Here, we report sub-diffraction resolution in two-photon excitation (TPE) fluorescence microscopy... more Here, we report sub-diffraction resolution in two-photon excitation (TPE) fluorescence microscopy achieved by merging this technique with continuous-wave (CW) stimulated-emission depletion (STED). We show an easy-to- implement and promising laser combination based on Ti:Sapphire ultrafast laser source for two-photon excitation and a commercial Leica TCS STED-CW microscope for resolution enhancement. Images of fluorescent nanoparticles produce comparative similar resolution to the one photon excitation. Two-photon excitation STED microscopy achieves approximately 3-4 fold improvement in resolution in the radial direction over conventional 2PELSM. Further improvements in resolution are theoretically achievable, suggesting that 2PE STED microscopy will permit nanoscale imaging, for instance, of neuronal structures located in relatively intact brain tissue.
Non-linear interactions between light and matter have been extensively used for spectroscopic ana... more Non-linear interactions between light and matter have been extensively used for spectroscopic analysis of biological, natural and synthetic samples [1–5]. In the 1990s, the development in laser technology allowed to apply these principles to the light microscopy field [6–8]. In this context multi-photon excitation (MPE) fluorescence microscopy and second harmonic generation (SHG) imaging are representative of the continuing growth of interest in optical microscopy. Although other modern imaging techniques like scanning near-field microscopy [9], scanning probe microscopy [10] or electron microscopy [11] provide higher spatial resolution, light microscopy techniques have unique characteristics for the three-dimensional (3D) investigation of biological structures in hydrated states, including the direct observation of living samples [12–14]. Multi-photon microscopy relies on the property of fluorescent molecules to simultaneously absorb two or more photons [15]. In this context, the advances in fluorescence labelling and the development of new fluorescent/luminescent probes like the so-called quantum dots [16] and the visible fluorescent proteins (VFPs), that can be expressed permanently bound to proteins of interest by genetically modified cells, allow the study of the complex and delicate relationships existing between structure and function in the four-dimensions (x-y-z-t) biological systems domain [17,18]. MPE shares with the confocal microscopy the intrinsical 3D exploration capability and provides some additional interesting features. First, MPE greatly reduces photo-interactions and permits to image living samples for long time periods. Second, it allows high sensitivity measurements due to the low background signal. Third, since most of the fluorescent molecules show a wide two-photon absorption spectrum, MPE allows simultaneous excitation of multiple fluorescent molecules with only one excitation wavelength, reducing the effects of chromatic abberations of the optical path. Fourth, two-photon microscopy can penetrate into thick and turbid media up to a depth of some 100 μm. Fifth, MPE can induce chemical rearrangement and photochemical reactions within a sub-femtoliter volume in solutions, culture cells and living tissues.
The ontogenetic cycle of the barnacle Balanus amphitrite (=Amphibalanus amphitrite) (Darwin, 1854... more The ontogenetic cycle of the barnacle Balanus amphitrite (=Amphibalanus amphitrite) (Darwin, 1854) includes a cyprid that binds submerged surfaces, metamorphosing into a sessile adult. γ-Aminobutyric acid (GABA) and GABA receptors have recently been located in its cyprid with a similar pattern to other crustaceans. Since NMDA R1 ionotropic glutamatergic receptors have been identified in crustacean neuromuscular junctions, we have investigated their presence in the B. amphitrite cyprid. The presence of NMDA R1 receptors might indicate a role for glutamate in neuromuscular control in B. amphitrite cyprids, therefore we studied the presence and distribution of the NMDA R1 by immunohistochemistry. Its distribution was observed in the peripheral nervous system and in non-neuronal elements. Actually, NMDA R1 immunoreactivity was detected in thoracic appendages, at the level of neuromuscular junctions, thus suggesting an involvement in motor control functions, as already demonstrated in other crustaceans. Immunoreactivity was also detected in ommatidia cells of the eye, in antennules, and in epidermal cells. The distribution pattern comparable to that of GABAergic molecules could indicate an interrelated agonistic/antagonistic role for these two systems, which could be considered as potential targets of combined antifouling strategies.▶ Glutamate receptor immunoreactivity was observed in neuromuscular junctions. ▶ Immunoreactivity was observed in ommatidia, epithelial cells and antennules. ▶ Glutamate could play a neuromuscular, sensory or non-neural role. ▶ Comparable glutamate/GABA receptors pattern distribution suggested their coordinated function.
