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