Teneo VolumeScope is a Scanning Electron Microscope (SEM) with an inbuild ultramicrotome for serial block-face scanning electron microscopy (SBF SEM). The Thermo Scientific VolumeScope with Multi-Energy Deconvolution (MED) is a state-of-the-art SBF SEM that combines physical and optical slicing technologies with 10 nm isotropic 3D datasets of resin embedded biological samples. This field-leading 10 nm isotropic resolution is possible through the use of MED-SEM technology, allowing optical sectioning to derive several virtual subsurface layers within each physical slice, thus dramatically improving resolution, particularly in the axial direction. Acquired volumes are typically larger than those collected with Focused Ion Beam SEM (FIB SEM) technology. 
The instrument can also be utilized as a stand-alone SEM and can be used to visualize the surface topology of samples. The microscope can be used in both high- and low-vacuum mode. In low-vaccum mode the sample may contain water and no metal coating.

Specification
Filament: Field emission gun (FEG)
Beam current range: 1 pA to 400 nA
Landing energy range: 20 eV – 30 keV*
Accelerating voltage range: 350 eV – 30 keV
Imaging modes: High Vacuum, Low Vacuum (10-50 Pa) 
Magnifications: up to 300.000 x
MapsTM software integration for wide-field tissue mapping (Tiling and Stitching).
On-stage compact microtome with easy installation for in-situ sectioning. Optimal imaging resolution at low voltages (1.0 nm at 1 keV using the immersion lens) for 2D SEM images. 
SBF SEM datasets capable of multiple ROI collection during the same job acquisition with 10-nm isotropic resolution.
Detectors:

• Everhart-Thornley SE detector (ETD)
• Trinity (T1 and T2) segmented lower in-lens detector
• VS-DBS: LoVac lens-mounted BSED
• Low-vacuum SE detector
• STEM 3+ – Retractable segmented detector
• IR-CCD

Tecnai 12 is a Transmission Electron Microscope (TEM) that fulfil the purpose of standard TEM ultrastructural investigations. The microscope is a high contrast TEM. Montaging pictures can be acquired using a digital Morada camera and the Radius software. 
The microscope is equipped with additional equipment for X-ray microanalysis (EDX) and scanning transmission (STEM), use for the elemental analysis or chemical characterization of a sample. Montaging pictures can be acquired using the TIA software.
Specification
Filament: LaB6 (Lanthanum Hexaboride) 
Accelerating voltage: up to 120 kV, operate at 80 kV
Imaging modes: Brightfield, Darkfield, STEM
Resolution: 0,2 nm
Magnifications: from 30 x
Image capture: MORADA CCD (3392 x 2248 x 16bit)
Detector: EDX
Software: Radius and TIA
 

JEM-1011 is a Transmission Electron Microscope (TEM) that fulfil the purpose of standard TEM ultrastructural investigations. The microscope is a high resolution TEM. Montaging pictures can be acquired using a digital Morada camera and the Radius software. 
Specification
Filament: Tungsten
Accelerating voltages: up to 100 kV, operate at 80 kV
Resolution: 0,2 nm
Magnifications: from 30 x 
Image capture: MORADA CCD (3392 x 2248 x 16bit)
Ditabis digital imaging plates
Software: Radius

Ultramicrotome for room temperature sectioning

Materials for TEM must be specially prepared to thicknesses which allow electrons to transmit through the sample, much like light is transmitted through materials in conventional optical microscopy. Because the wavelength of electrons is much smaller than that of light, the optimal resolution attainable for TEM images is many orders of magnitude better than that from a light microscope. 
Ultramicrotomes are instruments commonly used to prepare TEM samples. Glass or diamond knife is use for sectioning of specimens embedded in epoxy. Room temperature sectioning can be done for semi thick sections up to 3µm and ultra-thin sections down to 50 nm.

Leica EM UC7
Leica EM UC6
RMC MT-X

Ultramicrotome for cryo temperature sectioning

The Leica EM UC6/FC6 provides easy preparation of semi- and ultra-thin sections as well as perfect, smooth surfaces of biological samples for TEM, SEM, AFM and LM examination. With cryo chamber FC6 mounted to the ultramicrotome, low temperature sectioning at temperature -15 C to -160 C can be done for cryosections (60 - 3000 nm) for TEM and smooth surface for SEM.

