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gloved hands preparing a specimen
Infrastructure_

Specimen preparation

The critical first step to great results
Our specialists are available to train and advise users on the most suitable techniques for preparing samples for electron microscopy.

Biological specimen preparation

For enquiries relating to biological specimen preparation, please contact:

Naveena Gokoolparsadh
Biological Specimen Preparation Specialist
02 9351 7642 | naveena.gokoolparsadh@sydney.edu.au

Available facilities

There are four Leica Ultracut ultramicrotomes, including the latest model (UC7), available to cut semi-thin (for light microscopy) and ultra-thin (for TEM) sections of resin-embedded biological and materials specimens. High quality glass knives for ultramicrotomy can easily be produced using our Leica KMR3 Knifemaker.

The cryo ultramicrotome enables semi-thin and ultra-thin sectioning of biological specimens and materials samples at a wide at sub-zero temperatures ranging between -15°C to -185°C. Biological specimens can be unfixed, cryoprotected, frozen and sectioned, a method which is particularly useful for immunolabelling of tissue. Often materials samples are too soft section at room temperature, but are able to be cut frozen using the cryo-ultramicrotome.

The Leica EMPACT2 High Pressure Freezer (HPF) is used to rapidly freeze small biological specimens for TEM. The omission of chemicals during fixation greatly improves the preservation of the specimen at the ultrastructural level. Specimens are subjected to very high pressure immediately before being injected with liquid nitrogen, which eliminates the formation of ice crystals, producing vitreous samples. Tissue pieces, liquid suspensions or cell monolayers can be frozen in this way to a depth up to 200 microns.The HPF is also equipped with the following tools:

  • A biopsy gun, for fresh tissue biopsies
  • A Rapid Transfer System (RTS), which allows rapid transfer of samples to the HPF.
  • A Live Cell kit, which is used in conjunction with the RTS to enable correlative imaging between the confocal and TEM.

The Automatic Freeze Substitution unit (AFS) is used in conjunction with the HPF. Frozen specimens are transferred to the pre-cooled AFS unit, where the ice is gradually replaced by solvent or solvent/fixative solutions (eg: acetone or acetone/osmium) at sub-zero temperatures. This process minimises extraction of cell contents during the course of the dehydration and also maintains antigenicity.An average AFS run takes 5 days. There are two Leica AFS units available, including the latest model which is equipped with a robotoic attachment that allows the entire FS process to be carried out fully automatically.

Also known as Vibrating Blade Microtome, this instrument is used to section soft to medium hard specimens without prior embedding in resin or paraffin. Section thickness can be set from 1 to 1000 microns. The vibrating blade reduces compression of the sample during sectioning, allowing samples as soft as brain material to be sectioned. Samples can be fresh or fixed and sections can be collected and stained or immunolabelled for confocal/light microscopy before possible further processing for EM.

The Vitrobot is an automated plunge-freezing instrument used to prepare samples in suspension for cryo-TEM imaging on the JEOL 2100. The sample is placed directly on a TEM grid in a humidity and temperature controlled chamber then rapidly plunge frozen in liquid ethane, preserving ultrastructure as close as possible to the native state. Samples are then viewed under the TEM in cryogenic conditions.

The Critical Point Dryer (CPD) uses liquid carbon dioxide to flush solvent from dehydrated biological specimens to dry them for SEM imaging. This process much gentler than either air-drying or chemical drying with Hexamethyldisilazane (HMDS) and is particularly recommended when processing plant samples for SEM. A typical CPD run takes approximately 2 hours.

Cell culture

This facility is designed to accommodate the culture of mammalian cell lines. We provide the media, components, serum supplements and reagents necessary to sustain cells and tissues in culture systems. There are biological safety cabinets for the sterile handling of cells, as well as several atmosphere-controlled incubators for growing cells. Phase-contrast, fluorescence, standard bright-field microscopes, as well as a cell counter are available for cell analysis.

For enquiries relating to cell culture, please contact:

Minh Huynh
Cell Culture Specialist
02 9351 7525 | minh.huynh@sydney.edu.au

Available facilities

The Class II biological safety cabinet provides protection for personnel, environment and products in work with microorganisms and where the work produces a significant quantity of aerosol. The cabinet provides protection by creating an air barrier at the work opening, recirculating HEPA-filtered laminar airflow downwards through the work zone and by HEPA filtration of exhaust air.

The cytotoxic drug safety cabinet is designed to provide safety protection to the operator, products and the environment against exposure to aerosols and vapours that can be generated in the preparation and manipulation of cytotoxic drugs.

The requirements for protection involve the following:

  • Protection of cabinet users and other staff from exposure to aerosols or vapors which may be generated in the preparation, manipulation and dispensing of cytotoxic drugs.
  • Protection of drug products, so that they may be prepared in an environment which is essentially free from particulate and biological contamination.
  • Protection of cabinet maintenance personnel from the residue of drug particles, which can contaminate filters, fans and other mechanical components.

The ES-315 Autoclave provides convenient processing of your laboratory sterilisation and media preparation needs. The autoclave offers three cycles of operation ranging from basic sterilisation cycles to automatic warming cycles.

The warming cycle may be used directly following a sterilisation cycle or independently to melt and keep sterile media ready for use. The cycles may be programmed up to 99 hours in advance using the Delayed Start Timer. This feature enables you to have sterile media on hand and ready for use when you need it.

To ensure safety and complete sterilisation, the autoclave is equipped with an independent temperature and pressure monitoring system.

This multilabel reader covers all of the main non-radioactive reading technologies. Technologies and reading modes include fluorometry, luminometry, fluorescence polarisation, time-resolved fluorometry (TRF), and photometry (including UV absorbance). The plate reader is also loaded with temperature control and shaking.

HEPA air filtration systems provide continuous protection against unwanted airborne contaminants. The incubators provide precise CO2control with excellent temperature uniformity for researchers seeking the benefits of complete contamination control and dependable long-term performance.

Materials specimen preparation

For enquiries relating to materials specimen preparation, please contact:

Jacob Byrnes
Materials Specimen Preparation Specialist
02 9351 7546 | jacob.byrnes@sydney.edu.au

  • 3D Micromac microPREP laser micromachining tool
  • JEOL Cryo Ion Slicer rocking broad ion beam system for TEM
  • Gatan PIPS ll broad ion beam miller for TEM specimens
  • Gatan PECS ll broad ion miller for SEM specimens
  • Gatan PIPS Model 691 broad ion beam miller for TEM
  • Leica EM TXP target surfacing system
  • Struers TegraPol-25 automated grinder and polisher
  • Struers RotoPol-22 automated grinder and polisher
  • Struers Cito-Press-10 hot mounting system
  • Struers Accutom-50 (x2) precision cut-off machines
  • Struers Discotom-10 precision cut-off machine (large samples)
  • Allied High Tech MultiPrep precision grinder and polisher
  • Allied High Tech tripods for TEM thin film preparation
  • Allied High-Tech Techprep8 precision grinder and polisher
  • Gatan Disc Grinder 623 (x2) for precision material removal
  • Struers TenuPol-5 Electropolishing Jet Thinner to prepare TEM specimens from metals and alloys 3 mm discs.