A portable extensional rheometer for measuring the viscoelasticity of pitcher plant and other sticky liquids in the field.

BACKGROUND: Biological fluids often have interesting and unusual physical properties to adapt them for their specific purpose. Laboratory-based rheometers can be used to characterise the viscoelastic properties of such fluids. This, however, can be challenging as samples often do not retain their natural properties in storage while conventional rheometers are fragile and expensive devices ill-suited for field measurements. We present a portable, low-cost extensional rheometer designed specifically to enable in situ studies of biological fluids in the field. The design of the device (named Seymour) is based on a conventional capillary break-up extensional rheometer (the Cambridge Trimaster). It works by rapidly stretching a small fluid sample between two metal pistons. A battery-operated solenoid switch triggers the pistons to move apart rapidly and a compact, robust and inexpensive, USB 3 high speed camera is used to record the thinning and break-up of the fluid filament that forms between the pistons. The complete setup runs independently of mains electricity supply and weighs approximately 1 kg. Post-processing and analysis of the recorded images to extract rheological parameters is performed using open source software. RESULTS: The device was tested both in the laboratory and in the field, in Brunei Darussalam, using calibration fluids (silicone oil and carboxymethyl cellulose solutions) as well as Nepenthes pitcher plant trapping fluids as an example of a viscoelastic biological fluid. The fluid relaxation times ranged from 1 ms to over 1 s. The device gave comparable performance to the Cambridge Trimaster. Differences in fluid viscoelasticity between three species were quantified, as well as the change in viscoelasticity with storage time. This, together with marked differences between N. rafflesiana fluids taken from greenhouse and wild plants, confirms the need for a portable device. CONCLUSIONS: Proof of concept of the portable rheometer was demonstrated. Quantitative measurements of pitcher plant fluid viscoelasticity were made in the natural habitat for the first time. The device opens up opportunities for studying a wide range of plant fluids and secretions, under varying experimental conditions, or with changing temperatures and weather conditions.The following financial support is gratefully acknowledged: a Henslow Research Fellowship from the Cambridge Philosophical Society and a Leverhulme Early Career Fellowship for UB; a visiting research fellowship (POS-A/2012/116) for MDT from Xunta de Galicia’s Consellería de Cultura, Educación e Ordenación Universitaria of Spain and the European Union’s European Social Fund; and a summer project grant for CC from Sidney Sussex College, Cambridge.This is the final version of the article. It first appeared at http://www.plantmethods.com/content/11/1/16

Fast in vivo imaging of SHG nanoprobes with multiphoton light-sheet microscopy

Two-photon light-sheet microscopy (2P-SPIM) provides a unique combination of advantages for fast and deep fluorescence imaging in live tissues. Detecting coherent signals such as second-harmonic generation (SHG) in 2P-SPIM in addition to fluorescence would open further imaging opportunities. However, light-sheet microscopy involves an orthogonal configuration of illumination and detection that questions the ability to detect coherent signals. Indeed, coherent scattering from micron-sized structures occurs predominantly along the illumination beam. By contrast, point-like sources such as SHG nanocrystals can efficiently scatter light in multiple directions and be detected using the orthogonal geometry of a light-sheet microscope. This study investigates the suitability of SHG light-sheet microscopy (SHG-SPIM) for fast imaging of SHG nanoprobes. Parameters that govern the detection efficiency of KTiOPO4 and BaTiO3 nanocrystals using SHG-SPIM are investigated theoretically and experimentally. The effects of incident polarization, detection numerical aperture, nanocrystal rotational motion, and second-order susceptibility tensor symmetries on the detectability of SHG nanoprobes in this specific geometry are clarified. Guidelines for optimizing SHG-SPIM imaging are established, enabling fast in vivo light-sheet imaging combining SHG and two-photon excited fluorescence. Finally, microangiography was achieved in live zebrafish embryos by SHG imaging at up to 180 frames per second and single-particle tracking of SHG nanoprobes in the blood flow

Monitoring the orientation of rare-earth-doped nanorods for flow shear tomography

