Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 58
Filtrar
Mais filtros










Base de dados
Intervalo de ano de publicação
2.
J Am Chem Soc ; 142(22): 10184-10197, 2020 Jun 03.
Artigo em Inglês | MEDLINE | ID: mdl-32368907

RESUMO

In addition to the already described ligand L4a, two pyclen-based lanthanide chelators, L4b and L4c, bearing two specific picolinate two-photon antennas (tailor-made for each targeted metal) and one acetate arm arranged in a dissymmetrical manner, have been synthesized, to form a complete family of lanthanide luminescent bioprobes: [EuL4a], [SmL4a], [YbL4b], [TbL4c], and [DyL4c]. Additionally, the symmetrically arranged regioisomer L4a' was also synthesized as well as its [EuL4a'] complex to highlight the astonishing positive impact of the dissymmetrical N-distribution of the functional chelating arms. The investigation clearly shows the high performance of each bioprobe, which, depending on the complexed lanthanide, could be used in various applications. Each presents high brightness, quantum yields, and lifetimes. Staining of the complexes into living human breast cancer cells was observed. In addition, in vivo two-photon microscopy was performed for the first time on a living zebrafish model with [EuL4a]. No apparent toxicity was detected on the growth of the zebrafish, and images of high quality were obtained.

3.
Chemphyschem ; 21(10): 1036-1043, 2020 05 18.
Artigo em Inglês | MEDLINE | ID: mdl-32176399

RESUMO

Cationic lanthanide complexes are generally able to spontaneously internalize into living cells. Following our previous works based on a diMe-cyclen framework, a second generation of cationic water-soluble lanthanide complexes based on a constrained cross-bridged cyclam macrocycle functionalized with donor-π-conjugated picolinate antennas was prepared with europium(III) and ytterbium(III). Their spectroscopic properties were thoroughly investigated in various solvents and rationalized with the help of DFT calculations. A significant improvement was observed in the case of the Eu3+ complex, while the Yb3+ analogue conserved photophysical properties in aqueous solvent. Two-photon (2P) microscopy imaging experiments on living T24 human cancer cells confirmed the spontaneous internalization of the probes and images with good signal-to-noise ratio were obtained in the classic NIR-to-visible configuration with the Eu3+ luminescent bioprobe and in the NIR-to-NIR with the Yb3+ one.

4.
Opt Express ; 27(20): 28384-28394, 2019 Sep 30.
Artigo em Inglês | MEDLINE | ID: mdl-31684592

RESUMO

Wavefront shaping is a powerful method to refocus light through a scattering medium. Its application to large spectral bandwidths or multiple wavelengths refocusing for nonlinear bio-imaging in-depth is however limited by spectral decorrelations. In this work, we demonstrate ways to access a large spectral memory of a refocus in thin scattering media and thick forward-scattering biological tissues. First, we show that the accessible spectral bandwidth through a scattering medium involves an axial spatio-spectral coupling, which can be minimized when working in a confocal geometry. Second, we show that this bandwidth can be further enlarged when working in a broadband excitation regime. These results open important prospects for multispectral nonlinear imaging through scattering media.

5.
J Cell Biol ; 218(10): 3415-3435, 2019 10 07.
Artigo em Inglês | MEDLINE | ID: mdl-31471457

RESUMO

Focal adhesion (FA) turnover depends on microtubules and actin. Microtubule ends are captured at FAs, where they induce rapid FA disassembly. However, actin's roles are less clear. Here, we use polarization-resolved microscopy, FRAP, live cell imaging, and a mutant of Adenomatous polyposis coli (APC-m4) defective in actin nucleation to investigate the role of actin assembly in FA turnover. We show that APC-mediated actin assembly is critical for maintaining normal F-actin levels, organization, and dynamics at FAs, along with organization of FA components. In WT cells, microtubules are captured repeatedly at FAs as they mature, but once a FA reaches peak maturity, the next microtubule capture event leads to delivery of an autophagosome, triggering FA disassembly. In APC-m4 cells, microtubule capture frequency and duration are altered, and there are long delays between autophagosome delivery and FA disassembly. Thus, APC-mediated actin assembly is required for normal feedback between microtubules and FAs, and maintaining FAs in a state "primed" for microtubule-induced turnover.


