Leica KMR3 Knifemaker
Contents
1. Operating Manual Version 16224032 04 2010 Living up to Life MICROSYSTEMS Important Note Leica reserves the right to change technical specifications as well as manufacturing processes without prior notice Only in this way is it possible to continuously improve the technology and manufacturing techniques used to provide our customers with excellent products Any copyrights of this document are retained by Leica Mikrosysteme GmbH Vienna Any reproduction of text and illustrations or any parts thereof by printing photocopy ing or other methods including electronic systems and media requires express prior permission in writing Issued by Leica Mikrosysteme GmbH Hernalser Hauptstrasse 219 A 1170 Vienna Operating Manual Leica EM KMR3 Serial Number Date of purchase For the instrument serial number please refer to the name type label on the back of the instrument Please read this instruction manual carefully before operating the instrument Table of contents I SALOU MIS Ch UCI Sci acac ce tacete cence ei rina Ernici 2 2 Principles of KiMiTeinaK iG E 3 2 1 The Balanced Break method Fig 1 eerste i ere 3 2 2 Scoring and BIeaking PINCIPES lalla sla 3 ZO JEE ee 5 e e OU NS sedere lia 6 d Uppacking and IN EE WE H 00 100 Le BE 7 Se ET Ee ii T 4 Description of the Leica EM KMR3 iii 8 De ONT Ce DEE 11 LS E 11 VANE RE RE EEA 13 5 3 Making Knives from a s2. The useful knife edge is 30 longer on knives produced from 8mm thick glass compared to 6 4mm thick glass zm Note When less force has been used to break the knife the stress line falls away rapidly from the knife edge and fewer saw teeth can be seen Resulting in a longer usable knife edge Page 6 Leica EM KMR3 Operating Manual 04 10 3 Unpacking and Installation 3 1 Unpacking The Leica EM KMR3 is packed in a single carton Before unpacking ensure that the box is the correct way up Carefully remove the Knifemaker together with the plastic foam Check all items against the packing list enclosed in the shipping and examine each item for transport damage 3 2 Installation Place the Leica EM KMR3 on a sturdy laboratory bench or a table where it will not be affected by movement from other laboratory equipment such as a centrifuge The Leica EM KMR3 is completely assembled in the factory so the instrument is ready to use out of the box Page 7 Leica EM KMR3 Operating Manual 04 10 4 Description of the Leica EM KMR3 The scoring and breaking mechanism for knife making are assembled on a heavy vibration absorbing base The parts are shown in Fig 5 Page 8 Leica EM KMR3 Operating Manual 04 10 1 Clamping lever used to lower clamp and raise scoring head The clamping leaver has a defined stop to ensure equal clamp force at any time 2 Breaking wheel is used to break the glass after scoring By t
3. 32 The attachment surfaces of the truf are coated with molten wax by putting it onto the wax bath as shown in Fig 32 The truf is then brought into contact with the pre warmed knife and slid up until the rim of the truf is at the same level as the knife edge Fig 33 The truf is held in this position for a second or two until the wax has hardened Additional wax is applied on the right side of the truf using the spatula supplied with the Leica EM MP for further protection against leaks Fig 34 For detailed description please see the user manual of the Leica EM MP Fig 31 Fig 32 p Fig 33 Page 23 Leica EM KMR3 Operating Manual 04 10 10 Accessories and consumables 16840031 Glass strips 6 4 x 400 x 25 4mm box of 30 strips 16840032 Glass strips 8 x 400 x 25 4mm box of 24 strips 16840079 Glass strips 10 x 400 x 25 4mm box of 18 strips 16840042 Trufs 6 4mm 500 pcs 16840045 Trufs 8mm 500 pcs 16895032 Dust cover 16705225 Knife boxes 3 pcs 16706980 Scoring wheel for EM KMR3 Recommended accessories 16705403 LEICA EM MP 115 230VAC complete with metal spatula and 500g dental wax Hotplate with 3 different temperatures for mounting TRUFS on glass knives and for staining and drying of semithin sections on glass slides Leica best solution for EM sample preparation www leica microsystems com products electron microscope sample preparation Leica EM AMW Automatic Microwave Tissue Processor for EM