We show that after two-photon excitation of the photo initiator we are able to inhibit the polyme... more We show that after two-photon excitation of the photo initiator we are able to inhibit the polymerization process with a second beam of different wavelength. For achieving sub-diffraction resolution this is one of the key elements. Remarkably is that these experiments can be performed with a commercially available STED microscope slightly modified for two-photon-excitation (TPE). First experiments featuring an enhanced resolution will be presented.
The application fluorescence to confocal and two-photon excitation (2PE) optical microscopy has l... more The application fluorescence to confocal and two-photon excitation (2PE) optical microscopy has led to terrific advances in the study of biological systems from the three-dimensional (3D) micro-spectroscopic level down to single molecule detection (SMD) schemes. Both techniques are particularly relevant for the study of the 3D and dynamic properties of biological molecules within their natural environment, cells or tissues. In particular the advent of 2PE mitigates overall photobleaching and phototoxicity problems, opening new perspectives by providing further attractive advantages. Optical schemes and architectures for confocal and two-photon excitation from microscopic level to SMD will be discussed. Examples of three-dimensional and multiple fluorescence imaging from cells to single fluorescent molecules will be given. Examples in the utilization of confocal and 2PE for specific GFP switching at single molecule level and for monitoring of TPE uncaging will be shown.
Accumulating evidence indicate that the neuropeptide urotensin II and urotensin II receptors are ... more Accumulating evidence indicate that the neuropeptide urotensin II and urotensin II receptors are expressed in subsets of mammal spinal motoneurons. In fact, a role for the peptide in the regulation of motoneuron function at neuromuscular junction has been suggested, while roles for urotensin II at central synapses in spinal cord have never been addressed. We found that urotensin II receptors were closely associated with cholinergic terminals apposed to a subset of motoneuron and non-motoneuron cell bodies in the ventral horn of the adult mouse cervical spinal cord; urotensin II receptor was also expressed on non-cholinergic nerve terminals. In particular, urotensin II receptor appeared associated with both large cholinergic C-boutons and standard cholinergic terminals contacting some motoneuron perikarya. Cholinergic nerve terminals from mouse cervical spinal cord were equipped with functional presynaptic urotensin II receptors linked to excitation of acetylcholine release. In fact, functional experiments conducted on cervical spinal synaptosomes demonstrated a urotensin II evoked calcium-dependent increase in [3H]acetylcholine release pharmacologically verified as consistent with activation of urotensin II receptors. In spinal cord these actions would facilitate cholinergic transmission. These data indicate that, in addition to its role at the neuromuscular junction, urotensin II may control motor function through the modulation of motoneuron activity within the spinal cord.
Layer-by-Layer or self-assembly techniques can be used to prepare Fluorescent polymer samples on ... more Layer-by-Layer or self-assembly techniques can be used to prepare Fluorescent polymer samples on glass coverslips serving as benchmark for two-photon excitation microscopy from conventional to 4Pi set-up, or more in general for sectioning microscopy. Layers can be realized as ultra-thin (<< 100 nm) or thin (approx. 100 nm) characteristics coupled to different fluorescent molecules to be used for different microscopy applications. As well, stacks hosting different fluorescent molecules can be also produce. Thanks to their controllable thickness, uniformity and fluorescence properties, these polymer layers may serve as a simple and applicable standard to directly measure the z-response of different scanning optical microscopes. In two-photon excitation microscopy z-sectioning plays a central role and uniformity of illumination is crucial due to the non-linear behaviour of emission. Since the main characteristics of a particular image formation situation can be efficiently summarized in a Sectioned Imaging property chart (SIPchart), we think that coupling this calibration sample with SIPchart is a very important step towards quantitative microscopy. In this work we use these polymer layers to measure the z-response of confocal, two-photon excitation and 4Pi laser scanning microscopes, selecting properly ultra-thin and thin layers. Due to their uniformity over a wide region, i.e. coverslip surface, it is possible to quantify the z-response of the system over a full field of view area. These samples are also useful for monitoring photobleaching behavior as function of the illumination intensity. Ultrathin layers are also useful to supersede the conventional technique of calculating the derivative of the axial edges of a thick fluorescent layer. Polymer layers can be effciently used for real time alignment of the microscope.
One of most promising applications of encapsulated living cells is as a protected transplanted ti... more One of most promising applications of encapsulated living cells is as a protected transplanted tissue into the human body. A method to induce capsules with tuneable properties is the Layer-by-Layer technique producing shells, named nanocapsules, by means of the adhesion of oppositely charged polyelectrolytes onto charged surfaces. Our investigation deals with cell encapsulation.
