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
- 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 SmartSEMÂ
- 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
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