User Manual
Contents
1. DMT Confocal wire myograph system Model 120CW 5 3 Normalization The importance of normalizing the preparation is three fold 1 Experiments with elastic preparations like vessels can only have meaning if they are performed under conditions where the size is clearly defined 2 Clearly defined conditions are required in pharmacological experiments as the sensitivity of preparations to agonists and antagonists is dependent on the amount of stretch 3 The active response of a preparation is dependent on the extent of stretch which makes it important to set the preparation to an internal circumference giving maximal response The aim of the normalization procedure is to stretch the segment to a so called normalized internal circumference IC defined as a set fraction of the internal cir cumference IC that a fully relaxed segment would have at a specified transmural pressure For small rat arteries the target transmural pressure is typically LOO mmHg 13 3 kPa Principles of the normalization procedure In practice the normalization is performed by distending the segment stepwise and measuring sets of micrometer and force readings fig 5 13 step 1 4 These data are converted into values of internal circumference um and wall tension T mN mm respectively Plotting wall tension against internal circumference reveals an exponential curve and by applying the isobar curve corresponding to 100 mmHg IC is calculated from the poi2. when applying the weight F is equal acceleration of gravity times the mass of the weight r is the length of the transducer arm and F is the force acting on the force trans ducer The net torque acting at center of gravity is constant for the weight calibration setup which makes equation 1 and 2 equal making it possible to calculate the force acting on the force transducer FEE g sind weigt FL fag Mg 8 sind sind As the length of the pan arm is 2 cm the length of the transducer arm is 4 cm the weight is 2 g both angles are 900 and the acceleration of gravity is 9 81 ms the force acting on the force transducer is F 20m 2 89 61 ms sing0 4cm sin90 F_ 9 81 g m s As 1N is equal 1kg m s F is equal to F 9 81 mN User manual 120CW Chapter 4 21 22 4 2 2 Weight calibration procedure The section contains a complete step by step description of how to weight calibrate the force transducer 1 Fill the myograph chamber with double distilled water and move the jaws apart Mount a 40 um stainless steel wire on the jaws connected to the force transduc ers Place the calibration bridge balance and weight randomly on the myograph unit allowing it to be warmed up together with the myograph unit Turn on the heating in the Main menu on the Myo Interface After approximately 20 30 minutes the whole system will have reached the target temperature normally 37 C Place the wa
3. Figure 4 1 Illustration of how to ene adjust the supports Figure 4 1 1 Illustration of the a lt J reverse supports Fr pp ft 3 4 2 Calibration of force transducer s a part of the general maintenance of the myograph DMT recommends that the myograph is weight calibrated at least once every month DMT also recommends that the myograph is weight calibrated every time the system has been moved or has not been used for a long period of time 4 2 1 Principles of weight calibration Weight calibrating the force transducer is based on simple physics the net torque acting on a balance when applying a certain amount of weight The magnitude of the torque T about a point of rotation P is defined by LJ T r F sing where r is the distance from the point of rotation to the point on the object where the force F is acting with the angle of 0 Applying the physics to the weight calibration setup is illustrated in fig 4 2 DMT Confocal wire myograph system Model 120CW Transducer Arm TA N Figure 4 2 Theoretical principle of the weight calibration Applying the weight on the pan arm creates a net torque acting at the center of grav ity resulting in a force FT acting on the force transducer The following two equations describe the forces working in the weight calibration system tO Tia tg Oe he M jg 8 SING 2 EEA where r is the length of the pan arm F is the force acting on the pan arm
4. 850 mL 4 Aerate the solution with carbogen 95 O 5 CO for about 20 minutes 5 Solution 2 is added and the graduated bottle is filled with additional double distilled HO to reach the final volume of 1000 mL Continue the carbogen bubbling until the pH of the buffer solution reaches 7 4 25x concentrated PSS To make 1 L concentrated PSS Solution 1 Chemical MW g mol Conc mmol L Conc g L NaCl 58 44 118 99 173 85 KCI 14 56 4 69 8 75 CaCl 2H O 147 02 2 50 9 20 Solution 2 Chemical MW g mol Conc mmol L Conc g L MgSO HO 246 48 1 17 7 23 KH2PO 136 09 1 18 4 02 Solution 3 Chemical MW g mol Conc mmol L Conc g L EDTA 3 2 24 0 03 0 25 DMT Confocal wire myograph system Model 120CW 1 Dissolve the chemicals for solution 1 in about 800 mL double distilled HO in a 1000 mL graduated bottle Dissolve the chemicals for solutions 2 and 3 in 75 mL double distilled HO in individually cylinders Gently heat solution 3 to dissolve the EDTA 2 Solution 2 and 3 is added to solution 1 and the graduated bottle is filled with additional double distilled H O to reach a final volume of 1000 mL Before use 3 Dilute the 25 x PSS stock solution 1 25 with double distilled H O 4 Add 1 091 g L Glucose 2 100 g L NaHCO 5 Aerate the solution with carbogen 95 O 5 CO for at least 20 minutes necessary wait further for the pH of the buffer to reach pH 7 4 f High potassium physiological saline solution K
5. The force reading on the Myo Interface should now be very close to 9 81 mN Carefully remove the weight and proceed from step 3 to calibrate the other myograph If the force reading is different from 9 81 mN then try to calibrate the force transduc er once again starting with step 3 9 After calibrating carefully remove weight balance and calibration bridge The myograph is now ready for use 4 3 Checking the force transducer The myograph force transducer is a strain gauge connected in a Wheatstone bridge The force transducer is placed in a separate compartment on top of the Wire myo graph unit The separate compartment provides some mechanical protection for the force transducer but the transducer is still very vulnerable to applied forces exceeding 1 newton 100 gram or fluid running into the transducer compartment due to insuf ficient greasing of the transducer pinhole This section describes how to check the force transducer for any kind of damage User manual 120CW Chapter 4 23 24 4 3 1 Simple force transducer check 1 Ifthe force reading on the Myo Interface appears unstable then first check that the Myo Interface and the Confocal Myograph Unit are properly connected through the 25 pin grey cable 2 If the force reading still appears unstable then perform a new weight calibration of the force transducer as described earlier in this chapter During the weight calibration monitor the relative force reading values in
6. and initializing after which the start er up message depicted to the right is shown MODEL 120CW i VERSION 3 30 The display automatically shows the Main menu after several sec 28 02 2008 onds Otherwise press F1 to proceed to the Main menu When the start up message is active the and V keys can be used to adjust the display contrast setting 3 2 Menus and submenus The layout and relationship between the various menus and associated submenus in the Myo Interface are depicted in the schematic fig 3 1 General navigation The following controls are used to display the various menus choose varying menu options and change values F1 Change to the next menu F2 F4 Have varying functions depending on the current active menu Info Push this key in any menu to display all specific associated F key functions Numeric keys Enter data or setting values Enter Confirm entered numeric values or change between available choices Esc Delete incorrectly entered numeric values In some submenus used to return to main menu A Y Scroll up and down through the display as only four lines are capable of being displayed at a time Being in the top line of a submenu use A to change to the previous main menu The active line in the menu is indicated by a gt symbol Main menu The Main menu displays the current values from the force transducer the actual temperature probe reading and heating setting in C the heating status as we