Here, we report sub-diffraction resolution in two-photon excitation (TPE) fluorescence microscopy... more Here, we report sub-diffraction resolution in two-photon excitation (TPE) fluorescence microscopy achieved by merging this technique with continuous-wave (CW) stimulated-emission depletion (STED). We show an easy-to- implement and promising laser combination based on Ti:Sapphire ultrafast laser source for two-photon excitation and a commercial Leica TCS STED-CW microscope for resolution enhancement. Images of fluorescent nanoparticles produce comparative similar resolution to the one photon excitation. Two-photon excitation STED microscopy achieves approximately 3-4 fold improvement in resolution in the radial direction over conventional 2PELSM. Further improvements in resolution are theoretically achievable, suggesting that 2PE STED microscopy will permit nanoscale imaging, for instance, of neuronal structures located in relatively intact brain tissue.
Non-linear interactions between light and matter have been extensively used for spectroscopic ana... more Non-linear interactions between light and matter have been extensively used for spectroscopic analysis of biological, natural and synthetic samples [1–5]. In the 1990s, the development in laser technology allowed to apply these principles to the light microscopy field [6–8]. In this context multi-photon excitation (MPE) fluorescence microscopy and second harmonic generation (SHG) imaging are representative of the continuing growth of interest in optical microscopy. Although other modern imaging techniques like scanning near-field microscopy [9], scanning probe microscopy [10] or electron microscopy [11] provide higher spatial resolution, light microscopy techniques have unique characteristics for the three-dimensional (3D) investigation of biological structures in hydrated states, including the direct observation of living samples [12–14]. Multi-photon microscopy relies on the property of fluorescent molecules to simultaneously absorb two or more photons [15]. In this context, the advances in fluorescence labelling and the development of new fluorescent/luminescent probes like the so-called quantum dots [16] and the visible fluorescent proteins (VFPs), that can be expressed permanently bound to proteins of interest by genetically modified cells, allow the study of the complex and delicate relationships existing between structure and function in the four-dimensions (x-y-z-t) biological systems domain [17,18]. MPE shares with the confocal microscopy the intrinsical 3D exploration capability and provides some additional interesting features. First, MPE greatly reduces photo-interactions and permits to image living samples for long time periods. Second, it allows high sensitivity measurements due to the low background signal. Third, since most of the fluorescent molecules show a wide two-photon absorption spectrum, MPE allows simultaneous excitation of multiple fluorescent molecules with only one excitation wavelength, reducing the effects of chromatic abberations of the optical path. Fourth, two-photon microscopy can penetrate into thick and turbid media up to a depth of some 100 μm. Fifth, MPE can induce chemical rearrangement and photochemical reactions within a sub-femtoliter volume in solutions, culture cells and living tissues.
The ontogenetic cycle of the barnacle Balanus amphitrite (=Amphibalanus amphitrite) (Darwin, 1854... more The ontogenetic cycle of the barnacle Balanus amphitrite (=Amphibalanus amphitrite) (Darwin, 1854) includes a cyprid that binds submerged surfaces, metamorphosing into a sessile adult. γ-Aminobutyric acid (GABA) and GABA receptors have recently been located in its cyprid with a similar pattern to other crustaceans. Since NMDA R1 ionotropic glutamatergic receptors have been identified in crustacean neuromuscular junctions, we have investigated their presence in the B. amphitrite cyprid. The presence of NMDA R1 receptors might indicate a role for glutamate in neuromuscular control in B. amphitrite cyprids, therefore we studied the presence and distribution of the NMDA R1 by immunohistochemistry. Its distribution was observed in the peripheral nervous system and in non-neuronal elements. Actually, NMDA R1 immunoreactivity was detected in thoracic appendages, at the level of neuromuscular junctions, thus suggesting an involvement in motor control functions, as already demonstrated in other crustaceans. Immunoreactivity was also detected in ommatidia cells of the eye, in antennules, and in epidermal cells. The distribution pattern comparable to that of GABAergic molecules could indicate an interrelated agonistic/antagonistic role for these two systems, which could be considered as potential targets of combined antifouling strategies.▶ Glutamate receptor immunoreactivity was observed in neuromuscular junctions. ▶ Immunoreactivity was observed in ommatidia, epithelial cells and antennules. ▶ Glutamate could play a neuromuscular, sensory or non-neural role. ▶ Comparable glutamate/GABA receptors pattern distribution suggested their coordinated function.
