Your browser doesn't support javascript.
loading
Show: 20 | 50 | 100
Results 1 - 17 de 17
Filter
Add more filters










Publication year range
1.
Front Cell Dev Biol ; 11: 1307502, 2023.
Article in English | MEDLINE | ID: mdl-38046667

ABSTRACT

In our study, we harnessed an original Enhanced Speed Structured Illumination Microscopy (Fast-SIM) imaging setup to explore the dynamics of mitochondrial and inner membrane ultrastructure under specific photo-oxidation stress induced by Chlorin-e6 and light irradiation. Notably, our Fast-SIM system allowed us to observe and quantify a distinct remodeling and shortening of the mitochondrial structure after 60-80 s of irradiation. These changes were accompanied by fusion events of adjacent inner membrane cristae and global swelling of the organelle. Preceding these alterations, a larger sequence was characterized by heightened dynamics within the mitochondrial network, featuring events such as mitochondrial fission, rapid formation of tubular prolongations, and fluctuations in cristae structure. Our findings provide compelling evidence that, among enhanced-resolution microscopy techniques, Fast-SIM emerges as the most suitable approach for non-invasive dynamic studies of mitochondrial structure in living cells. For the first time, this approach allows quantitative and qualitative characterization of successive steps in the photo-induced oxidation process with sufficient spatial and temporal resolution.

2.
J Biomed Opt ; 28(6): 066501, 2023 06.
Article in English | MEDLINE | ID: mdl-37334209

ABSTRACT

Significance: Adaptive optics (AO) has been implemented on several microscopy setups and has proven its ability to increase both signal and resolution. However, reported configurations are not suited for fast imaging of live samples or are based on an invasive or complex implementation method. Aim: Provide a fast aberration correction method with an easy to implement AO module compatible with light-sheet fluorescence microscopy (LSFM) for enhanced imaging of live samples. Approach: Development of an AO add-on module for LSFM based on direct wavefront sensing without requiring a guide star using an extended-scene Shack-Hartmann wavefront sensor. The enhanced setup uses a two-color sample labeling strategy to optimize the photon budget. Results: Fast AO correction of in-depth aberrations in an ex-vivo adult Drosophila brain enables doubling the contrast when imaging with either cell reporters or calcium sensors for functional imaging. We quantify the gain in terms of image quality on different functional domains of sleep neurons in the Drosophila brain at various depths and discuss the optimization of key parameters driving AO. Conclusion: We developed a compact AO module that can be integrated into most of the reported light-sheet microscopy setups, provides significant improvement of image quality and is compatible with fast imaging requirements such as calcium imaging.


Subject(s)
Calcium , Drosophila melanogaster , Animals , Microscopy, Fluorescence , Drosophila , Neuroimaging , Brain/diagnostic imaging
3.
Opt Express ; 30(9): 15250-15265, 2022 Apr 25.
Article in English | MEDLINE | ID: mdl-35473251

ABSTRACT

Deep fluorescence imaging in mammalian brain tissues remains challenging due to scattering and optical aberration-induced loss in signal and resolution. Correction of aberrations using adaptive optics (AO) requires their reliable measurement in the tissues. Here, we show that an extended-source Shack-Hartmann wavefront sensor (ESSH) allows quantitative aberration measurements through fixed brain slices with a thickness up to four times their scattering length. We demonstrate in particular that this wavefront measurement method based on image correlation is more robust to scattering compared to the standard centroid-based approach. Finally, we obtain a measurement of the tissue scattering length taking advantage of the geometry of a Shack-Hartmann sensor.