Rare-earth phosphors exhibit unique luminescence polarization features originating from the anisotropic symmetry of the emitter ion's chemical environment. However, to take advantage of this peculiar property, it is necessary to control and measure the ensemble orientation of the host particles with a high degree of precision. Here, we show a methodology to obtain the photoluminescence polarization of Eu-doped LaPO4 nano rods assembled in an electrically modulated liquid-crystalline phase. We measure Eu3+ emission spectra for the three main optimal configurations ({\sigma}, {\pi} and {\alpha}, depending on the direction of observation and the polarization axes) and use them as a reference for the nano rod orientation analysis. Based on the fact that flowing nano rods tend to orient along the shear strain profile, we use this orientation analysis to measure the local shear rate in a flowing liquid. The potential of this approach is then demonstrated through tomographic imaging of the shear rate distribution in a microfluidic system.Comment: 8 pages, 3 figures + supplementary files for experimental and numerical method

Development of polarized nanoemitters as probes for orientation measurements

Les nanoparticules luminescentes sont particulièrement étudiées pour leur application dans les systèmes d’éclairages ou comme sondes en bio-imagerie. Parmi elles, les nanoparticules anisotropes de matrices cristallines dopées par des ions lanthanides présentent une émission polarisée, qui dépend de la symétrie des sites des ions émetteurs. Le lien entre direction de polarisation et axes cristallins des nanocristaux permet de déterminer leur orientation, et peut donc être exploité pour suivre l'orientation d’objets ou pour caractériser la déformation de milieux hôtes.Les objectifs de ce doctorat ont été de s’intéresser aux origines fondamentales de l’émission polarisée de nanobâtonnets de phosphate de lanthane dopés par des ions europium trivalents (LaPO4:Eu) et d’utiliser la luminescence polarisée à des mesures d’orientation.Dans une première partie, les nanobâtonnets de LaPO4:Eu ont été synthétisés puis alignés sous forme des films orientés. La luminescence de ces films a permis de suivre avec précision la transition de phase de la matrice hôte, de sa structure hexagonale à une structure monoclinique ; et de mettre en évidence la présence de défauts structuraux. La polarisation des spectres de luminescence a ensuite été étudiée. Les taux de polarisation mesurés sont plus élevés pour la phase monoclinique que pour la phase hexagonale. La sensibilité du spectre de polarisation au milieu diélectrique qui les entoure a été mise en évidence.La seconde partie de cette étude porte sur l’utilisation de la polarisation des nanobâtonnets de LaPO4:Eu pour déterminer leur orientation. La connaissance des spectres polarisés des films parfaitement alignés a permis de déterminer le paramètre d’ordre d’une suspension de nanobâtonnets désordonnés en écoulement dans un canal microfluidique puis d’estimer le taux de cisaillement de cet écoulement. Notre étude a permis de préciser quantitativement les conditions dans lesquelles l’utilisation de la luminescence polarisée comme sonde locale du taux de cisaillement d’un écoulement est valide.Luminescent nanoparticles have been studied for their applications in lighting devices or as probes in biology. Among these nanoparticles, the anisotropic crystals doped with lanthanides ions emit linearly polarized light. The relation between the polarized directions and the crystallographic axis of the nanocrystals allow determining their 3D orientation, which could be an asset to track objects or to characterize flows.The purposes of this thesis were to investigate the origin of the polarized light of nanorods of lanthanum phosphate doped with europium ions (LaPO4:Eu) and to apply this polarized light to determine their orientation.First, nanorods of LaPO4:Eu are synthesized and aligned to prepare oriented films. The phase transition of the LaPO4 matrix is investigated, from the hexagonal to the monoclinic structure. The luminescence is used to track precisely the transition and show the presence of structural defects. Then the polarized spectra are observed. The polarization degrees of the monoclinic phase are higher than those of the hexagonal one. The sensitivity of the polarization with the dielectric medium is also shown.Then, the polarized light is used to determine the orientation of the nanorods. The knowledge of the polarized spectra along he nanorods axis and perpendicularly to it is used to calculate the order parameter of disoriented nanorods in a microfluidic channel and then to estimate the shear rate of the flow. Our study allows quantifying the conditions in which the nanorods can be used as probes to measure the local shear rate

