Camilla Luccardini - Academia.edu (original) (raw)
Papers by Camilla Luccardini
Organic Letters, 2007
]) indicator dye (579/598 nm peak excitation/emission) with a side arm for conjugation via EDC or... more ]) indicator dye (579/598 nm peak excitation/emission) with a side arm for conjugation via EDC or click chemistry. Its large molar extinction and high quantum yield rank it among the brightest long-wavelength Ca 2+ indicators. Calcium Ruby is a promising alternative to existing dyes for imaging [Ca 2+ ] in multicolor fluorescence applications or in the presence of yellow-green cellular autofluorescence.
Frontiers in Cellular Neuroscience, 2015
Citation: Luccardini C, Leclech C, Viou L, Rio J-P and Métin C (2015) Cortical interneurons migra... more Citation: Luccardini C, Leclech C, Viou L, Rio J-P and Métin C (2015) Cortical interneurons migrating on a pure substrate of N-cadherin exhibit fast synchronous centrosomal and nuclear movements and reduced ciliogenesis. Front. Cell. Neurosci. 9:286.
Sensors (Basel, Switzerland), 2015
Semiconductor nanocrystals (NCs) or quantum dots (QDs) are luminous point emitters increasingly b... more Semiconductor nanocrystals (NCs) or quantum dots (QDs) are luminous point emitters increasingly being used to tag and track biomolecules in biological/biomedical imaging. However, their intracellular use as highlighters of single-molecule localization and nanobiosensors reporting ion microdomains changes has remained a major challenge. Here, we report the design, generation and validation of FRET-based nanobiosensors for detection of intracellular Ca(2+) and H⁺ transients. Our sensors combine a commercially available CANdot(®)565QD as an energy donor with, as an acceptor, our custom-synthesized red-emitting Ca(2+) or H⁺ probes. These 'Rubies' are based on an extended rhodamine as a fluorophore and a phenol or BAPTA (1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetra-acetic acid) for H⁺ or Ca(2+) sensing, respectively, and additionally bear a linker arm for conjugation. QDs were stably functionalized using the same SH/maleimide crosslink chemistry for all desired reactants...
Journal of Peptide Science
Methods in Enzymology, 2006
Single quantum dot imaging is a powerful approach to probe the complex dynamics of individual bio... more Single quantum dot imaging is a powerful approach to probe the complex dynamics of individual biomolecules in living systems. Due to their remarkable photophysical properties and relatively small size, quantum dots can be used as ultrasensitive detection probes. They make possible the study of biological processes, both in the membrane or in the cytoplasm, at a truly molecular scale and with high spatial and temporal resolutions. This chapter presents methods used for tracking single biomolecules coupled to quantum dots in living cells from labeling procedures to the analysis of the quantum dot motion.
Colloidal Quantum Dots for Biomedical Applications, 2006
Colloidal semiconductor quantum dots (QDs) have become common fluorescent probes in biology. Thei... more Colloidal semiconductor quantum dots (QDs) have become common fluorescent probes in biology. Their optical properties not only facilitate spectrally multiplexed detection but also enable single molecule measurements with high signal to noise ratio. This is of particular interest in cell biology since it allows individual QD-tagged biomolecules to be tracked with good spatial and temporal resolution over long durations. Recent
Journal of Neuroscience, 2013
In the developing brain, cortical GABAergic interneurons migrate long distances from the medial g... more In the developing brain, cortical GABAergic interneurons migrate long distances from the medial ganglionic eminence (MGE) in which they are generated, to the cortex in which they settle. MGE cells express the cell adhesion molecule N-cadherin, a homophilic cell-cell adhesion molecule that regulates numerous steps of brain development, from neuroepithelium morphogenesis to synapse formation.
Organic Letters, 2007
]) indicator dye (579/598 nm peak excitation/emission) with a side arm for conjugation via EDC or... more ]) indicator dye (579/598 nm peak excitation/emission) with a side arm for conjugation via EDC or click chemistry. Its large molar extinction and high quantum yield rank it among the brightest long-wavelength Ca 2+ indicators. Calcium Ruby is a promising alternative to existing dyes for imaging [Ca 2+ ] in multicolor fluorescence applications or in the presence of yellow-green cellular autofluorescence.
