Checklist for ultramicrotomy and tSEM training


LM imaging of embedded tissue

  • block images before and after trimming

block storage

ultramicrotome basics

  • binoculars
  • block movement knobs
  • electronic controls (illumination, knife advance, auto functions)
  • water level adjustment knob
  • enclosure and air table
  • video
  • diamond knives - handling
  • maintenance/cleaning
  • safety issues - compressed nitrogen gas cylinder

making UA and lead citrate stain solutions

  • safety issues - handling UA and lead, using hot plate, sharps/syringe needles

coating grids with PEI (or Pioloform)

  • preparation - glassware, PEI solution, grids, glass microscope slides
  • film casting and grid coating
  • film quality check - basic tSEM operation
  • safety issues - handling nitric acid and 1,2-dichroloethane, using a sonicator

block trimming for test-thins

  • orienting tissue block in chuck
  • rough trimming with razor blades and/or files
  • fine trimming with a diamond trim knife
  • note taking
  • safety issues -- sharps, plastic debris/dust containment

test thin sectioning

  • knife alignment against block face
  • breaking a ribbon of test thin sections
  • collecting test thin sections onto grids (or glass microscope slides for semi-thins)
  • collecting grids in wax-filled dish
  • post-section staining with UA and lead citrate
  • toluidine blue stain of semi-thins (for LM)
  • taking care of tools - eyelash, forceps, diamond knife
  • note taking
  • safety issues - sharps, UA, lead, tol blue

tSEM imaging of test thins

  • basic tSEM operation
  • SmartSEM interface, control knobs, joysticks
  • things to check before use: error messages, vac, beam status, stage location, transmission detector position
  • loading specimen, stage navigation
  • beam optimization (alignment, stigmation, focus)
  • image optimization (transmission detector setting, scan speed and averaging, etc.)
  • measurement tools
  • saving images
  • low mag images (transmission, SE2, or in-lens) to figure out tissue orientation in the block and to target ROI, measurement tools in SmartSEM 
  • high mag image to assess tissue quality (transmission, with or without ATLAS)
  • safety issues - compressed nitrogen gas cylinder

block trimming for serial sections

  • measurement function on ultramicrotome
  • trimming final block face
  • note taking
  • safety issues -- sharps, plastic debris/dust containment

serial sectioning

  • cut at least 200 serial sections at 45 nm thickness into a straight ribbon
  • break the ribbon into segments short enough to fit into slot grids
  • collect the segments onto grids
  • note taking

tSEM imaging of serial thin sections

  • low mag whole-grid images to count serial sections and check for large-scale flows (debris, holes, wrinkles, folds, etc.)
  • high-resolution large-field acquisition with ATLAS
  • code assignment
  • note taking