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1.
Nat Methods ; 21(1): 132-141, 2024 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-38129618

RESUMEN

Multiphoton microscopy can resolve fluorescent structures and dynamics deep in scattering tissue and has transformed neural imaging, but applying this technique in vivo can be limited by the mechanical and optical constraints of conventional objectives. Short working distance objectives can collide with compact surgical windows or other instrumentation and preclude imaging. Here we present an ultra-long working distance (20 mm) air objective called the Cousa objective. It is optimized for performance across multiphoton imaging wavelengths, offers a more than 4 mm2 field of view with submicrometer lateral resolution and is compatible with commonly used multiphoton imaging systems. A novel mechanical design, wider than typical microscope objectives, enabled this combination of specifications. We share the full optical prescription, and report performance including in vivo two-photon and three-photon imaging in an array of species and preparations, including nonhuman primates. The Cousa objective can enable a range of experiments in neuroscience and beyond.


Asunto(s)
Colorantes , Microscopía de Fluorescencia por Excitación Multifotónica , Animales , Microscopía de Fluorescencia por Excitación Multifotónica/métodos
2.
Proc Natl Acad Sci U S A ; 113(31): 8729-34, 2016 08 02.
Artículo en Inglés | MEDLINE | ID: mdl-27439876

RESUMEN

Concentration gradients of soluble proteins are believed to be responsible for control of morphogenesis of subcellular systems, but the mechanisms that generate the spatial organization of these subcellular gradients remain poorly understood. Here, we use a newly developed multipoint fluorescence fluctuation spectroscopy technique to study the ras-related nuclear protein (Ran) pathway, which forms soluble gradients around chromosomes in mitosis and is thought to spatially regulate microtubule behaviors during spindle assembly. We found that the distribution of components of the Ran pathway that influence microtubule behaviors is determined by their interactions with microtubules, resulting in microtubule nucleators being localized by the microtubules whose formation they stimulate. Modeling and perturbation experiments show that this feedback makes the length of the spindle insensitive to the length scale of the Ran gradient, allows the spindle to assemble outside the peak of the Ran gradient, and explains the scaling of the spindle with cell size. Such feedback between soluble signaling pathways and the mechanics of the cytoskeleton may be a general feature of subcellular organization.


Asunto(s)
Microtúbulos/metabolismo , Mitosis , Transducción de Señal , Proteína de Unión al GTP ran/metabolismo , Algoritmos , Línea Celular Tumoral , Retroalimentación Fisiológica , Humanos , Microscopía Confocal , Unión Proteica , Interferencia de ARN , Huso Acromático/metabolismo , Proteína de Unión al GTP ran/genética
3.
Biophys J ; 106(8): 1578-87, 2014 Apr 15.
Artículo en Inglés | MEDLINE | ID: mdl-24739157

RESUMEN

The spatial organization of microtubule polarity, and the interplay between microtubule polarity and protein localization, is thought to be crucial for spindle assembly, anaphase, and cytokinesis, but these phenomena remain poorly understood, in part due to the difficulty of measuring microtubule polarity in spindles. We develop and implement a method to nonperturbatively and quantitatively measure microtubule polarity throughout spindles using a combination of second-harmonic generation and two-photon fluorescence. We validate this method using computer simulations and by comparison to structural data on spindles obtained from electron tomography and laser ablation. This method should provide a powerful tool for studying spindle organization and function, and may be applicable for investigating microtubule polarity in other systems.


Asunto(s)
Polaridad Celular , Simulación por Computador , Microtúbulos/metabolismo , Huso Acromático/metabolismo , Animales , Caenorhabditis elegans/citología , Caenorhabditis elegans/embriología , Caenorhabditis elegans/metabolismo , Extractos Celulares , Embrión no Mamífero/citología , Embrión no Mamífero/metabolismo , Xenopus laevis
4.
bioRxiv ; 2024 Jan 23.
Artículo en Inglés | MEDLINE | ID: mdl-38328224

RESUMEN

The goal of this protocol is to enable better characterisation of multiphoton microscopy hardware across a large user base. The scope of this protocol is purposefully limited to focus on hardware, touching on software and data analysis routines only where relevant. The intended audiences are scientists using and building multiphoton microscopes in their laboratories. The goal is that any scientist, not only those with optical expertise, can test whether their multiphoton microscope is performing well and producing consistent data over the lifetime of their system.