7. as needed around the vessels to distin guish between the artery and vein The artery can easily be identified by the fol lowing characteristics fig 5 3 28 DMT Confocal wire myograph system Model 120CW Second Branch Figure 5 1 Branching of the mesenteric arteries Figure 5 2 Removal of adipose tissue around the area of interest Figure 5 3 Distinguishing between artery and vein User manual 120CW Chapter 4 29 e The branch points of arteries are V shaped whereas those of veins are more U shaped e The arterial wall contains a thick layer of smooth muscle cells compared to the vein wall which only contains a single or a few layers of smooth muscle cells The histological difference is clearly visible in the stereomicroscope e If you still have difficulty and the vein and artery still contain some blood then try to move the blood forward by very gently squeezing the vessels with a for ceps In the artery the blood will run back quickly whereas in the vein the blood will run back very slowly if it even does so Note it is important that you per form this on vessels other than those you will use as this procedure damages the vessels 8 Dissect away the vein using scissors to cut the adipose and connective tissue between the artery and vein One method is to cut the vein in one position and af terwards gently to pull the vein away from the artery In this way a fine membrane of connective tissue becomes visible betw
8. instructions are followed at all times 4 5 1 Myograph chamber pipes To prevent the pipes from being blocked by buffer salt deposits after an experiment use the chamber cover to remove the cleaning solutions used and described later in this Chapter Afterwards remove the cover from the myograph chamber and turn on the vacuum pump for about 10 seconds Wait to turn off the oxygen supply until turn ing off the vacuum pump Wipe off any buffer remaining on the outside of the pipes using a piece of paper DMI Confocal wire myograph system Model 120CW 4 5 2 Force transducer The force transducer is the most delicate and fragile component of the myograph system Therefore careful handling is necessary One of the jaws is connected to the transducer pin To prevent the buffer from running into the transducer house the hole is filled with high vacuum grease AS part of the daily maintenance it is very important to inspect the greasing of the transducer hole before starting any experiment Insufficient greasing causes damage and malfunction of the force transducer Important e DMT recommends that the high vacuum grease sealing up the transducer hole be changed at least once a week e DMT takes no responsibilities for the use of any other kinds of high vacuum grease than the one to be purchased from DMT e DMT takes no responsibilities for any kind of damage applied to the force trans ducer Figure 4 6 Transducer pin hole to be sealed
9. of division above the horizontal line represents 1 mm where each fifth line is marked by a longer line and a number which designates the length in mm Each part of division below the horizontal line is placed between each 1 mm mark scale above the horizontal line and represents 0 5 mm Thimble scale The thimble is divided into fifty equal parts and one complete rotation of the thimble is indicated by the smallest division on the sleeve which equals 0 5 mm Each divi son on the thimble scale is 10 um If the thimble scale falls between two lines then a number between 0 and 10 um must be approximated Example 1 1 Note that the thimble has stopped at a point beyond 10 on the sleeve indicat ing 10000 um 10 mm 2 Note that there is no mark completely visible between the 10 mm mark and the thimble 3 Read the value on the thimble corresponding to the intersection with the hori zontal line on the sleeve A Reading on sleeve 10000 um B No additional mark visible O um ETER C Thimble reading 380 um LET ULL Total reading 10380 um i Figure A8 2 Examp reading 10380 um Example 2 1 Note that the thimble has stopped at a point be yond 16 on the sleeve indicating 16000 um 16 mm 2 Note that this time a mark is visible between the 16 mm mark and the thimble indication 500 um 3 Read the value on the thimble corresponding to the intersection with the horizontal line on the sleeve A Reading on
10. sleeve 16000 um B One additional mark visible 500 um C Thimble reading 280 um Figure A8 3 Example 2 Total reading 16780 um reading 16780 um DMT Confocal wire myograph system Model 120CW Appendix 9 System specifications Vessel size gt 60 um Vessel alignment Manually X Y amp Z Chamber shape Single bath conical Chamber volume Max 10 mL Chamber material Acid resistant stainless steel Chamber cover With connection for suction and gassing Force range 100 200 mN Force resolution 0 01 mN Micrometers Manual precision Weight calibration Manual Heating Built into chamber independent of superfusion Temp range Ambient temp to 50 C Temp resolution 0 1 C Temp probe External Output reading Force MN Analog output Up to four outputs 1 0 V full scale for all acquired signals user de fined Serial output Serial interface RS232 RS485 Voltage 100 to 240 VAC auto 50 60 Hz Optional accessories pH meter enabled in the interface Range pH O 14 Temp correction O 50 C User manual 120CW Appendix 9 52 Notes DMT Confocal wire myograph system Model 120CW DANISA M10 T CHANOLOGSS DMT A S Skejbyparken 152 DK 8200 Aarhus N Denmark Tel 45 87 41 11 00 Fax 45 87 41 11 01 www dmt dk sales dmt dk Ssupport dmt dk DMT Asia Everwin Gardens Rm 502 Block B 521 Wanping Nan Lu Shanghai 200030 China Tel 86 0 21 64869685 Fax 86 0 21 6
11. the Calibra tion menu on the Myo Interface e Ifthe value is O or above 6500 then the force transducer is broken and needs to be changed e If the reading is between 1 499 or 3001 6250 then contact Danish Myo Technology for further instructions 4 4 Changing myograph window glass The glass in the myograph chamber window is fixed in placed and kept waterproof by a thin layer of high vacuum grease on the circular edge between the glass and the myograph chamber base Daily use of the myograph objectives that require wa ter or other immersion solvents will mean that the glass needs to be replaced daily prior to mounting The following procedure describes how to change the myograph window glass 1 Carefully loosen the glass from the myograph chamber edge using small forceps or similar tool 2 Clean the edge at the bottom of the myograph chamber to remove any remain ing old grease 3 Carefully apply a small amount of high vacuum grease around the edge using a wood stick or similar equipment Take a new window glass using forceps and gently push it into place Before starting a new experiment make sure that the complete edge is sealed up with high vacuum grease to keep the myograph window waterproof 4 5 Myograph maintenance The Confocal Wire Myograph System 120CW is a very delicate and sophisticated piece of research equipment In order to keep it working at its best DMT recommend that the following sections are read carefully and the
12. the wires are just separated and the force reading is approximately zero Then given that tension on the vessel is equal to force divided by wall length the wall tension at the i th mi crometer reading is calculated by T on ae 28 a a where is the microscope eyepiece reticule calibration factor in mm per division and a and a are the vessel end points when measuring the length of the mounted vessel segment The internal circumference of the mounted vessel at the i th reading is calculated by EE HANNEN where IC is the internal circumference of the mounted vessel when the wires are just separated and is given by IC 2 7 ed where d is the wire diameter For 40 um wires IC 205 6 um Using the Laplace relation the effective pressure P is calculated for each pair of readings The effective pressure is an estimate of the internal pressure which is nec essary to extend the vessel to the measured internal circumference T ge The stepwise distension is continued until the calculated effective pressure exceeds the target transmural pressure The target value needs to be optimized for the indi vidual tissue preparation optimal active force as determined by the length tension relationship for that tissue For rat mesenteric arteries the target transmural pres sure is normally 100 mmHg 13 3 kPa 100 mmHg IC T 100 mmHg 27 DMT Confocal wire myograph system Model 120CW An exponential curve
