Serial Cryomicrotomy of Saccharomyces cerevisiae for Serial Electron Cryotomography

Materials and Reagents

1. Parafilm

2. 50 ml Falcon tube

3. 1 ml pipette tip

4. S. cerevisiae cells

5. 0.1 mg/ml bovine serum albumin (Sigma-Aldrich)

6. 10-nm gold nanoparticles (BBI Solutions, EM.GC10 )

7. 50% (v/v) ethanol in distilled H₂O, 0.2 µm filtered (500 ml)

8. 18 MΩ H₂O, 0.2 µm filtered (1 L)

   Ensure that there are no debris in both H₂O and ethanol before using as debris can damage the diamond knife.

9. Dextran (M_r ~6000) (Sigma Aldrich, catalog number: 31388 )

10. Pressurized ethane gas

11. Liquid nitrogen

Equipment

Tools (Standard)

1. Parallel-bar EM grids coated with continuous carbon film (Electron Microscopy Sciences, catalog number: G150PB-CU )

2. EM grid boxes

3. K100X Glow Discharge (Quorum Emitech)

   Note: Product has been discontinued in 2010. The current equivalent is the SC7620 Mini Sputter Coater/Glow Discharge System.

4. Fine-tipped and self-locking tweezers for handling EM grids (Ted Pella, Inc.)

5. Metal chuck (included with UC7/FC7)

6. Chuck adaptor (0.45 mm) (included with UC7/FC7)

7. Plastic FC7 cover (included with UC7/FC7)

8. UC7/FC7 (Leica Microsystems)

   Note: An alternative cryomicrotome by RMC Products is commercially available. However, we do not have any experience with that product.

9. Diamond trimming and cutting knives (Diatome)

10. Crion (Leica Microsystems)

11. Centrifuge

12. Diamond knife

13. Forceps

Tools (Non-standard)

1. Fibre tool (Figure 1A)

2. Guinea pig hair (available from pet shops or animal husbandry facilities)

3. Micromanipulator (Narishige Scientific Instrument Lab, catalog number: MN-151S [6 mm joystick travel]) (Figure 1B) (Inspired by Ladinsky et al., 2006 ; Ladinsky, 2010; Studer et al., 2014 )

   Note: Any commercially available micromanipulator can be adapted for use in cryomicrotomy.

4. Copper tubes (0.45 mm inner diameter, 0.65 mm outer diameter) and wire tool for drawing samples into copper tubes (Part 733-1, Engineering Office M Wohlwend GmbH) (Figure 1C)

5. Flat-nosed pliers (Figure 1D)

6. Copper tube cutting device (Part. 732, Engineering Office M Wohlwend GmbH) (Figure 1E)

7. Steel bucket for storing copper tubes (Figure 1F) (design based on Zuber lab’s cryosectioning tools)

8. Aluminium mount (base and insert pieces) (Figure 1G)

9. Slotted plastic FC7 covers [modified from (Studer et al., 2014) to minimize ice contamination] (Figure 1H)

10. Air dust blower

    
    

    
    Figure 1. Tools for enabling serial cryomicrotomy. A. Custom-made fibre tool for manipulating the cryoribbon during cryomicrotomy. B. Custom micromanipulator for fine control of the fibre tool. During cryomicrotomy, the micromanipulator is secured to a heavy steel block with a magnetic stand. C. Copper tubes (0.45 mm inner diameter) and wire tool (upper) for drawing samples into the copper tubes for self-pressurized freezing. D. Pliers for clamping shut the end of the copper tubes before freezing. E. Device for cutting off flat ends of copper tube along the cyan dotted lines. Note that the rust, which cannot be completely removed, does not affect the performance of the cutting device. Users can also consider a cutter made from a rust-prone material. F. Custom steel bucket for storing copper tubes containing frozen samples. Steel bucket can easily fit into a 50 ml Falcon tube. G. Base and insert pieces of the aluminium mount that can be assembled on a diamond knife. H. Slotted plastic FC7 cover with cutouts for the (i) forceps, (ii) Crion, (iii) fibre tool, and (iv) for observing during cryomicrotomy. The translucency of the cover does not affect cryomicrotomy workflow.

Software

1. IMOD (Mastronarde, 1997) (https://bio3d.colorado.edu/imod/)

Procedure