Cryo and Room Temperature Sample Processing

There are three possible ways we can process samples in the IGC EM Facility

Method 1: Conventional Processing

Conventional sample processing entails using chemicals to fix the sample. Typically we begin the processing with a buffered aldehyde fixation, then we fix the sample further using osmium tetroxide before we dehydrate the sample and embed it in a plastic resin. However, it is important to note that each conventional fixation protocol is adapted to help address the biological questions of the research project. For example, we will change the buffer to alter the amount of cellular extraction, and we will introduce additional chemicals, such as tannic acid, or uranyl acetate to aid in the preservation and staining of the sample.

  • Duration of protocol: 12 hours over 2 – 3 days
  • Advantages: Protocol adaptable for many different kinds of samples; technique is relatively easy to do and to optimize.
  • Disadvantages: Penetration time of fixatives can be slow so the outer edge of a sample will fix earlier than the center of the sample; protocols can be very long; dehydration artifacts are unavoidable

Method 2: Microwave Chemical Processing

Microwave chemical processing is very similar to conventional processing with the modification that we use a specialized microwave to improve the penetration rate of the chemicals. Many times the chemicals used in microwave processing are the same as the chemicals used in conventional processing. The difference is that most fixation steps can be reduced from hours or minutes, down to minutes or seconds respectively. We still infiltrate the sample in resin overnight, but even with this long step, the overall protocol is noticeably faster when compared to the conventional processing protocols.

  • Duration of protocol: 3 - 4 hours over the course of 1 - 2 days
  • Advantages: Microwaves increase penetration time of chemicals, reducing protocol length, and hopefully improve overall fixation.
  • Disadvantages: Dehydration artifacts are unavoidable; membrane movement has been observed on some samples thus this protocol may not be optimal for all sample types.

Method 3: High Pressure Freezing Followed by Automatic Freeze Substitution

For the virtually instantaneous immobilization of small samples, high pressure freezing is the method of choice. Sample thickness should be as thin as possible, and can not exceed 200 µm.

Our Wohlwend Compact 2 High Pressure Freezer was installed in the EM Facility in May 2014. It is used in combination with the Leica AFS 2 for the freeze substitution and cold chemical fixation of the frozen samples. Just like conventional chemical fixation, different combinations of chemicals in the freeze substitution cocktail are selected for different technical and/or biological aspects of the study.

The high pressure freezer is located adjacent to an Olympus IX81 equipped with long working distance objectives. The two instruments are configured so that samples can be imaged by fluorescence microscopy just prior to high-pressure freezing for Correlative Light and Electron Microscopy (CLEM) techniques.

  • Duration of sample freezing: 10 msecs per sample plus sample loading time. Typically we need 1-2 hours to freeze an adequate number of samples.
  • Duration of freeze substitution: 3 hours to 7 days – it all depends on the protocol and the sample being processed.
  • Advantages: Samples are immobilized in ice almost instantaneously and fixation is done in a manner that reduces dehydration artifacts and variable fixation times.
  • Disadvantages: Samples must be very small, protocols can be long and can be expensive due to the amount of liquid nitrogen used and the high cost of the consumables.

If you have further questions please contact one of the technicians in the IGC Electron Microscopy Facility.