Assuntos
Actinas/metabolismo , Proteína da Polipose Adenomatosa do Colo/metabolismo , Adesões Focais/metabolismo , Microtúbulos/metabolismo , Humanos , Células Tumorais Cultivadas
6.
Opt Lett ; 44(9): 2137-2140, 2019 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-31042167

RESUMO

The measurement of the transmission matrix (TM) of a scattering medium is of great interest for imaging. It can be acquired directly by interferometry using an internal reference wavefront. Unfortunately, internal reference fields are scattered by the medium, which results in a speckle that makes the TM measurement heterogeneous across the output field of view. We demonstrate how to correct for this effect using the intrinsic properties of the TM. For thin scattering media, we exploit the memory effect of the medium and the reference speckle to create a corrected TM. For highly scattering media where the memory effect is negligible, we use complementary reference speckles to compose a new TM, not compromised by the speckled reference anymore. Using this correction, we demonstrate large field of view second harmonic generation imaging through thick biological media.

7.
J R Soc Interface ; 16(150): 20180611, 2019 01 31.
Artigo em Inglês | MEDLINE | ID: mdl-30958161

RESUMO

Type II collagen fibril diameters in cartilage are beneath the diffraction limit of optical microscopy, which makes the assessment of collagen organization very challenging. In this work we use polarization sensitive second harmonic generation (P-SHG) imaging to map collagen organization in articular cartilage, addressing in particular its behaviour under strain and changes which occur in osteoarthritis. P-SHG yields two parameters, molecular order and orientation, which provide measures of the degree of organization both at the molecular scale (below the diffraction limit) and above a few hundred nanometres (at the image pixel size). P-SHG clearly demonstrates the zonal collagen architecture and reveals differences in the structure of the fibrils around chondrocytes. P-SHG also reveals sub-micron scale fibril re-organization in cartilage strips exposed to tensile loading, with an increase in local organization in the superficial zone which weakly correlates with tensile modulus. Finally, P-SHG is used to investigate osteoarthritic cartilage from total knee replacement surgery, and reveals widespread heterogeneity across samples both microscale fibril orientations and their sub-micron organization. By addressing collagen fibril structure on scales intermediate between conventional light and electron microscopy, this study provides new insights into collagen micromechanics and mechanisms of degradation.


Assuntos
Cartilagem Articular , Condrócitos , Colágeno/metabolismo , Matriz Extracelular/metabolismo , Animais , Cartilagem Articular/citologia , Cartilagem Articular/metabolismo , Bovinos , Condrócitos/citologia , Condrócitos/metabolismo , Microscopia
8.
Opt Express ; 27(4): 5620-5640, 2019 Feb 18.
Artigo em Inglês | MEDLINE | ID: mdl-30876161

RESUMO

We report a detailed investigation on the second harmonic generation (SHG) emission from single 150 nm diameter non-centrosymmetric gold nanoparticles. Polarization-resolved analysis together with scanning electron microscopy images shows that these nanostructures exhibit a unique polarization-sensitive SHG that depends strongly on the particle's shape. An analytical approach based on multipolar analysis is introduced to link SHG properties to the nanoparticles' shape. Those multipolar modes can be probed using polarization-resolved SHG. This multipolar analysis offers a physical picture of the relation between shape (size, symmetries, defects, etc.) and nonlinear polarized optical efficiency.

9.
Biomed Opt Express ; 10(3): 1329-1338, 2019 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-30891349

RESUMO

Brillouin spectroscopy is an emerging analytical tool in biomedical and biophysical sciences. It probes viscoelasticity through the propagation of thermally induced acoustic waves at gigahertz frequencies. Brillouin light scattering (BLS) measurements have traditionally been performed using multipass Fabry-Pérot interferometers, which have high contrast and resolution, however, as they are scanning spectrometers they often require long acquisition times in poorly scattering media. In the last decade, a new concept of Brillouin spectrometer has emerged, making use of highly angle-dispersive virtually imaged phase array (VIPA) etalons, which enable fast acquisition times for minimally turbid materials, when high contrast is not imperative. The ability to acquire Brillouin spectra rapidly, together with long term system stability, make this system a viable candidate for use in biomedical applications, especially to probe live cells and tissues. While various methods are being developed to improve system contrast and speed, little work has been published discussing the details of imaging data analysis and spectral processing. Here we present a method that we developed for the automated retrieval of Brillouin line shape parameters from imaging data sets acquired with a dual-stage VIPA Brillouin microscope. We applied this method for the first time to BLS measurements of collagen gelatin hydrogels at different hydration levels and cross-linker concentrations. This work demonstrates that it is possible to obtain the relevant information from Brillouin spectra using software for real-time high-accuracy analysis.