Développement de nanoémetteurs polarisés pour leur application comme sondes d'orientation

Luminescent nanoparticles have been studied for their applications in lighting devices or as probes in biology. Among these nanoparticles, the anisotropic crystals doped with lanthanides ions emit linearly polarized light. The relation between the polarized directions and the crystallographic axis of the nanocrystals allow determining their 3D orientation, which could be an asset to track objects or to characterize flows.The purposes of this thesis were to investigate the origin of the polarized light of nanorods of lanthanum phosphate doped with europium ions (LaPO4:Eu) and to apply this polarized light to determine their orientation.First, nanorods of LaPO4:Eu are synthesized and aligned to prepare oriented films. The phase transition of the LaPO4 matrix is investigated, from the hexagonal to the monoclinic structure. The luminescence is used to track precisely the transition and show the presence of structural defects. Then the polarized spectra are observed. The polarization degrees of the monoclinic phase are higher than those of the hexagonal one. The sensitivity of the polarization with the dielectric medium is also shown.Then, the polarized light is used to determine the orientation of the nanorods. The knowledge of the polarized spectra along he nanorods axis and perpendicularly to it is used to calculate the order parameter of disoriented nanorods in a microfluidic channel and then to estimate the shear rate of the flow. Our study allows quantifying the conditions in which the nanorods can be used as probes to measure the local shear rate.Les nanoparticules luminescentes sont particulièrement étudiées pour leur application dans les systèmes d’éclairages ou comme sondes en bio-imagerie. Parmi elles, les nanoparticules anisotropes de matrices cristallines dopées par des ions lanthanides présentent une émission polarisée, qui dépend de la symétrie des sites des ions émetteurs. Le lien entre direction de polarisation et axes cristallins des nanocristaux permet de déterminer leur orientation, et peut donc être exploité pour suivre l'orientation d’objets ou pour caractériser la déformation de milieux hôtes.Les objectifs de ce doctorat ont été de s’intéresser aux origines fondamentales de l’émission polarisée de nanobâtonnets de phosphate de lanthane dopés par des ions europium trivalents (LaPO4:Eu) et d’utiliser la luminescence polarisée à des mesures d’orientation.Dans une première partie, les nanobâtonnets de LaPO4:Eu ont été synthétisés puis alignés sous forme des films orientés. La luminescence de ces films a permis de suivre avec précision la transition de phase de la matrice hôte, de sa structure hexagonale à une structure monoclinique ; et de mettre en évidence la présence de défauts structuraux. La polarisation des spectres de luminescence a ensuite été étudiée. Les taux de polarisation mesurés sont plus élevés pour la phase monoclinique que pour la phase hexagonale. La sensibilité du spectre de polarisation au milieu diélectrique qui les entoure a été mise en évidence.La seconde partie de cette étude porte sur l’utilisation de la polarisation des nanobâtonnets de LaPO4:Eu pour déterminer leur orientation. La connaissance des spectres polarisés des films parfaitement alignés a permis de déterminer le paramètre d’ordre d’une suspension de nanobâtonnets désordonnés en écoulement dans un canal microfluidique puis d’estimer le taux de cisaillement de cet écoulement. Notre étude a permis de préciser quantitativement les conditions dans lesquelles l’utilisation de la luminescence polarisée comme sonde locale du taux de cisaillement d’un écoulement est valide

Metrology of Multiphoton Microscopes Using Second Harmonic Generation Nanoprobes

International audienceIn multiphoton microscopy, the ongoing trend toward the use of excitation wavelengths spanning the entire near‐infrared range calls for new standards in order to quantify and compare the performances of microscopes. This article describes a new method for characterizing the imaging properties of multiphoton microscopes over a broad range of excitation wavelengths in a straightforward and efficient manner. It demonstrates how second harmonic generation (SHG) nanoprobes can be used to map the spatial resolution, field curvature, and chromatic aberrations across the microscope field of view with a precision below the diffraction limit and with unique advantages over methods based on fluorescence. KTiOPO4 nanocrystals are used as SHG nanoprobes to measure and compare the performances over the 850–1100 nm wavelength range of several microscope objectives designed for multiphoton microscopy. Finally, this approach is extended to the post‐acquisition correction of chromatic aberrations in multicolor multiphoton imaging. Overall, the use of SHG nanoprobes appears as a uniquely suited method to standardize the metrology of multiphoton microscopes

