Image 2D


Image 2D Data Files

Image, 512x512 image, Electron micrograph of cultured Drosophila Dl1 cells infected with flock house virus, prepared using chemical fixation and high pressure freezing followed by freeze substitution. This cell was prepared as part of an experiment to investigate different protocols for high pressure freezing.
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Image 2D Data Files, Full sized tiff image (CAFHPF_rec.tif) of the insect cells processed using conventional aldehyde fixation high pressure freezing. Image corresponds to Fig. 1B from the publication.
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Licensing

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Description

Electron micrograph of cultured Drosophila Dl1 cells infected with flock house virus, prepared using chemical fixation and high pressure freezing followed by freeze substitution. This cell was prepared as part of an experiment to investigate different protocols for high pressure freezing. This image has been downsampled from the raw data image which can be accessed using the link provided to the Cell Centered Database.

Technical Details

Fixation: The media was removed and saved. Then, cell pellets were fixed in 2% glutaraldehyde in 100 mM cacodylate buffer for 30 minutes on ice. The fixed pellet was resuspended in media and centrifuged again. Cells were loaded into the 100 mm well of a type A brass planchette (Ted Pella, Inc. Redding, CA) and fast frozen in the Bal-Tec HPM010 (Bal-Tec, Liechtenstein). Freeze substitution: After freezing, samples (2) and (3) were placed into a Leica EM AFS Freeze substitution (FS) machine (Leica Microsystems, Bannockburn, IL) and incubated at -90 deg C for 24 hours in 0.1 percent tannic acid in acetone. Samples were washed three times with cold acetone (cooled to -90 degrees C) over 5 minutes, and placed in 1 percent OsO4 and 0.1% UA in cold acetone for 72 hours and held at -90 degrees C. After slowly warming to room temperature at 5 degrees C per hour, the specimens were rinsed in pure acetone three times (10 min. at room temperature). Infiltration and embedding in Durcupan resin was subsequently performed at room temperature.Images were acquired using a JEOL4000EX IVEM, magnification: 30000.0; accelerating voltage: 80.0 keV.

Biological Sources

NCBI Organism Classification
Drosophila melanogaster
(Common fruit fly)
Cell Type
embryonic cell
Cellular Component
cell body

Attribution

Names
Mark Ellisman
Gina Sosinsky
Ying Jones
Link
CCDB 3939

Grouping

This image is part of a group.

Imaging

Image Type
recorded image
Imaging Mode
FBbi:00000623
Parameters Imaged
electron density
Data Qualifiers
processed data

Sample Preparation

Methods
osmium tetroxide fixed tissue
glutaraldehyde fixed tissue
Relation To Intact Cell
dispersed cells in vitro

Dimensions

Spatial Axis Image Size Pixel Size
X 512px ——
Y 512px ——

Detailed Project Information                           hide

Project ID: P1243
Leader: Mark Ellisman, Gina Sosinsky, Ying Jones
Collaborators:  
Description: This project is designed to achieve ultimate ultrastructure of animal tissues.
Experimental purpose: Testing new high pressure freezing techniques on cultured cells
Species: fruitfly
Cell type: Drosophila DL1 cell
Product type: confocal
Instrument: Biorad Radiance 2000 Confocal
Publications: Sosinsky GE, Crum J, Jones YZ, Lanman J, Smarr B, Terada M, Martone ME, Deerinck TJ, Johnson JE, Ellisman MH. The combination of chemical fixation procedures with high pressure freezing and freeze substitution preserves highly labile tissue ultrastructure for electron tomography applications. J Struct Biol. 2007 Sep 14; PMID:17962040
Start/End Dates: 0/4/2004 - unknown

Specimen Preparation                                     hide

Microscopy Product ID: 3939
Image Basename: CAFHPF
Protocol Description: Fixation: The media was removed and saved. Then, cell pellets were fixed in 2% glutaraldehyde in 100 mM cacodylate buffer for 30 minutes on ice. The fixed pellet was resuspended in media and centrifuged again. Cells were loaded into the 100 mm well of a type A brass planchette (Ted Pella, Inc. Redding, CA) and fast frozen in the Bal-Tec HPM010 (Bal-Tec, Liechtenstein).

Freeze substitution: After freezing, samples (2) and (3) were placed into a Leica EM AFS Freeze substitution (FS) machine (Leica Microsystems, Bannockburn, IL) and incubated at -90 deg C for 24 hours in 0.1 percent tannic acid in acetone. Samples were washed three times with cold acetone (cooled to -90 degrees C) over 5 minutes, and placed in 1 percent OsO4 and 0.1% UA in cold acetone for 72 hours and held at -90 degrees C. After slowly warming to room temperature at 5 degrees C per hour, the specimens were rinsed in pure acetone three times (10 min. at room temperature). Infiltration and embedding in Durcupan resin was subsequently performed at room temperature.

Detailed Imaging Parameters                          hide

Electron microscopy product
Magnification: 30000.0
Recording medium: No recording medium provided
Accelerating voltage: 80.0 keV
*CIL – Cell Image Library accession number. Please use this to reference an image.