosis. Following a 15 min treatment with a euplotin C (0.5 microg/ml), the latex particle uptake was inhibited up to 25%. Furthermore, the pretreatment of cells with taxol strongly counteracted euplotin C effect. The amount of extracellularly provided dextran, which is internalized exclusively by fluid-phase uptake, was quantified in cells whose phagocytic activity was blocked by trifluoperazine. The amount of the internalized dextran was about 50% of that in controls after 15 min incubation in the presence of euplotin C. Fluorescence confocal images showed that no endosomes were formed on the surface of these cells. The effect of euplotin C on the food vacuole formation and fluid-phase endocytosis is apparently mediated by a modification of microtubule network.

Abstract: Gamma-amino butyric acid (GABA) is believed to be the principal inhibitory neurotransmitter in the mammalian central nervous system, a function that has been extended to a number of invertebrate systems. The presence of GABA in the marine demosponge Chondrilla nucula was verified using immunofluorescence detection and high-pressure liquid chromatography. A strong GABA-like immunoreactivity (IR) was found associated with choanocytes, exopinacocytes, endopinacocytes lining inhalant, and exhalant canals, as well as in archaeocytes scattered in the mesohyl. The capacity to synthesize GABA from glutamate and to transport it into the vesicles was confirmed by the presence in C. nucula of glutamate decarboxylase (GAD) and vesicular GABA transporters (vGATs), respectively. GAD-like and vGAT-like IR show the same distribution as GABA-like IR. Supporting the similarity between sponge and mammalian proteins, bands with an apparent molecular weight of about 65-67 kDa and 57 kDa were detected using antibodies raised against mammalian GAD and vGAT, respectively. A functional metabotropic GABA(B)-like receptor is also present in C. nucula. Indeed, both GABA(B) R1 and R2 isoforms were detected by immunoblot and immunofluorescence. Also in this case, IR was found in choanocytes, exopinacocytes, and endopinacocytes. The content of GABA in C. nucula amounts to 1225.75 +/- 79 pmol/mg proteins and GABA is released into the medium when sponge cells are depolarized. In conclusion, this study is the first indication of the existence of the GABA biosynthetic enzyme GAD and of the GABA transporter vGAT in sponges, as well as the first demonstration that the neurotransmitter GABA is released extracellularly.

Abstract: In this study we report for the first time the localization of a photoreceptor pigment in the cilia of the colored heterotrich ciliates Blepharisma japonicum red and blue form, Fabrea salina, and Stentor coeruleus, as result of a confocal microscopy investigation. Optical sectioning confocal microscopy has been used for studying the spatial distribution of the pigment in the cell body, surprisingly showing that, besides its expected presence in the cortical region immediately below the cell membrane, it is located in the cilia too. In order to ascertain possible differences in the pigment fluorescence properties along the cell body, we have measured emission spectra from different parts of it (anterior, posterior, and cilia). Our results clearly indicate that in all cases the spectra are the same, within experimental errors. Finally, we have evaluated the pigment relative fluorescence efficiency of these ciliates. In an ordered scale from lower to greater efficiency, we have S. coeruleus, B. japonicum blue, B. japonicum red, and F. salina. The possible implications of our findings for the process of photosensory transduction are discussed.

Abstract: A theoretical microscopic technique is proposed that may reduce multiphoton interaction in the excitation volume of a two-photon microscope. Since higher-order photobleaching is common in two-photon excitation microscopy, the study of thin samples is limited by increased photobleaching and photodamage. This limitation is elevated by using even coherent state light. The advantage of even coherent state light is that only excitation due to an even number of photons can survive. The very first nonzero even excitation (two-photon) can be isolated from the nearby one- and three-photon excitation. Hence the photobleaching due to one- and three-photon excitation can be eliminated and higher-order processes can be minimized owing to their small molecular cross section.

Abstract: Multi-photon excitation (MPE) microscopy plays a growing role among microscopical techniques utilized for studying biological matter. In conjunction with confocal microscopy it can be considered the imaging workhorse of life science laboratories. Its roots can be found in a fundamental work written by Maria Goeppert Mayer more than 70 years ago. Nowadays, 2PE and MPE microscopes are expected to increase their impact in areas such biotechnology, neurobiology, embryology, tissue engineering, materials science where imaging can be coupled to the possibility of using the microscopes in an active way, too. As well, 2PE implementations in noninvasive optical bioscopy or laser-based treatments point out to the relevance in clinical applications. Here we report about some basic aspects related to the phenomenon, implications in three-dimensional imaging microscopy, practical aspects related to design and realization of MPE microscopes, and we only give a list of potential applications and variations on the theme in order to offer a starting point for advancing new applications and developments.

Abstract: GABAB receptors modulate swimming behavior in Paramecium by inhibiting dihydropyridine-sensitive Ca2+ channels via G-proteins. Prolonged occupancy of GABAB receptors by baclofen results in a decrease in GABAB receptor functions. Since changes in the number of cell-surface GABAA receptors have been postulated to be of importance in modulating inhibitory synaptic transmission in neurons, we have studied the cell-surface expression and maintenance of GABAB receptors in P. primaurelia. In this study, we use immunostaining in electron and confocal microscopy to demonstrate that constitutive internalization of GABAB receptors in P. primaurelia is mediated by clathrin-dependent and -independent endocytosis. Indeed, GABAB receptors colocalize with the adaptin complex AP2, which is implicated in the selective recruitment of integral membrane proteins to clathrin-coated vesicles, and with caveolin 1, which is associated with uncoated membrane invaginations. Furthermore, when endocytosis is blocked with hypertonic medium, cytosol acidification, filipin or with a peptide that disrupts the association between amphiphysin and dynamin, the effect of baclofen on swimming is increased. These results suggest that GABAB receptor endocytosis into clathrin-coated and -uncoated vesicles represents an important mechanism in the modulation of swimming behavior in Paramecium.

Abstract: Glial subcellular re-sealed particles (referred to as gliosomes here) were purified from rat cerebral cortex and investigated for their ability to release glutamate. Confocal microscopy showed that the glia-specific proteins glial fibrillary acidic protein (GFAP) and S-100, but not the neuronal proteins 95-kDa postsynaptic density protein (PSD-95), microtubule-associated protein 2 (MAP-2) and beta-tubulin III, were enriched in purified gliosomes. Furthermore, gliosomes exhibited labelling neither for integrin-alphaM nor for myelin basic protein, which are specific for microglia and oligodendrocytes respectively. The Ca2+ ionophore ionomycin (0.1-5 microm) efficiently stimulated the release of tritium from gliosomes pre-labelled with [3H]d-aspartate and of endogenous glutamate in a Ca(2+)-dependent and bafilomycin A1-sensitive manner, suggesting the involvement of an exocytotic process. Accordingly, ionomycin was found to induce a Ca(2+)-dependent increase in the vesicular fusion rate, when exocytosis was monitored with acridine orange. ATP stimulated [3H]d-aspartate release in a concentration- (0.1-3 mm) and Ca(2+)-dependent manner. The gliosomal fraction contained proteins of the exocytotic machinery [syntaxin-1, vesicular-associated membrane protein type 2 (VAMP-2), 23-kDa synaptosome-associated protein (SNAP-23) and 25-kDa synaptosome-associated protein (SNAP-25)] co-existing with GFAP immunoreactivity. Moreover, GFAP or VAMP-2 co-expressed with the vesicular glutamate transporter type 1. Consistent with ultrastructural analysis, several approximately 30-nm non-clustered vesicles were present in the gliosome cytoplasm. It is concluded that gliosomes purified from adult brain contain glutamate-accumulating vesicles and can release the amino acid by a process resembling neuronal exocytosis.

Abstract: We have studied the unfolding of single molecules of GFP-mut2 mutant trapped in wet silica gels in a wide range of GuHCl concentration. After the addition of denaturant, the number of fluorescent molecules decreases with unfolding rates (of the order of 0.01 min(-1)) that are in very good agreement with bulk fluorescence and circular dichroism data. Unexpectedly, single molecule experiments show rare fluctuations in the number of fluorescent proteins at equilibrium. On the other hand, although a first approximate description of the number decays can be reasonably performed by single exponential functions, the distributions of the single molecule unfolding times show a maximum at times congruent with 50-100 min up to the denaturation midpoint concentration of [GuHCl] congruent with 2.5 M. A theoretical analysis of the distributions indicates that this feature is a fingerprint of the competition between unfolding and refolding processes when the protein is very far from the midpoint denaturant concentration.

