Services

Ultramicrotomy

  • Conventional ultramicrotomy

    Conventional TEM sample preparation

    Introduction

    The sample preparation is suitable for tissues and cell cultures, both adhering and non-adhering. The procedure is divided into two significant steps

    1. Fixation, dehydration and embedding in resin blocks
    2. The ultramicrotomy of resin blocks. 
    • Modus operandi

       

      The embedding of biological samples in plastic blocks is subdivided into three chapters:

      • Fixation
      • Dehydration
      • Resin embedding, or polymerization

      Pilot studies and proof-of-principle requests are generally accepted after initial discussions with users. Such pre-studies allow us to tweak existing protocols to demonstrate the feasibility of the techniques for the project. Once the protocol is adjusted and the results show the required potential, the project leader is informed, and a designated person attached to the project is trained. Importantly, this way, the scientist has complete control over their sample(s) and the treatment (both before and after fixation), which increases the accuracy of interpreting the results. This approach is designed to ensure cost efficiency, enhance accessibility to these techniques, and create a fair pooling of consumables. 

      The preparation of TEM samples is conducted at regular intervals. Note: safety protocol prohibits users from accessing the laboratory facilities. The SNFS-eligible cost for resin embedding of biological samples (includes fixation, dehydration, and polymerization) are outlined below. 

    • SNSF eligible cost breakdown

      • Scientific support: The preparation protocol requires an expert’s attention for 10 hours to be completed. A maximum of 10 samples can be processed in one batch (i.e., 1 or 10 samples equals the same costs).  

        Consumables: For each batch, 100 CHF is charged to cover the cost of consumables.  

          Cost  Total   Total costs 
        Scientific support  40 CHF/h  10 h  400 CHF 
        Consumables  100 CHF per batch  1-10 samples  100 CHF 
       

      Summary: The biological resin embedding of up to 10 samples costs 400 CHF in scientific support and 100 CHF in consumables per batch.  

      Output: Cured resin blocks are ready for ultramicrotomy. Such blocks can be stored for extended periods, are not toxic, archived, and even revisited years later.

       

    Administrator

    Use of OsO4

    Summary 

    The biological resin embedding of up to 10 samples costs 400 CHF in scientific support and 100 CHF in consumables per batch.

    Output

    Cured resin blocks are ready for ultramicrotomy. Such blocks can be stored for extended periods, are not toxic, archived, and even revisited years later.

  • Ultramicrotomy of resin blocks
    • Modus operandi

      Ultramicrotomy is performed in the ultramicrotomy lab of the section Medicine (Other direct costs of infrastructure use). If users have their own sample preparation station, they can prepare the cured resin blocks themselves. The cost for ultrathin sectioning is detailed below.

    • SNSF eligible cost breakdown

      Scientific support: The sectioning (trimming and ultramicrotomy) of one sample requires one hour.

      Consumables: TEM grids are charged at CHF per grid. Typically, 2-3 grids are produced per sample.

        Hourly cost  Total   Total costs 
      Scientific support  40 CHF/h  1 h/sample  40 CHF/sample 
      Other direct costs of infras-tructure use  15 CHF/h  1 h/sample  15 CHF/sample 
      Consum-ables  2 CHF / grid  2-3 grids/sample  5 CHF/sample 

    Output

    Standard TEM grids with ultrathin sections of the samples, ready to be imaged by a transmission electron microscope.

    Summary

    the costs amount to 60 CHF per resin block. Note: There is no guarantee that the ultramicrotomy will be successful. Some samples cannot be ultramicrotomed because of their physical properties.

  • CryoTEM sample preparation
    CryoTEM use

    Cryofixation is an add-on for transmission electron microscopy, suitable for the fixation of nano-objects or near-nano-objects that depend on water as a structural component, such as liposomes. They are thereby kept hydrated without chemical fixatives, dehydration, embedding, or ultramicrotopy). Furthermore, cryofixation can image nanoparticles in a hydrated state, i.e., in a condition preceding the occurrence of drying effects that can lead to aggregation. It is not suitable for the observation of entire cells. Note: single particle reconstruction, which involves 3D reconstruction at subnanometer resolution of proteins, is not included in our service portfolio. 

    What samples can be processed by cryoTEM

    It is imperative that the samples are in an aqueous solution; the entire concept of cryoTEM is based on the physical effect of vitrification, which is unique to water. It is imperative that the samples are not viscous or dissolved in any other medium than water. Some examples of this process include the use of nanoparticles in aqueous solution (in order to test for aggregation state) and liposomes.

    • Modus operandi

      Cryofixation is always performed in conjunction with transmission electron microscopy (then operating at about -170°C), and booking the TEM is mandatory to book the cryofixation equipment. Cryofixed samples are not stored longer than a few hours. 

      Pilot studies and proof-of-principle requests are generally accepted after initial discussions with users. Such pre-studies allow us to tweak existing protocols to demonstrate the feasibility of the techniques for the project. Once the protocol is adjusted and the results show the required potential, the project leader is informed, and a designated person attached to the project is trained. Importantly, this way, the scientist has complete control over their sample(s) and the treatment (both before and after fixation), which increases the accuracy of interpreting the results. This approach is designed to ensure cost efficiency, enhance accessibility to these techniques, and create a fair pooling of consumables. 

      The preparation of TEM samples is conducted at regular intervals. Note: safety protocol prohibits users from accessing the laboratory facilities. The SNFS-eligible cost for resin embedding of biological samples (includes fixation, dehydration, and polymerization) are outlined below. 

    • SNSF eligible cost breakdown

      CryoTEM usage includes the use of the TEM, the Leica GP2 cryo-plunger, the Gatan 626 cryo-holder and all its accessories (the pumping system, the transfer station, the heating controller), and the cryogens (liquid nitrogen and liquid ethane). The user must obtain their own cryoTEM grids. Further information see Rates.

        CryoTEM  
      Scientific support (TEM)  25.26 CHF/h 
      Consumables  8.44 CHF/h 
               – Cryogen (Ethane, LN2)  6.70 CHF/h 
               – LaB6 Gun  2.04 CHF/h 
        Cost  Total   Total costs 
      Scientific support  40 CHF/h  10 h  400 CHF 
      Consumables  100 CHF per batch  1-10 samples  100 CHF 

    Summary

    a one-hour use of the TEM for cryo (including access to all the tools for cryofixation and cryo-TEM imaging) is charged at 33.70 CHF/h.

    Output

    Cryo-micrographs of your nanoparticles or liposomes

Image processing