Neuroscience Letters, 2004
The role of the microfilaments and microtubules cytoskeleton in the stability of the subcellular ... more The role of the microfilaments and microtubules cytoskeleton in the stability of the subcellular distribution and function of GABAA receptors has been studied in rat cerebellar granule cells in culture. The disruption of either the microfilaments or the microtubules structures did not result in detectable changes in the receptors distribution, as assessed by immunocytochemistry, or in their function, as assessed by the whole-cell patch-clamp approach. A distinct disruption of both the subcellular distribution and the function of the GABAA receptors was found only if both microfilaments and microtubules were destroyed. The results suggest that, in the short term, the plasma membrane localization/stabilization and function of these receptors in granule cells are largely independent from microfilaments and microtubules individually, although they obviously depend on the presence of an organized cellular framework.
Nano Letters, 2006
We report a simple method using semiconductor quantum dots (QDs) to track the motion of intracell... more We report a simple method using semiconductor quantum dots (QDs) to track the motion of intracellular proteins with a high sensitivity. We characterized the in vivo motion of individual QD-tagged kinesin motors in living HeLa cells. Single-molecule measurements provided important parameters of the motor, such as its velocity and processivity, as well as an estimate of the force necessary to carry a QD. Our measurements demonstrate the importance of single-molecule experiments in the investigation of intracellular transport as well as the potential of single quantum-dot imaging for the study of important processes such as cellular trafficking, cell polarization, and division.
Nano Letters, 2007
A fluorescence resonance energy transfer pair consisting of a colloidal quantum dot donor and mul... more A fluorescence resonance energy transfer pair consisting of a colloidal quantum dot donor and multiple organic fluorophores as acceptors is reported and the photophysics of the system is characterized. Most nanoparticle-based biosensors reported so far use the detection of specific changes of the donor/acceptor distance under the influence of analyte binding. Our nanoparticle design on the other hand leads to sensors that detect spectral changes of the acceptor (under the influence of analyte binding) at fixed donor/acceptor distance by the introduction of the acceptor into the polymer coating. This approach allows for short acceptor−donor separation and thus for high-energy transfer efficiencies. Advantageously, the binding properties of the hydrophilic polymer coating further allows for addition of poly(ethylene glycol) shells for improved colloidal stability.
Langmuir, 2006
Semiconductor colloidal quantum dots (QDs) are promising fluorescent probes for biology. Initiall... more Semiconductor colloidal quantum dots (QDs) are promising fluorescent probes for biology. Initially synthesized in organic solvents, they can be dispersed in aqueous solution by noncovalent coating with amphiphilic macromolecules, which renders the particles hydrophilic and modifies their interactions with other biological compounds. Here, after coating QDs with an alkyl-modified polyacrilic acid, we investigated their colloidal properties in aqueous buffers by electrophoresis, electron microscopy, light scattering, and rate zonal centrifugation. Despite polymer dispersity and variation of the size of the inorganic nanoparticles, the polymer-dot complexes appeared relatively well-defined in terms of hydrodynamic radius and surface charge. Our data show that these complexes contain isolated QD surrounded by a polymer layer with thickness 8-10 nm. We then analyzed their interaction with giant unilamellar vesicles, either neutral or cationic, by optical microscopy. At neutral pH, we found the absence of binding of the coated particles to lipid membrane, irrespective of their lipid composition. An abrupt surface aggregation of the nanoparticles on the lipid membrane was observed in a narrow pH range (6.0-6.2), indicative of critical binding triggered by polymer properties. The overall features of QDs coated with amphiphilic polymers open the route to using these nanoparticles in vivo as optically stable probes with switchable properties.