4. EM KMR3 Operating Manual 04 10 6 Knife shoulder fine adjustment The adjustments shown in Fig 22 and 23 are used to adjust the size of the knife shoulder As mentioned earlier in this user manual the sharpest knife edge is found opposite a very small knife shoulder The fine adjustment allows setting the knife shoulder size according to the application For resin sections knife shoulder should be between 0 5mm and 1mm For cryo sections the knife shoulder should be smaller than 0 3mm Knife shoulder adjustment of the left knife Fig 23 Use a 2mm Allen key and turn the adjusting knob Counter clockwise knife shoulder becomes bigger Turn it clockwise knife shoulder becomes smaller Knife shoulder adjustment of the right knife Fig 24 Raise the scoring head by using the clamping lever and remove the head Use a 2mm Allen key and turn the adjusting knob Counter clockwise knife shoulder becomes bigger Turn it clockwise knife shoulder becomes smaller Note Fine adjustments are very sensitive only a very small turn within the scale is necessary to change the size of the knife shoulder Page 19 Leica EM KMR3 Operating Manual 04 10 Fig 25 Fig 26 Sliding surfaces Fig 27 Fig 28 Page 20 Leica EM KMR3 Operating Manual 04 10 7 Care and maintenance The Leica EM KMR3 should always be kept clean The breaking pins and steel hemispheres glass strip support area must be free of any gla
5. Manual 04 10 5 4 Evaluation of the knife edge After making a pair of knives evaluation of the quality can be carried out with the Ultramicrotome Leica Ultracut UC7 Using the backlight illumination and setting the clearance angle to maximum a fine white line can be seen Fig 17 The image of the line indicates the quality of the knife edge which must be straight free of any dirt such as dust grease and finger prints and free of glass splinters The top light of the ultramicrotome can also be used for checking knife quality Fig 18 An example of a knife which should not be used is shown in Fig 19 This has been picked up incorrectly leaving a finger print over the knife edge In Fig 20 a pair of knives is shown broken and placed side by side according to chapter 5 3 The detail Fig 21 shows the knife shoulder of both knives The right hand knife edge was opposite the left hand knife shoulder during breaking and the left hand knife edge was opposite the right hand knife shoulder Note the sharpest knife edge is the one opposite the smaller knife shoulder The knife edge opposite a small knife shoulder lt 0 2mm has a knife angle very close to 45 as recommended for cryo ultramicrotomy Fig 22 Griffiths et al 1983 Tokuyasu 1986 For resin sectioning a larger knife shoulder approx 1mm is recommended Fig 22 Knife angle close to 45 Page 17 Leica EM KMR3 Operating Manual 04 10 Page 18 Leica
6. Samples Leica EM UC7 Ultramicrotome Leica EM AC20 Automatic Contrasting Page 24 Leica EM KMR3 Operating Manual 04 10 11 Technical specifications Method of Operation Score Marks Scoring Angle Glass Strips Scoring Wheel Dimensions and Weight Page 25 Leica EM KMR3 Operating Manual 04 10 Balanced break method Two pre set 45 Special glass for Ultramicrotomy Cryo ultramicrotomy and Histology Length 400mm Width 25 4mm Thickness 6 4mm and 8mm 10mm glass break only possible with the upgrade kit for 10mm order number 16706911110 Tungsten carbide Length 427mm Depth 260mm Height 245mm Weight 7kg OLOZ 7O ZEOPZZ9L UOIsJAA CHWY WA 29197 enue Bunesedo Uonipa au 10 ou ep MESS HWS YI 29187 Jo Sue Dags ase sobo e9197 94 pue VO 09 66887 Cr XC 6688 7 IAL UsMy LUUA DILL 617 assensidneH J sjeusaH Hus aggies 20197 1UBUA dog MICROSYSTEMS www leica microsystems com
7. ass knives It also holds the front fork with the knife shoulder adjustment 9 Rear fork is used to hold and position the glass square against the front fork It also holds the knife shoulder adjustment Page 9 Leica EM KMR3 Operating Manual 04 10 Page 10 Leica EM KMR3 Operating Manual 04 10 5 Knifemaking 5 1 Hints For the best Leica EM KMR3 performance it is recommended that glass strips supplied by Leica are always used Leica glass strips for ultramicrotomy are produced from specially selected glass the thickness and quality of which is precisely controlled Only strict tolerances ensured by careful quality control allow breaking of two high quality knives from one square All strips supplied by Leica are 400mm long and 25 4mm wide They are available in thicknesses of 6 4mm 8mm and 10mm Leica glass strips are washed individually wrapped in tissue paper and packed in a strong carton When handling the strips avoid contacting the narrow edges as these may finally form the knife edge The strips and squares can be held by the wide upper and lower surfaces Wear lint free cotton gloves to avoid finger prints on the glass surface Finger prints grease can negatively influence the score Never touch the corners of a partly broken strip or square as these will form the knife edge Cleaning the glass strips is in general not necessary because the strips are pre cleaned wrapped in tissue paper and kept away from dust in th
