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