Know Thyself
- When and/or under what conditions are your hands most stable?
- Make sure you get enough sleep.
- Make sure you are hydrated and fed enough.
- Do you feel calm enough to handle ultrathin sections?
- Establish a habit of keeping everything clean and neat.
- Always be aware of your surroundings, especially when handling a diamond knife.
- Practice using forceps, eyelash, and ultramicrotome until you feel like they are extensions of your fingers.
- Manipulate grids with forceps under different light conditions. You need to be able to pick one up, flip it over, hold it still, insert it into a slot on wax surface, etc. Also, practice using a grid on forceps to move and orient ultrathin sections on water at will.
- Figure out the most stable way to hold an eyelash. Use an eyelash under binoculars at different magnifications to manipulate ultrathin sections on water surface. You need to be able to tap on to a section without breaking it. Can you use it to move around a section on water surface, or stop one from moving? Try using different eyelashes and get yourself familiar with their properties (e.g., tip shape, flexibility, etc.). Figure out which one works the best for you.
- Ultramicrotome – know which knob to turn to get your block face aligned to the knife without thinking or looking. Get to know the illumination system by trying different settings. Make sure you know how to adjust binoculars to your eyesight. Get to know the field of view at different magnification – at the highest mag, you can see more details, but the field is too small to work with an eyelash effectively and efficiently. Figure out the best ergonomics for you – you need to be able to hold an eyelash steady without straining your shoulders and back.
Workflow
- Make grids coated with PEI film.
- If post-section staining is required, clean with nitric acid and air dry glassware for making lead citrate.
- Plan how your tissue block will be sectioned.
- Rough trim with razor blades
- Fine trim with a cryotrim knife
- Semi-thin and/or test-thin sections
- Evaluate quality of ultrastructure from test-thin sections
- Determine orientation and dimensions of the tissue in semi-thin or test-thin sections - use this information for final block-face trimming
- Trim the block face for ultrathin section series.
- Cut ultrathin sections.
- Break a ribbon of ultrathin sections into smaller fragments that fit into the grid.
- Collect the ribbon fragments onto the grid.
- Air dry the grids inserted on the surface of dental wax.
- If post-section staining is required, make a new batch of lead citrate and stain the sections on the day they are cut.
Trimming a block
Semi-thin and test-thin sections
Cutting series of ultrathin sections