Crystal engineering of nutraceutical phytosterols: new cocrystal solid solutions?

A cocrystal screening conducted with solid solutions of three phytosterols (β-sitosterol, campesterol and stigmasterol) and a set of coformers with strong hydrogen bond donors reveals that multicomponent solid solutions are preferentially formed instead of pure cocrystals and are much enriched with β-sitosterol with respect to stigmasterol, a natural product with cytotoxicity concerns

A Novel, Extremely Bioavailable Cocrystal of Pterostilbene

New multicomponent solid forms of the nutraceutical pterostilbene have been discovered and characterized through experimental cocrystal screening. Among the coformers tested, picolinic acid formed a cocrystal with a 10-fold enhancement of oral bioavailability in rats, which converts the new cocrystal into a very promising candidate for new formulations of pterostilbene with improved performance

Advanced optical microscopy for life sciences: from optical sections to optical nanodissection

La microscòpia òptica ha patit una gran revolució en les darreres tres dècades amb l’arribada dels làsers i les proteïnes fluorescents. Sent capaços de marcar individualment molècules i proteïnes en cèŀlules i organismes vius, ara els científics poden observar múltiples components ceŀlulars i proteïnes simultàniament, al llarg dels diferents compartiments i teixits, i també veure la interacció d’aquests entre si. Com que la microscòpia de fluorescència moderna està esdevenint una veritable microscòpia molecular amb tècniques específiques per a l’estudi de dinàmica de proteïnes com el FRAP o el FRET, la capacitat dels microscopis òptics de generar imatges altament resolutives, tant en la dimensió espacial com en la temporal, ha millorat dràsticament. En aquest capítol revisem els principis bàsics de la fluorescència, la preparació de mostres biològiques i els conceptes òptics darrere els quals es troba la capacitat de la microscòpia confocal de generar imatges tridimensionals a partir de seccions òptiques. També abordem tècniques basades en la manipulació amb làsers com la nanocirurgia per làser i en discutim aplicacions modernes dins de la biologia ceŀlular i del desenvolupament, en especial l’ablació ceŀlular i la cirurgia intraceŀlular, les quals han esdevingut avui dia eines importants per interactuar amb teixits pluriceŀlulars, ja que ens permeten avaluar forces i estats biomecànics de les cèŀlules durant processos importants del desenvolupament dels organismes. Finalment, expliquem els principis d’un dels últims avenços en l’adquisició d’imatges de fluorescència, la microscòpia en full de llum, que permet als científics esquivar les principals limitacions de les microscòpies convencional i confocal, ja que ofereix les capacitats d’adquirir imatges en profunditat en llargs períodes de temps.Paraules clau: microscòpia òptica, microscopi confocal, làser, fluorescència, full de llum.Optical microscopy has undergone several revolutions in the past three decades with the advent of lasers and fluorescent proteins. By being able to label single molecules and proteins in living cells and organisms, scientists can now observe multiple cellular components and proteins simultaneously, moving across compartments and tissues and interacting with each other. As modern fluorescence microscopy is turning into truly molecular microscopy with specific techniques for the study of protein dynamics like FRAP or FRET, the capability of optical microscopes to generate highly resolved images, both in spatial and temporal dimensions, is also dramatically improving. In this chapter, we review the basic principles of fluorescence, the preparation of biological samples and the optical concepts behind the capability of confocal microscopy to generate threedimensional images on the basis of optical sections. We also approach laser based manipulation techniques such as laser nanosurgery and discuss modern applications in cell and development biology, in particular how cell ablation and intracellular surgery have now become important tools for interacting with multicellular tissues since they allow the probing of forces and biomechanical states of cells during important development processes of organisms. Lastly, we set forth the principles of one of the latest advances in fluorescence imaging, light sheet microscopy, which allows experimentalists to circumvent several major limitations of conventional and confocal microscopy by offering the capability of imaging deeply and for very long periods of time.Keywords: optical microscopy, confocal microscope, laser, fluorescence,light sheet

LOBSTER: an environment to design bioimage analysis workflows for large and complex fluorescence microscopy data