10.
Sci Rep ; 9(1): 1670, 2019 02 08.
Artigo em Inglês | MEDLINE | ID: mdl-30737436

RESUMO

Ferroelectric nanocrystals have considerable interest for applications in nanophotonics, optical memories and bio-imaging. Their crystalline nature at the nanoscale remains however poorly known, mostly because structural investigation tools on single nanocrystals are lacking. In this work we apply polarization resolved second harmonic generation (P-SHG) imaging on isolated Barium Titanate (BaTiO3) nanocrystals to unravel their crystalline nature, exploiting the sensitivity of polarized SHG to local non-centrosymmetry and nanocrystals surface responses. We evidence crystalline heterogeneities in BaTiO3 nanocrystals manifested by a centrosymmetric shell around the tetragonal core of the crystals, corroborating hypotheses from previous ensemble structural investigations. This study shows that in contrast to bulk materials, nanocrystals exhibit a complex composition, which is seen to be reproducible among nanocrystals. P-SHG appears furthermore as a powerful methodology that reports structural behaviors in nanoscale dielectrics materials, at the individual nanoparticle scale.

11.
Opt Express ; 26(8): 9866-9881, 2018 Apr 16.
Artigo em Inglês | MEDLINE | ID: mdl-29715932

RESUMO

Fluorescence microscopy is widely used in biological imaging, however scattering from tissues strongly limits its applicability to a shallow depth. In this work we adapt a methodology inspired from stellar speckle interferometry, and exploit the optical memory effect to enable fluorescence microscopy through a turbid layer. We demonstrate efficient reconstruction of micrometer-size fluorescent objects behind a scattering medium in epi-microscopy, and study the specificities of this imaging modality (magnification, field of view, resolution) as compared to traditional microscopy. Using a modified phase retrieval algorithm to reconstruct fluorescent objects from speckle images, we demonstrate robust reconstructions even in relatively low signal to noise conditions. This modality is particularly appropriate for imaging in biological media, which are known to exhibit relatively large optical memory ranges compatible with tens of micrometers size field of views, and large spectral bandwidths compatible with emission fluorescence spectra of tens of nanometers widths.

12.
PLoS Biol ; 16(4): e2004718, 2018 04.
Artigo em Inglês | MEDLINE | ID: mdl-29702642

RESUMO

Sarcomeres are stereotyped force-producing mini-machines of striated muscles. Each sarcomere contains a pseudocrystalline order of bipolar actin and myosin filaments, which are linked by titin filaments. During muscle development, these three filament types need to assemble into long periodic chains of sarcomeres called myofibrils. Initially, myofibrils contain immature sarcomeres, which gradually mature into their pseudocrystalline order. Despite the general importance, our understanding of myofibril assembly and sarcomere maturation in vivo is limited, in large part because determining the molecular order of protein components during muscle development remains challenging. Here, we applied polarization-resolved microscopy to determine the molecular order of actin during myofibrillogenesis in vivo. This method revealed that, concomitantly with mechanical tension buildup in the myotube, molecular actin order increases, preceding the formation of immature sarcomeres. Mechanistically, both muscle and nonmuscle myosin contribute to this actin order gain during early stages of myofibril assembly. Actin order continues to increase while myofibrils and sarcomeres mature. Muscle myosin motor activity is required for the regular and coordinated assembly of long myofibrils but not for the high actin order buildup during sarcomere maturation. This suggests that, in muscle, other actin-binding proteins are sufficient to locally bundle or cross-link actin into highly regular arrays.


Assuntos
Citoesqueleto de Actina/ultraestrutura , Actinas/metabolismo , Drosophila melanogaster/ultraestrutura , Miofibrilas/ultraestrutura , Pupa/ultraestrutura , Sarcômeros/ultraestrutura , Citoesqueleto de Actina/metabolismo , Actinas/ultraestrutura , Animais , Fenômenos Biomecânicos , Conectina/metabolismo , Conectina/ultraestrutura , Drosophila melanogaster/crescimento & desenvolvimento , Drosophila melanogaster/metabolismo , Voo Animal/fisiologia , Microscopia de Polarização/métodos , Miofibrilas/metabolismo , Miosinas/metabolismo , Miosinas/ultraestrutura , Pupa/crescimento & desenvolvimento , Pupa/metabolismo , Sarcômeros/metabolismo
13.
Sci Rep ; 8(1): 4966, 2018 03 21.
Artigo em Inglês | MEDLINE | ID: mdl-29563576