Subject(s)
Optical Imaging , Optics and Photonics , Animals , Brain/diagnostic imaging , Mammals , Mice
4.
Cancers (Basel) ; 12(12)2020 Dec 11.
Article in English | MEDLINE | ID: mdl-33322532

ABSTRACT

The efficient intraoperative identification of cancers requires the development of the bright, minimally-toxic, tumor-specific near-infrared (NIR) probes as contrast agents. Luminescent semiconductor quantum dots (QDs) offer several unique advantages for in vivo cellular imaging by providing bright and photostable fluorescent probes. Here, we present the synthesis of ZnCuInSe/ZnS core/shell QDs emitting in NIR (~750 nm) conjugated to NAVPNLRGDLQVLAQKVART (A20FMDV2) peptide for targeting αvß6 integrin-rich head and neck squamous cell carcinoma (HNSCC). Integrin αvß6 is usually not detectable in nonpathological tissues, but is highly upregulated in HNSCC. QD-A20 showed αvß6 integrin-specific binding in two-dimension (2D) monolayer and three-dimension (3D) spheroid in vitro HNSCC models. QD-A20 exhibit limited penetration (ca. 50 µm) in stroma-rich 3D spheroids. Finally, we demonstrated the potential of these QDs by time-gated fluorescence imaging of stroma-rich 3D spheroids placed onto mm-thick tissue slices to mimic imaging conditions in tissues. Overall, QD-A20 could be considered as highly promising nanoprobes for NIR bioimaging and imaging-guided surgery.

5.
Biomaterials ; 219: 119357, 2019 10.
Article in English | MEDLINE | ID: mdl-31351245

ABSTRACT

In the last few years, zwitterionic polymers have been developed as antifouling surface coatings. However, their ability to completely suppress protein adsorption at the surface of nanoparticles in complex biological media remains undemonstrated. Here we investigate the formation of hard (irreversible) and soft (reversible) protein corona around model nanoparticles (NPs) coated with sulfobetaine (SB), phosphorylcholine (PC) and carboxybetaine (CB) polymer ligands in model albumin solutions and in whole serum. We show for the first time a complete absence of protein corona around SB-coated NPs, while PC- and CB-coated NPs undergo reversible adsorption or partial aggregation. These dramatic differences cannot be described by naïve hard/soft acid/base electrostatic interactions. Single NP tracking in the cytoplasm of live cells corroborate these in vitro observations. Finally, while modification of SB polymers with additional charged groups lead to consequent protein adsorption, addition of small neutral targeting moieties preserves antifouling and enable efficient intracellular targeting.


Subject(s)
Coated Materials, Biocompatible/chemistry , Nanoparticles/chemistry , Polymers/chemistry , Protein Corona/chemistry , Betaine/analogs & derivatives , Betaine/chemistry , Biotin/chemistry , Hydrodynamics , Ligands , Phosphorylcholine/chemistry , Quantum Dots/chemistry
6.
Opt Lett ; 44(10): 2514-2517, 2019 May 15.
Article in English | MEDLINE | ID: mdl-31090720

ABSTRACT

We propose an adaptive optics light-sheet fluorescence microscope (AO-LSFM) for closed-loop aberrations' correction at the emission path, providing intrinsic instrumental simplicity and high accuracy when compared to previously reported schemes. The approach is based on direct wavefront sensing, i.e., not on time-consuming iterative algorithms, and does not require the use of any guide star, thus reducing instrumental complexity and/or sample preparation constraints. The design is based on a modified Shack-Hartmann wavefront sensor providing compatibility with extended sources such as images from optical sectioning microscopes. We report an AO-LSFM setup based on such sensors, including characterization of the sensor performance, and demonstrate for the first time to the best of our knowledge a significant contrast improvement on neuronal structures of the ex vivo adult drosophila brain in depth.

7.
ACS Nano ; 13(3): 3125-3131, 2019 03 26.
Article in English | MEDLINE | ID: mdl-30835434

ABSTRACT

Whereas in vivo fluorescence imaging of cells immobilized within tissues provides a valuable tool to a broad range of biological studies, it still lacks the sensitivity required to visualize isolated cells circulating fast in the bloodstream due, in particular, to the autofluorescence from endogenous fluorophores. Time-gated imaging of near-infrared emitting ZnCuInSe/ZnS quantum dots (QDs) with fluorescence lifetimes in the range of 150-300 ns enables the efficient rejection of fast autofluorescence photons and the selection of QD fluorescence photons, thus significantly increasing sensitivity. We labeled model erythrocytes as well as lymphoma cells using these QDs coated with a stable zwitterionic polymer surface chemistry. After reinjection in the bloodstream, we were able to image and count individual QD-labeled cells circulating at mm·s-1 velocities in blood vessels.


