machines that cut extremely thin sections from a samplefor applications in histology or pathology use special metal, glass, or diamond blades, depending onthe type of specimen and the desired thickness. consists of a blade holding unit with a blade holder andclamped blade, the object clamp, an advancingmechanism, and a mechanism for adjusting sectionthickness.
Glass microtome blades used in sample preparation activities for lightmicroscopy and electron microscopyapplications. Diamond blades may be industrial-grade or gem-quality.i. Industrial-grade blades are used to slicehard materials such as bone and teeth.ii. Gem-quality blades for microtomes areused mainly in electron microscopyapplications
Base Sledge microtomes place the sample in a fixed holder or shuttle whichmoves back and forth across a knife or blade the sled rests upon a linear bearing and permits angularadjustment used with biological materials and samples that areembedded in paraffin cut thickness ranges from 1 -60 µm
Rotary microtomes used to cut thin sections of samples visible onlywith the aid of a light microscope. They areoften operated manually but can be motorized.The sample is cut upon the rotary action of ahand wheel which advances the sample towardsa blade. Rotary microtomes are used with hardmaterials, such as samples embedded in resins orparaffin. The thickness for manual operationranges from 0.5.µm to 60µm
Type of Microtome Type of Sample Sample Cut Thickness ApplicationSaw microtome Hard and brittle material > 30µm.Samples such as boneand teethSled microtome Embedded samples 1 – 60µmSamples sliced at anangleRotary microtome Embedded samples 0.5 µm - 60 µmThin samples. ManualcontrolVibrating microtomeDifficult/soft, Fresh/fixedsamplesFixed >10 µmFresh > 30 µmLess pressure andsample disruptionLaser microtome All samples >1 µmNo sample contact andno sample prepCryomicrotomes Frozen samples Very specific thicknessUltramicrotomesTEM 40-100nmSBFSEM 30-50nmExtremely thin cuts foranalysis with specialtymicroscopes
This device operates with a staged rotary action such thatthe actual cutting is part of the rotary motion. In a rotarymicrotome, the knife is typically fixed in a horizontalposition
Microtome base plate or stage: A platformwhich has rails that secure the knife holderbase. Knife holder base: A part that anchors theknife holder to the microtome stage. Theknife holder base can be moved toward oraway from the block, but MUST bestationary and locked during microtomy. Knife holder: comprised of severalcomponents including the: blade clamp that holds the blade knife tilt for adjusting the knife angle the face plate that guides that ribbonsaway from the blade and towards theoperator. Cassette clamp or block holder: Holds theparaffin block in place moves up and down with each revolutionwhile the blade is stationary may have knobs that allow the user tomanipulate the block face in variousdirections to bring the tissue inalignment with the blade.
Coarse handwheel: Moves theblock holder either toward theknife or away from the knife. Advancement handwheel: Turnsin one direction and advances theblock toward the knife at thespecified microns Most handwheels are equippedwith a safety lock to preventthe wheel from releasing andhaving the block holder comedown towards the blade whilea block is inserted or removed.The safety lock should be usedanytime the microtomist is notactively sectioning paraffinblocks. Micron adjustment: Micronsettings for section thickness canrange from 1 to 60 microns onmost microtomes.
Planar concave extremely sharp, but arealso very delicate and aretherefore only used withvery soft samples. wedge profile knives more stable and find usein moderately hardmaterials, such as inepoxy or cryogenicsample cutting chisel profile with its bluntedge, raises the stabilityof the knife, whilstrequiring significantlymore force to achievethe cut.
Used in Botanical Microtomy Techniquewhere hard materials like wood, bone andleather require a sledge microtome These microtomes have heavier blades andcannot cut as thin as a regular microtome
Operating handle Semi automatic advance – when pushed towards the knife the sledge willmove along the runners and will also advance the specimen the selectedamount (thickness). When pulled back the specimen retractionmechanism is activated. Thickness control A knob, calibrated in microns, is used to select the thickness of sectionsto be cut from 1μm – 40μm, in 1μm steps. Always set the thickness byturning the knob anticlockwise. If the thickness has to be reduced thenturn clockwise past the required thickness, then anticlockwise. Coarse advance/trim control Quickly raises or lowers the specimen 450μm per turn. Specimen holder Specimens are clamped in the vice or alternative holder if fitted can be raised and lowered after releasing the clamp lever
Trimming the Specimen Fit the specimen securely into the appropriate specimenholder on the microtome.To trim excess material from thesurface of the specimen block:a. First move the sledge towards the knife and adjust thespecimen height using the coarse advance so that thespecimen is just below the knife.b. Either set the thickness control to cut thick sections, eg:20μm, and move the sledge back and forth repeatedly totrim the specimen.c. Or set the thickness control to zero and alternately raise thespecimen slightly using the coarse advance and move thesledge forward then backwards. This method isquicker butshould only be used if the operator is experienced in theuse of base sledge microtomes.
Knife clamps Two screws, turned by a lever , are used to fix the knife in the knifeclamp and the knife clamp in the knife block The cutting angle of the knife is adjustable from 0-40º. Slew angles of theknife can be obtained by loosening the knife block clamp levers and theknife clamp levers and moving knife to slew angle required. Knife guards Each knife guard can be moved to the central position to cover theworking area of the knife, or to the outer position while section cutting. The black knobs secure the guards in the chosen position. Side pillars In normal use, these pillars slope inwards slightly. For large area sectionsthey can be unbolted from the base, swapped left to right and refitted, sothat they slop outwards, so providing more space.
Fitting the Object Holder Pull the sledge away from the knife holders.Secure the required object holder by placingit in the holder in the sledge slide.Set tocorrect height and lock by tightening clamplever Note that the clamp lever has a ratchet withits movement limited to prevent the leverbeing left in positions where it could impacton other parts during sectioning.
Fitting and Adjusting the Knife NB: The term ‘knife’ used here also includes disposable blade holder.a. Slacken the knife clamp levers just enough to let the knife slide into theknife clamps from the side.b. Insert the knife, taking care to avoid touching the edge against themetalwork.c. Check that the knife lies flat on the pad pieces in the knife clamps andthat the heel of the knife is not caught up on the slot in the knifeclamps.d. Screw the knife clamp levers down just sufficiently to steady the knife inposition.e. Slacken the knife block clamp levers . Set the knife holders to the desiredslew angle, ensure the knife clamps still cover the ends of the knife. Setthe knife to the desired cutting angle and tighten the knife clampinglevers.f. Check that the knife is still at the desired slew angles and tighten theknife block clamp levers. NB: To ensure that the knife clamps are correctly aligned in theholders, the knife block clamp levers must always be slackened beforethe knife clamping levers are tightened.
Cleaning Wipe off sectioning debris etc with a papertowel. Alcohols (industrial methylatedspirits, ethanol or methanol) may be used.Xylene may be used sparingly to remove built-upwax, but ensure surplus Xylene is wiped off. Avoid harsh solvents such as acetone Lubrication After cleaning, oil both the sliding surfaces ofthe sledge base with Microtome Sledge Oil. Nowmove the sledge several times over the fulllength of travel to distribute oil
Decontamination It is the responsibility of the user to ensure thata decontamination procedure is employed whichis appropriate to the nature of the work carriedout.
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