Vibratome uses a vibrating razor blade to cut sections from 1-999 µm under physiological conditions without freezing or embedding, and thus ultrastructure is well preserved.  The tissue can be fixed or fresh when using a vibratome.  The vibratome maintains cell morphology, enzyme activity and cell activity.  The microtome is well used for immunocytochemistry and histoenzymology. Ultimately, vibratome tissue sections can be trimmed down and used for high pressure freezing (HPF).

Leica EM KMR3 makes glass knives for perfect ultrathin sections for EM and LM applications. The balanced break method of the EM KMR3 ensures perfect glass knives in three thicknesses; 6.4 mm, 8 mm and 10 mm. The EM KMR3 is easy to use combined with the automatic reset of the breaking wheel and scoring mechanism to "default" after a breaking cycle, avoids handling errors.

Leica ACE600 is an easy operating desktop coater, designed to produce homogeneous coatings of conductive metal or carbon as required for electron microscopy. The fully automated instrument can be configured either as a sputter coater, a carbon thread evaporation coater or a glow discharger (to make TEM grids hydrophilic). Includes Quartz crystal measurement for reproducible layers, plain or planetary rotary stage for evenly distribution of the coating on the sample.

Sputter coating: When a target is bombarded with fast heavy particles, erosion of the target material occurs. The process, when occurring in the conditions of a gaseous glow discharge between an anode and cathode is termed sputtering. Enhancement of this process for scanning electron microscopy (SEM) sample coating is obtained by the choice of a suitable ionization gas and target material. Sputtered metal coatings offer the following benefits for SEM samples:

• Reduced microscope beam damage.
• Increased thermal conduction.
• Reduced sample charging (increased conduction).
• Improved secondary electron emission.
• Reduced beam penetration with improved edge resolution.
• Protects beam sensitive specimens.

Carbon coating: Formvar film is useful for the support of ultrathin sections on the finer mesh grids. Using of support film are ideal for those applications requiring large viewing areas without grid bar interference. These films must be strong, clean and remain attached to the specimen grids during specimen preparation. A Formvar film covered with a "light" layer of carbon will help to stabilize the film when the film is exposed to the electron beam.

Glow discharge: To ensure a smooth, even spread of sample and stain, the support film must be hydrophilic. A freshly carbon-coated grid will provide good spreading, but older grids may not. Methods for returning hydrophobic grids to a state where they will provide an acceptable stain is to glow discharge the grids with support films prior to adsorbing the sample suspension.

Bulk biological specimen (along with hydrated materials) must be dried rapidly, without introducing artefacts, before they can be imaged in a SEM. The critical point dryer is the most efficient instrument for quickly drying specimens without the shrinking or cracking that accompanies other methods.
The procedure of critical point drying is an efficient method for drying delicate samples. It preserves the surface structure of a specimen which could otherwise be damaged due to surface tension when changing from the liquid to gaseous state. Before drying, many biological samples are commonly prepared through fixation and dehydration steps.

Biowave pro+ is a microwave oven for biological sample processing. The microwave oven has a built-in vacuum, a connected ColdSpot Pro water load and processing surface, plus Pelco SteadyTemp pro which controls the processing temperature in the oven. The system makes it possible to process tissue and cells in ½ a day ready for sectioning, with high quality result.

From Ted Pellas page:
"The new design of the PELCO BioWave® Pro+ introduces features that improve efficiency and streamline functionality. It is built on trusted technology and adds enhancements for efficient laboratory processing. This new tissue processor further enhances processing of tissue for diagnostic and research TEM, immunofluorescence and confocal specimen processing."

TEM is a microscopy technique whereby a beam of electrons is transmitted through an ultrathin specimen, interacting with the specimen as it passes through it. An image is formed from the electrons transmitted through the specimen, magnified and focused by an objective lens and appears on an imaging screen, a fluorescent screen in most TEMs, plus a monitor, or on a layer of imaging plate, or to be detected by a sensor such as a CCD camera. 

Biological materials contain large quantities of water. To be able to view it in the electron microscopy, the first stage in preparing is the fixation, one of the most important and most critical stages. We need to fixed it in a way that the ultrastructure of the cells or tissues remain as close to the living material as possible. The sample is then dehydrated through an acetone or ethanol series, passed through a “transition solvent” such as propylene oxide and then infiltrated and embedded in a liquid resin such as epoxy and LR White resin. After embedding the resin block is then thin sectioned by a process known as ultramicrotomy, sections of 50 - 70 nm thickness are collected on metal mesh 'grids' and stained with electron dense stains before observation in the TEM.  Sectioning the sample allows us to look at cross-sections through samples to view internal (ultra)structure.  Many modifications to the basic protocol can be applied to achieve many different goals, immunogold labeling for example; the in situ localization of specific tissue constituents using antibody and colloidal gold marker systems. 

