Electron Microscopy Node

• Transmission Electron Microscopy (TEM)

TEM enables you to obtain high-resolution images of specimens due to the short wavelenght of the electrons. It is used to study the ultrastructure of tissues, cells, virus, organelles and macromolecule complexes.

The image in a TEM is generated when a beam of electrons beam pass through a thin section of the specimen and undergo scattering events. Regions with elements of high atomic number scatter more than regions with low atomic number.

• Chemical fixation

In conventional sample preparation, the specimens are generally fixed with aldehydes, contrasted/fixed with osmium tetroxide, dehydrated and embedded in a hard resin. This is followed by cutting of ultrathin sections (~50-60 nm) which are placed on grids covered with a thin support film. Sections are contrasted with heavy metals, usually uranyl acetate and lead citrate, and air-dried before imaging.

• Negative staining

Negative staining is an easy and fast method. It is used to visualize small particulates like proteins, exosomes, lipids, virus, filaments and more.

A drop of the sample in solution is put on a glow discharged grid. Excess solution is removed by a filter paper followed by the addition of a contrasting reagent, usually uranyl acetate. After removing the contrasting reagent by a filter paper, the grid with the samples is left to air dry before imaging.

It is possible to combine immunogold labeling and negative staining.

• Immunogold labeling

Immunogold labeling is a technique used to identify and localize protein antigens in e.g. tissues, cells and suspension.

Primary antibodies are used to target specific antigens of interest, which are subsequently detected by secondary antibodies conjugated to a colloidal gold particle. Due to the high scattering property of gold, the particles are easily detected in the microscope as dark spots.

BioVis can provide its customers with goat anti-mouse and goat anti-rabbit secondary antibodies. All other antibodies will be charged extra.

Image: Negative stain TEM image of bacteriophage. Magnification: 105 000X, Kindly provided by Marcelo Gutierrez-Valverde, IMBIM, Uppsala University.

SEM enables you to obtain high resolution images of the sample surfaces as well as intra-cellular ultrastructures.

The image in the SEM is generated mainly by scattered secondary electrons that are released from the sample surface as a thin electron beam rasters across it.

• Chemical fixation

In conventional SEM sample preparation, the specimens are generally fixed with aldehydes, contrasted/fixed with osmium tetroxide and dehydrated.

To study sample surfaces the specimen is dried in a critical point dryer and sputtered with a thin layer of gold/palladium prior imaging.

Image: SEM image of crodile skin, 1,14 x magnification, we gratefully thank Helge Raks-Anderssen for this image.

• HT7800 TEM

The BioVis EM node offers its customers a Hitachi HT7800 Transmission electron microscope. The instrument is easily operated and provides high-contrast imaging, ideal for biological samples.

Specifications of HT7800

• 80-120kV (Lab6 filament)
• Resolution: 0.2 nm minimum
• Magnification: up to 600.000x
• Multiholder available (can hold up to 3 samples)
• Camera: Xarosa EMSIS, 20 MP

We encourage our customers to sit independently and acquire images to ”get to know” their samples and the advantages/limitations of the microscope. We provide an introduction to the basic operation for imaging. Please note that we charge for introduction and using the instrument.

BioVis gratefully acknowledges the financial support from MedFarm FISK, IGP department och Beijer Stiftelsen which has co-financed the purchase of instrument.

• Glow Discharger

The glow discharger is an essential tool for electron microscopy sample preparation. It removes contaminants from the surface of electron microscopy grids and renders them hydrophilic, allowing aqueous samples to spread evenly across the grid surface. This treatment improves sample adsorption and is routinely used prior to negative staining and cryo-electron microscopy experiments. The Glow Discharger Pelco easiGlow is used routinely in negative staining to clean the grid surface and render it hydrophilic.

Image: TEM image of an short inner hair cell in the inner ear of an Cuban crocodile. Magnification: 9900X
Kindly provided by Helge Rask-Andersen, Pubcare, Uppsala University Hao Li et al., Front. Cell Dev. Biol, 2022

The BioVis EM node provides the following equipment for scanning electron microscopy (SEM) preparation:

• INFO SEM instrument

• Critical Point Dryer

The critical point dryer Leica EM CPD 300 is used to prepare delicate specimens for scanning electron microscopy (SEM). During the process, water within the sample is replaced by ethanol and subsequently by liquid carbon dioxide. By drying the specimen above the critical point of CO₂, surface tension forces are eliminated, preventing collapse or deformation of fragile structures and preserving the specimen's native morphology.

• Sputter Coater

Most biological and other non-conductive specimens accumulate electrical charge under the electron beam, which can distort images and reduce resolution. The sputter coater deposits an ultrathin conductive metal layer, typically gold, platinum, or a gold-palladium alloy, onto the specimen surface. This coating minimizes charging effects, improves signal quality, and enables high-resolution SEM imaging. Biovis is using the Leica EM ACE600 High Vacuum Sputter Coater.

Image: SEM image of crodile skin, 12170x magnification, we gratefully thank Helge Raks-Anderssen for this image.

SEM imaging is provided in cooperation with Ångström. As the cost is influenced by multiple factors managed outside BioVis, pricing may vary and cannot be established in advance.

Overview about estimated duration of services

Additional costs: Carbon coated grid 80 SEK

Note: The estimated time can vary depending on the difficulty of the sample. E.g. if it is necessary to search for an unusual structure, many sections may be required and thus the estimated time might increase.

After fixation store samples at 4 degrees Celcius before brining them to the EM core facility. Please contact staff before drop-off!

LOCATION of EM Facility

 Please fill in a orderform TEM or orderform SEM prior project start and send to us by mail. Links are found in main text on top of the page.