研究目的
To develop a method for ultrastructural visualization of single neurons in vivo by combining light and electron microscopy to map synaptic connections in neural circuits.
研究成果
The CoLSSEM method enables high-resolution correlated imaging of single neurons, improving the accuracy of tracing dendritic and axonal structures and synaptic connections, and holds promise for advancing connectomics studies in mammalian brains.
研究不足
The DAB staining from APEX2 can diffuse and obscure ultrastructural details in small cytoplasmic volumes like axons and spines, and the method requires optimization of fixative concentrations and staining times.
1:Experimental Design and Method Selection:
The study designed a fusion protein APEX2-Venus-CAAX (AVC) for membrane targeting, used in utero electroporation for sparse labeling, and combined confocal microscopy with ATUM-SEM for correlated imaging.
2:Sample Selection and Data Sources:
Mouse cortical pyramidal neurons were labeled sparsely using in utero electroporation at embryonic day
3:5, and brain sections were processed for imaging. List of Experimental Equipment and Materials:
Equipment includes Picospritzer III for injection, ECM 830 electroporation system, Leica VT1200S vibratome, Nikon SMZ18 stereoscope, Nikon Ti-e confocal microscope, Zeiss Sigma Field Emission Scanning Electron Microscope, and ATUM for section collection. Materials include plasmids (e.g., pAAV-Ef1a-DIO-AVC), fixatives (paraformaldehyde, glutaraldehyde), DAB substrate, and resins for embedding.
4:Experimental Procedures and Operational Workflow:
Steps involve DNA construct preparation, in utero electroporation, brain fixation and sectioning, APEX development with DAB, ROTO staining, resin embedding, ultrathin sectioning with ATUM, SEM imaging, and data reconstruction using Fiji software.
5:Data Analysis Methods:
Images were aligned and reconstructed using TrackEM2 plugin in Fiji for 3D visualization and manual tracing of neuronal structures.
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Vibratome
VT1200S
Leica
Used for sectioning brain tissue into 100 μm thick slices.
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Scanning Electron Microscope
Sigma
Zeiss
Used for obtaining electron micrographs of serial sections.
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Ultramicrotome
UC7
Leica
Used for ultrathin sectioning of resin-embedded samples.
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Camera
ORCA Flash 4.0
Hamamatsu Photonics
Used for capturing images on the stereoscope.
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Picospritzer III
III
Parker
Used for injecting DNA mixtures into the neocortical lateral horn of mouse embryos.
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Electroporation System
ECM 830
BTX
Used to deliver electric pulses for introducing plasmids into neural progenitor cells.
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Stereoscope
SMZ18
Nikon
Used for low-resolution fluorescence imaging of brain sections.
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Confocal Microscope
Ti-e
Nikon
Used for high-resolution imaging of labeled neurons.
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Diamond Knife
Ultra 35°
DiATOME
Used for sectioning in the ultramicrotome.
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