13. up with high vacuum grease 4 5 3 Linear slides Check the linear slides underneath the black cover for grease at least once a week In case of insufficient lubrication grease the slides with the original enclosed grease for linear slides at the places marked by the arrows in fig 4 7 User manual 120CW Chapter 4 25 26 Figure 4 7 Greasing points on the linear slides note only use the grease marked with Grease for linear slides 4 5 4 Myograph cleaning DMT strongly recommends that the myograph chamber and surroundings be cleaned after each experiment After an experiment use the following procedure to clean the myograph chamber and supports E Fill the myograph chamber to the edge with an 8 acetic acid solution and allow it to stand for a few minutes to dissolve calcium deposits and other salt build up Use a swab stick to mechanically clean all chamber surfaces Remove the acetic acid and wash the myograph chamber and jaws several times with double distilled water If any kind of hydrophobic reagent have been used which might be difficult to remove using step 1 and 2 then try incubating the chamber and jaws with 96 ethanol or a weak detergent solution To remove more resistant or toxic chemicals incubate the myograph chamber and glass cannulas with 1M HCI for up to 1 hour In exceptional cases incubate the chamber and supports with a up to 3M HNO solution for about 15 minutes Wash the myograph chamber and suppo
14. 1 Shanghai 200030 USA China Tel 45 87 411100 Tel 86 0 21 64869685 Tel 1 770 612 8014 Fax 45 87 41 11 O1 Fax 86 0 21 64280591 Fax 1 678 302 7013 www dmt dk www dmt asia com www dmt usa com sales dmt dk sales dmt asia com sales dmt usa com support dmt dk support dmt asia com Support dmt usa com User manual 120CW Trademarks Introduction The 120CW confocal wire myograph is specially designed to provide very close optical access to the mounted artery segment High resolution images of fluorescent dyes or markers within the living tissues such as those by laser scanning microscopy LSM become possible Combining LSM technology with myography allows simultaneous measurement of isometric force and dynamic intracellular events such as changes in intracellular Ca or pH occurring in living cells in the wall of isolated blood vessels The unique design of the myograph combines the precision and stability of our con ventional wire myographs with the added feature of precise Z axis movement with a micrometer This optimizes the use with different LSM s and various high magnifica tion and high numerical aperture objective lenses The conical bath design on the underside of the chamber allows the high numerical aperture lenses used on inverted microscopes and also direct immersion lenses used on standard upright microscopes to come directly in contact with the coverslip of the chamber window Also by using special mounting su
15. 2 Appendix 4 Myograph accessories and spare parts nranrnnnnnnnnnnnnnnnnnnnnnennn 43 A4 1 General myograph equipment Luanda 43 A4 2 Myograph 120CW system accesSorlieS rrsnnrrrnnnnnrnnnnnnnnnnnnnnnnnnennnnsnnnnnrennnnsnennnsee 44 A4 3 Wire Myograph 120CW system spare partS rrnannrrennnnnnnnnnrnnnnnnennnnnnnnnnnennnnnnennneee 44 Appendix 5 Fuse replacement nnnannnnnnannnnnnnnnnnnnnnennnnnnennnennennnennennnennnnnnennennn 45 Appendix 6 Normalization theory rrsanvannnanvnnnnannnnnnnnennnnnnennnnnnennnennennnennennn 46 Appendix 7 Calibration of eyepiece reticule rrnnrvannvnnnvnnnnnanenanenanennuennnenn 48 Appendix 8 How to read a millimeter micrometer scenerne 50 Appendix 9 System specifications nrnannnnnnvnnnnnnnnnnnnnnnnnnnennvennnennnennnennnennnennn 51 NOLES EET re 52 User Manual 120CW Contents 10 About this manual This manual contains a complete list of procedures describing how to install maintain and get started using the confocal wire myograph system model 120CW version 3 3 Chapter 1 provides an overview of the construction and basic features of the Myo In terface and the confocal wire myograph unit Chapter 2 describe step by step how to set up a complete confocal wire myograph system including connection to data acquisition Chapter 3 is a complete manual to the Myo Interface The chapter describes in detail the construction of the menu system and how to use all the feat
16. 4280591 www dmt asia com sales dmt asia com Support dmt asia com DMT USA Inc 1201 Peachtree Street 400 Colony Square Suite 200 630 Atlanta GA 30361 USA Tel 1 770 612 8014 Fax 1 678 302 7013 www dmt usa com sales dmt usa com Support dmt usa com
17. Confocal Wire Myograph System Model 120CW User Manual Version 3 3 DANISA MIO T amp T M LLE BES DANISA M10 TEaHnNOEolDdDGt DMT Confocal wire myograph system Model 120CW Confocal Wire Myograph System Model 120CW User Manual Trademarks PowerLab and LabChart are registered trademarks of ADInstruments Pty Ltd The names of specific recording units such as PowerLab 4 25 are trademarks of ADInstruments Pty Ltd Pentium is a registered trademark of the Intel Corporation Windows Windows 95 Windows 98 Windows ME Windows NT Windows 2000 Windows XP and Vista are registered trademarks of Microsoft Corporation All other trademarks are the properties of their respective owners DMT reserves the right to alter specifications as required This document was as far as possible accurate at the time of printing Changes may have been made to the software and hardware it describes since then New information may be supplied separately This documentation is provided with the DMT confocal wire myograph system Model 120CW v 3 3 Document Number 120CW UG3 3A No part of this document may be reproduced by any means without the prior written permission of DMT Copyright O 2008 DMT A S M DANISH MYO TECHNOLOG DMT A S DMT Asia DMT USA Inc Skejbyparken 152 Everwin Gardens 1201 Peachtree Street DK 8200 Aarhus N Rm 502 Block B 400 Colony Square Suite 200 630 Denmark 521 Wanping Nan Lu Atlanta GA 3036
18. PSS To make 1 L of KPSS Use the recipe for regular PSS but replace the desired concentration of NaCl with KCI For example to make 60 mM KPSS Solution 1 Chemical MW g mol Conc mmol L Conc g L NaCl 58 44 64 86 3 19 KCI 74 56 58 82 4 39 MgSO HO 246 48 1 17 0 29 KH PO 136 09 1 18 0 16 Calcium free physiological saline solution Ca free PSS To make 1 L of Ca free PSS Use the recipe for regular PSS but omit the CaCl and add EGTA to buffer the residual Ca in solution The concentration of EGTA in the PSS should be 1 5 mM to ensure sufficient buffering User manual 120CW Chapter 5 39 40 Appendix 1 Terms of warranty Warranty DMT A S warrants to the original user that myograph systems manufactured by DMT A S will be free from defects in materials and workmanship for a period of three years after the date of delivery DMT A S will repair or replace any defective part subject to the conditions limitations and exclusions Exclusions Force and pressure transducers separately or part of myograph systems manufac tured by DMT A S are disclaimed from any warranty Limitations This warranty shall not apply to equipment subjected to accidental damage improper use alteration or deterioration Warranty on third party products will be as determined by their respective manufac turer DMT A S shall not be liable for consequential incidental special or other direct or indirect damages resulting fro