Abstract:

Abstract: We propose a maximum a posteriori image restoration approach to 3D confocal microscopy. The image field is suitably modeled as a Markov random field, resulting in a Gibbs distributed image. A fuzzy-logic-based potential is employed in the Gibbs prior. Unlike other potentials, the fuzzy potential distinguishes intensity variation due to genuine edges and noise. The proposed approach has generated artifact-free restored confocal microscopy images.

Abstract: To date only peptidergic innervation has been described in the alimentary tract of barnacles. In the present work the presence and distribution of choline acetyltransferase (ChAT), the acetylcholine (ACh) synthesizing enzyme, was investigated by immunohistochemistry in the alimentary tract of the adult barnacle Balanus amphitrite. Numerous ChAT-immunoreactive (IR) cells and a net of ChAT-IR cytoplasmic processes were localized inside the epithelium of the posterior midgut, close to the basement membrane; no IR nerve endings were detected in the midgut longitudinal and circular muscle bundles. Epithelial neurons or endocrine cells in the gut epithelium have been described in some invertebrate species belonging to different taxa and their peptidergic features are reported in the literature. Our results point out the presence of neuroepithelial cells also in the gut epithelium of barnacles; moreover, for the first time, a cholinergic feature is suggested for this cell type. These data seem to indicate the involvement of ACh in the gut functions of barnacle and suggest that the barnacle alimentary tract is more complex than previously thought and requires further study.

Abstract: Immune protection of artificial tissue by means of pancreatic islet microencapsulation is a very ambitious new approach to avoid life-long immune suppression. But the success in the utilization of the alginate-beads with incorporated islets is unfortunately limited. Some of the problems cannot be solved by a two-component system, so polymer encapsulation of the microbeads was tested to improve the properties. In the present paper a pure nanoencapsulation multilayer approach was tested in order to reduce the size of the capsule and possibly apply in the future a multilayer capsule with individual properties in each layer or region of the capsule. Different polycations were attached in a self-assembly process. The advantage in using the surface charge of islets as binding site for the polyions is the guarantee of complete coverage after the second layer. Release of insulin was determined to characterize the function of the islets after encapsulation as well as the permeability of the capsule. Fluorescence microscopy was used to visualize the polyelectrolyte layers. Finally by means of an immune assay, the protection capability of the capsule was proved. In these first measurements the encapsulation with a multilayer nanocapsule was shown to be a possible alternative to the more space-consuming and random islet-trapping microencapsulation.

Abstract: We report about two-photon activation of a photoactivatable derivative of the Aequorea Victoria green fluorescent protein (paGFP). This special form of the molecule increases its fluorescence intensity when excited by 488 nm after irradiation with high intensity light at 413 nm. The aim in this work was to evaluate the use of two-photon interactions for confining the molecular switching of pa-GFP in the bright state. Therefore experiments were performed using fixed and living cells which were expressing the paGFP fluorophore and microspheres whose surface was modified by specific adsorption of the chromophores. The molecular switches were activated in a range of wavelength from 720 nm to 840 nm. The optimal wavelength for activation was then chosen for cell imaging. A comparison between the conventional activation and two-photon mode demonstrates clearly the better three- dimensional (3D) confinement and the possibility of selection of cell volumes of interest. This enables molecular trafficking studies at high signal to noise ratio.

Abstract: Dimethyl-pepep (D-pepep), a newly developed and very efficient two-photon absorber, has been tested here for two-photon excitation (TPE) cellular imaging. The spectral characteristics of the dye following one-photon excitation (OPE) and TPE (excitation and emission spectra, fluorescence lifetime, molecular brightness, saturation intensity) are reported. In vitro interaction studies with biomolecules show that dimethyl-pepep has a large affinity for DNA. A comparison with a widely used DNA stainer, 4-6-diamidino-2-phenylindole (DAPI) bound to DNA shows that the D-pepep brightness is one order of magnitude higher than that of DAPI, making this dye suitable for microscopy and imaging applications. TPE images taken from double-stained yeast Saccharomyces cerevisiae cells have revealed that D-pepep localizes mainly in the nucleus, similarly to DAPI, and in mitochondria, although to a minor extent. Preliminary tests have shown that the dye cellular toxicity is negligible.

Abstract: The effects of gamma-aminobutyric acid (GABA) on the release of glutamate from mouse spinal cord nerve endings have been studied using superfused synaptosomes. GABA elicited a concentration-dependent release of [3H]D-aspartate ([3H]D-ASP; EC50= 3.76 microM). Neither muscimol nor (-)baclofen mimicked GABA, excluding receptor involvement. The GABA-evoked release was strictly Na+ dependent and was prevented by the GABA transporter inhibitor SKF89976A, suggesting involvement of GAT-1 transporters located on glutamatergic nerve terminals. GABA also potentiated the spontaneous release of endogenous glutamate; an effect sensitive to SKF89976A and low-Na+-containing medium. Confocal microscopy shows that the GABA transporter GAT-1 is coexpressed with the vesicular glutamate transporter vGLUT-1 and with the plasma membrane glutamate transporter EAAT2 in a substantial portion of synaptosomal particles. The GABA effect was external Ca2+ independent and was not decreased when cytosolic Ca2+ ions were chelated by BAPTA. The glutamate transporter blocker DL-TBOA or dihydrokainate inhibited in part (approximately 35%) the GABA (10 microM)-evoked [3H]D-ASP release; this release was strongly reduced by the anion channel blockers niflumic acid and NPPB. GABA, up to 30 microM, was unable to augment significantly the basal release of [3H]glycine from spinal cord synaptosomes, indicating selectivity for glutamatergic transmission. It is concluded that GABA GAT-1 transporters and glutamate transporters coexist on the same spinal cord glutamatergic terminals. Activation of these GABA transporters elicits release of glutamate partially by reversal of glutamate transporters present on glutamatergic terminals and largely through anion channels.

Abstract: The presence of FMRFamide-related peptides (FaRPs) was investigated, by immunohistochemical methods with a polyclonal FMRFamide antiserum, in the sea-fan Eunicella cavolini (Van Koch 1887), a representative of the cnidarians (octocorallians). The identification of FaRP-immunoreactive elements as neuronal cells and a nerve net was performed by double immunohistochemical methods with the monoclonal anti-beta-tubulin antibody. A strong and widely distributed FaRPs immunoreactivity was detected: FaRPs-immunoreactive nerve cells were observed among and underlying gastrodermal epithelial cells, epidermal cells lining tentacles, muscular septs and gonophores. A diffuse FaRPs-immunoreactive nerve net was also found between epithelia and mesoglea and in the stalk of the gonophore. These results improve our knowledge of the gorgonian nervous system and demonstrate that most of the immunoreactive cells belong to neural elements.

Abstract: The immobilization of cells in defined arrays (cell patterning) is a key step towards cell-based biosensors or other cell-based devices. While cell patterning is usually achieved by modifying the surface on which only the cells should adhere and leaving the cells unmodified, we present here a different approach in which cells are first coated with polyelectrolytes and subsequently immobilized on patterned surfaces. By coating, the cells are protected and their interactions with the substrate are modified such that patterning is simplified. We used microcontact printing of polyelectrolytes to structure surfaces such that regions of opposite charges and the same charge as the cell coating were present and found that we can thus achieve patterning of the coated yeast cells. In accordance with prior work, we find that coating does not kill the cells and coated GFP-expressing cells still function after immobilization, which we checked by fluorescence microscopy.

Abstract: This report covers the two-photon activation and excitation properties of the PA-GFP, a photoactivatable variant of the Aequorea victoria green fluorescent protein in the spectral region from 720 to 920 nm. It is known from this special form of the molecule that it has an increased level of fluorescence emission when excited at 488 nm after irradiation at lambda approximately 413 nm, under single-photon excitation conditions. Here, we show that upon two-photon irradiation, PA-GFP yields activation in the spectral region from 720 to 840 nm. After photoactivation, the excitation spectrum shifts maintaining the very same emission spectrum of the single-photon case for the native and photoactivated protein. Additionally, when comparing the conventional photoactivation at lambda = 405 nm with a two-photon one, a sharper and better controllable three-dimensional volume of activation is obtained.