5.
Opt Express ; 21(25): 31604-14, 2013 Dec 16.
Artículo en Inglés | MEDLINE | ID: mdl-24514733

RESUMEN

In this paper, we examine the performance of a Blu-ray disk (BD) aspheric lens as the objective of a miniaturized scanning nonlinear optical microscope. By combining a single 2D micro-electro mechanical system (MEMS) mirror as the scanner and with different tube lens pairs, the field of view (FOV) of the studied microscope varies from 59 µm × 93 µm up to 178 µm × 280 µm, while the corresponding lateral resolution varies from 0.6 µm to 2 µm for two-photon fluorescence (2PF) signals. With a 34/s video frame rate, in vivo dynamic observation of zebrafish heartbeat through 2PF of the excited green fluorescence protein (GFP) is demonstrated.


Asunto(s)
Discos Compactos , Aumento de la Imagen/instrumentación , Lentes , Microscopía de Fluorescencia por Excitación Multifotónica/instrumentación , Diseño de Equipo , Análisis de Falla de Equipo , Miniaturización
6.
bioRxiv ; 2023 Oct 26.
Artículo en Inglés | MEDLINE | ID: mdl-37961478

RESUMEN

Recent studies have highlighted the significance of the spindle midzone - the region positioned between chromosomes - in ensuring proper chromosome segregation. By combining advanced 3D electron tomography and cutting-edge light microscopy we have discovered a previously unknown role of the regulation of microtubule dynamics within the spindle midzone of C. elegans. Using Fluorescence recovery after photobleaching and a combination of second harmonic generation and two-photon fluorescence microscopy, we found that the length of the antiparallel microtubule overlap zone in the spindle midzone is constant throughout anaphase, and independent of cortical pulling forces as well as the presence of the microtubule bundling protein SPD-1. Further investigations of SPD-1 and the chromokinesin KLP-19 in C. elegans suggest that KLP-19 regulates the overlap length and functions independently of SPD-1. Our data shows that KLP-19 plays an active role in regulating the length and turn-over of microtubules within the midzone as well as the size of the antiparallel overlap region throughout mitosis. Depletion of KLP-19 in mitosis leads to an increase in microtubule length in the spindle midzone, which also leads to increased microtubule - microtubule interaction, thus building up a more robust microtubule network. The spindle is globally stiffer and more stable, which has implications for the transmission of forces within the spindle affecting chromosome segregation dynamics. Our data shows that by localizing KLP-19 to the spindle midzone in anaphase microtubule dynamics can be locally controlled allowing the formation of a functional midzone.

7.
Elife ; 112022 07 01.
Artículo en Inglés | MEDLINE | ID: mdl-35775393

RESUMEN

The hippocampus consists of a stereotyped neuronal circuit repeated along the septal-temporal axis. This transverse circuit contains distinct subfields with stereotyped connectivity that support crucial cognitive processes, including episodic and spatial memory. However, comprehensive measurements across the transverse hippocampal circuit in vivo are intractable with existing techniques. Here, we developed an approach for two-photon imaging of the transverse hippocampal plane in awake mice via implanted glass microperiscopes, allowing optical access to the major hippocampal subfields and to the dendritic arbor of pyramidal neurons. Using this approach, we tracked dendritic morphological dynamics on CA1 apical dendrites and characterized spine turnover. We then used calcium imaging to quantify the prevalence of place and speed cells across subfields. Finally, we measured the anatomical distribution of spatial information, finding a non-uniform distribution of spatial selectivity along the DG-to-CA1 axis. This approach extends the existing toolbox for structural and functional measurements of hippocampal circuitry.


