Resources

INSTRUMENTATION
TEM BIOLOGICAL PREP
SEM BIOLOGICAL PREP
THEORY
ADDITIONAL INFORMATION

INSTRUMENTATION

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TEM BIOLOGICAL PREP

Preparation of grids coated with formvar films is easy and inexpensive. You can make a couple of month’s supply in a short time and just store until needed.
Formvar Film Prep

This is fool-proof method for mounting serial sections on single hole grids:
Serial-sections_single-hole -grids

Microwave technology not only shortens the preparation of hydrated samples for TEM but also produces superior results with many sample types. The following are some protocols that have worked well for us:

TEM Preparation

  • Thick sections (1-3µm) are invaluable as a way to identify locations for thin sectioning and TEM examination. Staining Thick Sections
  • Negative staining: for any small samples such as viruses, isolated organelles, collagen fibers, etc. The stain, an electron scatterer,  surrounds the sample making the sample particles standout against the darker background.Negative Staining Basics
  • Stock Solutions:  This handout contains commonly used regents and stock solutions for sample fixation.  Fixation stock solutions
  • Cell culture fixation: Routine fixation that works for most cell types. Cultured Cell Fix for TEM
  • Karnovsky’s Fixative: This is a very good fixative for hard to penetrate material such as plants, bacteria, and dense animal tissue.  Karnovsky’s Fixative
  • AGC fix: This is a great fix for difficult samples if all else fails.  It contains Acrolein (tear gas) so handle with care.  AGC Fixation_Bacteria-Yeast
  • Van Tuinen Fix: This fix is a variation on the AGC without using acrolein. It is ideal for bacteria and yeast but easily adaptable for other samples. Van Tuinen Fix
  • Ruthenium Red-PATO technique: This technique stains polysaccharides to help identify their location in the cell.  Polysaccharide Fixation_RR-PATO
  • Lowacryl HM-20 Embedding: This resin works great for low temperature embedding and ICC. Lowicryl_HM20_method
  • Staining Thin Sections: Uranyl Acetate and Reynold’s Lead Citrate.  Staining Thin Sections

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SEM BIOLOGICAL PREPARATION

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THEORY

  • Magnification Calibration.  Catalase is a useful internal calibration standard for viruses and other small particles. Catalase Calibration

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ADDITIONAL INFORMATION

Energy Dispersive Spectroscopy (XEDS) is a chemical microanalysis technique that detects x-rays emitted from the sample during bombardment by an electron beam to characterize the elemental composition of the analyzed volume.  The technique can provide simple sum spectrum, point identification, and compositional mapping.A primer  in XEDS by Bob Hafner at U. of Minn is: http://www.charfac.umn.edu/instruments/eds_on_sem_primer.pdf

This is a set of slides developed by Charles Lyman, Lehigh University from a semester course given at Lehigh as well as for the Lehigh Microscopy School.  http://www.slideserve.com/ashton/compositional-mapping

Electron Microprobe. Electron microprobe analysis (EMPA) is a non-destructive method for determining chemical composition of small amounts of solid material. This is a link to the Northern Arizona University Electron Microprobe lab.  It has basic information about use of this technology.   http://www4.nau.edu/microanalysis/Microprobe/Probe.html

Electron Backscatter Diffraction (EBSD).  EBSD is a nano-crystallographc technique used to used to examine the crystallographic orientation of materials. Oxford Instruments EBSD site has a basic introduction to this technology.    http://www.ebsd.com/

Transmission EBSD.  This technique makes use of a standard EBSD detector in an SEM to analyze thin films and nano particles with resolutions as small as 10nm, significantly smaller than the ~100nm typically resulting from traditional EBSD. An introduction to the technique can be found at: http://c3379894.workcast.net/hall_11.pdf

Wavelength Dispersive Spectrometry (WDS)  A wavelength-dispersive spectrometer uses the characteristic X-rays generated by individual elements to enable quantitative analyses. A couple of introductions to this technique are on-line posts:

SERC project at Carlton College: http://serc.carleton.edu/research_education/geochemsheets/wds.html Northern Arizona University: http://www4.nau.edu/microanalysis/Microprobe-SEM/Signal_Detection.html

Monte Carlo simulation. DTSA-II is a multi-platform software package for quantitative x-ray microanalysis.   It is a Monte Carlo simulation of electron and x-ray transport in solid materials designed by NIST scientists to encourage standards-based analysis by the novice user.    http://www.cstl.nist.gov/div837/837.02/epq/

Microsocopy Virtual Training.  Program for virtual use and training of microscopy (electron, light, and scanning probe) produced by the Beckman laboratory at the University of Illinois at Urbana-Champagne.   http://virtual.itg.uiuc.edu/

Training for SEM, TEM, and confocal and analytical techniques is available from the Australian Microscopy & Microanalysis Research Facility. The tutorials include very good  automations and tests at the end. http://www.ammrf.org.au/myscope/