© The Author(s) 2019.Open source software such as ImageJ and CellProfiler greatly simplified the quantitative analysis of microscopy images but their applicability is limited by the size, dimensionality and complexity of the images under study. In contrast, software optimized for the needs of specific research projects can overcome these limitations, but they may be harder to find, set up and customize to different needs. Overall, the analysis of large, complex, microscopy images is hence still a critical bottleneck for many Life Scientists. We introduce LOBSTER (Little Objects Segmentation and Tracking Environment), an environment designed to help scientists design and customize image analysis workflows to accurately characterize biological objects from a broad range of fluorescence microscopy images, including large images exceeding workstation main memory. LOBSTER comes with a starting set of over 75 sample image analysis workflows and associated images stemming from state-of-the-art image-based research projects

Neurogenesis redirects β-catenin from adherens junctions to the nucleus to promote axonal growth

Here, we show that, in the developing spinal cord, after the early Wnt-mediated Tcf transcription activation that confers dorsal identity to neural stem cells, neurogenesis redirects β-catenin from the adherens junctions to the nucleus to stimulate Tcf-dependent transcription in a Wnt-independent manner. This new β-catenin activity regulates genes implicated in several aspects of contralateral axon growth, including axon guidance and adhesion. Using live imaging of ex-vivo chick neural tube, we showed that the nuclear accumulation of β-catenin and the rise in Tcf-dependent transcription both initiate before the dismantling of the adherens junctions and remain during the axon elongation process. Notably, we demonstrated that β-catenin activity in post-mitotic cells depends on TCF7L2 and is central to spinal commissural axon growth. Together, our results reveal Wnt-independent Tcf/β-catenin regulation of genes that control the growth and guidance of commissural axons in chick spinal cord.A.H. was supported by a Juan de la Cierva fellowship from Ministerio de Ciencia e Innovación (FJCI-2015-26175). A.O. was supported by a Ministerio de Economía y Competitividad pre-doctoral fellowship (BES-2015-072035). The work in S.P.’s laboratory was supported by Ministerio de Ciencia e Innovación (BFU2017-83562-P and PID2020-116806GB-I00). Open Access funding provided by Consejo Superior de Investigaciones Científicas. Deposited in PMC for immediate release.Peer reviewe

AutoScanJ : A Suite of ImageJ Scripts for Intelligent Microscopy

We developed AutoscanJ, a suite of ImageJ scripts enabling to image targets of interest by automatically driving a motorized microscope at the corresponding locations. For live samples, our software can sequentially detect biological events from their onset and further image them at high resolution, an action that would be impractical by user operation. For fixed samples, the software can dramatically reduce the amount of data acquired and the acquisition duration in situations where statistically few targets of interest are observed per field of view. AutoScanJ is compatible with motorized fluorescence microscopes controlled by Leica LAS AF/X or Micro-Manager. The software is straightforward to set up and new custom image analysis workflows to detect targets of interest can be simply implemented and shared with minimal efforts as independent ImageJ macro functions. We illustrate five different application scenarios with the system ranging from samples fixed on micropatterned surfaces to live cells undergoing several rounds of division. The target detection functions for these applications are provided and can be used as a starting point and a source of inspiration for new applications. Overall, AutoScanJ helps to optimize microscope usage by autonomous operation, and it opens up new experimental avenues by enabling the real-time detection and selective imaging of transient events in live microscopy

MosaicExplorerJ: Interactive stitching of terabyte-size tiled datasets from lightsheet microscopy [version 2; peer review: 2 approved]

© 2020 Tosi S et al.We introduce MosaicExplorerJ, an ImageJ macro to stitch 3D tiles from terabyte-size microscopy datasets. As opposed to existing software, stitching does not require any prior information on the actual positions of the tiles, sample fiducials, or conversion of raw TIFF images, and the stitched images can be explored instantly. MosaicExplorerJ was specifically designed to process lightsheet microscopy datasets from optically cleared samples. It can handle multiple fluorescence channels, dual-side lightsheet illumination and dual-side camera detection.The preparation of some of the datasets that were used to test MosaicExplorerJ was partially funded by project TEC2016-78052-R from the Spanish Ministry of Economy and Competitiveness and RTC2017-6600-1 from Ministry of Science, Innovation and Universities, as well as project PI17/01766 from Ministerio de Ciencia, Innovación y Universidades, Instituto de Salud Carlos III (co-financed by European Regional Development Fund (ERDF), “A way of making Europe")

