Instrument Instructions |
Sorvall (RMC) MT-5000 MicrotomeFeatures
|
Setup
- Turn on power by depressing red button on upper left side of microtome.
- Loosen arc segment mount by unscrewing mount locking screw and remove arc segment mount from cantilever arm on microtome.
- Place specimen block in specimen holder (chuck) and tighten by rotating ring on specimen holder.
- Rotate specimen holder with specimen rotation adjustment screw to expose set screw and insert hex key.
- Turn ring on specimen holder clockwise until block is secure in specimen holder.
- Place arc segment mount in cantilever arm with micrometer on top and arc in horizontal position.
- Tighten mount locking screw.
- Set arc micrometer at 0° with specimen slide adjustment screw on arc segment mount.
- Rotate specimen with specimen rotation adjustment screw zero mark on specimen holder is aligned with mark on top of arc segment mount.
- Place glass knife in knife holder and secure with knife clamping thumbscrew on right side of holder.
- Adjust knife to appropriate angle by loosening knife stage locking screw (small screw on right side of knife stage) and moving silver clearance angle screw on left side of knife stage until clearance angle scale is set at desiredangle (usually 2-5°).
- Holding clearance angle screw, tighten knife stage locking screw.
- Adjust binoculars and light. Binoculars should be set at the lowest magnification and focused on block when block is level with knife edge.
- Light should be pulled toward front of microtome until it is shining on the block face when block is level with knife edge.
Block Trimming
To trim face of block:
- Move block to level of knife with handweel (right side of microtome).
- Center block in middle of knife with lateral movement knob on lower stage (black knob of right side). Increase magnification of binoculars if desired and adjust position of binoculars so that block is visible.
- If knife is far from block, move knife toward block by unlocking knife stage with lever on right side of lower stage and sliding knife stage slowly toward block. Do not hit block with knife.
- Move knife close to block by turning the coarse advance micromenter (large knob on left side of lower stage) clockwise toward back of microtome.
- Each mark on the coarse advance micrometer represents 10 mm.
- Move specimen past knife by turning handweel clockwise toward back of microtome.
- Advance knife by turning the coarse micrometer knob clockwise a short distance.
- Continue until excess plastic has been removed from block face and area of the specimen to be sectioned has been exposed.
To trim left and right sides of block:
- Move knife away from specimen by turning coarse micrometer knob counter-clockwise.
- Rotate knife with stage rotation micrometer (large silver knob on front of knife stage) to the left as far as it will go so that the knife approaches the block at an angle.
- Rotate specimen holder with specimen rotation adjustment screw to 2 marks to right of zero mark on specimen holder.
- Adjust knife position with lateral movement knob until edge of block is roughly at center of knife.
- Trim block edge by moving specimen past knife with hnadweel and advancing knife toward block with coarse micrometer until block face is reduced by desired amount.
- Move knife away from block by turning coarse micrometer knob counter-clockwise.
- Rotate specimen holder with specimen rotation adjustment screw until mark on arc segment mount is aligned with the position 2 marks to left of the 180° mark on specimen holder.
- Center block edge in middle of knife.
- Repeat procedure to trim other edge of block.
To trim top and bottom sides of block:
- Move knife away from specimen by turning coarse micrometer knob counter-clockwise.
- Rotate specimen holder with specimen rotation adjustment screw until mark on arc segment mount is aligned with the position 90° to left of original or zero position.
- Center block in middle of knife.
- Move knife toward block.
- Trim edge of block as above.
- Rotate specimen holder with specimen rotation adjustment screw until mark on arc segment mount is aligned with the position 90° to right of original or zero position.
- Center block in middle of knife.
- Move knife toward block.
- Trim remaining edge of block.
- Rotate specimen holder with specimen rotation adjustment screw until mark on arc segment mount is aligned with the zero mark on the specimen holder.
- The top and bottom edges of the block should be parallel to each other and to knife edge.
- Rotate knife stage with knife stage rotation micrometer until to zero mark on micrometer is aligned with lower mark.
Notes on trimming:
- Amount of trimming depends on size of section desired.
- In general, the smaller the block, the easier it will be to section.
- The most desirable block face does not exceed 0.5 mm.
- The above procedure yields a block in the shape of a trapezoid.
- The top and bottom edges of the block must be parallel.
- The left and right sides of the block are angled so that the top edge is shorter than the bottom edge.
- It will be necessary to use several glass knives during the trimming process.
- Change knives when they develop deep nicks or become dull so that it is difficult to force the knife through the block. Using brush (not canned air), dust plastic shavings from microtome.
Sectioning
- Place new glass trimming knife in knife holder.
- Remove 1-2 sections from block face.
- Move knife away from block a short distance by turning the coarse micrometer knob counter-clockwise toward front of microtome.
- Place glass or diamond sectioning knife in holder.
- Move knife holder with lateral movement micrometer knob until block is positioned at desired sectioning area of knife.
- Lower the block to the lowest position with the handwheel.
- Microtome will beep indicating that arm is in retracted position.
- Lower the magnification of the binoculars to the lowest magnification.
- Move the light toward the microtome to a position just behind the block.
- Fill boat on knife with Millipore-filtered ddH2O until meniscus is convex.
- Draw lens paper across water surface from front to back of knife to remove dirt from surface of water.
- arefully remove H2O from the boat with a syringe until a white (or silvery) reflection appears on the surface of the water at edge of knife.
- If water will not adhere to knife edge, draw eyelash brush across edge.