Polarized Luminescence of Anisotropic LaPO 4 :Eu Nanocrystal Polymorphs

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Measurement of intraneuronal transport in vivo in zebrafish larvae brain by tracking nanocrystal-labelled endosomes with fast non-linear microscopy

International audienceMotor proteins are responsible for the intracellular transport of critical cargoes such as organelles, vesicles, protein complexes and other materials within the cell, along the cytoskeleton. This transport is an essential molecular process participating in cell homeostasis, especially in neurons [1]. Axonal transport deficits are found in several neurological disorders and are a hallmark of neurodegenerative diseases [2]. Over the past few decades, much progress has been made in developing tools and methods to visualize and measure intracellular transport, by tracking the movement of molecular motors or the transported cargoes. In particular, fluorescent proteins (FP) fused to motors or cargo proteins [3] is largely used to investigate axonal transport, as it is a versatile tool with target specificity that is used in vivo in small organisms. These include Drosophila and Danio rerio (Zebrafish, Zf) larvae, which offers the advantage for optical microscopy of being transparent, and which are also amenable to genetic modifications. For instance, the motion of mitochondria was investigated in Zf larvae with genetically engineered fluorescent reporters [4] in the context of neurodegenerative disease [5] or axonal regeneration [6], using wide-field microscopy and more recently using a real-time 3D single particle tracking setup [7]. Such studies were also conducted in axons of motor-neurons of live mice, but these observations require a complex and invasive preparation consisting in surgically exposing the nerves. These various methods have reached a high degree of sophistication but to date the vast majority of axonal transport studies have only been conducted in sensory-motor neurons. Measurements in central nervous system neurons, where this transport also plays a key role, especially in disease conditions, are still lacking. Takihara et al [8] developed a low invasive surgical procedure allowing them to record by two-photon fluorescence microscopy bidirectional mitochondria motions in axons of retinal ganglion cells of a live mouse. Using two-photon microscopy imaging of the motor cortex, Knabbe et al [9] measured axonal transport properties of dense core vesicles labeled by a virally induced fluorescent reporter. While they addressed more complex systems, these live mouse observations [8,9] were carried out at moderate temporal resolutions of a maximum of one frame per second (corresponding to a dwell time of a few μs) with a spatial resolution of about 200 nm and for a maximum field-of-view size of 200 μm. Such a time resolution prevents the observation of transient events of short duration (<1 s) like short pauses induced by microtubule associated proteins obstacles.These limitations stem from the photobleaching of the FP reporter that prevents using the more intense laser excitation necessary to lower the dwell time. Therefore, in order to measure with a higher sensitivity axonal transport parameters in the brain of live organisms, there is a need for novel methodologies capable of achieving larger spatiotemporal resolution while maintaining a high throughput recording of ≈100 μm large field-of-views. To this aim, photostable optically active nanocrystals were used to measure the intraneuronal transport of endosomal compartments that they labeled from inside after their spontaneous internalization by endocytosis. Following the seminal work of Cui et al [10] using semiconductor nanocrystals as fluorescent labels to measure nerve growth factor retrograde transport, we used fluorescent diamond nanocrystals and evidenced subtle changes in endosomal transport in cultured neurons of transgenic mouse bearing