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1.
Nat Methods ; 15(9): 677-680, 2018 09.
Artigo em Inglês | MEDLINE | ID: mdl-30171236

RESUMO

As biomedical imaging datasets expand, deep neural networks are considered vital for image processing, yet community access is still limited by setting up complex computational environments and availability of high-performance computing resources. We address these bottlenecks with CDeep3M, a ready-to-use image segmentation solution employing a cloud-based deep convolutional neural network. We benchmark CDeep3M on large and complex two-dimensional and three-dimensional imaging datasets from light, X-ray, and electron microscopy.


Assuntos
Computação em Nuvem , Aprendizado Profundo , Processamento de Imagem Assistida por Computador/métodos
2.
J Microsc ; 283(2): 127-144, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-33844293

RESUMO

The technique of colour EM that was recently developed enabled localisation of specific macromolecules/proteins of interest by the targeted deposition of diaminobenzidine (DAB) conjugated to lanthanide chelates. By acquiring lanthanide elemental maps by energy-filtered transmission electron microscopy (EFTEM) and overlaying them in pseudo-colour over the conventional greyscale TEM image, a colour EM image is generated. This provides a powerful tool for visualising subcellular component/s, by the ability to clearly distinguish them from the general staining of the endogenous cellular material. Previously, the lanthanide elemental maps were acquired at the high-loss M4,5 edge (excitation of 3d electrons), where the characteristic signal is extremely low and required considerably long exposures. In this paper, we explore the possibility of acquiring the elemental maps of lanthanides at their N4,5 edge (excitation of 4d electrons), which occurring at a much lower energy-loss regime, thereby contains significantly greater total characteristic signal owing to the higher inelastic scattering cross-sections at the N4,5 edge. Acquiring EFTEM lanthanide elemental maps at the N4,5 edge instead of the M4,5 edge, provides ∼4× increase in signal-to-noise and ∼2× increase in resolution. However, the interpretation of the lanthanide maps acquired at the N4,5 edge by the traditional 3-window method, is complicated due to the broad shape of the edge profile and the lower signal-above-background ratio. Most of these problems can be circumvented by the acquisition of elemental maps with the more sophisticated technique of EFTEM Spectrum Imaging (EFTEM SI). Here, we also report the chemical synthesis of novel second-generation DAB lanthanide metal chelate conjugates that contain 2 lanthanide ions per DAB molecule in comparison with 0.5 lanthanide ion per DAB in the first generation. Thereby, fourfold more Ln3+ per oxidised DAB would be deposited providing significant amplification of signal. This paper applies the colour EM technique at the intermediate-loss energy-loss regime to three different cellular targets, namely using mitochondrial matrix-directed APEX2, histone H2B-Nucleosome and EdU-DNA. All the examples shown in the paper are single colour EM images only.


Assuntos
Elementos da Série dos Lantanídeos , Microscopia Eletrônica de Transmissão por Filtração de Energia , Diagnóstico por Imagem , Elétrons , Coloração e Rotulagem
3.
Bioinformatics ; 33(19): 3145-3147, 2017 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-28957496

RESUMO

SUMMARY: To expedite the review of semi-automated probability maps of organelles and other features from 3D electron microscopy data we have developed Probability Map Viewer, a Java-based web application that enables the computation and visualization of probability map generation results in near real-time as the data are being collected from the microscope. Probability Map Viewer allows the user to select one or more voxel classifiers, apply them on a sub-region of an active collection, and visualize the results as overlays on the raw data via any web browser using a personal computer or mobile device. Thus, Probability Map Viewer accelerates and informs the image analysis workflow by providing a tool for experimenting with and optimizing dataset-specific segmentation strategies during imaging. AVAILABILITY AND IMPLEMENTATION: https://github.com/crbs/probabilitymapviewer. CONTACT: mellisman@ucsd.edu. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.


