Biological Specimen Preparation

Ultramicrotomes
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.  Diamond knives are available for experienced users.

 

Cryoultramicrotome
The cryoultramicrotome 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. There are two Leica Ultracut cryo-ultramicrotomes are available, including the latest model (UC7) which is equipped with a cryo-manipulator.

  cryo-ultramicrotome

High-Pressure Freezer
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.
  hpf

 Automatic Freeze Substitution Unit
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.

   AFS

 Vibratome
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.

   vibratome

Vitrobot
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.

   vitrobot

 Critical Point Dryer (CPD)
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.

   cpd