Subject(s)
Optical Imaging , Quantum Dots/chemistry , Single-Cell Analysis , Animals , Cell Line, Tumor , Erythrocytes/cytology , Fluorescence , Healthy Volunteers , Humans , Infrared Rays , Male , Rats , Rats, Sprague-Dawley , Spectrometry, Fluorescence , Surface Properties , Time Factors
8.
J Biomed Opt ; 21(3): 36006, 2016 Mar.
Article in English | MEDLINE | ID: mdl-26968001

ABSTRACT

We present an implementation of a sensorless adaptive optics loop in a widefield fluorescence microscope. This setup is designed to compensate for aberrations induced by the sample on both excitation and emission pathways. It allows fast optical sectioning inside a living Drosophila brain. We present a detailed characterization of the system performances. We prove that the gain brought to optical sectioning by realizing structured illumination microscopy with adaptive optics down to 50 µm deep inside living Drosophila brain.


Subject(s)
Brain/diagnostic imaging , Image Processing, Computer-Assisted/methods , Microscopy, Fluorescence/methods , Neuroimaging/methods , Algorithms , Animals , Animals, Genetically Modified , Brain Chemistry , Drosophila , Equipment Design , Luminescent Proteins/chemistry , Luminescent Proteins/metabolism , Microscopy, Fluorescence/instrumentation , Recombinant Proteins/chemistry , Recombinant Proteins/metabolism
9.
ACS Appl Mater Interfaces ; 7(48): 26904-13, 2015 Dec 09.
Article in English | MEDLINE | ID: mdl-26551755

ABSTRACT

Distinctive optical properties of inorganic quantum dot (QD) nanoparticles promise highly valuable probes for fluorescence-based detection methods, particularly for in vivo diagnostics, cell phenotyping via multiple markers or single molecule tracking. However, despite high hopes, this promise has not been fully realized yet, mainly due to difficulties at producing stable, nontoxic QD bioconjugates of negligible nonspecific binding. Here, a universal platform for antibody binding to QDs is presented that builds upon the controlled functionalization of CdSe/CdS/ZnS nanoparticles capped with a multidentate dithiol/zwitterion copolymer ligand. In a change-of-paradigm approach, thiol groups are concomitantly used as anchoring and bioconjugation units to covalently bind up to 10 protein A molecules per QD while preserving their long-term colloidal stability. Protein A conjugated to QDs then enables the oriented, stoichiometrically controlled immobilization of whole, unmodified antibodies by simple incubation. This QD-protein A immobilization platform displays remarkable antibody functionality retention after binding, usually a compromised property in antibody conjugation to surfaces. Typical QD-protein A-antibody assemblies contain about three fully functional antibodies. Validation experiments show that these nanobioconjugates overcome current limitations since they retain their colloidal stability and antibody functionality over 6 months, exhibit low nonspecific interactions with live cells and have very low toxicity: after 48 h incubation with 1 µM QD bioconjugates, HeLa cells retain more than 80% of their cellular metabolism. Finally, these QD nanobioconjugates possess a high specificity for extra- and intracellular targets in live and fixed cells. The dithiol/zwitterion QD-protein A nanoconjugates have thus a latent potential to become an off-the-shelf tool destined to unresolved biological questions.


Subject(s)
Antibodies/metabolism , Molecular Imaging/methods , Nanoconjugates/chemistry , Quantum Dots/chemistry , Cadherins/metabolism , Dynamic Light Scattering , Endocytosis , HeLa Cells , Humans , Immobilized Proteins/metabolism , Ligands , MCF-7 Cells , Particle Size , Receptor, Cannabinoid, CB1/metabolism , Staphylococcal Protein A/metabolism
10.
ACS Nano ; 9(11): 11479-89, 2015 Nov 24.
Article in English | MEDLINE | ID: mdl-26505527