Abstract: We report the two-photon excitation and emission or a recently developed green fluorescent protein (GFP) mutant, E(2)GFP. Two main excitation bands are found at 780 and 870 nm. Blinking and irreversible and reversible bleaching were observed. Fluorescence blinking occurs in the millisecond range and has been ascribed to conversions between the neutral, anionic and dark zwitterionic states. Bleaching is observed after approximately 10 to 400 ms depending on the excitation power, and it is probably due to a conversion to a dark state. The striking feature of this GFP mutant is that the fluorescence can be recovered with very high efficiency only upon irradiation at 720 +/- 10 nm. This GFP mutant therefore seems promising as an almost permanent chromophore for two-photon excitation (TPE) microscopy or for applications in single-molecule memory arrays.

Abstract: The unfolding and refolding kinetics of >600 single GFPmut2 molecules, entrapped in wet nanoporous silica gels, were followed by monitoring simultaneously the fluorescence emission of the anionic and neutral state of the chromophore, primed by two-photon excitation. The rate of unfolding, induced by guanidinium chloride, was determined by counting the number of single molecules that disappear in fluorescence images, under conditions that do not cause bleaching or photoinduced conversion between chromophore protonation states. The unfolding rate is of the order of 0.01 min(-1), and its dependence on denaturant concentration is very similar to that previously reported for high protein load gels. Upon rinsing the gels with denaturant-free buffer, the GFPmut2 molecules refold with rates >10 min(-1), with an apparently random distribution between neutral and anionic states, that can be very different from the preunfolding equilibrium. A subsequent very slow (lifetime of approximately 70 min) relaxation leads to the equilibrium distribution of the protonation states. This mechanism, involving one or more native-like refolding intermediates, is likely rate limited by conformational rearrangements that are undetectable in circular dichroism experiments. Several unfolding/refolding cycles can be followed on the same molecules, indicating full reversibility of the process and, noticeably, a bias of denaturated molecules toward refolding in the original protonation state.

Abstract: This review is concerned with two-photon excited fluorescence microscopy (2PE) and related techniques, which are probably the most important advance in optical microscopy of biological specimens since the introduction of confocal imaging. The advent of 2PE on the scene allowed the design and performance of many unimaginable biological studies from the single cell to the tissue level, and even to whole animals, at a resolution ranging from the classical hundreds of nanometres to the single molecule size. Moreover, 2PE enabled long-term imaging of in vivo biological specimens, image generation from deeper tissue depth, and higher signal-to-noise images compared to wide-field and confocal schemes. However, due to the fact that up to this time 2PE can only be considered to be in its infancy, the advantages over other techniques are still being evaluated. Here, after a brief historical introduction, we focus on the basic principles of 2PE including fluorescence correlation spectroscopy. The major advantages and drawbacks of 2PE-based experimental approaches are discussed and compared to the conventional single-photon excitation cases. In particular we deal with the fluorescence brightness of most used dyes and proteins under 2PE conditions, on the optical consequences of 2PE, and the saturation effects in 2PE that mostly limit the fluorescence output. A complete section is devoted to the discussion of 2PE of fluorescent probes. We then offer a description of the central experimental issues, namely: choice of microscope objectives, two-photon excitable dyes and fluorescent proteins, choice of laser sources, and effect of the optics on 2PE sensitivity. An inevitably partial, but vast, overview of the applications and a large and up-to-date bibliography terminate the review. As a conclusive comment, we believe that 2PE and related techniques can be considered as a mainstay of the modern biophysical research milieu and a bright perspective in optical microscopy.

Abstract: Since the layer-wise polyelectrolyte deposition offers the opportunity to modify surfaces for biomedical applications, interactions and toxicity between polyelectrolytes and living cells become interesting. The aim of the present work is to determine the different factors such as contact area, charge, and transplantation site that influence the cell reaction to a specific polymer. We found that toxicity is influenced by all these factors and cannot be tested easily in a model.

Abstract: The presence in Paramecium of gamma-aminobutyric acid A-type receptors (GABA(A)) and the capability of the protozoon to synthesize and release the GABA neurotransmitter into the environment have already been demonstrated. This study investigates the involvement of the GABA(A) complex in the swimming control of the ciliated protozoon. The GABA(A) receptors were pharmacologically activated by the selective agonist muscimol and the effect on Paramecium primaurelia swimming behavior was analyzed. Paramecium normally swims forward, but the activation of GABA(A) receptors induced a peculiar response, characterized by alternate periods of whirling and forward swim. This effect was inhibited by the GABA(A) selective antagonists bicuculline and picrotoxin in a dose-dependent manner. Moreover, the application of benzodiazepines did not enhance the agonist action but only reduced it. Response to muscimol was also suppressed by nimodipine, a selective antagonist of dihydropyridine-sensitive calcium channels. This inhibition suggests that an inflow of calcium ions through L-type channels mediates the muscimol-induced swimming behavior.

Abstract: The number of neurotransmitter receptors on the plasma membrane is regulated by the traffic of intracellular vesicles. Golgi-derived vesicles provide newly synthesized receptors to the cell surface, whereas clathrin-coated vesicles are the initial vehicles for sequestration of surface receptors, which are ultimately degraded or recycled. We have previously shown that GABAB receptors display a punctuate vesicular pattern dispersed on the cell surface and throughout the cytoplasm and are internalized via clathrin-dependent and -independent endocytosis. Here we have studied constitutive GABAB receptor trafficking after internalization in Paramecium primaurelia by confocal laser scanning microscopy and multiple immunofluorescence analysis. After internalization, receptors are targeted to the early endosomes characterized by the molecular markers EEA1 and rab5. Some of these receptors, destined for recycling back to the plasma membrane, traffic from the early endosomes to the endosomal recycling compartment that is characterized by the presence of rab4-immunoreactivity (IR). Receptors that are destined for degradation exit the endosomal pathway at the early endosomes and traffic to the late endosome-lysosome pathway. In fact, some of the GABAB-positive compartments were identified as lysosomal structures by double staining with the lysosomal marker LAMP-1. GABAB vesicle structures also colocalize with TGN38-IR and rab11-IR. TGN38 and rab11 are proteins found in association with post-Golgi and recycling endosomes, respectively.

Abstract: "PowerUp Your Microscope" is a software package designed and realized for the optimization of 3D optical microscopy image quality using the Internet and inverse problems computational approaches. The package is mainly devoted to 3D microscopy users, being operative for wide-field, confocal, and multiphoton microscopy. It provides the microscopy community with an extremely easy and comparatively powerful access to advanced image restoration methods. The core of the computational section is the optical system modeling and inverse deconvolution implementation, which is strongly linked to Web-based software and technology. This project constitutes a real and effective migration to the Web, extending computational approaches to image restoration to the whole microscopy user community, regardless of their background.

Abstract: The presence of opioid, beta-adrenergic and cholinergic receptors has been demonstrated in ciliated protozoa, but little is known about gamma-aminobutyric acid (GABA) receptors. In this study we have analyzed the distribution of GABA(A)-type receptor subunits in Paramecium. Confocal laser microscopy using antibodies specific for alpha(1)-, alpha(2)-, alpha(3)-, alpha(6)-, beta(2/3)-, gamma(2)-, epsilon-, lambda-, and theta-subunits showed that most receptors are aggregated in clusters and are distributed both on cell surface and in the cytoplasm. The intensity of labelling of the alpha(6)-, beta(2/3)- and gamma(2)-subunits was more intense than the alpha(1)-, epsilon-, and theta-subunits, suggesting that the former are present in higher concentrations than the latter.

Abstract:

Abstract:

Abstract: This report deals with the fundamental quantum physics behind two-photon excitation also providing a link to the experimental consequences exploited in microscopy. The optical sectioning effect is demonstrated as well as the distribution of excitation and of fluorescence emission.

Abstract: The confocal microscope can image a specimen in its natural environment forming a 3D image of the whole structure by scanning it and collecting light through a small aperture (pinhole), allowing in vivo and in vitro observations. So far, the confocal fluorescence microscope (CFM) is considered a true volume imager because of the role of the pinhole that rejects information coming from out-of-focus planes. Unfortunately, intrinsic imaging properties of the optical scheme presently employed yield a corrupted image that can hamper quantitative analysis of successive image planes. By a post-image collection restoration, it is possible to obtain an estimate, with respect to a given optimization criterium, of the true object, utilizing the impulse response of system or Point Spread Function (PSF). The PSF can be measured or predicted so as to have a mathematical and physical model of the image-formation process. Further modelling and recording noise as an additive Gaussian process has used the regularized Iterative Constrained Tykhonov Miller (ICTM) restoration algorithm for solving the inverse problem. This algorithm finds the best estimate iteratively searching among the possible positive solutions; in the Fourier domain, such an approach is relatively fast and elegant. In order to compare the effective improvement in the quantitative image information analysis, we measured the volume of reference objects before and after image restoration, using the isotropic Fakir method.