19. S Window in the base of the cham ber for imaging Figure 1 1 The confocal wire myograph unit with close up detail of the chamber DMT Confocal wire myograph system Model 120CW 1 2 Myo Interface Up and down arrow keys Scroll up and Analog recorder output Connection to exter donni the menis nal data acquisition HE optional Myo Interface display e F Keys Have different but specific func Numeric keys Enter data or setting values tions dependent of the active main menu ere Enter key Confirm entered numeric val INFO Key Push this key in any menu ues or change between available choices to display all specific F key functions i e ESC key Delete incorrectly entered numeric values In some submenus used to return to main menu Figure 1 2 Myo Interface front panel 115 230 V 50 60 Hz automatic voltage selector e SES SEES SEES SEES SES SSEESSESSSESSSESSSEESSEESEEESEEES e ON OFF switch A EENE IE EA ER E TE A EET ER An e Power connector DE DER J REGULATOR RS232 MYOGRAPH Connection port for 120CW unit RS 232 port for serial connection to PC Connection port for pH meter optional USB port ET RE IR RE e12 V DC port Figure 1 3 Myo Interface rear panel User manual 120CW Chapter 1 13 Chapter 2 Setting up 2 1 The complete myograph 120CW system PC data acquisition and software Power supply PowerLab data acquisition sys
20. actor EDHF can contribute more or less than EDRF and in other vessels the same stimulation with ACh can promote release of endothelium derived contracting factor EDCF Therefore it is important to check the existing literature in order to determine the expected response in your particular vessel with the given concentration of agonist Step 1 PSS 3 uM NA Stimulate for 3 minutes Step 2 Add 10 pl 5 x 103 M ACh Wait 2 minutes Step 3 Wash out 4 x with PSS Ready for experiment User manual 120CW Chapter 5 37 38 5 6 Buffer recipes Physiological saline solution PSS To make 1 L of PSS Solution 1 Chemical MW g mol Conc mmol L Conc g L NaCl 58 44 118 99 6 95 KCI 74 56 4 69 0 35 MgSO 7H 0 246 48 LAT 0 29 KH PO 136 09 1 18 0 16 Solution 2 Chemical MW g mol Conc mmol L Conc g L CaCl 2H 0 147 02 2 50 0 37 Solution 3 Chemical MW g mol Conc mmol L Conc g L NaHCO 84 01 25 00 2 10 EDTA 372 24 0 03 0 01 Glucose 198 77 5 50 1 09 1 Dissolve the chemicals in approximately 100 mL double distilled H O as three individual solutions as described in the table above Gently heat solution 3 to dissolve the EDTA Solution 1 is added to a graduated bottle and the bottle is filled with double distilled HO to a final volume of 500 mL 3 Solution 3 is added to the graduated bottle which afterwards is filled with additional double distilled HO to a final volume of about
21. at the mounting heads are carefully adjusted and aligned so that there are no disturbances in the force recording when the heads are close together The alignment procedure is described elsewhere in the manual Chapter 4 For the mounting procedure the following equipment is used a stereomicroscope magnification 4 40x and a fibre light source two pairs of fine forceps a pair of fine scissors trabecular type a small screwdriver and wire 5 To have as much space as possible move the supports up with the vertical mi crometer screw 1 3 before you start the mounting procedure User manual 120CW Chapter 5 31 6 Place the myograph in front of you with the micrometer support to your left The head with the shortest distance between the pegs should be attached to the micrometer arm 7 Close heads partly 8 Hold a wire with the forceps one third from the top Place the wire between the heads un derneath the top left peg and over the lower right peg 9 When the wire is in position the heads are moved together and the top end of the wire is bent up and fixed by the top left screw Re member always to place the wires clockwise under the screw heads so that tightening of the screws will tighten the wires too 10 The protruding wire should be straight and long enough to reach the lower left screw Excess wire can be cut off cold PSS 11 Fill the chamber with cold PSS and transfer the vessel to the m
22. boratory use EMC requirements Part 2 6 Particular requirements In vitro diagnostic IVD medical equipment With reference to regulations in the following direc tives 2006 95 EC 89 336 EEC User manual 120CW Certificate of Conformity Contents TREERE EN Er eres 3 Fade oe I og EE EE EN Se EDER RE DEERE 4 SUNN 5 EMC EM 6 PAD TE EEE EE 6 Certificate of Conformity arnnnxnnnnnnnnnnnnnnnnnnannnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnvennvennnennnene 7 About this manual css Enns 10 Unpacking the myograph system anrnanvnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnennnnnennnennennn 11 Chapter 1 System overview anrnannnnnnnnnnnnnnnnnannnnnnannnnnnnnennnnnnennnnnnennnnnnennnnnnnn 12 EE VENN Narve 12 LNR 13 Chapter 2 Setting UD wis vnccsesadccnnannicesecesiondveanetecenaucnssuavesasensussdeudenuwaseussvuansuawenhs 14 2 1 The complete myograph 1200W system rvsassunennnvnvnnsunnnnnnnnnnnunvnnnvnnnnsuvnnnnunennununene 14 2 2 Setting up step by step cncesacssstuntesancdbseancdesadenstsdencesudeneceadanatadpacaneeboctesdeeatnodsncsanesnceaas 14 STENEN 15 Chapter 3 The Myo Interface anrnannnnnnnnnnnnnnnnnnnnnnennnennnennnennvennnennnennnennnennnennnnn 16 3 1 Turning on the Myo Interface rrennnnnnnnnnnnnnnvnnnnnnnnnnnnnnnnnnnnnnnnennnnnnennnnnensnnnnnnnnennnnr 16 3 2 Menus and submenus dreicdisinnturedomanatannauatawswaddcannaedeadomevdedsamadinaeebadinawhemtchnunaveriousancaavsauws 16 Chapter 4 The confocal wire
23. ct type See the table below Line plug according to standard 110 125 V UL817 and CSA C22 2 No 42 220 230 V CEE 7 page VII SR section 107 2 D1 IEC 83 page C4 240 V BS 1363 of 1984 Specification for 13A fused plugs and switched and unswitched socket outlets Protect the power cord from being walked on or pinched particularly at power plugs and the point where they connect to the apparatus Refer all servicing to qualified service personnel Servicing is required when the apparatus has been damaged in any way such as the power supply cord or plug is damaged liquid has spilled onto or objects have fallen into the apparatus the apparatus has been exposed to rain or moisture does not operate normally or has been dropped User manual 120CW Safety EMC EMI This equipment has been tested and found to comply with the limits for a Class B Digital device pursuant to part 15 of the FCC rules These limits are designed to provide reasonable protection against harmful interference in residential installations This equipment generates uses and can radiate radio frequency energy and if not installed and used in accordance with the instructions may cause harmful interference to radio communications However there is no guarantee that interference will not occur in a particular installation If this equipment does cause harmful interference to radio or television reception which can be determined by monitoring the interferenc
24. e typically consisting of 50 100 divi sions to measure the length of an object in terms of reticular divisions spanned by the object Having the conversion factor specific for the eyepiece reticule and used magnification the length of the object in mm is easily calculated All reticules need to be calibrated in order to determine the specific conversion factor characteristic for the particular eyepiece reticule and the used magnification For such purpose a special devise called a stage micrometer is needed A stage microm eter is simply a microscope glass slide having a scale engraved on the surface A typi cal micrometer scale is 2 00 mm long engraved with divisions of 0 01 mm equalling 10 um per division However micrometer glass slides with less fine divisions are also useful for calibrating a stereomicroscope to be used with the wire myograph Ocular calibration procedure 1 Decide which microscope magnification is to be used for the segment length measurements Use the largest possible fixed magnification where the eyepiece reticule scale still covers the whole gap of the myograph jaws 2 Place the stage micrometer on the microscope stage and focus on it Fit one of the division lines on the stage micrometer to one of the division lines of the reticule scale very precisely While keeping the stage micrometer absolutely fixed on the microscope stage find another position on both scales where the division lines also fit precisely Read t