a genetic risk factor of a neuropsychiatric disease [11]. These experiments were conducted in cultured neurons.Here we extend such nanoparticle-based assay to measure the endosomal transport parameters in neurons of the brain of zebrafish larvae. To this aim we use size ≈ 120 nm nanocrystals exhibiting large second-order non-linear optical properties, composed of potassium titanate phosphate (KTiOPO4, KTP), injected in Zf larvae optical tectum (OT) where they are subsequently endocyted by neurons. Indeed, in a previous work, we showed that these KTP nanocrystals (nanoKTP) are spontaneously internalized in 2D cultured primary neuron and can be imaged by collecting their second harmonic generation (SHG) signal un- der infrared (IR) pulsed laser excitation [13]. Key advantages of SHG over fluorescence are non-saturation and non-bleaching. In the present in vivo work, we harnessed these properties combined with fast raster scanning of the IR laser beam to achieve a large, up to 20 frames/s, frame rate identical to the one employed in our in vitro intraneuronal transport assay [11]. Such large frame rate allows the detection of short pausing duration underpinning complex molecular environments otherwise smeared out by too low temporal resolutions. Moreover, due to a dominant coefficient of the nonlinear suceptibility tensor [13], SHG from KTP behaves like a dipole and therefore has a direction emission, that we can further exploit to reveal rotational dynamics. Incidentally, the ability to image nanoKTP in live Zf larvae blood circulation at high frame rate was recently reported in a wide-field configuration with light-sheet illumination [14].In our experiment, we showed strong evidence that the nanoKTP move within axons of periventricular neurons (PVN), which cell bodies are located between the ventricle and the neuropil. These axons project radially inside the neuropil where they establish synaptic contacts with retinal ganglion cells dendrites. As microtubule orientation is polarized far enough from the axonal initial segment, we are then able to separate the retrograde phases of motion from the anterograde ones. To this aim, we developed a novel pipeline of video analysis, which identifies the direction of transport and directly yields as the outputs of a set of data, the statistical distributions of various intraneuronal transport metrics for anterograde and retrograde motions, in normal and perturbed situations.While in our previous in vitro study we investigated the impact of microtubule disruption or of concentration in associated proteins, here we address the complementary aspects of the modifications of specific molecular motor concentrations, either by applying dynapyrazole [15], a recently developed retrograde motor dynein inhibiting drug, or by using transgenic Zf engineered to bear loss-of-function alleles of the anterograde motor protein Kif5aa [16]. Dynapyrazole induces a reduction of 41% of the retrograde runlength, accompanied with a trend of 31% reduction of the mobile fraction of nanoKTP. In kif5aa mutant we confirm the previously reported increase of the mobile fraction of vesicles (45% increase for synaptophysin vesicles in moto-neuron axons [16]), here of +25%. This “release” of motion upon decrease of Kif5aa motor concentration is consistent with the tug-of-war model of axonal transport [21]. This interpretation is further supported by dose-dependent increases of retrograde motion directionality within individual trajectories, and of retrograde run length (+42% compared to wild-type) that could not be detected in the previous work [16].The high sensitivity of our nonlinear nanoparticle-based axonal transport measurement assay in revealing small molecular concentration changes opens prospects in screening the functional impacts of neurodegenerative disease genetic factors in the whole animal model of zebrafish larvae for which genetic tools are largely available.References:1.1. Hirokawa, N.; Niwa, S.; Tanaka, Y. Molecular Motors in Neurons: Transport Mechanisms and Roles in Brain Function, Development, and Disease. Neuron 2010, 68, 610–638.2.Millecamps, S.; Julien, J.