SEM is primarily useful for giving a three-dimensional image of the surface of the specimen and is for viewing large objects.

As with TEM, specimens are imaged with a beam of electrons, but instead of the electrons being transmitted through the specimen, the beam is "scanned" across, creating an image of the surface of the sample, with exceptional depth of field. This image is achieved via the detection of "secondary" electrons that are released from the specimen as a result of it being scanned by very high energy "primary" electrons (ie. those emitted from the electron "gun" in the SEM). As most biological specimens are made up of non-dense material the amount of secondary electrons produced is too low to be of much use in creating an image and therefore they are usually coated with a very fine layer of a metal which readily produces secondary electrons. The large depth of field achievable can produce an image of great visual depth with a three-dimensional appearance.

The operating environment of a standard scanning electron microscope dictates that specialist preparation techniques are used.  Typically, a biological specimen is chemically fixed, dehydrated through an acetone or ethanol series and then dried at the critical point - a method used to minimize specimen distortion due to drying tensions. The samples are mounted on a stub of metal with adhesive, coated with 40 - 60 nm of metal such as Gold/Palladium and then observed in the microscope. For dry samples, this process is not necessary. SEM can also be used to investigate smooth surfaces of industrial samples.

To generate good quality SBF-SEM images, the tissue must be rendered electron-dense, producing image contrast and increasing conductivity to prevent charging artefacts which distort the images. The stages of sample processing are similar to those used in preparation for TEM imaging.

Osmium tetroxide has long been used as a TEM fixative and tissue stain. While sufficient to provide contrast in TEM imaging, tissue stained with osmium tetroxide alone does not impart enough contrast in SBF-SEM imaging where low (2-5 kV) accelerating voltages and a BSE detector are used. Thus, protocols have been developed to increase the impregnation of heavy metals, including the osmium-thiocarbohydrazide-osmium (OTO) method, where thiocarbohydrazide acts as a bridging reagent allowing more osmium to bind to the tissue, and the ferrocyanide-reduced osmium tetroxide methods (rOTO). Pyrogallol can also be replaced with thiocarbohydrazide to improve the penetration. En block staining with uranyl acetate and lead aspartate is important to increase the yield of BSEs during imaging.

Biological samples for SBF-SEM need to be embedded in resin which supports the tissue, creates uniform hardness across tissue and resin, allows the block to remain stable, resist shrinkage and maintain integrity in the electron beam. At present, all commercially available resins for EM are non-conductive. However, recent developments have suggested that the addition of materials, such as carbon nanotubes or carbon black filler, can produce a conductive resin which reduces charging artefacts and improves spatial resolution.

The principles of TEM specimen preparation apply equally to SBF-SEM with the added demands that the tissue should be as conductive as possible in order to allow scanning of the block face without build-up of surface charge. In addition to enhancing image contrast, heavy metals make tissue more conductive, which reduces charging, reduces the breakdown of resin and thus improves sectioning and image quality. The tissue should also exhibit enhanced electron density to allow for a strong high-contrast BSE signal to be delivered. This in turn facilitates image visualization, segmentation and quantification.

Details in light microscope samples can be enhanced by stains that absorb light; similarly TEM samples of biological tissues can utilize high atomic number stains to enhance contrast. The stain absorbs electrons or scatters part of the electron beam which otherwise is projected onto the imaging system. Uses heavy metals such as lead and uranium to scatter imaging electrons and thus give contrast between different structures, since many (especially biological) materials are nearly "transparent" to electrons (weak phase objects). 

Heavy metal salts attach to various organelles or macromolecules within the sections to increase their electron density and they appear dark against a lighter background. Uranyl ions react strongly with phosphate and amino groups so that nucleic acids and certain proteins are highly stained. Lead ions bind to negatively charged components and osmium-reacted areas (membranes).

Positive staining stains the specimen such that the structures are dark against a lighter background. Grids are stained with heavy metals, such as uranyl acetate and lead citrate. 