25. e the excised intestine section in a Petri dish about 9 cm in diameter coated with a 5 mm thick layer of Sylgard at the bottom to hold the fixing pins Immedi ately fill the Petri dish with cold PSS which has been well aerated with carbogen The dissection is performed without further oxygenation of the PSS The cold tem perature ensures the artery is relaxed during the mounting procedure 4 Pin down the proximal end of the intestine section on the left hand side of the Petri dish without stretching the vessels Pin down the remaining of the intestine section in an anti clockwise direction In this configuration proximal end at the left side distal end at the right side and running anti clockwise from proximal to distal side the feeding vasculature is on the far side of the intestine and the veins are usually uppermost 5 Select the vessel segment to be investigated fig 5 1 First time myograph users are recommended to start dissecting and mounting vessel segments from the first or second branch from the superior mesenteric artery internal diameter 200 300 um 6 Use high quality forceps and ocular dissection scissors to dissect the vessel segment of interest Start cutting through the mesenteric membrane along both sides of the vessel about 1 2 mm from the vessel To avoid accidentally cutting the artery always cut along the length of the vessels and never perpendicular to them fig 5 2A B 7 Dissect away as much adipose tissue
26. e while turning the equipment off and on the user is encouraged to correct the interference by one or more of the following measures e Reorient or relocate the receiving antenna e Increase the separation between the equipment and receiver e Connect the equipment into an outlet on a circuit different to that which the receiver is connected to e Consult the dealer or an experienced radio TV technician for help Approvals Complies with the EMC standards EMC 89 336 EEC EN 61326 2 6 2005 EN 61000 3 2 Certified with the safety standards Directive 2006 95 EC EN 61010 1 2001 EN 61010 1 Corr 1 2003 EN 61010 1 Corr 1 2003 EN 61010 2 101 2003 DMI Confocal wire myograph system Model 120CW Certificate of Conformity DMT A S Skejbyparken 152 8200 Aarhus N Denmark hereby declares its responsibility that the following product Confocal Wire Myograph System Model 120CW version 3 3 is covered by this certificate and marked with CE label conforms with the following standards EN 61010 1 2001 Safety requirements for electrical equipment for EN61010 1 C0rr 1 2003 measurement control and laboratory use EN 61010 1 Corr 1 2003 Part 1 General requirements EN 61010 2 101 2003 Safety requirements for electrical equipment for measurement control and laboratory use Part 2 101 Particular requirements for in vitro diagnostic IVD medical equipment EN 61326 2 6 2005 Electrical equipment for measurement control and la
27. een the adipose tissue and the artery Carefully cut the fine membrane to remove the vein and adipose tissue while avoiding any direct contact between the scissor and artery fig 5 4 A B Figure 5 4 Removal of vein Figure 5 5 A B and C Cutting free the artery to be studied 30 DMT Confocal wire myograph system Model 120CW 9 Clean the artery by removing any remaining adipose or connective tissue Gently pull away adipose or connective tissue to make the connective tissue membrane become visible Cut the membrane to remove the tissue 10 Cut the distal end of the artery section to be investigated Afterwards cut the proximal end while ensuring that the vessel segment has the correct length fig 5 5A C 5 2 Mounting protocol for small arteries The difficulty of mounting ring preparations in any wire myograph mostly depends on the lumen size The myograph technique is usable for ring preparations with internal diameter gt 100 um using 40 um stainless steel wires 25 um tungsten wires are avail able for ring preparations with an internal diameter as low as 60 um For large diam eter vessels mounting pins 200 um and larger are an available option The confocal myograph reverse mounting jaws are shaped with pegs at the bottom to position the vessel as close as possible to the objective of the inverted confocal microscope The special jaws with a vessel mounted are depicted in fig 6 2 The pegs make the mounting of the ve
28. equili brated and normalized The present procedure is suitable for rat mesenteric arter ies Another procedure may be needed for other animal species and tissue or vessel types Principles of the standard start procedure The standard start procedure consists of a series of five stimuli and washout periods The first two stimuli are performed using a mixture of KPSS and 10 uM noradrena line to give a maximum contractile response The third stimulus is performed using a mixture of PSS and 10 uM noradrenaline to give a maximum pure agonist mediated a adrenoceptor contraction The fourth stimulus is performed using KPSS to give a depolarising contractile response this stimulus also includes a component from neurally released noradrenaline The final stimulus is performed using a mixture of PSS and 10 uM noradrenaline All solutions are preheated to 37 C and aerated with a mixture of 95 O and 5 CO before use Instructions for making the necessary solutions are described at the end of this chapter Repeat 1 x Stimulus 1 amp 2 Wash out KPSS 10 uM NA 4 x with PSS Stimulate for 3 minutes Wait 5 minutes Stimulus 3 Wash out PSS 10 uM NA 4 x with PSS Stimulate for 3 minutes Wait 5 minutes Stimulus 4 Wash out KPSS 4 x with PSS Stimulate for 3 minutes Wait 5 minutes Stimulus 5 Wash out KPSS 10 uM NA 4 x with PSS Stimulate for 3 minutes Ready for experiment DMI Confocal
29. er layer User manual 120CW Appendix 4 43 A4 2 Myograph 120CW system accessories This section contains a list of special accessories available for the Confocal Wire Myo graph System 120CW e ADI PowerLab data acquisition system Including Chart data acquisition and analysis software e DMT Normalization module DMT Item Chart add on software pH meter DMT Item Including pH electrode and Myo Interface pH software flash update e 25 um tungsten wire DMT Item For mounting of ring preparations with internal diameter down to 60um e Electronic vacuum valve DMT Item VV 9937 e Standard PC system DMT Item e DMT CS 100 2 Channel amp CS 200 4 Channel Current Stimulators DMT Item CS 100 amp CS 200 Combined pulse and train generator A4 3 Wire Myograph 120CW system spare parts This section contains a complete list of standard available spare parts for the myo graph 120CW For parts not listed in this section or for special parts which may need to be custom made please contact DMT for further information e Force transducer DMT Item FI 12110 Roll of 40 um stainless steel wire DMT Item SSW 123456 High vacuum grease and grease for linear slides DMT Item HVG 1000 e Reverse mounting jaws stainless steel DMT Item RJ 120 e Reverse mounting jaws plastic with in built platinum stimulation electrodes Mounting pins 200 um wire thickness e Calibration kit DMT Item GK 1050 44 DMT Confocal wire my
30. he position of the two fit points on both scales and fill in the values in the ocular calibration sheet Repeat the procedure twice DMI Confocal wire myograph system Model 120CW Ocular calibration sheet Date Operators O Microscope type and ff Ocular type Magnification Front lens Stage micrometer Reticular scale Position 1 Position 2 Length Position 1 Position2 Length 1 div div mm 2 div div mm 3 div div mm Mean Calculations 1 Calculate the length between the two positions on the stage micrometer by sub tracting the value of position 1 from the value of position 2 Multiply the length in divisions with the length of each division to get the length in mm 2 Calculate the length between the two positions on the reticule scale by subtracting the value of position 1 from the value of position 2 3 Calculate the mean length value of both the stage micrometer and the reticular Scale 4 Calculate the conversion factor 5 Mean Stage Micrometer Length mm Mean Reticule Scale div User manual 120CW Appendix 7 49 50 Appendix 8 How to read a millimeter micrometer Sleeve scale Thimble scale e e F i H ME NA et soy E PIG ed EES AS i DA Figure A8 1 Overview of the micrometer parts actual reading 20000 um 20 mm Sleeve scale The micrometer sleeve scale has a total length of 25 mm divided into fifty equal parts Each part