Assuntos
Processamento de Imagem Assistida por Computador/métodos , Microscopia Eletrônica/métodos , Software , Organelas/ultraestrutura , Probabilidade , Fluxo de Trabalho
4.
Microsc Microanal ; 21(1): 231-8, 2015 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-25392009

RESUMO

The recently developed three-dimensional electron microscopic (EM) method of serial block-face scanning electron microscopy (SBEM) has rapidly established itself as a powerful imaging approach. Volume EM imaging with this scanning electron microscopy (SEM) method requires intense staining of biological specimens with heavy metals to allow sufficient back-scatter electron signal and also to render specimens sufficiently conductive to control charging artifacts. These more extreme heavy metal staining protocols render specimens light opaque and make it much more difficult to track and identify regions of interest (ROIs) for the SBEM imaging process than for a typical thin section transmission electron microscopy correlative light and electron microscopy study. We present a strategy employing X-ray microscopy (XRM) both for tracking ROIs and for increasing the efficiency of the workflow used for typical projects undertaken with SBEM. XRM was found to reveal an impressive level of detail in tissue heavily stained for SBEM imaging, allowing for the identification of tissue landmarks that can be subsequently used to guide data collection in the SEM. Furthermore, specific labeling of individual cells using diaminobenzidine is detectable in XRM volumes. We demonstrate that tungsten carbide particles or upconverting nanophosphor particles can be used as fiducial markers to further increase the precision and efficiency of SBEM imaging.


Assuntos
Encéfalo/ultraestrutura , Microscopia Eletrônica de Varredura/métodos , Animais , Imageamento Tridimensional , Camundongos , Microscopia Eletrônica de Varredura/instrumentação
5.
Microsc Microanal ; 20(3): 706-14, 2014 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-24641915

RESUMO

Energy filtered transmission electron microscopy techniques are regularly used to build elemental maps of spatially distributed nanoparticles in materials and biological specimens. When working with thick biological sections, electron energy loss spectroscopy techniques involving core-loss electrons often require exposures exceeding several minutes to provide sufficient signal to noise. Image quality with these long exposures is often compromised by specimen drift, which results in blurring and reduced resolution. To mitigate drift artifacts, a series of short exposure images can be acquired, aligned, and merged to form a single image. For samples where the target elements have extremely low signal yields, the use of charge coupled device (CCD)-based detectors for this purpose can be problematic. At short acquisition times, the images produced by CCDs can be noisy and may contain fixed pattern artifacts that impact subsequent correlative alignment. Here we report on the use of direct electron detection devices (DDD's) to increase the signal to noise as compared with CCD's. A 3× improvement in signal is reported with a DDD versus a comparably formatted CCD, with equivalent dose on each detector. With the fast rolling-readout design of the DDD, the duty cycle provides a major benefit, as there is no dead time between successive frames.


Assuntos
Astrócitos/ultraestrutura , Células Epiteliais/ultraestrutura , Microscopia Eletrônica de Transmissão por Filtração de Energia/instrumentação , Microscopia Eletrônica de Transmissão por Filtração de Energia/métodos , Razão Sinal-Ruído , Coloração e Rotulagem/métodos , Animais , Encéfalo/patologia , Células HeLa , Humanos , Camundongos Endogâmicos C57BL
6.
bioRxiv ; 2024 Jun 06.
Artigo em Inglês | MEDLINE | ID: mdl-38895398

RESUMO

We demonstrate limited-tilt, serial section electron tomography (ET), which can non-destructively map brain circuits over large 3D volumes and reveal high-resolution, supramolecular details within subvolumes of interest. We show accelerated ET imaging of thick sections (>500 nm) with the capacity to resolve key features of neuronal circuits including chemical synapses, endocytic structures, and gap junctions. Furthermore, we systematically assessed how imaging parameters affect image quality and speed to enable connectomic-scale projects.

7.
J Struct Biol ; 161(3): 352-8, 2008 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-18054249

RESUMO

A prototype direct detection device (DDD) camera system has shown great promise in improving both the spatial resolution and the signal to noise ratio for electron microscopy at 120-400 keV beam energies (Xuong et al., 2007. Methods in Cell Biology, 79, 721-739). Without the need for a resolution-limiting scintillation screen as in the charge coupled device (CCD), the DDD camera can outperform CCD based systems in terms of spatial resolution, due to its small pixel size (5 microm). In this paper, the modulation transfer function (MTF) of the DDD prototype is measured and compared with the specifications of commercial scientific CCD camera systems. Combining the fast speed of the DDD with image mosaic techniques, fast wide-area imaging is now possible. In this paper, the first large area mosaic image and the first tomography dataset from the DDD camera are presented, along with an image processing algorithm to correct the specimen drift utilizing the fast readout of the DDD system.