RESUMO

Phosphoinositides (PIs) play important roles in numerous membrane-based cellular activities. However, their involvement in the mechanism of T cell receptor (TCR) signal transduction across the plasma membrane (PM) is poorly defined. Here, we investigate their role, and in particular that of phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] in TCR PM dynamics and activity in a mouse T-cell hybridoma upon ectopic expression of a PM-localized inositol polyphosphate-5-phosphatase (Inp54p). We observed that dephosphorylation of PI(4,5)P2 by the phosphatase increased the TCR/CD3 complex PM lateral mobility prior stimulation. The constitutive and antigen-elicited CD3 phosphorylation as well as the antigen-stimulated early signaling pathways were all found to be significantly augmented in cells expressing the phosphatase. Using state-of-the-art biophotonic approaches, we further showed that PI(4,5)P2 dephosphorylation strongly promoted the CD3ε cytoplasmic domain unbinding from the PM inner leaflet in living cells, thus resulting in an increased CD3 availability for interactions with Lck kinase. This could significantly account for the observed effects of PI(4,5)P2 dephosphorylation on the CD3 phosphorylation. Our data thus suggest that PIs play a key role in the regulation of the TCR/CD3 complex dynamics and activation at the PM.


Assuntos
Complexo CD3/metabolismo , Membrana Celular/metabolismo , Fosfatidilinositóis/metabolismo , Receptores de Antígenos de Linfócitos T/metabolismo , Linfócitos T/metabolismo , Animais , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Humanos , Hibridomas , Células Jurkat , Camundongos , Monoéster Fosfórico Hidrolases/genética , Monoéster Fosfórico Hidrolases/metabolismo , Fosforilação , Linfócitos T/citologia
14.
Biophys J ; 113(7): 1520-1530, 2017 Oct 03.
Artigo em Inglês | MEDLINE | ID: mdl-28978445

RESUMO

Myelin around axons is currently widely studied by structural analyses and large-scale imaging techniques, with the goal to decipher its critical role in neuronal protection. Although there is strong evidence that in myelin, lipid composition, and lipid membrane morphology are affected during the progression of neurodegenerative diseases, there is no quantitative method yet to report its ultrastructure in tissues at both molecular and macroscopic levels, in conditions potentially compatible with in vivo observations. In this work, we study and quantify the molecular order of lipids in myelin at subdiffraction scales, using label-free polarization-resolved coherent anti-Stokes Raman, which exploits coherent anti-Stokes Raman sensitivity to coupling between light polarization and oriented molecular vibrational bonds. Importantly, the method does not use any a priori parameters in the sample such as lipid type, orientational organization, and composition. We show that lipid molecular order of myelin in the mouse spinal cord is significantly reduced throughout the progression of experimental autoimmune encephalomyelitis, a model for multiple sclerosis, even in myelin regions that appear morphologically unaffected. This technique permits us to unravel molecular-scale perturbations of lipid layers at an early stage of the demyelination progression, whereas the membrane architecture at the mesoscopic scale (here ∼100 nm) seems much less affected. Such information cannot be brought by pure morphological observation and, to our knowledge, brings a new perspective to molecular-scale understanding of neurodegenerative diseases.


Assuntos
Encefalomielite Autoimune Experimental/metabolismo , Lipídeos , Bainha de Mielina/metabolismo , Microscopia Óptica não Linear , Animais , Progressão da Doença , Encefalomielite Autoimune Experimental/patologia , Adjuvante de Freund , Lipídeos/química , Membranas Artificiais , Camundongos Endogâmicos C57BL , Bainha de Mielina/química , Bainha de Mielina/patologia , Glicoproteína Mielina-Oligodendrócito , Fragmentos de Peptídeos , Medula Espinal/química , Medula Espinal/metabolismo , Medula Espinal/patologia
15.
Appl Opt ; 56(16): 4827, 2017 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-29047620

RESUMO

This publisher's note amends the author list and Acknowledgments in Appl. Opt.56, 2589 (2017)APOPAI0003-693510.1364/AO.56.002589.

