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Electron Microscopy Resources & Training

Electron Microscopy basics, techniques and more.

Meet EM Staff

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

HPF Methods - C. Elegans

HPF Methods - Immunolabeling

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

HPF Methods - Overview

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

HPF Methods - Solutions

HPF Methods - Substitution, Embedding & Immunocytochemistry

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

HPF Methods - Common Fillers

HPF Methods - Cryoprep for Model Systems

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