Langmuir, 2009
Colloidal nanocrystal (NC) donors wrapped with a polymer coating including multiple organic accep... more Colloidal nanocrystal (NC) donors wrapped with a polymer coating including multiple organic acceptor molecules are promising scaffolds for fluorescence resonance energy transfer (FRET)-based nanobiosensors. Over other self-assembling donor-acceptor configurations, our preloaded polymers have the virtue of producing compact assemblies with a fixed donor/acceptor distance. This property, together with the possibility of stoichiometric polymer loading, allowed us to directly address how the FRET efficiency depended on the donor/acceptor. At the population level, nanoprobes based on commercial as well as custom CdSe/ZnS donors displayed the expected dose-dependent rise in transfer efficiency, saturating from about five ATTO dyes/NC. However, for a given acceptor concentration, both the intensity and lifetime of single-pair FRET data revealed a large dispersion of transfer efficiencies, highlighting an important heterogeneity among nominally identical FRET-based nanoprobes. Rigorous quality check during synthesis and shell assembly as well as postsynthesis sorting and purification are required to make hybrid semiconductor-organic nanoprobes a robust and viable alternative to organic or genetically encoded nanobiosensors.
Journal of Biomedicine and Biotechnology, 2007
Semiconductor nanocrystals (NCs) are increasingly being used as photoluminescen markers in biolog... more Semiconductor nanocrystals (NCs) are increasingly being used as photoluminescen markers in biological imaging. Their brightness, large Stokes shift, and high photostability compared to organic fluorophores permit the exploration of biological phenomena at the single-molecule scale with superior temporal resolution and spatial precision. NCs have predominantly been used as extracellular markers for tagging and tracking membrane proteins. Successful internalization and intracellular labelling with NCs have been demonstrated for both fixed immunolabelled and live cells. However, the precise localization and subcellular compartment labelled are less clear. Generally, live cell studies are limited by the requirement of fairly invasive protocols for loading NCs and the relatively large size of NCs compared to the cellular machinery, along with the subsequent sequestration of NCs in endosomal/lysosomal compartments. For long-period observation the potential cytotoxicity of cytoplasmically loaded NCs must be evaluated. This review focuses on the challenges of intracellular uses of NCs.
Cell Calcium, 2009
The limited choice and poor performance of red-emitting calcium (Ca(2+)) indicators have hampered... more The limited choice and poor performance of red-emitting calcium (Ca(2+)) indicators have hampered microfluorometric measurements of the intracellular free Ca(2+) concentration in cells expressing yellow- or green-fluorescent protein constructs. A long-wavelength Ca(2+) indicator would also permit a better discrimination against cellular autofluorescence than the commonly used fluorescein-based probes. Here, we report an improved synthesis and characterization of Calcium Ruby, a red-emitting probe consisting of an extended rhodamine chromophore (578/602 nm peak excitation/emission) conjugated to BAPTA and having an additional NH(2) linker arm. The low-affinity variant (K(D,Ca) approximately 30 microM) with a chloride in meta position that was specifically designed for the detection of large and rapid Ca(2+) transients. While Calcium Ruby is a mitochondrial Ca(2+)probe, its conjugation, via the NH(2) tail, to a 10,000 MW dextran abolishes the sub-cellular compartmentalization and generates a cytosolic Ca(2+) probe with an affinity matched to microdomain Ca(2+) signals. As an example, we show depolarization-evoked Ca(2+) signals triggering the exocytosis of individual chromaffin granules. Calcium Ruby should be of use in a wide range of applications involving dual- or triple labeling schemes or targeted sub-cellular Ca(2+) measurements.
Science, 2003
Plasma scattering measurement is effective technique to observe low frequency density fluctuation... more Plasma scattering measurement is effective technique to observe low frequency density fluctuations excited in plasma. The spatial and wave number resolutions and the S/N ratio of measurement depend on the wavelength range, the size and the intensity of a probe beam. A well-collimated, submillimeter wave beam is suitable for improving the spatial and wave number resolutions. Application of high frequency gyrotron is effective in improving the S/N ratio of the measurement because of its capacity to deliver high power. Unlike the molecular vapor lasers, the gyrotrons generate diverging beam of radiation with TEmn mode structure. It is therefore necessary to convert the output radiation into a Gaussian beam. A quasi-optical antenna is a suitable element for the conversion system under consideration since it is applicable to several TE 0n and TE 1n modes. In order to apply the gyrotron to plasma scattering measurement, we have stabilized the output (P=110W, f=354GHz) of gyrotron up to the level (∆P/P<1 %, ∆f<10 kHz). The gyrotron output can be stabilized by decreasing the fluctuation of the cathode potential.