MosaicExplorerJ: Interactive stitching of terabyte-size tiled datasets from lightsheet microscopy

© 2021 Tosi S et al.We introduce MosaicExplorerJ, an ImageJ macro to stitch 3D tiles from terabyte-size microscopy datasets organized on a regular 2D grid. As opposed to existing software, stitching does not require any prior information on the actual positions of the tiles, or conversion of raw TIFF images to a multi-resolution format for interactive exploration and fast processing. MosaicExplorerJ was specifically designed to process lightsheet microscopy datasets from optically cleared samples. It can handle multiple fluorescence channels, dual-sided lightsheet illumination and dual-sided camera detection.This publication was supported by COST Action NEUBIAS (CA15124), funded by COST (European Cooperation in Science and Technology). MJB acknowledges the support of Jérôme Lejeune Foundation. : The preparation of some of the datasets that were used to test MosaicExplorerJ was partially funded by project TEC2016-78052-R from the Spanish Ministry of Economy and Competitiveness and RTC2017-6600-1 from Ministry of Science, Innovation and Universities, as well as project PI17/01766 from Ministerio de Ciencia, Innovación y Universidades, Instituto de Salud Carlos III (cofinanced by European Regional Development Fund (ERDF), “A way of making Europe")

Reversible silencing of endogenous receptors in intact brain tissue using 2-photon pharmacology

The physiological activity of proteins is often studied with loss-of-function genetic approaches, but the corresponding phenotypes develop slowly and can be confounding. Photopharmacology allows direct, fast, and reversible control of endogenous protein activity, with spatiotemporal resolution set by the illumination method. Here, we combine a photoswitchable allosteric modulator (alloswitch) and 2-photon excitation using pulsed near-infrared lasers to reversibly silence metabotropic glutamate 5 (mGlu5) receptor activity in intact brain tissue. Endogenous receptors can be photoactivated in neurons and astrocytes with pharmacological selectivity and with an axial resolution between 5 and 10 μm. Thus, 2-photon pharmacology using alloswitch allows investigating mGlu5-dependent processes in wild-type animals, including synaptic formation and plasticity, and signaling pathways from intracellular organelles. © 2019 National Academy of Sciences. All rights reserved.ACKNOWLEDGMENTS. We thank Jordi Hernando (Autonomous University of Barcelona) for useful discussions on 2-photon excitation; Pere Català (Utrecht University) for help with GCaMP; Francisco Ciruela (University of Barcelona) for mGlu5-eYFP plasmid; Erin Schuman and Stephan Junek (Max Planck Institute for Brain Research, Frankfurt) for preliminary 2-photon excitation experiments; and Ashraf Muhaisen (University of Barcelona) for help with slicing. This research received funding from European Union Research and Innovation Programme Horizon 2020 [Human Brain Project SGA2 Grant Agreement 785907 (WaveScalES)], European Research ERA-Net SynBio programme (Modulightor project), and financial support from Agency for Management of University and Research Grants/Generalitat de Catalunya (CERCA Programme; 2017-SGR-1442 project), Fonds Européen de Développement Économique et Régional (FEDER) funds, Ministry of Economy and Competitiveness (MINECO)/FEDER (Grant CTQ2016-80066-R), and the Fundaluce foundation. S.P. was supported by an FI fellowship from the Agency for Management of University and Research Grants/Generalitat de Catalunya (2014FI_B2 00160). H.L. was supported by an Institute for Bioengineering of Catalonia Severo Ochoa International PhD Programme fellowship from MINECO. M.B. was supported by a H2020-MSCA-IF Reintegration Grant. K.E.P. receives support from NIH/National Institute of Neurological Disorders and Stroke Grant R01NS099254 and NSF Biophotonics Grant 1604544. E.S. receives support from MINECO (Grant SAF2016-7426).Peer reviewe