31. hese purposes nor for the prevention diagnosis curing treatment or alleviation of disease injury or handicap e Do not open the unit the internal electronics pose a risk of electric shock e Do not use this apparatus near water e To reduce the risk of fire or electric shock do not expose this apparatus to rain or moisture Objects filled with liquids should not be placed on the apparatus e Do not block any ventilation openings Install in accordance with the manufacturer s instructions e Do not install near any heat sources such as radiators heat registers stoves or other apparatus that produce heat e Only use attachments and accessories specified by the manufacturer e Unplug this apparatus during lightning storms or when unused for long periods of time e This apparatus must be grounded e Use a three wire grounding type cord similar to the one supplied with the product e Do not defeat the safety purpose of the polarized or grounding type plug A polarized plug has two flat blades one being wider than the other A grounding type plug has two blades and a third round grounding pin The wide blade or the third prong is provided for your safety If the provided plug does not fit into your outlet consult an electrician for replacement of the obsolete outlet Be advised that different operating voltages require the use of different types of line cord and attachment plugs Check the voltage in your area and use the corre
32. is fitted to the internal circumference pressure data as illus trated in fig 5 14 in Chapter 5 Now the isobar corresponding to 100 mmHg is used to calculate the IC value from the point of interception between the function of the exponential curve and the function of the 100 mmHg isobar The normalized internal circumference IC is calculated by multiplying the internal circumference corresponding to 100 mmHg IC po by a factor k The factor is for rat mesenteric arteries 0 9 Again this value should be optimized for the particular tissue preparation being used by a length tension curve IC k IC The normalized internal lumen diameter is then calculated by _ IC 1 TU The micrometer reading X at which the internal circumference of the normalized ves sel is set to is calculated by IC IC 2 User manual 120CW Appendix 6 47 Appendix 7 Calibration of eyepiece reticule Principles of ocular calibration The purpose of calibrating the eyepiece reticule is to determine a conversion factor 6 allowing the microscope to be used for measuring vessel seg ment lengths mounted in the wire myograph D 40 50 60 l 90 y im Several types of eyepiece reticules are available for Such a purpose The most simple and yet very useful type is a horizontal scale as illustrated in fig AT 1 Figure A7 1 Horizontal eyepiece reticule scale The basic principle is to use the eyepiece reticul
33. ist of special myograph 120CW accessories and spare parts Besides the main focus on development and manufacturing DMT has specialised in offering our customers first class laboratory equipment needed for a confocal wire myograph set up at very competitive prices Please contact DMT Sales Department for further product information and prices A4 1 General myograph equipment This section contains a complete and yet very useful checklist of laboratory equip ment needed when setting up a basic wire myograph system e Zeiss Axiovert 40CFL microscope that is ready for future fluorescence upgrade Item MZA 8000 e Dissection stereo microscope Item DSM 9953 Including ocular micrometer and stage micrometer DMT recommends the Zeiss Stemi 2000 StereoMicroscope e 2 Dissection and mounting forceps Item DF 3000 DMT recommends Dumont Medical No 5 tip 0 10 mm x 0 06 mm e Pipettes DMT recommends CappAero pipettes e Light source Item LS 9955 DMT recommends Schott Cold Light Source either Model KL 200 or Model KL 1500 Water bath including heater Item WBH 9951 DMT recommends Julabo SL e 1 pair of ocular Scissors Item DS 1000 DMT recommends the Geuder G 19745 8cm straight scissors e Glass bottle 2 L Vacuum pump litem VP 9952 DMT recommends a membrane vacuum pump having a volume of at least 6 L min e Dissection petri dish Item PD 2000 DMT recommends a 9 cm glass Petri dish coated with a 5 mm Sylgard polym
34. linear slide support in all vertical and horizontal planes using the four Allen screws D Use the four Allen screws to make the final horizontal and vertical fig 4 1 adjustments to match the linear slide support to the force transducer support The correct matching of the supports is illustrated in fig 4 1 1 Important Be careful not only to continuously tighten the Allen screws when doing the final adjustments Always remember to loosen the Allen screw placed diagonal to the Allen screw being tightened otherwise there is a high risk of damaging the myograph frame Special designed jaws The myograph are supplied with jaws for inverted scopes but can be used with normal stainless steel jaws to mount small vessels on an upright system For experimental work requiring electrical field stimulation a set of plastic jaws with attached platinum electrodes is available Chamber The chamber has a conical shape and can thus be used with a small amount of buffer liquid The chamber can be customized to operate with volumes of 3 mL or less The conical milling on the bottom outside of the chamber allows objectives on inverted microscopes to be raised to the thin glass window to accommodate very low working distance objectives User manual 120CW Chapter 4 19 20 A Screw on support connected to grenene e C Screw on the linear slide the force transducer Q pe D Four Allen screws apanan B Screw on support connected
35. ling with very delicate equipment and therefore care must be taken DMT recommends that each part of the myograph system be wrapped individually i e with bubble wrap and placed together in a large box preferable the box you once received the Myo graph system in Place the wrapped items in the middle of the box and fill out the surroundings with chips of expanded polystyrene Important Before closing the box make sure that no enclosed items can be shaken around as transport by road or air from time to time can be quite roughly Address the box to DMT A S Skejbyparken 152 DK 8200 Aarhus N Denmark Make sure that all four sides of the box are marked fragile or similar Make an indi cation on the top of the box that it contains goods returned for repair service Customers outside the EC must further enclose a pro forma invoice stating that the box contains goods being returned for repair or service If arranging transportation through a courier please keep in mind the high value of the myograph system and that a standard insurance provided by the courier in most cases is insufficient to cover damage or loss of the myograph system In most cases an additional insurance coverage is needed DMT Confocal wire myograph system Model 120CW Appendix 4 Myograph accessories and spare parts This section contains a complete register of equipment needed to set up a Confocal Wire Myograph System In addition the chapter contains a l
36. ll as the pH probe reading optional Temperature To change the temperature setting move the Set Temp line to the top of the display the gt symbol is displayed in that line Use the numeric keys to enter a new temperature setting and press Enter to confirm Heating To turn on the myograph heating move the Heat is line to the top of the display and use the Enter key to switch between on and off Press the Info key and the display shows the F key options associated with the Main menu Choose one of the F keys to proceed with the following options or press Info again to return to the Main menu F1 Change to the next main menu Recorder F2 Opens the Valve Activation submenu An optional electronic vacuum valve is available for the system which can be activated here F3 Opens the Zero Force submenu press F1 to zero the force or F4 to cancel DMT Confocal wire myograph system Model 120CW F1 MAIN MENU RECORDER MENU Rec 1 FORCE Rec 1lo 20 Rec 1hi 20 Rec 2 TEMP Rec 2lo 0 Rec2hi 50 Rec 3 No use Rec 3lo Rec 3 hi Rec 4 No use Rec 4lo Rec4 hi SETUP Calibrate FORCE Valve dly 5 Contrast 35 Baudrate 9600 Option 0 Force 0 15 mN Act temp 36 8 C Settemp 37 0 C Heatis ON Change menu Start timer Stop Pause Reset F1 Change menu Change menu F2 Valve ON F3 Zero force F4 Condensed me