Organic Letters, 2007
]) indicator dye (579/598 nm peak excitation/emission) with a side arm for conjugation via EDC or... more ]) indicator dye (579/598 nm peak excitation/emission) with a side arm for conjugation via EDC or click chemistry. Its large molar extinction and high quantum yield rank it among the brightest long-wavelength Ca 2+ indicators. Calcium Ruby is a promising alternative to existing dyes for imaging [Ca 2+ ] in multicolor fluorescence applications or in the presence of yellow-green cellular autofluorescence.
Frontiers in Cellular Neuroscience, 2015
Citation: Luccardini C, Leclech C, Viou L, Rio J-P and Métin C (2015) Cortical interneurons migra... more Citation: Luccardini C, Leclech C, Viou L, Rio J-P and Métin C (2015) Cortical interneurons migrating on a pure substrate of N-cadherin exhibit fast synchronous centrosomal and nuclear movements and reduced ciliogenesis. Front. Cell. Neurosci. 9:286.
Sensors (Basel, Switzerland), 2015
Semiconductor nanocrystals (NCs) or quantum dots (QDs) are luminous point emitters increasingly b... more Semiconductor nanocrystals (NCs) or quantum dots (QDs) are luminous point emitters increasingly being used to tag and track biomolecules in biological/biomedical imaging. However, their intracellular use as highlighters of single-molecule localization and nanobiosensors reporting ion microdomains changes has remained a major challenge. Here, we report the design, generation and validation of FRET-based nanobiosensors for detection of intracellular Ca(2+) and H⁺ transients. Our sensors combine a commercially available CANdot(®)565QD as an energy donor with, as an acceptor, our custom-synthesized red-emitting Ca(2+) or H⁺ probes. These 'Rubies' are based on an extended rhodamine as a fluorophore and a phenol or BAPTA (1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetra-acetic acid) for H⁺ or Ca(2+) sensing, respectively, and additionally bear a linker arm for conjugation. QDs were stably functionalized using the same SH/maleimide crosslink chemistry for all desired reactants...
Journal of Peptide Science
Methods in Enzymology, 2006
Single quantum dot imaging is a powerful approach to probe the complex dynamics of individual bio... more Single quantum dot imaging is a powerful approach to probe the complex dynamics of individual biomolecules in living systems. Due to their remarkable photophysical properties and relatively small size, quantum dots can be used as ultrasensitive detection probes. They make possible the study of biological processes, both in the membrane or in the cytoplasm, at a truly molecular scale and with high spatial and temporal resolutions. This chapter presents methods used for tracking single biomolecules coupled to quantum dots in living cells from labeling procedures to the analysis of the quantum dot motion.
Colloidal Quantum Dots for Biomedical Applications, 2006
Colloidal semiconductor quantum dots (QDs) have become common fluorescent probes in biology. Thei... more Colloidal semiconductor quantum dots (QDs) have become common fluorescent probes in biology. Their optical properties not only facilitate spectrally multiplexed detection but also enable single molecule measurements with high signal to noise ratio. This is of particular interest in cell biology since it allows individual QD-tagged biomolecules to be tracked with good spatial and temporal resolution over long durations. Recent
Journal of Neuroscience, 2013
In the developing brain, cortical GABAergic interneurons migrate long distances from the medial g... more In the developing brain, cortical GABAergic interneurons migrate long distances from the medial ganglionic eminence (MGE) in which they are generated, to the cortex in which they settle. MGE cells express the cell adhesion molecule N-cadherin, a homophilic cell-cell adhesion molecule that regulates numerous steps of brain development, from neuroepithelium morphogenesis to synapse formation.
Organic Letters, 2007
]) indicator dye (579/598 nm peak excitation/emission) with a side arm for conjugation via EDC or... more ]) indicator dye (579/598 nm peak excitation/emission) with a side arm for conjugation via EDC or click chemistry. Its large molar extinction and high quantum yield rank it among the brightest long-wavelength Ca 2+ indicators. Calcium Ruby is a promising alternative to existing dyes for imaging [Ca 2+ ] in multicolor fluorescence applications or in the presence of yellow-green cellular autofluorescence.