ABSTRACT

Long-term inspection of biological phenomena requires probes of elevated intra- and extracellular stability and target biospecificity. The high fluorescence and photostability of quantum dot (QD) nanoparticles contributed to foster their promise as bioimaging tools that could overcome limitations associated with traditional fluorophores. However, QDs' potential as a bioimaging platform relies upon a precise control over the surface chemistry modifications of these nano-objects. Here, a zwitterion-vinylimidazole block copolymer ligand was synthesized, which regroups all anchoring groups in one compact terminal block, while the rest of the chain is endowed with antifouling and bioconjugation moieties. By further application of an oriented bioconjugation approach with whole IgG antibodies, QD nanobioconjugates were obtained that display outstanding intra- and extracellular stability as well as biorecognition capacity. Imaging the internalization and intracellular dynamics of a transmembrane cell receptor, the CB1 brain cannabinoid receptor, both in HEK293 cells and in neurons, illustrates the breadth of potential applications of these nanoprobes.


Subject(s)
Betaine/analogs & derivatives , Diagnostic Imaging/methods , Imidazoles/chemistry , Polymers/chemistry , Quantum Dots/chemistry , Betaine/chemical synthesis , Betaine/chemistry , Colloids , Fluorescein/chemistry , HEK293 Cells , HeLa Cells , Humans , Hydrogen-Ion Concentration , Imidazoles/chemical synthesis , Kinetics , Polymerization , Polymers/chemical synthesis , Receptor, Cannabinoid, CB1/metabolism , Surface Properties
11.
J Biomed Opt ; 19(5): 051208, 2014 May.
Article in English | MEDLINE | ID: mdl-24395624

ABSTRACT

Fluorescence imaging is a promising technique for the detection of individual cell migration. Its sensitivity is, however, limited by a high tissue autofluorescence and a poor visible light penetration depth. In order to solve this problem, the fluorescence signal peak wavelength should lie in an absorption and diffusion free region and should be distinguishable, either spectrally or temporally, from the autofluorescence background. We present, here, the synthesis and characterization of low toxicity Zn-Cu-In-Se/ZnS core/shell quantum dots. Their fluorescence emission wavelength peaks around 800 nm, where the absorption and scattering of tissues are minimal. They are coated with a new ligand, which yields small, stable, and bright individual probes in the live cell cytoplasm, even 48 h after the labeling. Furthermore, these near-infrared-emitting quantum dots have a long fluorescence lifetime component (around 150 ns) compared to autofluorescence (<5 ns). Taking the advantage of this property and coupling these probes to a time-gated detection, we demonstrate efficiently the discrimination between the signal and short lifetime fluorescence such as the autofluorescence. This technique is supported by a method we developed, to massively stain cells that preserves the quantum dot stability and brightness for 48 h.


Subject(s)
Optical Imaging/methods , Quantum Dots , Spectroscopy, Near-Infrared/methods , Animals , Cattle , Cytological Techniques/methods , HeLa Cells , Humans , Muscles/chemistry
12.
Small ; 8(7): 1029-37, 2012 Apr 10.
Article in English | MEDLINE | ID: mdl-22378567

ABSTRACT

The in vivo labeling of intracellular components with quantum dots (QDs) is very limited because of QD aggregation in the cell cytoplasm and/or QD confinement into lysosomal compartments. In order to improve intracellular targeting with QDs, various surface chemistries and delivery methods have been explored, but they have not yet been compared systematically with respect to the QD intracellular stability. In this work, the intracellular aggregation kinetics of QDs for three different surface chemistries based on ligand exchange or encapsulation with amphiphilic polymers are compared. For each surface chemistry, three delivery methods for bringing the nanoparticles into the cells are compared: electroporation, microinjection, and pinocytosis. It is concluded that the QD intracellular aggregation behavior is strongly dependent on the surface chemistry. QDs coated with dihydrolipoic acid-sulfobetaine (DHLA-SB) ligands diffuse freely in cells for longer periods of time than for QDs in the other chemistries tested, and they can access all cytoplasmic compartments. Even when conjugated to streptavidin, these DHLA-SB QDs remain freely diffusing inside the cytoplasm and unaggregated, and they are able to reach a biotinylated target inside HeLa cells. Such labeling was more efficient when compared to commercial streptavidin-conjugated QDs, which may be due to the smaller size of DHLA-SB QDs and/or to their superior intracellular stability.