8. ber 3 8mm glass will break approx at indication number 5 10mm glass will break approx at indication number 8 Upgrade kit for 10mm is optional Oder number 16706911110 After the break the breaking wheel is automatically reset to its home position Raise the scoring head by using the clamping lever The scoring device is also automatically reset to its home position Pull the knife drawer towards you until it reaches the click stop after a few mm of moving Remove the right glass strip and place it onto the foam glass storage area Turn the left glass strip and place it with the fresh broken end facing the click stop Slide the click stop to the right to its next click position Press the glass strip against it and move the drawer towards the glass until it stops Repeat steps as described above halving the glass strip each time until two 25mm Squares are produced Fig 9 Do not use the right end square of both 200mm glass strips as the ends are machine broken and therefore geometrically not perfect Fig 10 Page 13 Leica EM KMR3 Operating Manual 04 10 Fig 11 Fig 12 Fig 13 Fig 14 Fig 15 Fig 16 Page 14 Leica EM KMR3 Operating Manual 04 10 5 3 Making knives from a square Move the click stop to the left as it is not used for breaking knives Raise the scoring head by using the clamping lever Rotate the square a quarter turn clockwise in order to produce the knife edges from the freshly broken side of
9. e break the glass sits on two steel hemispheres and is held from above by two pins The design of the EM KMR3 allows a simultaneous movement of the two breaking pins to apply exactly the same force to each side of the glass strip ore square The break follows the score line as far as it goes and then a free break occurs The direction of this free break is determined by the mass of the glass on either side of the break and the breaking forces Page 3 Leica EM KMR3 Operating Manual 04 10 The free break curves to the edge of the square resulting in one knife and one flat edge counterpiece knife shoulder opposite the knife edge Fig 2 When the score runs centrally through a square a very small counterpice is obtained and the knife angle is very close to 45 Fig 2a This is the optimal result for cryo knives For resin sectioning the user sets the knife shoulder adjustment a little larger 0 5mm to produce a larger knife angle which is more stable for resin sectioning Fig 2 Knife edge of the left knife Counterpiece Knife shoulder Page 4 Leica EM KMR3 Operating Manual 04 10 Counterpiece Knife shoulder of the right Fig 2a Knife edge Knife edge knife Counterpiece Knife shoulder 2 3 The real knife angle When scoring the square all scores stop some distance from the corner When pressure is applied under the score the fracture is initiated and is seen first as a deepening of the score The
10. e carton However if there is a need to clean the glass wash each strip separately in cool tap water which has a low concentration of a mild laboratory detergent added to it Completely rinse off all detergent then rinse thoroughly with distilled water Dry gently with a clean soft and lint free cloth or place it in a drying oven Note Wear always lint free cotton gloves when handling the glass A N Warning Glass knives and glass strips have sharp edges handle with great care Page 11 Leica EM KMR3 Operating Manual 04 10 Fig 6 Fig 7 Fig 9 Fig 10 Page 12 Leica EM KMR3 Operating Manual 04 10 5 2 Making squares Raise the scoring head by using the clamping lever Fig 5 No 1 Set the scoring mark selector to the glass strip symbol Fig 5 No 3 Press down on the click stop Fig 5 No 5 and slide it to the far left hole until it clicks in Press the 400mm glass strip against it and push the knife drawer Fig 5 No 8 away from you until it reaches the end stop this will align the glass strip against the reference guide Lower the scoring head and clamp it to its defined stop Fig 6 The weight of the scoring head holds the glass strip in position Press the white knob of the scoring device Fig 7 to sore the glass Move the click stop away from the glass this will allow a free break Turn the breaking wheel slowly to initiate the break Fig 8 6 4mm glass will break approx at indication num