Neuroscience Letters, 2004
The role of the microfilaments and microtubules cytoskeleton in the stability of the subcellular ... more The role of the microfilaments and microtubules cytoskeleton in the stability of the subcellular distribution and function of GABAA receptors has been studied in rat cerebellar granule cells in culture. The disruption of either the microfilaments or the microtubules structures did not result in detectable changes in the receptors distribution, as assessed by immunocytochemistry, or in their function, as assessed by the whole-cell patch-clamp approach. A distinct disruption of both the subcellular distribution and the function of the GABAA receptors was found only if both microfilaments and microtubules were destroyed. The results suggest that, in the short term, the plasma membrane localization/stabilization and function of these receptors in granule cells are largely independent from microfilaments and microtubules individually, although they obviously depend on the presence of an organized cellular framework.
Nano Letters, 2006
We report a simple method using semiconductor quantum dots (QDs) to track the motion of intracell... more We report a simple method using semiconductor quantum dots (QDs) to track the motion of intracellular proteins with a high sensitivity. We characterized the in vivo motion of individual QD-tagged kinesin motors in living HeLa cells. Single-molecule measurements provided important parameters of the motor, such as its velocity and processivity, as well as an estimate of the force necessary to carry a QD. Our measurements demonstrate the importance of single-molecule experiments in the investigation of intracellular transport as well as the potential of single quantum-dot imaging for the study of important processes such as cellular trafficking, cell polarization, and division.
Nano Letters, 2007
A fluorescence resonance energy transfer pair consisting of a colloidal quantum dot donor and mul... more A fluorescence resonance energy transfer pair consisting of a colloidal quantum dot donor and multiple organic fluorophores as acceptors is reported and the photophysics of the system is characterized. Most nanoparticle-based biosensors reported so far use the detection of specific changes of the donor/acceptor distance under the influence of analyte binding. Our nanoparticle design on the other hand leads to sensors that detect spectral changes of the acceptor (under the influence of analyte binding) at fixed donor/acceptor distance by the introduction of the acceptor into the polymer coating. This approach allows for short acceptor−donor separation and thus for high-energy transfer efficiencies. Advantageously, the binding properties of the hydrophilic polymer coating further allows for addition of poly(ethylene glycol) shells for improved colloidal stability.
Langmuir, 2006
Semiconductor colloidal quantum dots (QDs) are promising fluorescent probes for biology. Initiall... more Semiconductor colloidal quantum dots (QDs) are promising fluorescent probes for biology. Initially synthesized in organic solvents, they can be dispersed in aqueous solution by noncovalent coating with amphiphilic macromolecules, which renders the particles hydrophilic and modifies their interactions with other biological compounds. Here, after coating QDs with an alkyl-modified polyacrilic acid, we investigated their colloidal properties in aqueous buffers by electrophoresis, electron microscopy, light scattering, and rate zonal centrifugation. Despite polymer dispersity and variation of the size of the inorganic nanoparticles, the polymer-dot complexes appeared relatively well-defined in terms of hydrodynamic radius and surface charge. Our data show that these complexes contain isolated QD surrounded by a polymer layer with thickness 8-10 nm. We then analyzed their interaction with giant unilamellar vesicles, either neutral or cationic, by optical microscopy. At neutral pH, we found the absence of binding of the coated particles to lipid membrane, irrespective of their lipid composition. An abrupt surface aggregation of the nanoparticles on the lipid membrane was observed in a narrow pH range (6.0-6.2), indicative of critical binding triggered by polymer properties. The overall features of QDs coated with amphiphilic polymers open the route to using these nanoparticles in vivo as optically stable probes with switchable properties.
