Electron Microscopy Resources & Training

Shared Resources' Cellular Imaging provides transmission and scanning electron microscopy. Services include a variety of sample preparations along with cryo techniques and capabilities.

Resources selected by: Bobbie Schneider, Former Manager
Electron Microscopy Core Facility, Shared Resources, Fred Hutchinson Cancer Research Center

Website: Cellular Imaging

Contact Info: (206) 667-4289 emsr@fredhutch.org

Bobbie Schneider's Notes:
from High Pressure Freezing for Preservation of High Resolution Fine Structure and Antigenicity for Immunolabeling

• Mix cell media (growth media) in 20% BSA to start with
• Use smallest chamber that will accommodate samples
• Take the cells from the bottom of eppendorf after spinning them down
• Can use dialysis tubing for cell suspensions • Cut a range of section thickness for HPF sections = 30-80nm
• May want to further enhance contrast with methanolic UA • May have to observe ice crystal damage sometimes at 100,000x
• Get dog nail clippers

Bobbie Schneider's Notes:
from Recent Advances in High-Pressure Freezing by Kent McDonald, Morphew, Verkade, Muller-Reichert (2007)

• With 2000 Bar pressure and lowering the freezing point of water by ~20 degrees C you can freeze to a greater depth
• Artifacts are from dehydration in conventional preparation and not fixation
• Use sapphire disks for cells
• Long tweezers = Ted Pella Cat#5306
• Scrape cells from the filter with a toothpick
• To wick off fluid in a planchett you can use 2 paper points on either side of the planchett
• With cells in an eppendorf you can spin them down and then take a pipette to take off the solution from the top and use dog nail clippers to cut off the tip.
• Freeze Substitution times from Kent:
  1. -90 C at 8 hr (but could be from 5-36 hr)
  2. -60 C at 5 C/hr and it remains at -60 C for 8-12 hr
  3. Then warmed to 0 C at 5 C/hr and held at 0 C until processing
  4. For more contrast hold at -20 C for 12 hr instead of -60 C

• or

  1. -90 C at 8 hr (5-36 hr)
  2. warm up rate of 5 C/hr and halt at -25 C for 12 hr (overnight) for more contrast
  3. and then warm to 0 C
  4. samples are warmed rapidly to RT and rinsed 3x 15 min with acetone for membrane contrast

• To make BSA:
  1. Weigh out 1 gm and add solution to make up to 5ml final volume. 
  2. You can warm to 37 C (oven) for a few min.s

Bobbie Schneider's Notes:
from Practical Methods in High-Pressure Freezing, Freeze –Substitution, Embedding and Immunocytochemistry for Electron Microscopy by Mary Morphew (Laboratory Manual)

• For a cell monolayer of mammalian cells for an LM/EM study – use thermanox plastic chips and push them to the bottom of a petri dish
• For animal tissue – use 2 microscope slides onto which are glued 2 thin razor blades and cover with hexadecane
• Freeze sub. is the process of dissolution of ice in a frozen specimen by an organic solvent at low temp and usually takes place in the presence of a secondary fixative.  It is carried out at temp below which secondary ice crystals may grow (below -70 C) – can now be warmed up because water is absent.
• FS Solutions:
  • 0.1-0.5% glut in acetone
  • 2-4% paraf +/- 0.1% glut
  • Acetone only – for very sensitive antigens.  Used with Lowicryl HM 20 at -50 C
  • 0.01%-0.1% OSO4 – soak on a drop of saturated sodium metaporiodate for 10 min to remove bound OSO4.  Do not use UV

Bobbie Schneider's Notes:
from Cryopreparation Methods for Electron Microscopy of Selected Model Systems by Kent McDonald (2007)

• Describes HPF well
• You must crack samples in which hexadecane was used as a filler because it forms a barrier to free exchange of solvents during FS and it will not dissolve
• For flies you can use yeast (Bakers) with 10% methanol or buffer – mix 1:1 to start and make it thick
• Forceps – EMS Cat#72919-ss
• Worms should be moving in the cup before freezing
• Overfill cup just slightly = best, so no air is trapped
• Worms = use 100um deep planchett
• Fly embryos = 200um deep planchett
• C. elegans Web site: http://elegans.swmed.edu/ and then use “electron microscopy “ as a search term in the literature search link
• FS = substitute organic solvent – usually acetone for the cell water at a low temp. -90 C.  Acetone is better then methanol (bad ultrastructure)
• Glut. Is active at ~ -50 C – gets into the tissue and OSO4 is active at ~ - 30 C
• Papers detailing FS fixatives  = McDonald 1999, McDonald and Miller-Reichert 2002, and McDonald et al.  2000
• Use spurs resin to embed yeast
• For Immuno – Kent uses LRW hard formula
  Lowicryl HM20 is better for preserving cell morphology and can polymerize it with UV irradiation at -50 C
• Section Thickness:
  • Yeast cells = 40-60nm
  • Worm and flies = vary thickness between 60-100nm depending on cytoplasmic features of interest
• Post Staining:
  • General = 2% UA in 70% methanol for 5 min. and lead for 3 min
  • For worms or flies = may go as long as 10 min methanolic UA and 5 min lead. If interested in cell walls or certain classes of membrane or endocytic vesicles
•  HPF Artifacts:
  • Ice damage occurs when the rate of freezing is too slow to prevent rearrangement of cellular water molecules into ice crystals
  • In the nucleus the nuclear envelope should have a smooth curve
  • You will find ice damage gradient from the surface toward the interior of cells and tissues
  • The HPF usually has a good depth of freezing before ice crystals appear
  • Steinbrecht (1982, 1985, 1993) = artifact papers to read
  • Chromatin and mitochondria show ice crystal damage first
  • Mitochondria show a clear “halo” around the outer membrane
  • Pressure related artifact is “eruption” of dense protein storage vacuoles that are often found in embryonic tissue