EMD-12729

Tomography
EMD-12729 Deposition: 07/04/2021
Map released: 09/06/2021
Last modified: 09/06/2021
Overview 3D View Sample Experiment Validation Volume Browser Additional data Links
Overview 3D View Sample Experiment Validation Volume Browser Additional data Links

EMD-12729

Tomogram of a Drosophila Malpighian tubule vitrified by plunge freezing upon incubation in 10% glycerol

EMD-12729

Tomography
EMD-12729 Deposition: 07/04/2021
Map released: 09/06/2021
Last modified: 09/06/2021
Overview 3D View Sample Experiment Validation Volume Browser Additional data Links
Method: Tomography
Aggregation State: Tissue
Specimen preparation [1]
Cryo protectant: 10% glycerol
Details: Dissected intact Drosophila Malpighian tubules with ureter, hind- and midgut were deposited on an EM grid.
Buffer
pH: 7.4
Buffer components [1]:
Name Formula Concentration ChEBI
PBS - - -
Grid
Mesh: 200
Model: Quantifoil R2/1
Material: MOLYBDENUM
Support Film [1]
Material Topology Thickness
CARBON HOLEY -
Vitrification
Cryogen name: ETHANE-PROPANE
Instrument: HOMEMADE PLUNGER
Details: The grids were mounted on a manual plunger, blotted from the back side using Whatman paper #1 (Sigma-Aldrich) and plunged into a 2:1 ethane:propane mixture cooled down by liquid nitrogen by the Martinsried-Plunger..
Sectioning (Focused ion beam)
Instrument: OTHER
Ion: OTHER
Voltage: 30 kV
Current: 0.05 nA
Duration: 3600 s
Temperature: 90 K
Initial thickness: 50000 nm
Final thickness: 160 nm
Details: To prepare thin electron transparent lamellae into the tissue, plunge-frozen grids were first mounted into Autogrid frames (FEI). The grids were then mounted into a dual-beam Quanta 3D FIB/SEM (FEI) using a custom-built transfer shuttle and a cryo-transfer system (PP3000T, Quorum). The samples were kept at -180 C throughout FIB milling by the cryo-stage. To improve SEM imaging, a thin layer of pure metallic Pt was sputtered onto the sample under cryo conditions in the PP3000T transfer system to increase its electrical conductivity. The following parameters were used: 10 mA sputtering current, 500 V between stage and sputtering target and 30 s of exposure at 4.5x10-2 mbar. To interpret and annotate the topographical anatomy of the tissue, overview maps of the EM grid were acquired by SEM at 10 kV at 100-250x magnification (object pixel size 1.1-0.4 um) and by secondary electrons induced by the Ga+ focused ion beam at 30 kV at 338x magnification (object pixel size 0.7 um). To protect the milling front of the lamellae, gaseous organic platinum was frozen on top of the grid using a gas injection system. To prevent bending of the lamella during the preparation, micro-expansion joints were milled left and right of the intended lamella preparation site. 10-20 um wide lamellae were prepared into the tissue with the ion beam at 30 kV at shallow angles (8-14 deg) in four consecutive steps: for the thicker tissue regions (e.g. VNC or skeletal muscle), the areas above and below the intended lamella were first
Microscopy [1]
Microscope: FEI TITAN KRIOS
Illumination mode: FLOOD BEAM
Imaging mode: BRIGHT FIELD
Electron source: FIELD EMISSION GUN
Acceleration voltage: 300 kV
Calibrated defocus: 0.5 µm -
Nominal magnification: 33000.0
Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Cooling holder cryogen: NITROGEN
Alignment procedure: BASIC
Details: Additional Volta phase plate alignment.
Specialist optics
Phase plate: VOLTA PHASE PLATE
Energy filter
Name: GIF Quantum LS
Slit width: 20 eV
Image Recording [1]
Detector model: GATAN K2 SUMMIT (4k x 4k)
Detector mode: COUNTING
Dimensions: 3868 pixel x 3868 pixel
Average electron dose per image: 3.0 e/Å2
Image processing [1]
Final reconstruction
Number of images used: 50
Algorithm: BACK PROJECTION
Software [1]
Name Version Details
IMOD 4.9.0 -
Tomography
Format: CCP4
Data type: IMAGE STORED AS SIGNED INTEGER (2 BYTES)
Annotation details: Tomogram of a Drosophila Malpighian tubule vitrified by plunge freezing upon incubation in 10% glycerol
Geometry
X Y Z
Dimensions 928 928 200
Origin 0 0 -100
Spacing 928 928 200
Voxel size 17.56 Å 17.56 Å 17.56 Å
Contour list
Primary Level Source
True - AUTHOR