Three-dimensional volume imaging with electron microscopy toward connectome

Ohno, Nobuhiko; Katoh, Mitsuhiko; Saitoh, Yurika; Saitoh, Shu‐ichi; Ohno, Shinichi

doi:10.1093/jmicro/dfu112

Cited by 36 publications

(18 citation statements)

References 57 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Due to the complexity of neural connections, three-dimensional (3D) reconstruction using electron microscope images is desired. Serial section transmission electron microscopy (ssTEM) has been used for 3D reconstruction, but ssTEM is time-consuming and technically demanding [ 22 , 23 ]. To overcome the disadvantages, focused ion beam/scanning electron microscopy (FIB/SEM) has been developed to acquire serial section SEM images, which have been shown to be sufficient for the reconstruction of 3D images of the synaptic connections and structures [ 22 – 25 ].…”

Section: Introductionmentioning

confidence: 99%

Chronic Fluoxetine Induces the Enlargement of Perforant Path-Granule Cell Synapses in the Mouse Dentate Gyrus

et al. 2016

View full text Add to dashboard Cite

A selective serotonin reuptake inhibitor is the most commonly prescribed antidepressant for the treatment of major depression. However, the mechanisms underlying the actions of selective serotonin reuptake inhibitors are not fully understood. In the dentate gyrus, chronic fluoxetine treatment induces increased excitability of mature granule cells (GCs) as well as neurogenesis. The major input to the dentate gyrus is the perforant path axons (boutons) from the entorhinal cortex (layer II). Through voltage-sensitive dye imaging, we found that the excitatory neurotransmission of the perforant path synapse onto the GCs in the middle molecular layer of the mouse dentate gyrus (perforant path-GC synapse) is enhanced after chronic fluoxetine treatment (15 mg/kg/day, 14 days). Therefore, we further examined whether chronic fluoxetine treatment affects the morphology of the perforant path-GC synapse, using FIB/SEM (focused ion beam/scanning electron microscopy). A three-dimensional reconstruction of dendritic spines revealed the appearance of extremely large-sized spines after chronic fluoxetine treatment. The large-sized spines had a postsynaptic density with a large volume. However, chronic fluoxetine treatment did not affect spine density. The presynaptic boutons that were in contact with the large-sized spines were large in volume, and the volumes of the mitochondria and synaptic vesicles inside the boutons were correlated with the size of the boutons. Thus, the large-sized perforant path-GC synapse induced by chronic fluoxetine treatment contains synaptic components that correlate with the synapse size and that may be involved in enhanced glutamatergic neurotransmission.

show abstract

Section: Introductionmentioning

confidence: 99%

Chronic Fluoxetine Induces the Enlargement of Perforant Path-Granule Cell Synapses in the Mouse Dentate Gyrus

et al. 2016

View full text Add to dashboard Cite

show abstract

“…Recent effort towards homogenous staining of bulk tissues enabled acquisition of images from the brain tissues for reconstruction of very large volume 14 15 . However, even with these approaches, imaging at higher magnifications and longer dwelling times for a better signal-to-noise ratio need higher electron doses and may be hampered by sample charging and damage, which are particularly prominent in SBF-SEM imaging 16 17 18 . It currently remains unclear whether increasing the conductivities of resins, which are typically non-conductive, reduces charging and sample damage and is beneficial for successful volume imaging with SEM.…”

mentioning

confidence: 99%

Conductive resins improve charging and resolution of acquired images in electron microscopic volume imaging

Nguyen

Thai

Saitoh

et al. 2016

Sci Rep

Self Cite

View full text Add to dashboard Cite

Recent advances in serial block-face imaging using scanning electron microscopy (SEM) have enabled the rapid and efficient acquisition of 3-dimensional (3D) ultrastructural information from a large volume of biological specimens including brain tissues. However, volume imaging under SEM is often hampered by sample charging, and typically requires specific sample preparation to reduce charging and increase image contrast. In the present study, we introduced carbon-based conductive resins for 3D analyses of subcellular ultrastructures, using serial block-face SEM (SBF-SEM) to image samples. Conductive resins were produced by adding the carbon black filler, Ketjen black, to resins commonly used for electron microscopic observations of biological specimens. Carbon black mostly localized around tissues and did not penetrate cells, whereas the conductive resins significantly reduced the charging of samples during SBF-SEM imaging. When serial images were acquired, embedding into the conductive resins improved the resolution of images by facilitating the successful cutting of samples in SBF-SEM. These results suggest that improving the conductivities of resins with a carbon black filler is a simple and useful option for reducing charging and enhancing the resolution of images obtained for volume imaging with SEM.

show abstract

“…Bars: 50 μm (B,C) , 12.5 μm (D) , 5 μm (E) or 500 nm ( E , inset). Images were adapted from Ohno et al ( 2015 ) with permission.…”

Section: Basic Methodology Of Sample Preparations and Data Acquisitiomentioning

confidence: 99%

Methodological Improvements With Conductive Materials for Volume Imaging of Neural Circuits by Electron Microscopy

Nguyen

Thai

Sui

et al. 2018

Front. Neural Circuits

Self Cite

View full text Add to dashboard Cite

Recent advancements in electron microscope volume imaging, such as serial imaging using scanning electron microscopy (SEM), have facilitated the acquisition of three-dimensional ultrastructural information of biological samples. These advancements help build a comprehensive understanding of the functional structures in entire organelles, cells, organs and organisms, including large-scale wiring maps of neural circuitry in various species. Advanced volume imaging of biological specimens has often been limited by artifacts and insufficient contrast, which are partly caused by problems in staining, serial sectioning and electron beam irradiation. To address these issues, methods of sample preparation have been modified and improved in order to achieve better resolution and higher signal-to-noise ratios (SNRs) in large tissue volumes. These improvements include the development of new embedding media for electron microscope imaging that have desirable physical properties such as less deformation in the electron beam and higher stability for sectioning. The optimization of embedding media involves multiple resins and filler materials including biological tissues, metallic particles and conductive carbon black. These materials alter the physical properties of the embedding media, such as conductivity, which reduces specimen charge, ameliorates damage to sections, reduces image deformation and results in better ultrastructural data. These improvements and further studies to improve electron microscope volume imaging methods provide options for better scale, quality and throughput in the three-dimensional ultrastructural analyses of biological samples. These efforts will enable a deeper understanding of neuronal circuitry and the structural foundation of basic and higher brain functions.

show abstract

Three-dimensional volume imaging with electron microscopy toward connectome

Cited by 36 publications

References 57 publications

Chronic Fluoxetine Induces the Enlargement of Perforant Path-Granule Cell Synapses in the Mouse Dentate Gyrus

Chronic Fluoxetine Induces the Enlargement of Perforant Path-Granule Cell Synapses in the Mouse Dentate Gyrus

Conductive resins improve charging and resolution of acquired images in electron microscopic volume imaging

Methodological Improvements With Conductive Materials for Volume Imaging of Neural Circuits by Electron Microscopy

Contact Info

Product

Resources

About