Abstract: Aging is believed to be a nonadaptive process that escapes the force of natural selection. Here, we challenge this dogma by showing that yeast laboratory strains and strains isolated from grapes undergo an age- and pH-dependent death with features of mammalian programmed cell death (apoptosis). After 90-99% of the population dies, a small mutant subpopulation uses the nutrients released by dead cells to grow. This adaptive regrowth is inversely correlated with protection against superoxide toxicity and life span and is associated with elevated age-dependent release of nutrients and increased mutation frequency. Computational simulations confirm that premature aging together with a relatively high mutation frequency can result in a major advantage in adaptation to changing environments. These results suggest that under conditions that model natural environments, yeast organisms undergo an altruistic and premature aging and death program, mediated in part by superoxide. The role of similar pathways in the regulation of longevity in organisms ranging from yeast to mice raises the possibility that mammals may also undergo programmed aging.

Abstract: One of the most promising tools for future applications in science and medicine is the use of nanotechnologies. Especially self-assembly systems, e.g., polyelectrolyte (PE) capsules prepared by means of the layer-by-layer technique with tailored properties, fulfill the requirements for nano-organized systems in a satisfactory manner. The nano-organized shells are suitable as coating for living cells or artificial tissue to prevent immune response. With these shells, material can be delivered to predefined organs. In this paper, some preliminary results are presented, giving a broad overview over the possibilities to use nano-organized capsules. Based on the observations that the cells while duplicating break the capsule a mutant yeast strain (Saccharomyces cerevisiae), which express GFP-tubulin under galactose promotion, was investigated by means of confocal laser scanning microscopy. The measurements reveal an increased surface charge in the region of buds developed prior encapsulation. In order to test the used PE pair for cytotoxicity, germinating conidia of the fungi Neurospora crassa were coated. The investigation with fluorescence microscopy shows a variation in the surface charge for the growing region and the conidium poles. The capsules exhibit interesting properties as valuable tool in science and a promising candidate for application in the field of medicine.

Abstract: Green Fluorescent Protein (GFP) mutants are extensively used in optical microscopy studies of in vivo biological processes in cells. Nonetheless, blinking and bleaching of the GFP chromophore at the single molecule level greatly limits its usefulness. We have worked out what we think are the best experimental conditions for the use of the GFP mutant, GFP-mut2, as a single molecule marker in two-photon excitation measurements. We have measured molecular brightness, excited state lifetime, blinking and photo-bleaching times versus the two-photon excitation intensity on proteins embedded in silica gel matrices versus the excitation wavelength in the range 700-1,000 nm. Our results indicate that GFPmut2 can be employed as a long-lived reporter of biological processes.

Abstract:

Abstract: Multi-photon driven photo-switching between dark and bright (fluorescent) states of a green fluorescent protein (GFP) mutant is demonstrated. A single-protein investigation shows the existence of two distinct bright states that display sharp two-photon cross-section bands peaked at 780 nm and at 870 nm. Fluorescence of these two species can be independently switched on and off. These results highlight a new photoconversion pathway for photochromic GFPs and can have significant applications in multi-photon confocal microscopy and in optical data-storage architectures.

Abstract: We report on the construction details of a compact autocorrelator set-up for the measurement of the width of infrared laser pulses at the focal plane of a microscope for two-photon excitation fluorescence imaging. One of the novelties of the set-up, which leads to an improved measurement accuracy, is the use of a modulation technique that is achieved by mounting one of the interferometer mirrors on a loudspeaker driven by a sinusoidal bias at low frequency. A non-linear least-square routine selects only that part of the fluorescence signal that is modulated at the same frequency as the loudspeaker bias. To further increase the accuracy, the laser pulse width is obtained from a series of measurements at different values of the modulation bias. The autocorrelator is a compact single bread-board (10 x 20 cm); it is PC-controlled both for the acquisition and the analysis of the data and can be coupled to different ports of the microscope. The increase in the pulse width measured for three different ports of the microscope is well accounted for by the group velocity dispersion and the glass thickness of the optics found along these paths.

Abstract: Signal transduction pathways inactivated during periods of starvation are implicated in the regulation of longevity in organisms ranging from yeast to mammals, but the mechanisms responsible for life-span extension are poorly understood. Chronological life-span extension in S. cerevisiae cyr1 and sch9 mutants is mediated by the stress-resistance proteins Msn2/Msn4 and Rim15. Here we show that mitochondrial superoxide dismutase (Sod2) is required for survival extension in yeast. Deletion of SOD2 abolishes life-span extension in sch9Delta mutants and decreases survival in cyr1:mTn mutants. The overexpression of Sods--mitochondrial Sod2 and cytosolic CuZnSod (Sod1)--delays the age-dependent reversible inactivation of mitochondrial aconitase, a superoxide-sensitive enzyme, and extends survival by 30%. Deletion of the RAS2 gene, which functions upstream of CYR1, also doubles the mean life span by a mechanism that requires Msn2/4 and Sod2. These findings link mutations that extend chronological life span in S. cerevisiae to superoxide dismutases and suggest that the induction of other stress-resistance genes regulated by Msn2/4 and Rim15 is required for maximum longevity extension.

Abstract: We have studied the fluorescence emission by two-photon excitation of four dyes widely used for bioimaging studies, rhodamine 6G, fluorescein, pyrene and indo-1 at the single molecule level. The single dye molecules, spread on a glass substrate by spin coating, show a constant fluorescence output until a sudden transition to a dark state very close to the background. The bleaching time that is found to vary in the series pyrene, indo-1, fluorescein and rhodamine 6G from the fastest to the slowest one respectively, has a Gaussian distribution indicating that the observed behavior is not due to photobleaching. Moreover, the bleaching time decreases with the glass substrate temperature reaching a vanishing nonmeasurable value for a limiting temperature whose value is found in the same series as for the bleaching time, from the lowest to the highest temperature respectively. The observed bleaching shows a clear correlation to the amount of absorbed power not reirradiated as fluorescence and to the complexity of the molecule. These observations are interpreted as thermal bleaching where the temperature increase is induced by the two-photon absorption of the single dyes as confirmed also by numerical simulations.

Abstract:

Abstract: In Paramecium, internal Ca(2+) concentration increase coupled to membrane depolarization induces a reversal in the direction of ciliary beating and, consequently, a reversal in swimming direction. The ciliary reversal (CR) duration is correlated to Ca(2+) influx, and the addition of drugs that block the Ca(2+) current leads to a reduction in the backward swimming duration. In this study we have examined the possible function of GABA(B) receptors in P. primaurelia swimming control. The presence of GABA(B) immunoanalogue in Paramecium was evidenced using SDS-PAGE, Western blotting, and confocal laser scanning microscopy. By applying the specific GABA(B) receptor agonist baclofen, a dose-dependent inhibition of the membrane depolarization-induced CR duration was observed. This inhibition was antagonized by phaclofen, persisted when K(+) channel blockers were applied, and disappeared after treatment with nifedipine and verapamil. Moreover, the action of baclofen on depolarization-induced CR was suppressed by treatment with pertussis toxin. Therefore, these experiments suggest that baclofen modulates CR by a G protein (G(0) or G(1)) mediated inhibition of dihydropyridine-sensible calcium channels. Finally, synthesis and release of GABA in the environment by Paramecium have been demonstrated by HPLC. Possible correlations between GABA(B) receptor activation and the regulation of intracellular Ca(2+) levels are discussed.

Abstract: Single-molecule spectroscopy and single-molecule detection are emerging areas that have many applications when combined with scanning, imaging, and spectroscopy techniques. We have combined a commercial confocal scanning head, to a Ti:sapphire laser and to an inverted microscope, for the detection of single molecule fluorescence of varies dyes by two-photon excitation. We collected spot images of fluorescent molecules that have been deposited on a substrate considering both blinking and photobleaching behavior of fluorescent spots. Here, we report data related to two-photon interactions that occur with the following fluorescent molecules: Indo-1, Rhodamine 6G, Fluorescein, and Pyrene. The choice of these specific dyes is based upon their wide use in biological and medical applications together with the varying complexity of their chemical structure that increases from Pyrene to Indo-1. Moreover, we report some data about single molecule studies related to denaturation of an enhanced green fluorescent protein, GFPmut2, under one photon excitation regime, that show a very similar trend to that observed for the already mentioned fluorescent molecules.