11. essure is correct when one faint smooth line without glass splinters is produced on the glass surface The pressure is too strong when a groove and glass splinters can be seen The pressure is too low when the scoring line is faint and interrupted Factory set scoring pressure for 6 4 and 8mm glass Fig 29 scale position approx at 0 5 User set scoring pressure for 10mm glass Fig 30 scale position approx at 1 5 Upgrade kit for 10mm is optional Order number 16706911110 Fig 29 Fig 30 8 Breaking 10mm thick glass strips When breaking 10mm glass make sure the upgrade kit for 10mm order number 16706911110 has been ordered together with your KMR3 This built in kit allows applying a clamping pressure suitable for breaking 10mm glass Set the scoring pressure to scale position 2 Fig 30 Follow the sequences described in chapter 5 2 and 5 3 10mm glass strip will break approx at indication number 8 scale indicator on the breaking wheel Page 22 Leica EM KMR3 Operating Manual 04 10 9 Mounting plastic boats trufs onto the knife Water boats trufs are used to hold distilled water where ultrathin sections can then float on the surface after room temperature sectioning Fitting a truf is simple using the Leica EM MP is used Fig 31 Wax is melted in the wax bath 2 3 full and the glass knives are gently warmed on the hotplate with the knife edge overhanging the edge of the hotplate as shown in Fig
12. fracture extends towards the corners of the square following the line of the score Where the score ends and the break is free the fracture deviates from the line of the score to curve away from the corner towards one of the edges of the square This results in the real angle of the knife being somewhat greater than the angle of scoring The real angle of the knife increases as the score is moved further from the diagonal This is when the knife shoulder becomes larger For example when preparing knives from a square the real angle of the knife is close to 45 when the knife shoulder is smaller lt 0 5mm Increasing the size of the knife shoulder gt 0 5mm results in an even larger knife angle which can be over 55 Fig 3 Fig 3 Scoring angle Real knife angle Page 5 Leica EM KMR3 Operating Manual 04 10 2 4 Length of useful edge When a glass knife edge is examined under darkfield illumination using a stereo microscope or on LEICA Ultramicrotomes using the back light chapter 5 4 it can be seen that the central part is most useful for ultrathin sectioning The right side of the edge has visible marks saw teeth which reduce the quality of the knife and the left corner is also unsuitable for sectioning because of the stress line Fig 4 The useful knife edge starts where the stress line moves away form the knife edge until the part where the stress marks saw teeth can be seen Fig 4 Stress line Note
13. quare iii 15 5 4 Evaluation of the knife edge EE 17 6 Knife shoulder TT E dr CT 19 i Care and maintenant EN 21 7 1 Replacement of the scoring wheel Cartridge ii 21 7 2 Adjusting the scoring PRESS EE 22 8 Breaking 10mm thick e UE d ln a 22 H Mounting plastic boats trufs onto the KNIFE iii 23 10 Accessories and consumables E 24 11 Technical EE 25 Page 1 Leica EM KMR3 Operating Manual 04 10 1 Safety instructions Prior to the operation of the Leica EM KMR3 please read the instruction manual carefully Always use safety goggles during operation to avoid eye injury Use lint free gloves thus to avoid finger prints onto the glass strip Clean the instrument regularly with a brush to remove glass splinters Store broken knives safely in the knife boxes provided Do not leave knives laying around or on the Leica EM KMR3 Used knives should be put into a marked sharps box for safe disposal Symbols in this manual and their meaning Warning take extra care Notes Important information for the user gt Identification Product Leica EM KMR3 Manufactured by Leica Mikrosysteme GmbH Hernalser Hauptstrasse 219 A 1170 Vienna Tel 43 1 488 99 0 Fax 43 1 488 99 350 Internet http www leica microsystems com Page 2 Leica EM KMR3 Operating Manual 04 10 2 Principles of Knifemaking 2 1 The Balanced Break method Fig 1 In
14. ss particles when a glass break is performed Use a brush or Dust off to clean after each glass break The sliding surfaces of the scoring and clamping head Fig 25 and Fig 26 must also be kept clean and dry Raise the scoring head using the clamping lever and remove the head Use a dry lint free cloth and clean the sliding surfaces After cleaning re place the scoring head No grease or oil should be applied 7 1 Replacement of the scoring wheel cartridge With the original Leica scoring wheel approx 5000 glass strips 6 4 and 8mm can be scored to produce glass knives if the scoring pressure is adjusted correctly To replace the scoring wheel raise the scoring head completely remove and place it on a table upside down Using the TORAX Allen Key provided with the spare scoring wheel shipment open the clamping screw for the scoring wheel shaft Fig 27 and pull out the shaft Fig 28 Insert a new scoring wheel re insert the shaft replace the screw and tighten it Replace the scoring head and make a test score on a piece of glass strip The scoring pressure with the new scoring wheel cartridge will probably be too strong or maybe not enough For adjustment see chapter 7 2 Page 21 Leica EM KMR3 Operating Manual 04 10 7 2 Adjusting the scoring pressure By turning the wheel beneath the pressure scale Fig 29 30 to the left pressure can be decreased Turning to the right the pressure can be increased The pr