Asunto(s)
Hipocampo , Células Piramidales , Animales , Dendritas/fisiología , Hipocampo/fisiología , Ratones , Neuronas/fisiología , Células Piramidales/fisiología
8.
Nat Commun ; 12(1): 6639, 2021 11 17.
Artículo en Inglés | MEDLINE | ID: mdl-34789723

RESUMEN

Imaging the activity of neurons that are widely distributed across brain regions deep in scattering tissue at high speed remains challenging. Here, we introduce an open-source system with Dual Independent Enhanced Scan Engines for Large field-of-view Two-Photon imaging (Diesel2p). Combining optical design, adaptive optics, and temporal multiplexing, the system offers subcellular resolution over a large field-of-view of ~25 mm2, encompassing distances up to 7 mm, with independent scan engines. We demonstrate the flexibility and various use cases of this system for calcium imaging of neurons in the living brain.


Asunto(s)
Microscopía de Fluorescencia por Excitación Multifotónica/métodos , Red Nerviosa/fisiología , Imagen Óptica/métodos , Encéfalo/citología , Encéfalo/fisiología , Calcio/metabolismo , Diseño de Equipo , Microscopía de Fluorescencia por Excitación Multifotónica/instrumentación , Red Nerviosa/citología , Neuronas/citología , Neuronas/fisiología , Imagen Óptica/instrumentación
9.
Elife ; 102021 06 11.
Artículo en Inglés | MEDLINE | ID: mdl-34114562

RESUMEN

Most female meiotic spindles undergo striking morphological changes while transitioning from metaphase to anaphase. The ultra-structure of meiotic spindles, and how changes to this structure correlate with such dramatic spindle rearrangements remains largely unknown. To address this, we applied light microscopy, large-scale electron tomography and mathematical modeling of female meiotic Caenorhabditis elegans spindles. Combining these approaches, we find that meiotic spindles are dynamic arrays of short microtubules that turn over within seconds. The results show that the metaphase to anaphase transition correlates with an increase in microtubule numbers and a decrease in their average length. Detailed analysis of the tomographic data revealed that the microtubule length changes significantly during the metaphase-to-anaphase transition. This effect is most pronounced for microtubules located within 150 nm of the chromosome surface. To understand the mechanisms that drive this transition, we developed a mathematical model for the microtubule length distribution that considers microtubule growth, catastrophe, and severing. Using Bayesian inference to compare model predictions and data, we find that microtubule turn-over is the major driver of the spindle reorganizations. Our data suggest that in metaphase only a minor fraction of microtubules, those closest to the chromosomes, are severed. The large majority of microtubules, which are not in close contact with chromosomes, do not undergo severing. Instead, their length distribution is fully explained by growth and catastrophe. This suggests that the most prominent drivers of spindle rearrangements are changes in nucleation and catastrophe rate. In addition, we provide evidence that microtubule severing is dependent on katanin.


Asunto(s)
Caenorhabditis elegans/metabolismo , Meiosis , Microtúbulos/metabolismo , Oocitos/metabolismo , Huso Acromático/metabolismo , Anafase , Animales , Teorema de Bayes , Proteínas de Caenorhabditis elegans/metabolismo , Segregación Cromosómica , Cromosomas/metabolismo , Tomografía con Microscopio Electrónico/métodos , Femenino , Katanina/metabolismo , Metafase , Modelos Teóricos
10.
Opt Express ; 18(23): 24085-91, 2010 Nov 08.
Artículo en Inglés | MEDLINE | ID: mdl-21164755

RESUMEN

Without cavity dumping or external amplification, we report a femtosecond Cr:forsterite laser with a 1.4 W output power and 2 W in continuous wave (CW) operated with a crystal temperature of 267 K. In the femtosecond regime, the oscillator generates Kerr-lens-mode-locked 84 fs pulses with a repetition rate of 85 MHz, corresponding to a high 16.5 nJ pulse energy directly from a single Cr:forsterite resonator. This intense femtosecond Cr:forsterite laser is ideal to pump varieties of high power fiber light sources and could be thus ideal for many biological and spectroscopy applications.


