Ultramicrtmy Operating Prcedure fr Leica UCT Ultramicrtme (rm temperature / cnventinal sectining) An ultramicrtme is used t prduce ultra-thin sectins (60-200 nm thick) fr the purpses f imaging/analysis in the transmissin electrn micrscpe. Cnceptually, the prcess is similar t a delimeat slicer in that yu pass a large blck f material against a knife, incrementing the blck slightly with each prgressive cycle. Cnventinal rm temperature sectining is cnsidered wet, sectins are flated atp a reservir f water prir t transfer nt a TEM grid. Whereas cry-sectining is dry, carefully transferring the sectin ribbn nt the grid and adhering with electrstatics. Generally, the prcess f rm temperature ultramicrtmy can be brken dwn int the fllwing steps: - Dehydratin/embedding sample int a resin blck - Trimming the resin blck t frm a pyramid fr cutting - Cutting ultra-thin sectins - Transfer sectins t a TEM grid DIRECTIONS FOR USE NOTE: instructins here assume yu have yur sample already embedded in a plymerized resin blck Start-up: Lg int the ultramicrtme thrugh the lgin cmputer Turn n switch in back f the cntrller unit Activate the abve and belw illuminatin lights Trimming the resin blck: Resin cntaining the sample f interest must be trimmed away (either with a straight-edge razr blade by hand r in a semi-autmated fashin with a diamnd trimming knife) t prduce a pyramid-shaped blck with a flat surface area fr specimen sectining. Bulk resin remval: Cut the surface dwn, remving resin, until the specimen is reached in the resin blck.
Munt sample n the stage (s that the newly expsed specimen in the blck can be seen thrugh the binculars. Using a razr, trim the expsed resin blck t frm a pyramid. Accmplish this by cutting resin slwly, mving frm the utside f the blck inwards. Final dimensins f any given side shuld be less than 0.5mm. The smaller the crsssectinal area cut per sectin, the better the sectins will cut. Cutting ultrathin sectins: Munt the trimmed resin blck nt the ultramicrtme arm. Munt either a glass knife r diamnd trimming knife Face-ff: using the glass r diamnd trimming knife, carefully remve the very tp surface f the pyramid. This will remve the rughness generated frm previus trimming steps and expses a perpendicularly-riented surface fr sectining (Cutting speed can be fairly quick fr this step, 10 mm/s. Remving 100-200 nm per cut) Switch t a diamnd knife (r well-cut glass knife with a water reservir attachment). Carefully fill the reservir with clean, freshly filtered DI water. NOTE: be careful nt t splatter any drplets f water nt the resin blck as this will cause prblems with sectining later. Adjust water level apprpriate fr resin type. Sme resins cut better with the water level flat with the knife edge. Others require the assistance f gravity, s require the water level t be slightly lwer, but ensure that the water tuches the knife edge. Cut ultrathin sectins (sectin thickness ranging frm 55 85 nm thick depending n applicatin. 65 r 70 nm is standard thickness). Make sure yur cutting windw is set prperly (using Cut-start and cut-stp selectins). The beginning f the cut shuld be just befre the resin blck makes cntact with yur knife edge. The stp pint shuld be after the blck has passed cmpletely thrugh the blck. Cnfirm that the cut speed during the windw f sectining is 1 mm/s r slwer. This ensures that the mvement is slw enugh fr a gd sectin. The slwer, the better. Hwever, the slwer, the lnger the sectining sessin will take. Cut such that the wider part f yur trapezidal shaped pyramid makes cntact with the diamnd knife first. This als enables better/smther cutting.
Sectins will cme ff the knife edge and if the tp and bttm f yur trapezid were trimmed well (parallel t each ther), the sectins shuld stick t the previus ne, frming a ribbn that flats ut nt the water reservir. If the sectins dn t seem t cme ff, r stick t the back-side f the knife edge, it is likely that yu have a water drplet n the surface f yur resin blck. Remve bth the knife and sample blck, remve water frm the reservir and air-dry the surface f the pyramid befre remunting and trying again.
Transfer f sectins t TEM grid: Sectins flat n the water (preferably as a ribbn) due t surface tensin. These sectins/ribbns can be manipulated t different regins f the reservir with an eyelash manipulatr (eyelash r fine hair f sme srt stuck nt a wden stick with nail plish). Prepare grids by placing them n 2 sheets f filter paper with the supprt film side facing up. Pick up sectins with a lp and transfer them t the TEM grid. Excess liquid shuld be wicked away by the filter paper and the grid will initially stick t the lp. Patiently cntinue t carefully blt the grid and wait until enugh liquid evaprates that the grid falls ff the lp. Allw grid t air dry further. Cleaning the diamnd knife: If yu are using a diamnd knife fr yur sectining, prper care must be taken t clean it. Otherwise dried-n sectins will quickly degrade the cutting capacity f the knife. Pur ut water frm the reservir. Using a squirt bttle with DI in it, thrughly rinse bth sides f the knife (this can be dne ver the trash can) Using a squirt bttle with ethanl in it, thrughly rinse bth sides f the knife (this can be dne ver the trash can) Take the diamnd knife plystyrene cleaning rd and cut a new brush edge (use a clean razr t d this as yu d nt want resin particulates embedded in yur cleaning rd) Dip the brush edge int 100% ethanl and run rthgnally alng the knife edge (as seen belw) Allw t air dry.
Shut-dwn: Clean diamnd knife Clean wrkspace area Shut dwn ultramicrtme cntrller unit Lg ut Remember: Sign the lgbk and LOGOUT frm the Lgin system cmputer. * Please cntact Paul Sha (8-3851. psha@princetn.edu) r Nan Ya (8-6394. nya@princetn.edu, 120 Bwen Hall) if yu have any questins. Emergency Infrmatin: Medical Emergencies: Cntact 911 and Public Safety (609) 258-1000 Rm / facility emergencies: Cntact Public Safety (609) 258-1000 Issues related t the instrument: 1. Cntact IAC Staff. 2. If unsure, leave system as is. 3. Try t pwer dwn the system. Audible/Siren Emergency Alerts: Fllw previus steps 2 & 3 and leave the building. Emergency Cntact Infrmatin: Nan Ya: Office (609)258-6394; Cell (908) 922-2236 Email: nya@princetn.edu Jhn Schreiber: Office(609)258-0034; Cell (215) 431-4670 Email: js51@princetn.edu Paul Sha : Office (609)258-3851; Cell (847) 721-086 Email: psha@princetn.edu