We show that after two-photon excitation of the photo initiator we are able to inhibit the polyme... more We show that after two-photon excitation of the photo initiator we are able to inhibit the polymerization process with a second beam of different wavelength. For achieving sub-diffraction resolution this is one of the key elements. Remarkably is that these experiments can be performed with a commercially available STED microscope slightly modified for two-photon-excitation (TPE). First experiments featuring an enhanced resolution will be presented.
The application fluorescence to confocal and two-photon excitation (2PE) optical microscopy has l... more The application fluorescence to confocal and two-photon excitation (2PE) optical microscopy has led to terrific advances in the study of biological systems from the three-dimensional (3D) micro-spectroscopic level down to single molecule detection (SMD) schemes. Both techniques are particularly relevant for the study of the 3D and dynamic properties of biological molecules within their natural environment, cells or tissues. In particular the advent of 2PE mitigates overall photobleaching and phototoxicity problems, opening new perspectives by providing further attractive advantages. Optical schemes and architectures for confocal and two-photon excitation from microscopic level to SMD will be discussed. Examples of three-dimensional and multiple fluorescence imaging from cells to single fluorescent molecules will be given. Examples in the utilization of confocal and 2PE for specific GFP switching at single molecule level and for monitoring of TPE uncaging will be shown.
Accumulating evidence indicate that the neuropeptide urotensin II and urotensin II receptors are ... more Accumulating evidence indicate that the neuropeptide urotensin II and urotensin II receptors are expressed in subsets of mammal spinal motoneurons. In fact, a role for the peptide in the regulation of motoneuron function at neuromuscular junction has been suggested, while roles for urotensin II at central synapses in spinal cord have never been addressed. We found that urotensin II receptors were closely associated with cholinergic terminals apposed to a subset of motoneuron and non-motoneuron cell bodies in the ventral horn of the adult mouse cervical spinal cord; urotensin II receptor was also expressed on non-cholinergic nerve terminals. In particular, urotensin II receptor appeared associated with both large cholinergic C-boutons and standard cholinergic terminals contacting some motoneuron perikarya. Cholinergic nerve terminals from mouse cervical spinal cord were equipped with functional presynaptic urotensin II receptors linked to excitation of acetylcholine release. In fact, functional experiments conducted on cervical spinal synaptosomes demonstrated a urotensin II evoked calcium-dependent increase in [3H]acetylcholine release pharmacologically verified as consistent with activation of urotensin II receptors. In spinal cord these actions would facilitate cholinergic transmission. These data indicate that, in addition to its role at the neuromuscular junction, urotensin II may control motor function through the modulation of motoneuron activity within the spinal cord.
Layer-by-Layer or self-assembly techniques can be used to prepare Fluorescent polymer samples on ... more Layer-by-Layer or self-assembly techniques can be used to prepare Fluorescent polymer samples on glass coverslips serving as benchmark for two-photon excitation microscopy from conventional to 4Pi set-up, or more in general for sectioning microscopy. Layers can be realized as ultra-thin (<< 100 nm) or thin (approx. 100 nm) characteristics coupled to different fluorescent molecules to be used for different microscopy applications. As well, stacks hosting different fluorescent molecules can be also produce. Thanks to their controllable thickness, uniformity and fluorescence properties, these polymer layers may serve as a simple and applicable standard to directly measure the z-response of different scanning optical microscopes. In two-photon excitation microscopy z-sectioning plays a central role and uniformity of illumination is crucial due to the non-linear behaviour of emission. Since the main characteristics of a particular image formation situation can be efficiently summarized in a Sectioned Imaging property chart (SIPchart), we think that coupling this calibration sample with SIPchart is a very important step towards quantitative microscopy. In this work we use these polymer layers to measure the z-response of confocal, two-photon excitation and 4Pi laser scanning microscopes, selecting properly ultra-thin and thin layers. Due to their uniformity over a wide region, i.e. coverslip surface, it is possible to quantify the z-response of the system over a full field of view area. These samples are also useful for monitoring photobleaching behavior as function of the illumination intensity. Ultrathin layers are also useful to supersede the conventional technique of calculating the derivative of the axial edges of a thick fluorescent layer. Polymer layers can be effciently used for real time alignment of the microscope.
One of most promising applications of encapsulated living cells is as a protected transplanted ti... more One of most promising applications of encapsulated living cells is as a protected transplanted tissue into the human body. A method to induce capsules with tuneable properties is the Layer-by-Layer technique producing shells, named nanocapsules, by means of the adhesion of oppositely charged polyelectrolytes onto charged surfaces. Our investigation deals with cell encapsulation.
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