Asunto(s)
Cromo/química , Electricidad , Rayos Láser , Compuestos de Silicona/química , Análisis Espectral , Factores de Tiempo
11.
Opt Express ; 18(16): 17382-91, 2010 Aug 02.
Artículo en Inglés | MEDLINE | ID: mdl-20721125

RESUMEN

With a micro-electro-mechanical system (MEMS) mirror, we successfully developed a miniaturized epi-third-harmonic-generation (epi-THG) fiber-microscope with a video frame rate (31 Hz), which was designed for in vivo optical biopsy of human skin. With a large-mode-area (LMA) photonic crystal fiber (PCF) and a regular microscopic objective, the nonlinear distortion of the ultrafast pulses delivery could be much reduced while still achieving a 0.4 microm lateral resolution for epi-THG signals. In vivo real time virtual biopsy of the Asian skin with a video rate (31 Hz) and a sub-micron resolution was obtained. The result indicates that this miniaturized system was compact enough for the least invasive hand-held clinical use.


Asunto(s)
Tecnología de Fibra Óptica/instrumentación , Aumento de la Imagen/instrumentación , Sistemas Microelectromecánicos , Microscopía/instrumentación , Fibras Ópticas , Fotones , Grabación de Cinta de Video/instrumentación , Diseño de Equipo , Humanos , Miniaturización
12.
Opt Express ; 18(7): 7397-406, 2010 Mar 29.
Artículo en Inglés | MEDLINE | ID: mdl-20389762

RESUMEN

Direct-backward third harmonic generation (DBTHG) has been regarded as negligible or even inexistent due to the large value of wave-vector mismatch. In the past, BTHG signals were often interpreted as back-reflected or back-scattered forward-THG (FTHG). In this paper, we theoretically and experimentally demonstrate that backward third harmonic waves can be directly generated, and that their magnitude can be comparable with FTHG in nanostructures. Experimental data of DBTHG from ZnO thin films, CdSe quantum dots and Fe(3)O(4) nanoparticles agree well with simulation results based on the Green's function. An integral equation was also derived for fast computation of DBTHG in nano films. Our investigation suggests that DBTHG can be a potentially powerful tool in nano-science research, especially when combined with FTHG measurements.


Asunto(s)
Nanoestructuras/química , Nanotecnología/métodos , Óptica y Fotónica , Algoritmos , Simulación por Computador , Compuestos Férricos/química , Procesamiento de Imagen Asistido por Computador , Modelos Estadísticos , Distribución Normal , Puntos Cuánticos
13.
Elife ; 92020 09 23.
Artículo en Inglés | MEDLINE | ID: mdl-32966209

RESUMEN

The spindle shows remarkable diversity, and changes in an integrated fashion, as cells vary over evolution. Here, we provide a mechanistic explanation for variations in the first mitotic spindle in nematodes. We used a combination of quantitative genetics and biophysics to rule out broad classes of models of the regulation of spindle length and dynamics, and to establish the importance of a balance of cortical pulling forces acting in different directions. These experiments led us to construct a model of cortical pulling forces in which the stoichiometric interactions of microtubules and force generators (each force generator can bind only one microtubule), is key to explaining the dynamics of spindle positioning and elongation, and spindle final length and scaling with cell size. This model accounts for variations in all the spindle traits we studied here, both within species and across nematode species spanning over 100 million years of evolution.


Asunto(s)
Caenorhabditis elegans , Tamaño de la Célula , Microtúbulos , Huso Acromático , Animales , Caenorhabditis elegans/citología , Caenorhabditis elegans/genética , Caenorhabditis elegans/fisiología , Evolución Molecular , Microtúbulos/química , Microtúbulos/genética , Microtúbulos/metabolismo , Modelos Biológicos , Fenotipo , Huso Acromático/química , Huso Acromático/genética , Huso Acromático/metabolismo
14.
J Biomed Opt ; 14(1): 014008, 2009.
Artículo en Inglés | MEDLINE | ID: mdl-19256696