-P. Axonal transport deficits and neurodegenerative diseases. Nature Reviews Neuroscience 2013, 14, 161–176. 3.Surana, S.; Villarroel-Campos, D.; Lazo, O. M.; Moretto, E.; Tosolini, A. P.; Rhymes, E. R.; Richter, S.; Sleigh, J. N.; Schiavo, G. The evolution of the axonal transport toolkit. Traffic 2020, 21, 13–33. 4.Mandal, A.; Pinter, K.; Drerup, C. M. Analyzing Neuronal Mitochondria in vivo Using Fluorescent Reporters in Zebrafish. Frontiers in Cell and Developmental Biology 2018, 6, 144. 5.Plucinska, G.; Paquet, D.; Hruscha, A.; Godinho, L.; Haass, C.; Schmid, B.; Misgeld, T. In Vivo Imaging of Disease-Related Mitochondrial Dynamics in a Vertebrate Model System. Journal of Neuroscience 2012, 32, 16203–16212. 6.Xu, Y.; Chen, M.; Hu, B.; Huang, R.; Hu, B. In vivo Imaging of Mitochondrial Trans- port in Single-Axon Regeneration of Zebrafish Mauthner Cells. Frontiers in Cellular Neuroscience 2017, 11, 4. 7.Wehnekamp, F.; Plucińska, G.; Thong, R.; Misgeld, T.; Lamb, D. C. Nanoresolution real-time 3D orbital tracking for studying mitochondrial trafficking in vertebrate axons in vivo. eLife 2019, 8, e46059. 8.Takihara, Y.; Inatani, M.; Eto, K.; Inoue, T.; Kreymerman, A.; Miyake, S.; Ueno, S.; Na- gaya, M.; Nakanishi, A.; Iwao, K. et al. In vivo imaging of axonal transport of mitochondria in the diseased and aged mammalian CNS. Proceedings of the National Academy of Sciences 2015, 112, 10515–10520.9.Knabbe, J.; Nassal, J. P.; Verhage, M.; Kuner, T. Secretory vesicle trafficking in awake and anaesthetized mice: differential speeds in axons versus synapses. The Journal of Physiology 2018, 596, 3759–3773. 10.Cui, B.; Wu, C.; Chen, L.; Ramirez, A.; Bearer, E. L.; Li, W.-P.; Mobley, W. C.; Chu, S. One at a time, live tracking of NGF axonal transport using quantum dots. Proceedings of the National Academy of Sciences 2007, 104, 13666–13671.11.Haziza, S.; Mohan, N.; Loe-Mie, Y.; Lepagnol-Bestel, A.-M.; Massou, S.; Adam, M.-P.; Le, X. L.; Viard, J.; Plancon, C.; Daudin, R. et al. Fluorescent nanodiamond tracking reveals intraneuronal transport abnormalities induced by brain-disease-related genetic risk factors. Nature nanotechnology 2016, 12, 322–328. 12.Mayer, L.; Slablab, A.; Dantelle, G.; Jacques, V.; Lepagnol-Bestel, A.-M.; Perruchas, S.; Spinicelli, P.; Thomas, A.; Chauvat, D.; Simonneau, M. et al. Single KTP nanocrystals as second-harmonic generation biolabels in cortical neurons. Nanoscale 2013, 5, 8466 8471. 13.Xuan, L. L.; Zhou, C.; Slablab, A.; Chauvat, D.; Tard, C.; Perruchas, S.; Gacoin, T.; Villeval, P.; Roch, J. Photostable Second-Harmonic Generation from a Single KTiOPO4 Nanocrystal for Nonlinear Microscopy. Small 2008-09, 4, 1332–1336. 14.Malkinson, G.; Mahou, P.; Chaudan, E.; Gacoin, T.; Sonay, A. Y.; Pantazis, P.; Beau- repaire, E.; Supatto, W. Fast In Vivo Imaging of SHG Nanoprobes with Multiphoton Light-Sheet Microscopy. ACS Photonics 2020, 7, 1036–1049. 15.Steinman, J. B.; Santarossa, C. C.; Miller, R. M.; Yu, L. S.; Serpinskaya, A. S.; Furukawa, H.; Morimoto, S.; Tanaka, Y.; Nishitani, M.; Asano, M. et al. Chemical structure-guided design of dynapyrazoles, cell-permeable dynein inhibitors with a unique mode of action. eLife 2017, 6, e25174.16.Auer, T. O.; Xiao, T.; Bercier, V.; Gebhardt, C.; Duroure, K.; Concordet, J.-P.; Wyart, C.; Suster, M.; Kawakami, K.; Wittbrodt, J. et al. Deletion of a kinesin I motor unmasks a mechanism of homeostatic branching control by neurotrophin-3. eLife 2015, 4, e05061.17.Encalada, S. E.; Goldstein, L. S. B. Biophysical Challenges to Axonal Transport: Motor- Cargo Deficiencies and Neurodegeneration. Annual Review of Biophysics 2014, 43, 141– 169

Advances in fast multiphoton microscopy using light-sheet illumination

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