研究目的
To present a protocol for performing correlative light and electron microscopy (CLEM) on Caenorhabditis elegans embryos to analyze the subcellular localization of GFP-tagged LC3 homologs (LGG-1 and LGG-2) during autophagy and phagocytosis.
研究成果
The protocol allows for easy and rapid correlation between electron and light microscopy, enabling the determination of subcellular localization of fluorescent proteins. It was successfully used to show that LGG-1 is enriched in vesicles inside apoptotic corpses, while LGG-2 is enriched in phagocytic cells in C. elegans embryos.
研究不足
The CLEM pictures have a lower contrast than classical transmission electron microscopy, and the approach may have difficulty in retrospective location of specific regions of interest compared to other methods.
1：Experimental Design and Method Selection:
The protocol involves cryofixation by high-pressure freezing to preserve structures, freeze substitution and inclusion in GMA resin to preserve GFP fluorescence, ultramicrotomy, and successive observations by light and electron microscopies, followed by correlation of images.
2：Sample Selection and Data Sources:
Caenorhabditis elegans embryos from specific strains (RD286 and ZH2333) expressing GFP::LGG-1 and GFP::LGG-2 are used.
3：List of Experimental Equipment and Materials:
Includes HPF apparatus EM PACT2, AFS2 system, ultramicrotome EM UC7, microscopes (AxioObserver Z1, Axioskop 2 Plus), TEM (JEOL 1400), various buffers, resins, and tools.
4：Experimental Procedures and Operational Workflow:
Steps include cryofixation, freeze substitution, resin embedding, sectioning, light microscopy, electron microscopy, and image correlation using software.
5：Data Analysis Methods:
Images are analyzed and superimposed using ImageJ, PowerPoint, and Photoshop software.
独家科研数据包，助您复现前沿成果，加速创新突破
获取完整内容-
Inverted microscope
AxioObserver Z1
Zeiss
Light microscopy for observing fluorescent and bright field images
-
Upright microscope
Axioskop 2 Plus
Zeiss
Light microscopy with Nomarski optics
-
Transmission electron microscope
JEOL 1400
JEOL
Electron microscopy for ultrastructural analysis
-
Flat carrier
200 μm deep flat carrier
Leica Microsystem
Holding samples during cryofixation
-
HPF apparatus
EM PACT2
Leica Microsystem
Cryofixation by high-pressure freezing to preserve sample structures
-
Automated freeze-substitution system
AFS2
Leica Microsystem
Freeze substitution process with integrated binocular lens and incubating chamber
-
Ultramicrotome
EM UC7
Leica Microsystems
Sectioning of resin-embedded samples for microscopy
-
Diamond knife
Histo, 2 mm, 45°
Diatome
Cutting thin sections for microscopy
-
Camera
Evolve EMCCD
Roper Scientific
Imaging for light microscopy
-
Camera
CoolSNAP
Roper Scientific
Imaging for light microscopy
-
Cell culture dish
35 mm cell view
Greiner Bio-One
Mounting grids for inverted microscope observations
-
Flat embedding insert
Leica Microsystem
Container for samples during freeze substitution
-
Specimen processing metal block
Leica Microsystem
Holding flat embedding insert during processing
-
Aclar sheet
Leica Microsystem
Used in resin embedding process
-
Finder grid
200-mesh copper formvar carbon-coated finder grid
Holding sections for microscopy
-
登录查看剩余13件设备及参数对照表
查看全部