Abstract: One of the most promising applications of encapsulated living cells is their use as protected transplanted tissue into the human body. A suitable system for the protection of living cells is the use of nano- or microcapsules of polyelectrolytes. These shells can be deposited easily on top of the cells by means of a layer-by-layer technique. An interesting feature of the capsules is the possibility to control their properties on a nanometre level, tuning their wall texture via the preparation conditions. Here we introduce a model system to test the protection ability of polyelectrolyte capsules. Common bakery yeast cells were encapsulated. They were coated with a fluorescently labelled shell at conditions known to guarantee cell survival, and the cell interior was stained with DAPI. The protozoan Paramecium primaurelia was incubated with this double-stained living yeast and visualized by means of two-photon excitation fluorescence microscopy. Cross-sections of the dye-stained material as well as autofluorescence of the fixed protozoan allowed us to follow the digestion of the coated yeast with time. Our investigation reveals that capsules prepared under these deposition conditions are permeable to lysosomal enzymes, leading to degradation of the yeast inside the intact capsules. Our preliminary results indicate the suitability of the introduced model as a test system of this permeability.

Abstract: Previously, we have identified aloe-emodin (AE) as a new type of anticancer agent, with activity that is based on apoptotic cell death promoted by a neuroectodermal tumor-specific drug uptake. We attempt to clarify the intracellular target of AE and the apoptosis-signaling pathway activated by AE in neuroblastoma cell lines. Two-photon excitation microscopy and spectroscopic titrations documented that AE is highly concentrated in susceptible cells and binds to DNA. One of the most important mediators of apoptotic response to genotoxic stimuli, such as anticancer agents, is the p53 tumor suppressor gene. To evaluate the role played by p53 in AE-induced apoptosis a p53 mutant cell line, which lacks transcriptional activity of p53 targeted genes, was tested. AE displayed a reduced growth inhibitory and pro-apoptotic activity in p53 mutant cells (SK-N-BE(2c)) with respect to the p53 wild-type line (SJ-N-KP). This effect was not caused by a reduced drug uptake in the mutant neuroblastoma cell line but was related to a different apoptotic cell phenotype. Whereas SJ-N-KP cells were susceptible to a p53 transcription-dependent pathway of apoptosis, SK-N-BE(2c) cells underwent apoptosis with up-regulation of p53 expression but not of p53-target genes. After AE treatment p53 translocates to the mitochondria inter-membrane space in both neuroblastoma cell lines. Due to its high accumulation in neuroectodermal tumor cells AE could also kill tumor cells harboring p53 mutant genes. This property would further contribute to AE specific anti-tumor activity and might be exploitable in the clinic.

Abstract: The effects of the refractive-index mismatch in confocal laser scanning microscopy were extensively studied. The axial aberration induced in the case of fluorescent microspheres was measured. The data were used to take into account the mismatch-induced aberrations and to consider object-size influence. Then we focused on the effect of refractive-index mismatch on the effective system's point-spread function under different mismatch conditions and on depth of focusing. We experimentally verified that the peak of the point-spread function intensity profile decreases and the point-spread function itself progressively broadens as a function of the combined effect of the refractive-index mismatch and of the penetration depth, leading to a worsening of the system's overall performances. We also performed these same measurements by embedding subresolution beads in an oocyte's cytoplasm, which can be considered a turbid medium. We found evidence consistent with the previously developed theoretical model; in particular we found a strong dependence of the intensity peak on the focusing depth.

Abstract: Single-molecule experiments are performed by investigating spectroscopic properties of molecules either diffusing in and out of the observation volume or fixed in space by different immobilization procedures. To evaluate the effect of immobilization methods on the structural and dynamic properties of proteins, a highly fluorescent mutant of the green fluorescent protein, GFPmut2, was spectroscopically characterized in bulk solutions, dispersed on etched glasses, and encapsulated in wet, nanoporous silica gels. The emission spectrum, the fluorescence lifetimes, the anisotropy, and the rotational correlation time of GFPmut2, encapsulated in silica gels, are very similar to those obtained in solution. This finding indicates that the gel matrix does not alter the protein conformation and dynamics. In contrast, the fluorescence lifetimes of GFPmut2 on glasses are two-to fourfold higher and the fluorescence anisotropy decays yield almost no phase shifts. This indicates that the interaction of the protein with the bare glass surface induces a significant structural perturbation and severely restricts the rotational motion. Single molecules of GFPmut2 on glasses or in silica gels, identified by confocal image analysis, show a significant stability to illumination with bleaching times of the order of 90 and 60 sec, respectively. Overall, these data indicate that silica gels represent an ideal matrix for following biologically relevant events at a single molecule level.

Abstract: A series of evidences suggests that enhanced susceptibility to programmed cell death (PCD) is a major pathogenetic factor in Alzheimer's disease (AD). We investigated this issue, analyzing amyloid beta-protein (A beta) production in a model of neuronal PCD, induced by staurosporine in a murine neuroblastoma cell line. When PCD was induced, a 280-290% secreted A beta occurred, in spite of a 20% metabolism and an unchanged A betaPP expression. The increased intracellular A beta reactivity largely colocalized with a marker of ER. Inhibition of caspases blocked the cleavage at the C-terminus of beta PP, but only partially rescued A beta overproduction caused by staurosporine treatment. Our findings suggest that PCD fosters the physiological pathways of A beta production characteristic of neuronal cells, and they confirm the theory that unbalance of PCD is a central event in AD pathogenesis. Moreover, our data indicate that still unidentified cellular mechanisms, other than caspases activation, are responsible of the specific alteration of A betaPP processing during PCD.

Abstract: Confocal laser scanning microscopy (CLSM) was used to examine molecules related to the cholinergic neurotransmission system and detected at all the larval stages of Paracentrotus lividus, by histochemical and immunohistochemical methods. CLSM, providing spatial resolution of the cells located both at the larval surface and at depth, allows a complete mapping in a three-dimensional volumetric frame. At early larval stages acetylcholinesterase- as well as choline acetyltransferase-like molecules were found mainly in the gut wall cells, and along the ciliary bands of the arms, together with muscarinic acetylcholine receptors. At perimetamorphic stages, cholinergic molecules were present in the ciliate strands along the arms, in the larval body and in the rudiment. At metamorphosis, positivity to cholinergic molecules translocated to the juvenile, where a high frequency of mAChR- and ChAT-like positive cells was found.

Abstract: Using quantitative immunoelectron microscopy we show here that when the nuclear matrix is isolated from rat hepatocytes in the presence of an inhibitor of RNase activity both lamins and the nuclear mitotic apparatus protein (NuMA) preferentially localize within the electron-dense domains of the internal nuclear matrix (INM). After RNA digestion NuMA undergoes a sharp depletion, while labeling by an antibody against lamins A and C within the electron-transparent regions increases, suggesting that a subset of lamin epitopes is masked by the interaction with RNA. We were able to explain this result by visualizing for the first time a thin web of lamin protofibrils which connects the electron-dense regions. Confirmation of these changes has been obtained by immunoblot analysis and confocal microscopy. As RNA digestion results both in the release of NuMA and in the collapse of the INM, we propose that a fraction of nuclear RNA brings about the association of NuMA islands with a lamin scaffold and that this interaction is required to maintain the latter in a state of high molecular dispersion.

Abstract: Nanocapsules, fuzzy assemblies of polyelectrolyte, represent a comparatively new class of colloids with controlled nanostructure and tunable properties. Due to the fact that the core as well as the dissolution influences the wall texture and the properties of the hollow capsules, the use of carbonate crystals as template is most convenient. Yeast cells constitute as a core candidate as well. They are a good system for testing the protective ability of shells and the permeability of the walls with respect to the needs of biological systems, namely, feeding and stability against attacks. The main features of the nanocapsules have been studied by two-photon, confocal, and atomic force microscopy. Nanocapsules are of biomedical interest because they can be used, for example, for the controlled release and targeting of drugs as well as for the protection of enzymes, proteins, and foreign cells.

Abstract: Three-dimensional confocal laser scanning microscopy (CLSM) was used as an essential investigation method to obtain information about the formation and morphological characteristics of nanocapsules. Nanocapsules are built by layer-by-layer deposition of alternatively charged polyelectrolytes on templates forming nanostructured hollow shells. CLSM is unique in allowing for monitoring of the core dissolution process in real time and for studying nanocapsule functioning in hydrated conditions within a three-dimensional and temporal framework. Since we are also interested in the identification of other possible templates, we briefly report on the use of yeast cells as biocolloidal cores monitored by means of two-photon microscopy. Here we focus our attention on the use of CdCO(3) crystals as template candidates for the preparation of stable capsules. Both cubic and spherical CdCO(3) cores have been produced. Cubic cores exhibit higher monodispersity and smaller size compared to spherical ones. Capsules templated on these cores have a higher surface-to-volume ratio that is valuable for applications related to drug delivery, functional properties of the shells and adsorption of proteins, and other biologically relevant molecules. Microsc. Res. Tech. 59:536-541, 2002.