Negative staining is a rapid procedure used for visualization of small particles, such as viruses, macromolecules, proteins, organelles, or bacteria, in fluids. The conventional protocol involves the absorption of the specimen to a glow discharged carbon-coated EM grid, which is washed with two drops of deionized water and subsequently stained with two drops of heavy metal solution. The specimen will be embedded in a layer of dried heavy metal solution that remain around the edges of particles, such that the details of the particles are clearly defined, producing a strong contrast between the background and the particle.

This technique uses antibodies to detect the intracellular location of structures of particular proteins by electron microscopy. Ultrathin sections are labelled with antibodies against the required antigen and then labelled with gold particles. Gold particles of different diameters enable two or more proteins to be studied.

EMLab can offer post-embedding immunogold labelling of frozen hydrated ultrathin sections (Tokuyasu-method) and samples embedded in resin (Epoxy and LR White).

The investigator must supply the primary and secondary antibodies. The investigator should do immunolabelling at the fluorescent light microscopy level before attempting it at the EM level.

Tokuyasu cryosectioning technique is one of the most reliable and sensitive immunolocalization techniques for detection of specific proteins in cells and different types of samples. By conjugating a sample specific antibody to a small colloidal gold particle (eg 1nm, 5nm, 10nm, 20nm) labelled structures can be identified using a transmission electron microscope.

The method follows a simple protocol: samples are lightly chemically fixed, after which they are frozen in the presence of sucrose and cut into 50-100 nm cryosections. The sections are then thawed and labeled with antibodies that specifically recognize the molecule of interest, while coupled to gold particles that are detectable in the microscope. These sections are ideal substrates for immunolabeling, since antigens are not exposed to organic solvent dehydration or masked by resin. Instead, the structures remain fully hydrated throughout the labeling procedure. Furthermore, target molecules inside dense intercellular structural elements, cells and organelles are accessible to antibodies from the section surface. By applying stereology, gold labeling can be quantified and evaluated for specificity. It complements fluorescent labelling by localizing immune reactions to specific intracellular structures at a resolution that cannot be achieved with fluorescence. For this reason it has become a powerful tool when it comes to prove co-localization of antigens. The technique is also used at scanning electron microscope level to provide details about the surface distribution of antigens.  

The EMLab provides basic to advanced training on our transmission electron microscopy (TEM) and scanning electron microscopy (SEM). We also provide training on small instruments. 

Training is available for a fee at an hourly rate for the instrument. Our training is tailored to individual users, however even experienced users must demonstrate they are proficient in using the microscope of their choice. Assistance for approved user will still be available by request but will be charged (technical assistance price/hour). If the user has been inactive for more than 3 months, his/her access to the instrument will be terminated. Prior to regaining access, the user must be retrained supervised by the lab staff. The user will be charged for the new training. 

For full-scale projects or to get data-on-demand for grants or publications, the EMLab provides fee-for-service full-service microscopy. Please use the Quotation form to request this service and ensure that you provide adequate detail on the form that the lab staff can respond appropriately. All quotations are non-binding fee estimates.

For any potential user, our staff provides free consultation services to help define how electron microscopy (EM) may be utilized for the proposed project.  In this consultation, users can expect the lab staff to help determine whether EM is appropriate for answering the given question of the project. The staff will assist in defining the scope of the EM to be used in the project. Further the staff will help identify what equipment (ancillary and microscope) the user would need to access in our facility to complete the EM data collection or specimen preparation for their project.   

You can contact us by email CMIC Electron Microscopy Lab.

Training on electron microscopes will be provided and approved by the lab staff. Training will be provided as needed. When the training is approved, the user will be given an unassisted access to the instruments during the working hours (8 AM – 4 PM). Working outside working hours needs to be approved by the lab staff.

All users must attend general laboratory safety and chemical management training prior to work in the EMLab. A user must request permission to bring a chemical into or out of the lab. Users are expected to follow policies and procedures defined in the chemical management for the lab, and safety protocols established during the training. Any user not adhering to policy, procedures, protocols or safety requirements will be suspended from the lab. Samples that have been embedded in EMLab will be stored in the lab. The user cannot remove the samples from the lab without clarifying with the lab staff in advance.

We can offer the possibility of temporary data storage on our server. The server has limited capacity and a tendency to quickly fill up. The data will be deleted after 3 months without notification!  Printers and computers are for lab use and research ONLY. They may not be used for any other personal, professional, or academic affairs.