Langmuir, 2009
Colloidal nanocrystal (NC) donors wrapped with a polymer coating including multiple organic accep... more Colloidal nanocrystal (NC) donors wrapped with a polymer coating including multiple organic acceptor molecules are promising scaffolds for fluorescence resonance energy transfer (FRET)-based nanobiosensors. Over other self-assembling donor-acceptor configurations, our preloaded polymers have the virtue of producing compact assemblies with a fixed donor/acceptor distance. This property, together with the possibility of stoichiometric polymer loading, allowed us to directly address how the FRET efficiency depended on the donor/acceptor. At the population level, nanoprobes based on commercial as well as custom CdSe/ZnS donors displayed the expected dose-dependent rise in transfer efficiency, saturating from about five ATTO dyes/NC. However, for a given acceptor concentration, both the intensity and lifetime of single-pair FRET data revealed a large dispersion of transfer efficiencies, highlighting an important heterogeneity among nominally identical FRET-based nanoprobes. Rigorous quality check during synthesis and shell assembly as well as postsynthesis sorting and purification are required to make hybrid semiconductor-organic nanoprobes a robust and viable alternative to organic or genetically encoded nanobiosensors.
Journal of Biomedicine and Biotechnology, 2007
Semiconductor nanocrystals (NCs) are increasingly being used as photoluminescen markers in biolog... more Semiconductor nanocrystals (NCs) are increasingly being used as photoluminescen markers in biological imaging. Their brightness, large Stokes shift, and high photostability compared to organic fluorophores permit the exploration of biological phenomena at the single-molecule scale with superior temporal resolution and spatial precision. NCs have predominantly been used as extracellular markers for tagging and tracking membrane proteins. Successful internalization and intracellular labelling with NCs have been demonstrated for both fixed immunolabelled and live cells. However, the precise localization and subcellular compartment labelled are less clear. Generally, live cell studies are limited by the requirement of fairly invasive protocols for loading NCs and the relatively large size of NCs compared to the cellular machinery, along with the subsequent sequestration of NCs in endosomal/lysosomal compartments. For long-period observation the potential cytotoxicity of cytoplasmically loaded NCs must be evaluated. This review focuses on the challenges of intracellular uses of NCs.
Cell Calcium, 2009
The limited choice and poor performance of red-emitting calcium (Ca(2+)) indicators have hampered... more The limited choice and poor performance of red-emitting calcium (Ca(2+)) indicators have hampered microfluorometric measurements of the intracellular free Ca(2+) concentration in cells expressing yellow- or green-fluorescent protein constructs. A long-wavelength Ca(2+) indicator would also permit a better discrimination against cellular autofluorescence than the commonly used fluorescein-based probes. Here, we report an improved synthesis and characterization of Calcium Ruby, a red-emitting probe consisting of an extended rhodamine chromophore (578/602 nm peak excitation/emission) conjugated to BAPTA and having an additional NH(2) linker arm. The low-affinity variant (K(D,Ca) approximately 30 microM) with a chloride in meta position that was specifically designed for the detection of large and rapid Ca(2+) transients. While Calcium Ruby is a mitochondrial Ca(2+)probe, its conjugation, via the NH(2) tail, to a 10,000 MW dextran abolishes the sub-cellular compartmentalization and generates a cytosolic Ca(2+) probe with an affinity matched to microdomain Ca(2+) signals. As an example, we show depolarization-evoked Ca(2+) signals triggering the exocytosis of individual chromaffin granules. Calcium Ruby should be of use in a wide range of applications involving dual- or triple labeling schemes or targeted sub-cellular Ca(2+) measurements.
Science, 2003
Plasma scattering measurement is effective technique to observe low frequency density fluctuation... more Plasma scattering measurement is effective technique to observe low frequency density fluctuations excited in plasma. The spatial and wave number resolutions and the S/N ratio of measurement depend on the wavelength range, the size and the intensity of a probe beam. A well-collimated, submillimeter wave beam is suitable for improving the spatial and wave number resolutions. Application of high frequency gyrotron is effective in improving the S/N ratio of the measurement because of its capacity to deliver high power. Unlike the molecular vapor lasers, the gyrotrons generate diverging beam of radiation with TEmn mode structure. It is therefore necessary to convert the output radiation into a Gaussian beam. A quasi-optical antenna is a suitable element for the conversion system under consideration since it is applicable to several TE 0n and TE 1n modes. In order to apply the gyrotron to plasma scattering measurement, we have stabilized the output (P=110W, f=354GHz) of gyrotron up to the level (∆P/P<1 %, ∆f<10 kHz). The gyrotron output can be stabilized by decreasing the fluctuation of the cathode potential.