Abstract: In Paramecium primaurelia the uptake and intracellular flow of cholesteryl ester was studied by fluorescence confocal laser scanning optical microscopy and by the fluorescent analogue cholesteryl-BODIPY FL C12 (BODIPY-CE). The BODIPY FL fluorophore has the characteristic of emitting green fluorescence, which is red-shifted as the probe concentrates. In cells incubated with 25 microm BODIPY-CE for 30 s, fluorescence is found in vesicles located around the cytopharynx in the posterior half of the cell. Successively, the lipid is internalized by food vacuoles, the fluorescent vesicles are distributed throughout the cell and the intracellular membranes are labelled. The food vacuole number is maximum after 10-15 min of continuous labelling, then it decreases until no food vacuoles are found in 30-min fed cells. BODIPY-CE accumulates in red-labelled cytoplasmic droplets located in the anterior half of the cell. When food vacuole formation is inhibited by trifluoperazine, fluorescence is found on cellular membranes and in small green-labelled vesicles at the apical pole. The inhibition of clathrin-mediated endocytosis does not interfere in P. primaurelia with BODIPY-CE intracellular flow: intracellular membranes and storage droplets in the cell anterior part are dyed. Conversely, the use of sterol-binding drugs prevents the lipid accumulation in droplets, stopping the lipid within the cytoplasmic membranes. Furthermore, the cells treated with monensin and cytochalasin B show a labelling of the cellular membranes and lipid droplets, whereas NH4Cl reduces the lipid storage. Low temperature (4 degrees C) does not prevent the internalization of BODIPY-CE that, however, is localized at the cytoplasmic membrane level and does not accumulate in storage droplets. In addition, BODIPY-CE inhibits phagocytosis, as evidenced by comparing the kinetics of food vacuole formation of control cells, only fed with latex particles, with that of cells fed with latex particles and BODIPY-CE. In conclusion, this study points out that in P. primaurelia the cholesteryl ester enters the cell via food vacuoles and through the plasma membrane and, inside the cell, it alters cell functions.

Abstract: We have implemented a simple program to solve three of the problems related to 3D reconstruction (3D-Rec) of soft tissues: alignment of sections, distortions, and estimation of the spatial position of elements of interest inside the tissues. As a model, we chose the distribution of FMRFamide-like immunopositive neurons in the ventral ganglion of the barnacle Balanus amphitrite collected during different seasonal periods. Images of immunostained sections were acquired by means of a CCD-camera-equipped microscope and a PC and the reference points were taken inside the sections. The FMRFamide-like immunopositive neurons detected in the barnacle ventral ganglion were grouped into four different classes according to size, shape and staining intensity. More numerous FMRFamide-like immunopositive neurons were detected in the autumn-collected barnacle than in the summer counterpart. The two 3D reconstructions obtained from transverse and longitudinal ventral ganglion sections were efficaciously compared after 90 degrees rotation of one of them. Comparison of these two 3D-Rec suggests the presence of at least two groups of FMRFamide-like immunopositive neurons that are seasonally-related and probably involved in reproduction.

Abstract: We have combined a confocal laser scanning head modified for TPE (two-photon excitation) microscopy with some spectroscopic modules to study single molecules and molecular aggregates. The behavior of the TPE microscope unit has been characterized by means of point spread function measurements and of the demonstration of its micropatterning abilities. One-photon and two-photon mode can be simply accomplished by switching from a mono-mode optical fiber (one-photon) coupled to conventional laser sources to an optical module that allows IR laser beam (two-photon/TPE) delivery to the confocal laser scanning head. We have then described the characterization of the two-photon microscope for spectroscopic applications: fluorescence correlation, lifetime and fluorescence polarization anisotropy measurements. We describe the measurement of the response of the two-photon microscope to the light polarization and discuss fluorescence polarization anisotropy measurements on Rhodamine 6G as a function of the viscosity and on a globular protein, the Beta-lactoglobulin B labeled with Alexa 532 at very high dilutions. The average rotational and translational diffusion coefficients measured with fluorescence polarization anisotropy and fluorescence correlation methods are in good agreement with the protein size, therefore validating the use of the microscope for two-photon spectroscopy on biomolecules.

Abstract: The expression of GABA(A) receptors in Xenopus oocytes injected with rat brain mRNA was studied by immunocytochemistry and evaluation of the distribution of fluorescent probes at the confocal microscope. The beta(2/3) subunit distributed exclusively on the membrane at the animal pole of the oocytes. Treatment of oocytes for 20 min with the protein tyrosine kinase inhibitor genistein, 200 microM, resulted in a lower presence of GABA(A) receptors on the membrane. The inactive genistein analogue daidzein, 200 microM, had no effect even with a 30 min treatment. Alkaline phosphatase but not a protein tyrosine phosphatase, when injected into oocytes, reduced GABA(A) receptor membrane expression. The data indicate that protein tyrosine phosphorylation modulates the expression on the plasma membrane of presynthesized GABA(A) receptors.

Abstract: In ciliated protozoa, most nutrients are internalized via phagocytosis by food vacuole formation at the posterior end of the buccal cavity. The uptake of small-sized molecules and external fluid through the plasma membrane is a localized process. That is because most of the cell surface is internally covered by an alveolar system and a fibrous epiplasm, so that only defined areas of the cell surface are potential substance uptake sites. The purpose of this study is to analyze, by fluorescence confocal laser scanning microscopy, the relationship between WGA (Triticum vulgaris agglutinin) and dextran internalization in Paramecium primaurelia cells blocked in the phagocytic process, so that markers could not be internalized via food vacuole formation. WGA, which binds to surface constituents of fixed and living cells, was used as a marker for membrane transport and dextran as a marker for fluid phase endocytosis. After 3 min incubation, WGA-FITC is found on plasma membrane and cilia, and successively within small cytoplasmic vesicles. After a 10-15 min chase in unlabeled medium, the marked vesicles decrease in number, increase in size and fuse with food vacuoles. This fusion was evidenced by labeling food vacuoles with BSA-Texas red. Dextran enters the cell via endocytic vesicles which first localize in the cortical region, under the plasma membrane, and then migrate in the cytoplasm and fuse with other endocytic vesicles and food vacuoles. When cells are fed with WGA-FITC and dextran-Texas red at the same time, two differently labeled vesicle populations are found. Cytosol acidification and incubation in sucrose medium or in chlorpromazine showed that WGA is internalized via clathrin vesicles, whereas fluid phase endocytosis is a clathrin-independent process.

Abstract: We have characterized a commercial confocal scanning head for the detection of single molecule fluorescence by two-photon excitation. We have verified that the distribution of the fluorescence emitted by dyes and labeled proteins on glass substrates is discrete with quanta proportional to a common reference signal. We describe and test a simple and quantitative tool to discriminate between single molecules and molecular aggregates on single snapshots based on the analysis of the intensity distribution. We have verified the square dependence of the fluorescence intensity vs. the excitation power, suggesting that no appreciable saturation and fast photo-damage of the chromophores takes place at the excitation power employed here.

Abstract: The fluorochrome 3,3'-dihexyloxacarbocyanine iodide [DiOC6(3)], a vital dye utilized to stain the endoplasmic reticulum (ER) of animal and plant cells, has been used to visualize the ER-type structures of Paramecium primaurelia under confocal laser scanning microscopy (CLSM). The morphology of the ER has been studied in paramecia in different physiological conditions. Cells are analysed in early and late logarithmic growth phases, in stationary and in death phases, during shift-up by refeeding after starvation and shift-down by using a starvation medium. In log-phase growing paramecia, the ER constitutes an anastomosing membrane system consisting of short tubules and flattened sacs forming a peripheral network, which is abundant in the cortical region around the trichocysts and the ciliary basal bodies. The tubular network and cytoplasmic membranes are reduced in stationary-phase cells; the original conditions are restored in starved cells after refeeding. The analysis of serial optical sections collected by CLSM at 0.5 microm intervals and three-dimensional reconstruction from these sections allow us to visualize differences between differently growing cells.