15. the balanced break method an original Leica glass strip 400mm in length 6 4 8 or 10mm thick is scored and broken into two equal halves each 200mm long With an equal mass of glass on each side of the score the break is balanced and the freshly fractured surfaces are plane By continuing to divide each piece produced into two equal halves up to 16 squares can be made All squares produced have straight sides and precise right angled corners unlike Squares produced from sequential breaking of a glass strip which have curved surfaces Fig 1 2 2 Scoring and breaking principles Producing good glass knives routinely depends on a supply of reproducible squares an accurately positioned score and controlled pressure precisely applied to make the break The LEICA EM KMR3 gives the user a choice of two different score lengths each one factory set to produce the optimum break The short score glass strip symbol is used to break the glass strips into squares The long score square symbol is used to break the final Knives As a general rule the knife edge is straighter and the counterpiece knife shoulder is small when the fracture occurs close to the corner long score Using a short score was in the past suggested for cryo knives as the free break is longer resulting in the sharpest longest useable knife edge Griffiths et al 1983 Tokuyasu 1986 Each score is preset and equidistant from the corners of the square During th
16. the glass Position the square into the notch of the front fork Fig 11 Push the knife drawer slowly direction rear fork Fig 12 and place the square into the notch of the rear fork Push the knife drawer until it reaches the end stop and the square is securely positioned Fig 13 Set the scoring mark selector to the square symbol Lower the scoring head score and break according description in chapter 5 2 To break the final knives it is recommended to break as slow as possible For a reproducible break use the numbers on the breaking wheel as force indication Raise the scoring head and pull the knife drawer towards you until it reaches the end stop Fig 14 With both hands remove left and right knife simultaneously Fig 15 Or press both together and remove with one hand Do not allow the knives to touch while removing Each of the knives has a knife edge and a knife shoulder Knife edge of the left hand knife Knife shoulder of the right hand knife Left hand knife Right hand knife Knife edge of the right hand knife Knife shoulder of the left hand knife Turn the right knife to bring the knife edge side by side with knife edge of the left knife Fig 16 Note The best knives are made using the lowest breaking pressure They may take a minute or more to break Page 15 Leica EM KMR3 Operating Manual 04 10 Fig 17 Fig 18 Fig 19 Fig 20 Page 16 Leica EM KMR3 Operating
17. urning the wheel slowly clockwise two breaking pins apply exactly the same force to each side of the glass The breaking wheel features defined positions and automatically resets after the break 3 Scoring mark selector can be set by rotating to two different scoring lengths The short score 12mm glass strip symbol is for scoring the glass strips to break squares The long score 22mm square symbol is used to score squares for making high quality knives 4 Reference guide acts generally as a stop for the glass strip The glass strip is pressed against the reference guide before it is clamped The left hand reference guide acts also as a track for the moveable stop with its four click stop positions 5 Moveable click stop with its four click positions is mounted on the left hand reference guide and is used to position the 400 mm long glass strips according the balanced break method 6 Scoring device the knob is pushed inwards until it reaches the magnetic end stop where it is held until after the break It is then automatically released The score itself is performed by a tungsten carbide scoring wheel 7 Scoring pressure adjustment is used to change the scoring pressure if necessary when 6 4 8mm or 10mm glass Strips are used A scale indicates the scoring pressure 8 Knife drawer is used to press the glass strip against the reference guide for positioning the Square and for convenient removal of the gl
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