- If water still does not adhere, lower water level. Wet the edge of a wedge-shaped of balsa wood with saliva (chew it gently).
- Draw moistened edge of balsa wood across edge of knife from one side to the other in one direction only. Fill boat on knife with millipore-filtered ddH2O until meniscus is convex.
- Draw lens paper across water surface from front to back of knife to remove dirt from surface of water.
- Carefully remove H2O from the boat with a syringe until a white (or silvery) reflection appears on the surface of the water at edge of knife.
- Move light back and forth a short distance until reflection on surface of water is of maximum size.
- Remove water until reflection on surface of water is present at edge of knife.
- Readjust light to obtain maximum reflection in boat while maintaining reflection at sectioning area of knife edge.
- Check alignment of knife and block:
- Press fiber optics light button (lower button on upper left panel of microtome) to turn on fiber optics light.
- Increase magnification of binoculars to highest magnification.
- Adjust position of binoculars if necessary.
- Move knife toward block until it is close to block and reflection of knife is present on block face.
- To align block face with knife edge along length of block:
- This adjustment should not be necessary if you have removed 1-2 sections from face of block with trimming knife before placing the sectioning knife in holder.
- Observe reflection while slowing moving block past knife edge by turning handweel.
- Shadow should maintain same height from bottom to top of block of block face.
- If shadow decreases in height, top of block face is closer to knife than the bottom of block face.
- If the shadow increases in height from the bottom to the top of the block, the bottom of the block face is closer to the knife than the top of the block face.
- To adjust, increase the distance between the block face and knife.
- Loosen the arc segment mount with mount locking screw and rotate holder so that arc is in the up-down position.
- Rotate specimen holder 90° so that parallel top and bottom edges of block are horizontal to knife edge.
- Rotate arc with specimen slide adjustment screw on arc segment mount until the shadow remains even in height along length of block.
- To align knife so that knife edge is parallel to block face across width of block:
- Observe height of reflection across width block.
- Shadow should be uniform in height across width of block face.
- If shadow is higher at one side than the other, the block face is farther away from knife on the side with the higher shadow.
- To adjust, carefully rotate knife holder toward this side of block with stage rotation micrometer knob until shadow is uniform in thickness across width of block. Be careful not to hit block face with the knife when loosening and rotating the holder.
- You may wish to increase the distance between the block face and the knife a small amount.
- To align block so that bottom edge of block face is parallel with knife edge:
- Move block slowly past knife with handwheel and observe shadow as it appears on bottom of block face and disappears at top of block.
- If top and bottom edges of the block are parallel to each other and to the knife edge, the shadow will appear across the entire width of the block at the lower edge and leave the top edge at the same time.
- If the shadow appears on the lower edge at one side of the block before the other side, the edge of the block is not parallel to the knife.
- To adjust, rotate holder with specimen rotation adjustment screw until shadow of knife appears on lower edge of block evenly across the block width. Be careful not to hit block face with the knife when loosening and rotating the holder. You may wish to increase the distance between the block face and the knife a small amount.
- When block face and knife edge are properly aligned, move block with handwheel until block face is level with knife edge.
- Set visutrac so that upper start light is 2-3 marks above the block position light and the stop light is 2-3 marks below the block position light.
- * Carefully move the knife toward the block by turning the coarse advance micrometer knob clockwise until the reflection of the knife edge on the block face is thin. If you wish, you may use the fine advance micrometer screw for this adjustment. To activate the fine advance micrometer (small micrometer on side of coarse micrometer), tighten silver thumbscrew on coarse micrometer. Each mark on the fine advance micrometer represents 0.5 mm.
- Move binoculars a position near upright maintaining reflction of knife toward block.
- Press jog button (upper button on lower left panel of microtome) and release. Jog button moves cantilever arm forward toward the block a distance equal to the distance set on the digital thickness control.
- Continue to press jog button until color of reflection on block face changes to dark blue or black.
- Press fiber optics light button to turn off fiber optics light.
- Move binoculars back to comfortable position. S
- elect appropriate cutting speed on digital cutting speed control marked millimeters/sec (upper left panel of microtome). Usual setting is 00.7-00.9 mm/sec.
- Select appropriate section thickness on digital section thickness control (lower left panel of microtome). Usual setting is 60-90 nm.
- Press motor button (lower button on lower left panel of microtome).
- Observe color of sections.
- Adjust section thickness until a silver or light gold section is cut each time the block moves past the knife.
Notes on sectioning:
- If a section is not cut each time the block moves past the knife, increase the section thickness.
- Sections should stick together to form a ribbon.
- If the ribbon curves, the top and bottom edges of the block face are not parallel.
- If sectioning has continued for a substantial length of time, ultrafine feed may reach the maximum limit of advancement, and sections will not be cut.
- Microtome will beep with each stroke when approaching maximium arm advancement.
- Turn off motor by pressing motor button.
- Press reset button (middle button, upper left panel of microtome). Lowest marks on visutrac will be lit.
- When arm is retracted light indicators on visutrac will return to set positions.
- Repeat procedures from *.
To turn off microtome:
- Press motor button to turn off motor.
- If using knife, make sure all sections are removed from knife edge and boat.
- Move knife away from block with coarse micrometer knob.
- Remove knife from holder by loosening knife clamping thumbscrew.
- If using diamond knife, dump water over back of boat, rinse with ddH2O and place in box.
- Remove specimen holder from arc segment mount.
- Remove specimen from specimen holder.
- Press reset button and wait until arm is fully retracted.
- Press power button to turn off microtome.
- Leave area clean and neat.