37. m economic loss or property damage sustained by you or any end user from the use of the products sold or services rendered hereunder Warranty returns A Return Material Authorisation RMA number is required for all returns This number should be clearly indicated on all returned myograph systems Products damaged due to improper or inadequate packaging when returned for RMA purposes are not granted warranty coverage DMI Confocal wire myograph system Model 120CW ppendix 2 Service check A myograph working at optimal performance is extremely important for success when studying small blood vessels or other small tubular tissues To make sure that our customers always are dealing with first class myographs DMT offers a Myograph Service Check at a very favourable price The Myograph Service Check includes a complete separation for inspection of all mechanical and electronic parts The myograph is then reassembled adjusted and finally all electronic and mechanical parts are tested Please note that the service does not include replacement of transducers or any other needed spare parts Please contact DMT for information about prices User manual 120CW Appendix 2 41 42 Appendix 3 Shipping instructions If the myograph system needs to be sending back for service or repair please read the following shipping instructions very carefully Before you start packing the myograph system please remember that you are dea
38. myograph unit renarnnanvnnnennnennnennnnnnnennnennnennnen 19 AL POSURE Supp 0 EE EN NE 19 AZ Calibration TN 20 4 2 1 Principles of weight calibration annnnrnnnnnnnnnnnnnnnnennnnnnnnnnnnnnnvnnnnnnrennnnnnrennnnnnnsnnnnn 20 4 2 2 Weight calibration procedure uer eemeemsnabnkdekenddjospeddsdsuntnsenena 22 4 3 Checking the force transducer sarecatsdnncesedaricocsaninmectasscoteasedeiseiniaeccsunancdanssatdansemiuoasonas 23 4 3 1 simple force transducer Check man as Eee 24 4 4 Changing myograph window glass assnrrennnnnrrvnnnnnnrennnnnnrennnnnnnnnnnnnnrennnnnnrennnnnnnnnnnnn 24 4 5 Myograph maintenance sere tee csittepPemteatesulncnatestensaidveumaedeasenaude teaicnalecieratlecehentevaeauiavesanantasacins 24 4 5 1 Myograph chamber Ol 9 EEE NE 24 28 FT 25 45 3 Linear gt EE EEE EEE EE 25 4 5 4 Myograph cleaning EEE TE VE 26 Chapter 5 Getting started rnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnennnnnnennnnnnennnnnnennnnnnen 28 5 1 Dissection protocol for small mesenteric arterieS m rrnnurnunnvsnnvnnnnnnnnnnnennnnnnnnennne 28 5 2 Mounting protocol for NNN 31 29 da 140 100 EEE 35 SS OC SA EE EN NE NN EN 36 i ECO MG eo EE E 37 DMI Confocal wire myograph system Model 120CW Appendix 1 Terms of warranty nraanvnanenannnnnnnnnnnnnnnnnnennnenanennnennnennnennnennnennnen 40 Appendix 2 Service check suser EEN 41 Appendix 3 Shipping instructionS rsar asrnanvnnnnannnnnnnnnnnnnnnennnnnnennnennennnennennr 4
39. niti ate the calibration process Press Esc to return to the Setup menu if you do not wish to calibrate Force calibration This procedure is described in detail in Chapter 4 pH calibration optional Having chosen to calibrate the pH settings press F4 to initiate the calibration In the Calibrate pH Offset display the first line shows the value of the offset buffer first buffer solution which is always 7 00 The second line shows the temperature of the buffer solution The temperature is an important parameter in the calibration formula and is obtained automatically by placing the myograph temperature probe in the buffer solution The third line shows the output from the pH probe as raw data from the A D converter Place the pH meter electrode and temperature probe in the offset buffer solution and turn on stirring When the relative pH output in the bottom line is stable press F3 to proceed The first line now displays the value of the slope buffer second buffer solution which is always 4 00 Place the pH and temperature probes in the slope buffer solution and turn on stirring When the relative pH output in the bottom line is stable press F3 to proceed The pH calibration is now finished The parameters are stored in the internal memory of the Myo Interface Press F3 to return to the Setup menu DMI Confocal wire myograph system Model 120CW Chapter 4 The confocal wire myograph unit Chapter 4 contains a complete explana
40. nt of intersection using the Laplace relation fig 5 14 IC is calculated from IC oo by multiplying a factor giving an internal circumference at which the active force production as well as the sensitivity to agonists of the segment is maximal For rat mesenteric arteries the factor is 0 9 but both this factor as well as the transmural pressure has to be optimized for each particular segment The normalized internal diameter is calculated by dividing IC with Appendix 6 contains a complete description of the mathematical rationale and calcu lations of the normalization procedure Force mN mm 100 200 300 40 500 600 700 800 900 Internal Circumference pm Time min Figure 5 13 Illustration of the stepwise Figure 5 14 Illustration of the expo normalization procedure nential curve fitting and determina tion of IC oo User manual 120CW Chapter 5 35 5 4 Standard start The purpose of performing a standard start is to 1 Re activate the mechanical and functional properties of the vessel segment 2 Check that responses to different types of stimuli are normal in appearance and thereby ensuring that the functionality of the vessel segment has not been dam aged during the dissection or mounting procedures 3 Ensure that the tension development gives an effective active pressure that is above the chosen accepted value usually 13 3 kPa 100 mmHg The standard start is performed after the vessel segment has been heated
41. nu Le Apply no force Activate Valve F3 Push F3 when stable Force 1715 Yes Fl No F4 Force 0 15 mN Act temp 36 8 C Settemp 37 0 C Heat ON Calibrate Force CALIBRATE FORCE F3 CALIBRATE FORCE Place 2 gram on pan Push F3 when stable Force 1880 Calibrate Force DONE Push Esc Force zero 17157 Force gain 168 9 Figure 3 1 Menu overview the green panels indicate the menu appearance options when the optional pH meter is installed F4 Displays a condensed Main menu Press F4 to toggle between the condensed and the normal view Recorder menu The 120CW Myo Interface has four analog output ports on the front panel for connec tion to a data acquisition system These default settings for these output ports are Rec 1 Myograph force FORCE output Rec 2 Temperature TEMP output Rec 3 pH reading output if installed Rec 4 No output The output order can be programmed to whatever order you desire Make the re corder line of interest active uppermost in display and use the Enter key to toggle between the available signals The full scale output from the Myo Interface is one volt In the Recorder menu it is possible to change the associated values for each analog output that correspond to O V lo and 1 V hi Timer menu The Timer menu makes it possible for the Myo Interface to act as a timer the inter face can sound an alarm after a predefined countdown To set the countdown