Abstract: Here we report that aloe-emodin (AE), a hydroxyanthraquinone present in Aloe vera leaves, has a specific in vitro and in vivo antineuroectodermal tumor activity. The growth of human neuroectodermal tumors is inhibited in mice with severe combined immunodeficiency without any appreciable toxic effects on the animals. The compound does not inhibit the proliferation of normal fibroblasts nor that of hemopoietic progenitor cells. The cytotoxicity mechanism consists of the induction of apoptosis, whereas the selectivity against neuroectodermal tumor cells is founded on a specific energy-dependent pathway of drug incorporation. Taking into account its unique cytotoxicity profile and mode of action, AE might represent a conceptually new lead antitumor drug.

Abstract: Techniques based on two-photon excitation (TPE) allow three-dimensional (3D) imaging in highly localized volumes, of the order of magnitude of a fraction of a femtolitre up to single-molecule detection. In TPE microscopy a fundamental advantage over conventional widefield or confocal 3D fluorescence microscopy is given by the use of infrared (IR) instead of ultraviolet (UV) radiation to excite those fluorophores requiring UV excitation, hence causing little damage to the specimen or to fluorescent molecules outside the volume of the TPE event and allowing a deeper penetration within the sample compared with conventional one-photon excitation of fluorescence. In our laboratory, within the framework of a national INFM project, we have realized a TPE fluorescence microscope, part of a multipurpose architecture also including lifetime imaging and fluorescence correlation spectroscopy modules. The core of the architecture is a mode-locked Ti:sapphire infrared pulsed laser pumped by a high-power (5 W, 532 nm) solid-state laser and coupled to an ultracompact scanning head. For the source we have measured a pulse width from 65 to 95 fs as a function of wavelength (690-830 nm). The scanning head allows conventional and two-photon confocal imaging. Point spread function measurements are reported with examples of applications to the study of biological systems.

Abstract: Sparse fluorescent pointlike subresolution objects have been imaged using a diffraction limited single-pinhole confocal fluorescence microscope. A Maximum likelihood image restoration algorithm has been used in conjunction with a measure of the experimental point spread function for improving the three-dimensional imaging of subresolution sparse objects. The experimental point-spread-function profiles have been improved by a factor of 1.95 in lateral direction and 3.75 in axial direction resulting in full-width half maximum (FWHM) values of 91 +/- 11 nm and 160 +/- 26 nm. This amounts to 1. 43 and 2.15 in optical units, respectively. The lateral and axial FWHM of the sparse pointlike subresolution objects is about 5 and 3 times smaller than the wavelength. This result points to the attractive possibility of utilising a compact confocal architecture for localising punctuate fluorescent objects having subresolution dimensions. The key resides in the utilisation of the measured point spread function coupled to an appropriate image restoration approach, and, of course, in the stability of the confocal system being used.

Abstract:

Abstract:

Abstract: BACKGROUND: In Paramecium primaurelia, an exconjugant cell can produce two lines with different mating capacities. Mating type II cells can form a higher food vacuole number and digest the nutrient taken up in a shorter time; thus, mating type II cells grow at a faster rate than do mating type I cells. The present study was done to determine whether cells that ingest more nutrients also have a larger amount of storage lipids. METHODS: Quantitative and qualitative determinations of neutral lipids were obtained by means of cytofluorometry and fluorescence confocal laser scanning microscopy (CLSM), respectively, by using nile red on cells in different physiologic states. RESULTS: Lipid droplet number and neutral lipid content were higher in mating type II cells than in mating type I cells in the early logarithmic growth phase (i.e., immature well-fed cells). These values were reversed during the middle and the late logarithmic phases and became equal in the stationary phase (i.e., mature starved cells). In well-fed cells maintained with food excess, differences in neutral lipid content between the two mating types also were present in mature cells. CONCLUSIONS: Although differences between mating type I and mating type II lines were not correlated to cell size, a relation was found between lipid content and food ingestion capacity. A depletion of bacteria in the culture medium could be responsible for the lack of differences in mature starved cells. CLSM allowed us to gather volume information about the lipid droplet distribution within the cell.

Abstract:

Abstract:

Abstract:

Abstract:

Abstract: Within the framework of a national National Institute of Physics of Matter (INFM) project, we have realised a two-photon excitation (TPE) fluorescence microscope based on a new generation commercial confocal scanning head. The core of the architecture is a mode-locked Ti:Sapphire laser (Tsunami 3960, Spectra Physics Inc., Mountain View, CA) pumped by a high-power (5 W, 532 nm) laser (Millennia V, Spectra Physics Inc.) and an ultracompact confocal scanning head, Nikon PCM2000 (Nikon Instruments, Florence, Italy) using a single-pinhole design. Three-dimensional point-spread function has been measured to define spatial resolution performances. The TPE microscope has been used with a wide range of excitable fluorescent molecules (DAPI, Fura-2, Indo-1, DiOC(6)(3), fluoresceine, Texas red) covering a single photon spectral range from UV to green. An example is reported on 3D imaging of the helical structure of the sperm head of the Octopus Eledone cirrhosa labelled with an UV excitable dye, i.e., DAPI. The system can be easily switched for operating both in conventional and two-photon mode.

Abstract:

Abstract:

Abstract:

Abstract:

Abstract: Trichocysts are secretory organelles located at the surface of several ciliates, docked at the plasma membrane. Their secretion is similar to other exocytic processes: the trichocyst membrane fuses with the plasma membrane, its content is released outside the cell, and the membrane is retrieved back into the cell. The fate of the trichocyst membrane in living Paramecium primaurelia was investigated by inducing massive synchronous exocytosis in the presence of fluorescein isothiocyanate-conjugated lectins or of cationized ferritin. The marker is trapped within the retrieved trichocyst membrane sac, and many regularly spaced, fluorescent ghosts are formed. As time proceeds, the number of labeled ghosts decreases, and few fluorescent vacuoles appear within the cell. The relationship between trichocyst ghosts and the vesicles of the phagosome-lysosome system was examined by labeling cells with Texas Red-conjugated bovine serum albumin, a fluorescent marker for phagocytosis. Starting from two confocal images of the same cell labeled with the two fluorescent probes, a new single image was generated by associating each image with a different red or green value. This multimodal analysis showed that trichocyst ghosts fuse with secondary lysosomes or are incorporated into digestive vacuoles. The vacuolar content is degraded and fluorescence is then found in the vesicles of the phagosome-lysosome system, and, at last, in small weakly labeled vesicles located on the cell surface.

Abstract: We have used a confocal laser scanning optical microscope imaging device and a bioimage-oriented workstation equipped for augmented reality to study the helical sperm head of the octopus Eledone cirrhosa. This approach allows us to study different complex organisational motifs due to the spatial arrangement of linear helical structures. We consider this helical specimen an enlarged copy of one of the most important biostructures governing cell functioning such as chromatin-DNA. Moreover, this very same sample is made of highly compacted chromatin that can be studied at higher resolution, i.e., by means of scanning force microscopy. Fluorescence optical sectioning has been used to enter the spatial organisation. Three-dimensional images of single, twisted, and folded fibers are shown.

Abstract:

Abstract: Two common fixatives--glutaraldehyde and ethanol/acetic acid mixture--were studied to understand their effects on DNA distribution inside the cell nucleus. Native calf thymocytes were analyzed by using a DNA selective fluorescent dye (DAPI) and computational optical sectioning microscopy on isolated cells before and after fixation. In order to estimate quantitatively the intranuclear DNA distribution, the stained calf thymocytes images were processed by removing the out-of-focus contributions present in each optical section. Although preliminary, the results show that within individual nuclei the frequency distribution of the fluorescence intensity appears significantly and differentially altered by the two fixatives. Namely with respect to the native unfixed preparation, the ethanol/acetic acid causes the complete disappearance of the higher intensity pixels, whereas glutaraldehyde fixation can be associated with the appearance of new ones of an even higher intensity. The quantitative analysis of the processed images allowed us to reconstruct the topological distribution of DNA inside the nucleus and to correlate the data with the results obtained by differential scanning calorimetry on similar samples.