RESUMEN

Atopic dermatitis (AD) is characterized by hyperkeratosis of epidermis and fibrosis within dermis in chronic skin lesions. Thus far, the histology of skin lesions has been evaluated only by examination of excised specimens. A noninvasive in vivo tool is essential to evaluate the histopathological changes during the clinical course of AD. We used Cr:forsterite laser-based multimodality nonlinear microscopy to analyze the endogenous molecular signals, including third-harmonic generation (THG), second-harmonic generation (SHG), and two-photon fluorescence (TPF) from skin lesions in AD. Significant differences in thickness of epidermis and stratum corneum (SC), and modified degrees of fibrosis in dermis (measured by THG signals and SHG signals, respectively), are clearly demonstrated in in vitro studies. Increased TPF levels are positively associated with the levels of the THG signals from the SC. Our in vitro observations of histological changes are replicated in the in vivo studies. These findings were reproducible in skin lesions from human AD. For the first time, we demonstrate the feasibility of preclinical applications of Cr:forsterite laser-based nonlinear microscopy. Our findings suggest that the optical signatures of THG, TPF, and SHG can be used as molecular markers to assess the pathophysiological process of AD and the effects of local treatment.


Asunto(s)
Dermoscopía/métodos , Hiperqueratosis Epidermolítica/patología , Microscopía Confocal/métodos , Microscopía de Fluorescencia por Excitación Multifotónica/métodos , Animales , Dermatitis Atópica , Femenino , Fibrosis , Hiperqueratosis Epidermolítica/complicaciones , Ratones , Ratones Endogámicos C57BL , Ratones Desnudos , Reproducibilidad de los Resultados , Sensibilidad y Especificidad
15.
Nat Phys ; 15(12): 1295-1300, 2019 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-32322291

RESUMEN

Cytoskeletal networks are foundational examples of active matter and central to self-organized structures in the cell. In vivo, these networks are active and densely crosslinked. Relating their large-scale dynamics to the properties of their constituents remains an unsolved problem. Here, we study an in vitro active gel made from aligned microtubules and XCTK2 kinesin motors. Using photobleaching, we demonstrate that the gel's aligned microtubules, driven by motors, continually slide past each other at a speed independent of the local microtubule polarity and motor concentration. This phenomenon is also observed, and remains unexplained, in spindles. We derive a general framework for coarse graining microtubule gels crosslinked by molecular motors from microscopic considerations. Using microtubule-microtubule coupling through a force-velocity relationship for kinesin, this theory naturally explains the experimental results: motors generate an active strain rate in regions of changing polarity, which allows microtubules of opposite polarities to slide past each other without stressing the material.

16.
Mol Biol Cell ; 30(19): 2503-2514, 2019 09 01.
Artículo en Inglés | MEDLINE | ID: mdl-31339442

RESUMEN

Spindle microtubules, whose dynamics vary over time and at different locations, cooperatively drive chromosome segregation. Measurements of microtubule dynamics and spindle ultrastructure can provide insight into the behaviors of microtubules, helping elucidate the mechanism of chromosome segregation. Much work has focused on the dynamics and organization of kinetochore microtubules, that is, on the region between chromosomes and poles. In comparison, microtubules in the central-spindle region, between segregating chromosomes, have been less thoroughly characterized. Here, we report measurements of the movement of central-spindle microtubules during chromosome segregation in human mitotic spindles and Caenorhabditis elegans mitotic and female meiotic spindles. We found that these central-spindle microtubules slide apart at the same speed as chromosomes, even as chromosomes move toward spindle poles. In these systems, damaging central-spindle microtubules by laser ablation caused an immediate and complete cessation of chromosome motion, suggesting a strong coupling between central-spindle microtubules and chromosomes. Electron tomographic reconstruction revealed that the analyzed anaphase spindles all contain microtubules with both ends between segregating chromosomes. Our results provide new dynamical, functional, and ultrastructural characterizations of central-spindle microtubules during chromosome segregation in diverse spindles and suggest that central-spindle microtubules and chromosomes are strongly coupled in anaphase.