16.
Sci Rep ; 7(1): 12482, 2017 10 02.
Artigo em Inglês | MEDLINE | ID: mdl-28970520

RESUMO

Thioflavin T (ThT) is standardly used as a fluorescent marker to detect aggregation of amyloid fibrils by conventional fluorescence microscopy, including polarization resolved imaging that brings information on the orientational order of the fibrils. These techniques are however diffraction limited and cannot provide fine structural details at the fibrils scales of 10-100 nm, which lie beyond the diffraction limit. In this work, we evaluate the capacity of ThT to photoswitch when bound to insulin amyloids by adjusting the redox properties of its environment. We demonstrate that on-off duty cycles, intensity and photostability of the ThT fluorescence emission under adequate buffer conditions permit stochastic super-resolution imaging with a localization precision close to 20 nm. We show moreover that signal to noise conditions allow polarized orientational imaging of single ThT molecules, which reveals ultra-structure signatures related to protofilaments twisting within amyloid fibrils.


Assuntos
Amiloide/ultraestrutura , Benzotiazóis/química , Corantes Fluorescentes/química , Insulina/química , Imagem Óptica/métodos , Imagem Individual de Molécula/métodos , Amiloide/química , Animais , Bovinos , Humanos , Hidrazinas/química , Imagem Óptica/instrumentação , Imagem Individual de Molécula/instrumentação , Soluções
17.
Sci Adv ; 3(9): e1600743, 2017 09.
Artigo em Inglês | MEDLINE | ID: mdl-28879230

RESUMO

The control and use of light polarization in optical sciences and engineering are widespread. Despite remarkable developments in polarization-resolved imaging for life sciences, their transposition to strongly scattering media is currently not possible, because of the inherent depolarization effects arising from multiple scattering. We show an unprecedented phenomenon that opens new possibilities for polarization-resolved microscopy in strongly scattering media: polarization recovery via broadband wavefront shaping. We demonstrate focusing and recovery of the original injected polarization state without using any polarizing optics at the detection. To enable molecular-level structural imaging, an arbitrary rotation of the input polarization does not degrade the quality of the focus. We further exploit the robustness of polarization recovery for structural imaging of biological tissues through scattering media. We retrieve molecular-level organization information of collagen fibers by polarization-resolved second harmonic generation, a topic of wide interest for diagnosis in biomedical optics. Ultimately, the observation of this new phenomenon paves the way for extending current polarization-based methods to strongly scattering environments.

18.
Chem Commun (Camb) ; 53(44): 6005-6008, 2017 May 30.
Artigo em Inglês | MEDLINE | ID: mdl-28516180

RESUMO

An Yb(iii) complex based on a dimethyl cyclen macrocyclic ligand functionalized by charge transfer antennae was prepared. This cationic [YbL3]+ complex is stable and soluble in water and presents interesting photophysical nonlinear properties. It is spontaneously internalized and accumulates in live cells. High quality images have been obtained both in a classical NIR-to-vis configuration and in the more challenging NIR-to-NIR one.


Assuntos
Complexos de Coordenação/química , Corantes Fluorescentes/química , Imagem Óptica , Fótons , Itérbio/química , Cátions/química , Linhagem Celular Tumoral , Sobrevivência Celular , Humanos , Raios Infravermelhos , Conformação Molecular
19.
J Opt Soc Am A Opt Image Sci Vis ; 33(7): 1353-62, 2016 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-27409693

RESUMO

Second-harmonic generation microscopy can provide estimation of some local molecule distribution properties. However, in order not to get erroneous conclusions, it is important to detect measurements with insufficient precision. Such a detection technique is developed considering an approximation of the ultimate precision provided by the Cramer-Rao bound. This method is characterized and a simple approximation of its detection and false alarm probabilities is developed.

20.
Nat Commun ; 7: 11562, 2016 05 18.
Artigo em Inglês | MEDLINE | ID: mdl-27189667

RESUMO

Nonlinear optical methods, such as coherent anti-Stokes Raman scattering and stimulated Raman scattering, are able to perform label-free imaging, with chemical bonds specificity. Here we demonstrate that the use of circularly polarized light allows to retrieve not only the chemical nature but also the symmetry of the probed sample, in a single measurement. Our symmetry-resolved scheme offers simple access to the local organization of vibrational bonds and as a result provides enhanced image contrast for anisotropic samples, as well as an improved chemical selectivity. We quantify the local organization of vibrational bonds on crystalline and biological samples, thus providing information not accessible by spontaneous Raman and stimulated Raman scattering techniques. This work stands for a symmetry-resolved contrast in vibrational microscopy, with potential application in biological diagnostic.

SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA