RESUMEN
Confocal microscopy is widely used to visualize gene expression patterns and developmental processes in plants. However, the imaging of plant tissue can be challenging due to its opacity, which often makes previous immersion in a clearing agent necessary. Many commonly-used chemicals suffer either from their incompatibility with fluorescent proteins or their complex and lengthy application. 2,2'-thiodiethanol (TDE) has recently been described as a clearing agent with an emphasis on high resolution microscopy due to its potential to adjust the refractive index. Here, we evaluate the use of TDE-based clearing for confocal as well as two-photon microscopy in various Arabidopsis thaliana tissue types. We demonstrate that tissue fixation is a mandatory prerequisite for the use of TDE, in order to preserve tissue integrity and fluorescent protein activity. TDE concentrations between 50-70% are a good compromise for imaging of technically challenging tissue to achieve good clearing without affecting fluorescent protein activity. TDE-based clearing is simple and rapid to use and allows for a flexible experimental setup while facilitating high quality imaging.
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Arabidopsis/metabolismo , Inhibidores Enzimáticos/química , Proteínas Fluorescentes Verdes/metabolismo , Microscopía Confocal/métodos , Compuestos de Sulfhidrilo/química , Arabidopsis/ultraestructura , Fluorescencia , Microscopía Confocal/instrumentación , Fotones , Fijación del TejidoRESUMEN
Core Facilities (CF) for advanced light microscopy (ALM) have become indispensable support units for research in the life sciences. Their organizational structure and technical characteristics are quite diverse, although the tasks they pursue and the services they offer are similar. Therefore, throughout Europe, scientists from ALM-CFs are forming networks to promote interactions and discuss best practice models. Here, we present recommendations for ALM-CF operations elaborated by the workgroups of the German network of ALM-CFs, German Bio-Imaging (GerBI). We address technical aspects of CF planning and instrument maintainance, give advice on the organization and management of an ALM-CF, propose a scheme for the training of CF users, and provide an overview of current resources for image processing and analysis. Further, we elaborate on the new challenges and opportunities for professional development and careers created by CFs. While some information specifically refers to the German academic system, most of the content of this article is of general interest for CFs in the life sciences. Microsc. Res. Tech. 79:463-479, 2016. © 2016 THE AUTHORS MICROSCOPY RESEARCH AND TECHNIQUE PUBLISHED BY WILEY PERIODICALS, INC.
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Instituciones de Salud , Laboratorios , Microscopía , Investigación Biomédica , Alemania , HumanosRESUMEN
BACKGROUND: Digital anatomical atlases are increasingly used in order to depict different gene expression patterns and neuronal morphologies within a standardized reference template. In evo-devo, a discipline in which the comparison of gene expression patterns is a widely used approach, such standardized anatomical atlases would allow a more rigorous assessment of the conservation of and changes in gene expression patterns during micro- and macroevolutionary time scales. Due to its small size and invariant early development, the annelid Platynereis dumerilii is particularly well suited for such studies. Recently a reference template with registered gene expression patterns has been generated for the anterior part (episphere) of the Platynereis trochophore larva and used for the detailed study of neuronal development. RESULTS: Here we introduce and evaluate a method for whole-body gene expression pattern registration for Platynereis trochophore and nectochaete larvae based on whole-mount in situ hybridization, confocal microscopy, and image registration. We achieved high-resolution whole-body scanning using the mounting medium 2,2'-thiodiethanol (TDE), which allows the matching of the refractive index of the sample to that of glass and immersion oil thereby reducing spherical aberration and improving depth penetration. This approach allowed us to scan entire whole-mount larvae stained with nitroblue tetrazolium/5-bromo-4-chloro-3-indolyl phosphate (NBT/BCIP) in situ hybridization and counterstained fluorescently with an acetylated-tubulin antibody and the nuclear stain 4'6-diamidino-2-phenylindole (DAPI). Due to the submicron isotropic voxel size whole-mount larvae could be scanned in any orientation. Based on the whole-body scans, we generated four different reference templates by the iterative registration and averaging of 40 individual image stacks using either the acetylated-tubulin or the nuclear-stain signal for each developmental stage. We then registered to these templates the expression patterns of cell-type specific genes. In order to evaluate the gene expression pattern registration, we analyzed the absolute deviation of cell-center positions. Both the acetylated-tubulin- and the nuclear-stain-based templates allowed near-cellular-resolution gene expression registration. Nuclear-stain-based templates often performed significantly better than acetylated-tubulin-based templates. We provide detailed guidelines and scripts for the use and further expansion of the Platynereis gene expression atlas. CONCLUSIONS: We established whole-body reference templates for the generation of gene expression atlases for Platynereis trochophore and nectochaete larvae. We anticipate that nuclear-staining-based image registration will be applicable for whole-body alignment of the embryonic and larval stages of other organisms in a similar size range.
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Under most physiological circumstances, monocytes are excluded from parenchymal CNS tissues. When widespread monocyte entry occurs, their numbers decrease shortly after engraftment in the presence of microglia. However, some disease processes lead to focal and selective loss, or dysfunction, of microglia, and microglial senescence typifies the aged brain. In this regard, the long-term engraftment of monocytes in the microglia-depleted brain remains unknown. Here, we report a model in which a niche for myeloid cells was created through microglia depletion. We show that microglia-depleted brain regions of CD11b-HSVTK transgenic mice are repopulated with new Iba-1-positive cells within 2 wk. The engrafted cells expressed high levels of CD45 and CCR2 and appeared in a wave-like pattern frequently associated with blood vessels, suggesting the engrafted cells were peripheral monocytes. Although two times more numerous and morphologically distinct from resident microglia up to 27 wk after initial engraftment, the overall distribution of the engrafted cells was remarkably similar to that of microglia. Two-photon in vivo imaging revealed that the engrafted myeloid cells extended their processes toward an ATP source and displayed intracellular calcium transients. Moreover, the engrafted cells migrated toward areas of kainic acid-induced neuronal death. These data provide evidence that circulating monocytes have the potential to occupy the adult CNS myeloid niche normally inhabited by microglia and identify a strong homeostatic drive to maintain the myeloid component in the mature brain.
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Sistema Nervioso Central/citología , Homeostasis , Microglía/citología , Adenosina Trifosfato/metabolismo , Animales , Sistema Nervioso Central/metabolismo , Ratones , Microglía/metabolismo , Timidina Quinasa/genéticaRESUMEN
In vivo imaging of pathological protein aggregates provides essential knowledge of the kinetics and implications of these lesions in the progression of proteopathies, such as Alzheimer disease. Luminescent conjugated oligothiophenes are amyloid-specific ligands that bind and spectrally distinguish different types of amyloid aggregates. Herein, we report that heptamer formyl thiophene acetic acid (hFTAA) passes the blood-brain barrier after systemic administration and specifically binds to extracellular ß-amyloid deposits in the brain parenchyma (Aß plaques) and in the vasculature (cerebral ß-amyloid angiopathy) of ß-amyloid precursor protein transgenic APP23 mice. Moreover, peripheral application of hFTAA also stained intracellular lesions of hyperphosphorylated Tau protein in P301S Tau transgenic mice. Spectral profiling of all three amyloid types was acquired ex vivo using two-photon excitation. hFTAA revealed a distinct shift in its emission spectra when bound to Aß plaques versus Tau lesions. Furthermore, a spectral shift was observed for Aß plaques versus cerebral ß-amyloid angiopathy, indicating that different amyloid types and structural variances of a specific amyloid type can be distinguished. In conclusion, by adding spectral signatures to amyloid lesions, our results pave the way for a new area of in vivo amyloid imaging, allowing in vivo differentiation of amyloid (sub)types and monitoring changes of their structure/composition over time.
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Amiloide/metabolismo , Encéfalo/patología , Luminiscencia , Microscopía de Fluorescencia por Excitación Multifotónica/métodos , Tiofenos/administración & dosificación , Enfermedad de Alzheimer/metabolismo , Enfermedad de Alzheimer/patología , Animales , Barrera Hematoencefálica/metabolismo , Barrera Hematoencefálica/patología , Encéfalo/metabolismo , Humanos , Inmunohistoquímica , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Coloración y Etiquetado , Tiofenos/químicaRESUMEN
Repetitive in vivo imaging in mice has become an indispensable tool for studying dynamic changes in structure and function of the brain. We describe a head fixation system, which allows rapid re-localization of previously imaged regions of interest (ROIs) within the brain. Such ROIs can be automatically relocated and imaged over weeks to months with negligible rotational change and only minor translational errors. Previously stored imaging positions can be fully automated re-localized within a few seconds. This automated rapid and accurate relocation simplifies image acquisition and post-processing in longitudinal imaging experiments. Moreover, as the laser is only used for data acquisition and not for finding previously imaged ROIs, the risk of laser induced tissue damage and photobleaching is greatly reduced. Thus, here described head fixation device appears well suited for in vivo repetitive long-term imaging in rodent brain.
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Microscopía/instrumentación , Neuroimagen/instrumentación , Restricción Física/instrumentación , Animales , Encéfalo/anatomía & histología , Encéfalo/fisiología , Ratones , Fotones , Factores de TiempoRESUMEN
Membrane lipid microdomains (lipid rafts) play an important role in T cell function by forming areas of high lipid order that facilitate activation. However, their role in regulating T cell differentiation and function remains controversial. In this study, by applying a new approach involving microscopy and flow cytometry, we characterize membrane lipid order in ex vivo primary human CD4(+) T cells. We reveal that differential membrane lipid order dictates the response to TCR stimulation. T cells with high membrane order formed stable immune synapses and proliferated robustly, intermediate order cells had reduced proliferative ability accompanied by unstable immune synapse formation, whereas low order T cells were profoundly unresponsive to TCR activation. We also observed that T cells from patients with autoimmune rheumatic disease had expanded intermediate order populations compared with healthy volunteers. This may be important in dictating the nature of the immune response since most IFN-γ(+)CD4(+) T cells were confined within intermediate membrane order populations, whereas IL-4(+)CD4(+) T cells were contained within the high order populations. Importantly, we were able to alter T cell function by pharmacologically manipulating membrane order. Thus, the results presented from this study identify that ex vivo CD4(+) T cells sustain a gradient of plasma membrane lipid order that influences their function in terms of proliferation and cytokine production. This could represent a new mechanism to control T cell functional plasticity, raising the possibility that therapeutic targeting of membrane lipid order could direct altered immune cell activation in pathology.
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Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD4-Positivos/metabolismo , Lípidos de la Membrana/metabolismo , Microdominios de Membrana/metabolismo , Adulto , Linfocitos T CD4-Positivos/citología , Células Cultivadas , Técnicas de Cocultivo , Femenino , Humanos , Sinapsis Inmunológicas/inmunología , Activación de Linfocitos/inmunología , Masculino , Lípidos de la Membrana/fisiología , Microdominios de Membrana/fisiología , Sondas Moleculares , Compuestos de Piridinio , Células TH1/citología , Células TH1/inmunología , Células TH1/metabolismo , Células Th2/citología , Células Th2/inmunología , Células Th2/metabolismoRESUMEN
Extracellular deposition of the amyloid-ß peptide (Aß) in the brain parenchyma is a hallmark lesion of Alzheimer's disease (AD) and a predictive marker for the progression of preclinical to symptomatic AD. Here, we used multiphoton in vivo imaging to study Aß plaque formation in the brains of 3- to 4-month-old APPPS1 transgenic mice over a period of 6 months. A novel head fixation system provided robust and efficient long-term tracking of single plaques over time. Results revealed an estimated rate of 35 newly formed plaques per cubic millimeter of neocortical volume per week at 4-5 months of age. At later time points (i.e., in the presence of increasing cerebral ß-amyloidosis), the number of newly formed plaques decreased. On average, both newly formed and existing plaques grew at a similar growth rate of 0.3 µm (radius) per week. A solid knowledge of the dynamics of cerebral ß-amyloidosis in mouse models provides a powerful tool to monitor preclinical Aß targeting therapeutic strategies and eases the interpretation of diagnostic amyloid imaging in humans.
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Péptidos beta-Amiloides/metabolismo , Amiloidosis/patología , Encéfalo/patología , Placa Amiloide/patología , Animales , Femenino , Gliosis/patología , Proteínas Fluorescentes Verdes/genética , Humanos , Masculino , Ratones , Ratones Transgénicos , Microglía/patología , Microscopía de Fluorescencia por Excitación Multifotónica , Coloración y EtiquetadoRESUMEN
Engrailed transcription factors regulate survival, cell fate decisions and axon pathfinding in central neurons. En-2 can also attenuate Purkinje cell (PC) maturation. Here, we use array analysis to scrutinize gene expression in developing PCs overexpressing Engrailed-2 (L7En-2). The majority (70%) of regulated genes was found down-regulated in L7En-2 cerebella, consistent with the known repressive function of Engrailed-2. Differential gene expression, verified by in situ hybridization or Western blotting, was particularly evident during the first postnatal week, when L7En-2 PCs display conspicuous deficits in dendritogenesis. Functional classification revealed clusters of genes linked to vesicle formation and transport. Consistently, Golgi stacks located at the axonal pole of wild type PC somata were rarely detected in L7En-2 PCs. In addition, long continuous stretches of endoplasmic reticulum typically found around the axonal pole of wild type PCs were less frequently observed in transgenic cells. Engrailed-2 might therefore orchestrate PC survival and process formation as a regulator of subcellular organization.
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Cerebelo/fisiología , Proteínas de Homeodominio/fisiología , Proteínas del Tejido Nervioso/fisiología , Células de Purkinje/fisiología , Vesículas Sinápticas/genética , Vesículas Sinápticas/metabolismo , Animales , Animales Recién Nacidos , Supervivencia Celular/genética , Cerebelo/citología , Regulación hacia Abajo/genética , Regulación de la Expresión Génica/fisiología , Proteínas de Homeodominio/antagonistas & inhibidores , Proteínas de Homeodominio/biosíntesis , Proteínas de Homeodominio/genética , Ratones , Ratones Transgénicos , Proteínas del Tejido Nervioso/antagonistas & inhibidores , Proteínas del Tejido Nervioso/biosíntesis , Proteínas del Tejido Nervioso/genética , Transporte de Proteínas/genética , Células de Purkinje/citologíaRESUMEN
The transcription factor Engrailed-2 is expressed in cerebellar Purkinje cells (PCs) throughout embryonic development but is downregulated in PCs after birth. Since the onset of PC differentiation coincides with this change of gene expression, we asked whether downregulation of Engrailed-2 is necessary for proper timing of PC differentiation. To investigate this, we used an L7En-2 transgenic mouse model in which Engrailed-2 expression in PCs is maintained beyond the day of birth. In these L7En-2 mice the onset of parvalbumin expression was delayed in all PCs by about 3 days; the spatial expression pattern, however, remained comparable to wildtype cerebella. Furthermore, parvalbumin expression resembled the known pattern of normal PC maturation, suggesting a direct link between parvalbumin expression and PC differentiation. Consistent with a delay of PC differentiation, we found that PCs of L7En-2 cerebella displayed a reduced tendency to align in the typical monolayer. The average size of L7En-2 PCs was reduced and the dendritic arbor developed more slowly than in wildtype PCs. In contrast, major morphological features of PCs were comparable in L7En-2 and wildtype cerebella after postnatal day 11. In addition, we observed a transient reduction of PC survival in organotypic slice cultures of L7En-2 cerebella in comparison with wildtype slice cultures. Since PC survival parallels PC differentiation in vitro, we propose that the observed delay in PC differentiation upon Engrailed-2 overexpression is an intrinsic property of Engrailed-2 activity, and that downregulation of Engrailed-2 in wildtype PCs around the day of birth is critical for the timing of distinct steps of PC differentiation.