Abstract: Vital fluorescent dyes (FITC-albumin, Texas Red-albumin, and acridine orange) were used together with a confocal laser scanning optical microscope (CLSM) to display and analyze formation, movement, and fusion of vesicles during the phagocytosis of Paramecium primaurelia, in the x-y-z-t space. By immobilizing living cells pulsed with a food vacuole marker at successive times after chasing in unlabeled medium, the intracellular movement of food vacuoles from their formation at the cytostome to their egestion at the cytoproct was visualized, and food vacuoles were selected in a specific digestion stage. Small pinocytic vesicles are shown to evaginate from the vacuoles and move in the cytoplasm. These vesicles are transported toward the cytopharynx where they enlarge the membrane of the nascent food vacuoles or fuse with stage II food vacuoles, when the vacuoles of stage II increase their size, changing from an acidic to an alkaline status. A multimodal analysis of confocal fluorescence images and the false-color technique were used to visualize vesicle movement vs. time. Starting from three images of the same cell at succeeding time points, a composite image was generated by associating with each originally acquired image a different color corresponding to each sampling point in time. The composite image shows that vesicles move away from the food vacuole in a scattered manner exhibiting changes in direction.

Abstract: We outline a method which uses differentially polarized light scattering to study the properties of bacterial cell suspensions, i.e., spores of Bacillus Subtilis. The identification of bacterial cells of different strains, with and without plasmid insertion, was performed by means of differential polarization light scattering (DPLS). The samples displayed distinct angular behaviors for the S14 and S34 normalized scattering parameters of the Mueller matrix depending on the strain and on a plasmid insertion in the chromosomal unit. These experiments, performed blindly, point out the possibility of achieving real time identification of micro-organisms by DPLS spectrometry.

Abstract: Synchronized CHO-K1 cells and their dibutyryl c-AMP treated counterparts have been characterized by means of static and flow fluorescence cytometry at the level of nuclear DNA and cytoplasmic microtubules. In order to confirm earlier findings on synchronized population, Carnoy fixed and hydrolyzed, several new findings are here reported at the level of single intact cell. The fluorescence intensity of DAPI-stained glutaraldehyde fixed 2C cells correlates well with the average absorbance of the corresponding Feulgen-stained cells, thereby appearing also to be a measure of chromatin condensation during the G1 phase. In the early part of G1, the drastic alteration in anti-beta tubulin immunostaining is shown to parallel microtubule depolymerization induced by calcium or colcemide. The known 1-2 h lengthening of the G1 period after reverse-transformation appears to correlate with a similar delay in the abrupt chromatin decondensation. The above results are discussed in terms of the role of microtubules and nuclear morphometry (and their coupling) in the control of cell cycle progression of transformed vs. fibroblast-like cells.

Abstract: Nucleosomes, chromatin and nuclei, extracted from rat hepatocytes, are studied by a new "in house" experimental configuration which measures circular intensity differential scattering (CIDS) and other elements of the polarized light scattering matrix. The Mueller matrix elements, S14 and S34, that are related to the geometric parameters of the superhelical arrangement of polynucleosomes point to the existence of a quaternary structure at low ionic strength for chromatin prepared by the cold-water method, which is lost by shearing, and is not found in the soluble chromatin prepared through the nuclease method. Only salt addition to a final concentration of 5 mM MgCl2, 150 mM NaCl and 10 mM Tris HCl (pH 7) yields a sizeable (S14 + S34) signal in the latter chromatin, which is however still different from the corresponding signal of native nuclei and of "cold-water" chromatin. Comfortingly, the (S14 + S34) signal from isolated nucleosomes is consistently very low (nearly zero) as predicted by multiple dipole simulation within the framework of classical electrodynamics. Results are discussed in terms of the topological constraints present in the native long chromatin fiber, which are lost after limited nuclease digestion and after shearing.

Abstract: Linear and circular lambda-DNA at different ethidium bromide concentrations have been studied by means of polarized light scattering, namely the S14, S34, S33 and S13 elements of Mueller matrix. While S33 at low angle appears well correlated with the total light scattering evaluated by optical density measurements at 632.8 nm for linear and circular DNA of the same mass, the magnitude and slope of the S14, S34 and S13 signals display significant changes for the circular lambda-DNA depending on the degree of negative superhelical density as induced by the different ethidium bromide concentrations. At the same time, for linear lambda-DNA the signal remains invariant, making explicit for the differential scattering of polarized light the possibility to obtain additional information by its angular dependence. Strikingly also the effect of 0.2% glutaraldehyde versus ethanol fixation on the native lambda-DNA structural properties appears to confirm earlier findings by other well-established probes. Results are discussed in terms of first physical principles and of their potential bearings towards our understanding of the mechanism controlling gene expression.

Abstract: This work describes IMAGO, an integrated bio-imaging system developed in our laboratory. The whole system consists of a personal computer, a commercially available frame grabber directly plugged into a personal computer, video input/output modules, specific hardware for z-axis movement and light shuttering, and a software package. IMAGO is user-friendly, menu driven and enables one to perform image acquisition with different methods: optical sectioning, flashing epifluorescence, transmitted and phase contrast microscopy. It makes various functions possible, including: image transfer, gray scale processing, conventional and advanced filtering, logical operations, look-up table management, three-dimensional (3D) editing, 3D representation and auto-correlation techniques. More than 100 image processing functions have been implemented and can be easily managed through IMAGO. Examples are given in the area of biophysical research, like 3D representation of nuclei and of electron microscopic images, in situ microscopy of living cells. IMAGO processes information in an x, y, z, t space.

Abstract: In this work, we describe a software package, MUCIDS, completely developed in our laboratory, for acquisition and processing of differential polarization light-scattering data from specimens of biophysical interest. MUCIDS is a C environment that manages the whole activity of an instrument used for measurements of Mueller matrix scattering elements. It allows one to capture, analyse, process and display data from this or from other similar light-scattering experiments. The entire system is suitable for routine measurements in a general biophysical (or microbiological) laboratory because of its easy handling and maintenance. The software was written in C lattice and will run on IBM personal computers and similar. It uses IBM/DAC and GPIB/IBM interface cards.

Abstract: The properties of an optical microscope are analyzed and analytically evaluated with a simple and effective model in order to understand the true meaning, limitations, and real capabilities of a defocusing technique. Major emphasis is given to the applications related to microscopic objects of biological interest using fluorescence and absorption light microscopy. A procedure for three-dimensional viewing is analyzed and discussed.

Abstract: In this paper we describe a computerized system for data recovery from differential scanning calorimetry of mammalian cells and their biopolymers. The 'in-house'-designed preamplifier, digital acquisition, control card and its real-time software provide us with a powerful workstation to acquire and analyze large quantities of data on-line and off-line. The final data are obtained after eliminating noise interference using both hardware and software filters. Fourier analysis is also performed for a more refined thermodynamic characterization. All software is written in Basic and Fortran 77 under the DOS 3.10 operating system on a personal computer.

Abstract: G0, G1, and mammalian cells and nuclei were shortly digested with either micrococcal nuclease or DNAse I, both before and after mild fixation, either before (G0) or after (G1) partial hepatectomy. Cells were Feulgen stained and examined by high resolution light microscopy. In metabolically active G1 nuclei, intranuclear DNA appears organized at least in two distinct domains, whereby the highly dispersed one is large enough to be detected at the resolution of the light microscope and appears preferentially attacked by limited DNAse I digestion. The action of the enzyme is readily apparent only in the nuclei that are first digested and then fixed. Spectroscopic characterization of the same nuclei reveals that the fixation causes a sizeable removal of proteins, mostly in the soluble chromatin subfraction. Results are discussed in terms of two control levels for gene expression and for higher order DNA structure.

Abstract: Differential scanning calorimetry, gel electrophoresis and polarized light scattering of chromatin prepared by different methods have been carried out at low and high ionic strength, before and after shearing. These noninvasive studies, when compared to the ones similarly conducted in the corresponding native nuclei, conclusively point to the artefactual nature of chromatin prepared by limited nuclease digestion, which has no resemblence with the in situ chromatin-DNA structure being instead preserved by lysis of native nuclei and by subsequent sedimentation and suspension of the viscous chromatin mass. Native nucleofilaments appear longer than 200 nucleosomes and yield, from thermodynamic and optical standpoints, a tight quaternary structure maintained even at 0.01 M.

Abstract: Circular intensity differential scattering (CIDS) has been proven a powerful method in determining the higher-order structure of large biopolymers, such as chromatin. Theoretical predictions of the expected differential light scattering of circularly polarized light have previously been made for chromatin, either within the Born approximation, treating nucleosomes as noninteracting, oblate ellipsoids, or within a multiple dipole approximation, treating nucleosomes as interacting spheres. In order to conduct a meaningful interpretation of the CIDS signal in terms of given geometric parameters of the chiral structure, we have in this paper combined the two approaches considering the mutual interactions of ellipsoidal nucleosomes. In the process we have also found a confirmation for the validity of the Born approximation itself.