Assuntos
Processamento de Imagem Assistida por Computador/métodos , Microscopia Eletrônica de Transmissão/instrumentação , Microscopia Eletrônica de Transmissão/métodos
8.
Adv Struct Chem Imaging ; 2(1): 11, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-27695667

RESUMO

Serial block-face scanning electron microscopy (SBEM) is quickly becoming an important imaging tool to explore three-dimensional biological structure across spatial scales. At probe-beam-electron energies of 2.0 keV or lower, the axial resolution should improve, because there is less primary electron penetration into the block face. More specifically, at these lower energies, the interaction volume is much smaller, and therefore, surface detail is more highly resolved. However, the backscattered electron yield for metal contrast agents and the backscattered electron detector sensitivity are both sub-optimal at these lower energies, thus negating the gain in axial resolution. We found that the application of a negative voltage (reversal potential) applied to a modified SBEM stage creates a tunable electric field at the sample. This field can be used to decrease the probe-beam-landing energy and, at the same time, alter the trajectory of the signal to increase the signal collected by the detector. With decelerated low landing-energy electrons, we observed that the probe-beam-electron-penetration depth was reduced to less than 30 nm in epoxy-embedded biological specimens. Concurrently, a large increase in recorded signal occurred due to the re-acceleration of BSEs in the bias field towards the objective pole piece where the detector is located. By tuning the bias field, we were able to manipulate the trajectories of the  primary and secondary electrons, enabling the spatial discrimination of these signals using an advanced ring-type BSE detector configuration or a standard monolithic BSE detector coupled with a blocking aperture.

9.
Stud Health Technol Inform ; 112: 100-9, 2005.
Artigo em Inglês | MEDLINE | ID: mdl-15923720

RESUMO

Through support from the National Institutes of Health's National Center for Research Resources, the Biomedical Informatics Research Network (BIRN) is pioneering the use of advanced cyberinfrastructure for medical research. By synchronizing developments in advanced wide area networking, distributed computing, distributed database federation, and other emerging capabilities of e-science, the BIRN has created a collaborative environment that is paving the way for biomedical research and clinical information management. The BIRN Coordinating Center (BIRN-CC) is orchestrating the development and deployment of key infrastructure components for immediate and long-range support of biomedical and clinical research being pursued by domain scientists in three neuroimaging test beds.


Assuntos
Pesquisa Biomédica/organização & administração , Diagnóstico por Imagem , Sistemas de Informação/instrumentação , Doenças do Sistema Nervoso , Sistemas Computacionais , Humanos , National Institutes of Health (U.S.) , Integração de Sistemas , Estados Unidos
10.
Ultramicroscopy ; 110(7): 744-7, 2010 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-20382479

RESUMO

The complete characterization of a novel direct detection device (DDD) camera for transmission electron microscopy is reported, for the first time at primary electron energies of 120 and 200 keV. Unlike a standard charge coupled device (CCD) camera, this device does not require a scintillator. The DDD transfers signal up to 65 lines/mm providing the basis for a high-performance platform for a new generation of wide field-of-view high-resolution cameras. An image of a thin section of virus particles is presented to illustrate the substantially improved performance of this sensor over current indirectly coupled CCD cameras.


Assuntos
Microscopia Eletrônica de Transmissão/instrumentação , Animais , Drosophila/virologia , Desenho de Equipamento , Microscopia Eletrônica de Transmissão/estatística & dados numéricos , Vírion/ultraestrutura
11.
J Clin Monit Comput ; 19(4-5): 279-94, 2005 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-16328943

RESUMO

The Cybermedia Center (CMC), Osaka University, is a research institution that offers knowledge and technology resources obtained from advanced researches in the areas of large-scale computation, information and communication, multimedia content and education. Currently, CMC is involved in Japanese national Grid projects such as JGN II (Japan Gigabit Network), NAREGI and BioGrid. Not limited to Japan, CMC also actively takes part in international activities such as PRAGMA. In these projects and international collaborations, CMC has developed a Grid system that allows scientists to perform their analysis by remote-controlling the world's largest ultra-high voltage electron microscope located in Osaka University. In another undertaking, CMC has assumed a leadership role in BioGrid by sharing its experiences and knowledge on the system development for the area of biology. In this paper, we will give an overview of the BioGrid project and introduce the progress of the Telescience unit, which collaborates with the Telescience Project led by the National Center for Microscopy and Imaging Research (NCMIR). Furthermore, CMC collaborates with seven Computing Centers in Japan, NAREGI and National Institute of Informatics to deploy PKI base authentication infrastructure. The current status of this project and future collaboration with Grid Projects will be delineated in this paper.


Assuntos
Redes de Comunicação de Computadores , Segurança Computacional , Informática , Telemedicina , Biologia Computacional , Japão , Integração de Sistemas
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