Subject(s)
Betaine/analogs & derivatives , Quantum Dots , Thioctic Acid/analogs & derivatives , Animals , Betaine/chemistry , Cytoplasm/metabolism , Electroporation , Embryo, Nonmammalian/metabolism , HeLa Cells , Humans , Microinjections , Thioctic Acid/chemistry , Xenopus laevis
13.
J Biomed Opt ; 16(7): 076019, 2011 Jul.
Article in English | MEDLINE | ID: mdl-21806280

ABSTRACT

We describe the implementation and use of an adaptive optics loop in the imaging path of a commercial wide field microscope. We show that it is possible to maintain the optical performances of the original microscope when imaging through aberrant biological samples. The sources used for illuminating the adaptive optics loop are spectrally independent, in excitation and emission, from the sample, so they do not appear in the final image, and their use does not contribute to the sample bleaching. Results are compared with equivalent images obtained with an identical microscope devoid of adaptive optics system.


Subject(s)
Microscopy, Fluorescence/instrumentation , Fluorescent Dyes , HeLa Cells , Humans , Microscopy, Fluorescence/statistics & numerical data , Optical Phenomena , Quantum Dots , Tubulin/metabolism
14.
Biophys J ; 100(11): 2810-9, 2011 Jun 08.
Article in English | MEDLINE | ID: mdl-21641327

ABSTRACT

We describe a wide-field fluorescence microscope setup which combines HiLo microscopy technique with the use of a two-color fluorescent probe. It allows one-shot fluorescence optical sectioning of thick biological moving sample which is illuminated simultaneously with a flat and a structured pattern at two different wavelengths. Both homogenous and structured fluorescence images are spectrally separated at detection and combined similarly with the HiLo microscopy technique. We present optically sectioned full-field images of Xenopus laevis embryos acquired at 25 images/s frame rate.


Subject(s)
Fluorescent Dyes/chemistry , Microscopy, Fluorescence/methods , Optical Phenomena , Animals , Color , Microscopy, Fluorescence/instrumentation , Microspheres , Xenopus laevis/embryology
15.
J Am Chem Soc ; 132(13): 4556-7, 2010 Apr 07.
Article in English | MEDLINE | ID: mdl-20235547

ABSTRACT

We have developed a novel surface coating for semiconductor quantum dots (QDs) based on a heterobifunctional ligand that overcomes most of the previous limits of these fluorescent probes in bioimaging applications. Here we show that QDs capped with bidentate zwitterionic dihydrolipoic acid-sulfobetaine (DHLA-SB) ligands are a favorable alternative to polyethylene glycol-coated nanoparticles since they combine small sizes, low nonspecific adsorption, preserved optical properties, and excellent stability over time and a wide range of pH and salinity. Additionally, these QDs can easily be functionalized with biomolecules such as streptavidin (SA) and biotin. We applied streptavidin-functionalized DHLA-SB QDs to track the intracellular recycling of cannabinoid receptor 1 (CB1R) in live cells. These QDs selectively recognized the pool of receptors at the cell surface via SA-biotin interactions with negligible nonspecific adsorption. The QDs retained their optical properties, allowing the internalization of CB1R into endosomes to be followed. Moreover, the cellular activity was apparently unaffected by the probe.


Subject(s)
Betaine/analogs & derivatives , Molecular Imaging/methods , Quantum Dots , Thioctic Acid/analogs & derivatives , Betaine/chemistry , Cell Line , Cell Survival , Humans , Ligands , Microscopy, Fluorescence , Particle Size , Receptor, Cannabinoid, CB1/chemistry , Surface Properties , Thioctic Acid/chemistry
16.
Appl Opt ; 42(34): 6880-8, 2003 Dec 01.
Article in English | MEDLINE | ID: mdl-14661799

ABSTRACT

Apertureless scanning near-field optical microscopy has been used to image fluorescent latex spheres with a resolution of a few tens of nanometers and good signal-to-noise ratio. The near-field fluorescence images reveal optical interference with several highly contrasted fringes located around the spheres. The origin of the interference is discussed in detail, and models are used to explain their formation. Spatial coherence is also discussed.

SELECTION OF CITATIONS
SEARCH DETAIL
...