Asunto(s)
Segregación Cromosómica/fisiología , Huso Acromático/metabolismo , Polos del Huso/metabolismo , Anafase/genética , Animales , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/genética , Línea Celular Tumoral , Segregación Cromosómica/genética , Cromosomas/genética , Cromosomas/fisiología , Humanos , Cinetocoros/metabolismo , Meiosis/genética , Microtúbulos/metabolismo , Huso Acromático/genética , Polos del Huso/genética
17.
Science ; 366(6467): 870-875, 2019 11 15.
Artículo en Inglés | MEDLINE | ID: mdl-31727834

RESUMEN

A van der Waals heterostructure built from atomically thin semiconducting transition metal dichalcogenides (TMDs) enables the formation of excitons from electrons and holes in distinct layers, producing interlayer excitons with large binding energy and a long lifetime. By employing heterostructures of monolayer TMDs, we realize optical and electrical generation of long-lived neutral and charged interlayer excitons. We demonstrate that neutral interlayer excitons can propagate across the entire sample and that their propagation can be controlled by excitation power and gate electrodes. We also use devices with ohmic contacts to facilitate the drift motion of charged interlayer excitons. The electrical generation and control of excitons provide a route for achieving quantum manipulation of bosonic composite particles with complete electrical tunability.

18.
Opt Express ; 16(13): 9534-48, 2008 Jun 23.
Artículo en Inglés | MEDLINE | ID: mdl-18575520

RESUMEN

We demonstrated that lipid-enclosed CdSe quantum dots (LEQDs) can function as versatile contrast agents in epi-detection third harmonic generation (THG) microscopy for biological applications in vivo. With epi-THG intensities 20 times stronger than corresponding fluorescence intensities from the same LEQDs under the same conditions of energy absorption, such high brightness LEQDs were proved for the abilities of cell tracking and detection of specific molecular expression in live cancer cells. Using nude mice as an animal model, the distribution of LEQD-loaded tumor cells deep in subcutaneous tissues were imaged with high THG contrast. This is the first demonstration that THG contrast can be manipulated in vivo with nanoparticles. By linking LEQDs with anti-Her2 antibodies, the expression of Her2/neu receptors in live breast cancer cells could also be easily detected through THG. Compared with fluorescence modalities, the THG modality also provides the advantage of no photobleaching and photoblinkin g effects. Combined with a high penetration 1230 nm laser, these novel features make LEQDs excellent THG contrast agents for in vivo deep-tissue imaging in the future.


Asunto(s)
Compuestos de Cadmio , Perfilación de la Expresión Génica/métodos , Aumento de la Imagen/métodos , Lípidos/química , Microscopía Fluorescente/métodos , Neoplasias/patología , Puntos Cuánticos , Compuestos de Selenio , Animales , Compuestos de Cadmio/química , Línea Celular Tumoral , Materiales Biocompatibles Revestidos/química , Medios de Contraste , Portadores de Fármacos/química , Humanos , Ratones , Ratones Desnudos , Técnicas de Sonda Molecular , Compuestos de Selenio/química
19.
Elife ; 72018 07 25.
Artículo en Inglés | MEDLINE | ID: mdl-30044223

RESUMEN

Proper kinetochore-microtubule attachments, mediated by the NDC80 complex, are required for error-free chromosome segregation. Erroneous attachments are corrected by the tension dependence of kinetochore-microtubule interactions. Here, we present a method, based on fluorescence lifetime imaging microscopy and Förster resonance energy transfer, to quantitatively measure the fraction of NDC80 complexes bound to microtubules at individual kinetochores in living human cells. We found that NDC80 binding is modulated in a chromosome autonomous fashion over prometaphase and metaphase, and is predominantly regulated by centromere tension. We show that this tension dependency requires phosphorylation of the N-terminal tail of Hec1, a component of the NDC80 complex, and the proper localization of Aurora B kinase, which modulates NDC80 binding. Our results lead to a mathematical model of the molecular basis of tension-dependent NDC80 binding to kinetochore microtubules in vivo.


Asunto(s)
Cinetocoros/metabolismo , Microtúbulos/metabolismo , Proteínas Nucleares/metabolismo , Aurora Quinasa B/metabolismo , Calibración , Línea Celular Tumoral , Simulación por Computador , Proteínas del Citoesqueleto , Transferencia Resonante de Energía de Fluorescencia , Humanos , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Metafase , Modelos Biológicos , Método de Montecarlo , Proteínas Serina-Treonina Quinasas/metabolismo , Tubulina (Proteína)/metabolismo
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