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