42. ograph system Model 120CW Appendix 5 Fuse replacement The main fuse of the myograph system is placed inside the power inlet on the Myo In terface If the fuse blows it is easily changed using the following procedure When a fuse blows and needs to be changed it is imperative that the replacement fuse is equal to the one blown The 310A system uses T1 6A 250 V 6 3 x 32 mm DMT recommends that both fuses in the fuse block are changed at the same time as it can be difficult to determine which fuse is blown To replace the fuses Use a small screwdriver to open the voltage selector block Remove the red fuse block Remove the existing fuses Insert the new fuses Replace the fuse block back into the voltage selector block Note ensure that the correct voltage for your country is displayed Se RO ES Voltage selector block User manual 120CW Appendix 5 45 46 Appendix 6 Normalization theory The importance of making a normalization before initiating an experiment with any tubular tissue segment is described in Chapter 5 In this appendix the mathematical rationale and calculations underlying the normalization procedure are described in detail Mathematical calculations Let X Y be the pair of values representing the micrometer reading and force read ing respectively characterising each step in the normalization procedure Y is the force reading at the start position of the normalization procedure where
43. pes on the myograph chamber cover to an adjustable oxy gen supply using small silicone tubing internal diameter 1 5 mm 4 Suction connection Connect the large pipe on the myograph chamber cover to a vacuum pump via a suction bottle and the vacuum valve as illustrated in fig 2 2 The internal diam eter of rubber tubes for connection to the pipes on the chamber cover is 2 mm An electronic vacuum valve is available from DMT 5 Electrode optional Connect the pH electrode to the pH port on the backside of the Myo Interface and install the software flash update as described in the provided procedure Perform a pH calibration as described in chapter 3 RENSEDE e Inlet for adding drugs Suction connection e Oxygen supply Spare connection for chamber e superfusion Figure 2 2 Chamber cover 2 3 The first weight calibration Prior to the shipment of the Confocal wire myograph system it has gone through two days of continuous testing including a weight calibration However in order to ensure that the myograph is operating at an optimum DMT recommends that a new weight calibration is performed before the confocal wire myograph system is used The weight calibration procedures are described in detail in chapter 4 User Manual 120CW Chapter 2 15 16 Chapter 3 The Myo Interface 3 1 Turning on the Myo Interface When the 120CW Myo Interface is switched on the display shows that the system is starting up
44. ports for individual cleaning to make sure that all surfaces are clean Figure 4 8 The top part of the myograph can be tilted for easier access when cleaning User manual 120CW Chapter 4 27 Chapter 5 Getting started Chapter 5 contains some practical protocols and procedures describing how to quickly get started using the Confocal wire myograph system The chapter mostly ad dresses users who are not yet familiar with the wire myograph technique However it may contain some hints that will also be useful for the experienced myograph user 5 1 Dissection protocol for small mesenteric arteries The wire myograph technique is versatile in that a large variety of physiological and pharmacological studies of ring preparations from different species are possible The wire myograph is predominantly used for investigation of small blood vessels and as an example this chapter describes the dissection of rat mesenteric arteries 1 A laboratory rat is euthanized in accordance to the local national law and regula tions midline laparotomy is performed to expose the mesenteric bed 2 Use scissors to remove about 10 cm of intestine together with its feeding vascu lature including part of the superior mesenteric artery Be careful not to damage the vasculature during this procedure The proximal end of the intestine section must be about 10 cm from pylorus Make a cut in the proximal end of the intes tine for later identification 3 Plac
45. pports designed specifically for vertical positioning an isolated blood vessel can be positioned directly above or on the chamber window This permits use of lenses with working distances smaller than 250 um on an inverted LSM This may be advantageous for simultaneous electrophysiological measurements The chambers are supplied with custom covers for inverted or upright microscope systems with connections for suction gassing or measurement electrodes pH NO O tension The myograph are supplied with jaws for inverted scopes but can be used with conventional jaws on an upright system For experimental work requiring electrical field stimulation a set of plastic jaws with at tached platinum electrodes is available During the experiment the circumference of the vessel is kept constant i e the ves sel is examined under isometric conditions Compounds are added directly to the chamber and the resulting contractile changes in vessel force tension are measured While on the LSM data acquired from the myograph such as force and temperature can be recorded continuously either through the serial interface output or with the Myo Interface analog outputs connected to an external data acquisition system DMI Confocal wire myograph system Model 120CW Safety The confocal wire myograph system has been designed for use only in teaching and research applications It is not intended for clinical or critical life care use and should never be used for t
46. rmed calibration bridge and balance on the myograph unit as illustrated in fig 4 3 Note the weight should not be placed on the balance yet Make sure that the tip of the transducer arm on the balance is placed in the gap between the wire and the jaw as illustrated in fig 4 4 Carefully move the calibra tion bridge until the tip of the transducer arm is placed freely in the gap which means it does not touch either the wire or the jaw Go to the Setup menu on the Myo Interface and choose to calibrate the myo graph force transducer Press F4 to start calibration Figure 4 3 Weight calibration setup showing placement of the calibration bridge and balance with weight in place DMT Confocal wire myograph system Model 120CW Figure 4 4 Illustration of how to fit the bal ance between the wire and the gap in the support 6 Make sure that absolutely no force is applied on the force transducer by check ing that the tip of the transducer arm is not touching either the wire or jaw Also check that the relative force reading in the display is stable Press F3 to proceed with calibrating T Carefully place the 2 g weight on the pan as illustrated in figs 4 3 and 4 4 The force applied on the force transducer should mimic the stretch created by the contraction of a mounted ring preparation Wait until the relative force reading is stable Press F3 to finish the calibration 8 Press Esc and go to the Main menu on the Myo Interface
47. rts several times with double distilled water Important Notes Be very careful using step 3 and 4 repeatedly times as strong reagents can cause extreme damage to the myograph unit Be very careful not to exert any force on the myograph jaws during the cleaning procedure After cleaning ALWAYS check that the greasing around the transducer pin is suf ficient to keep out the buffer solution from the transducer compartment DMT Confocal wire myograph system Model 120CW In cases of red or brown discolorations appearing on the chamber sides the following cleaning procedure will work in most cases 1 Incubate the myograph chamber for 30 minutes with 20ul of a 2 mM T 1210 Tetrakis 2 pyridylmethyl ethylenediamine solution dissolved in double distilled water 2 Use aswab stick to mechanically clean all the affected surfaces during the last 15 minutes of the incubation period 3 Wash the myograph chamber several times with double distilled water 4 Incubate the myograph chamber with 96 ethanol for 10 minutes while continu ing the mechanical cleaning with a swab stick 5 Remove the ethanol solution and wash a few times with double distilled water Incubate the myograph chamber with an 8 acetic acid solution for 10 minutes and continue the mechanical cleaning with a swab stick 6 Wash the myograph chamber several times with double distilled water Important Notes e In exceptional cases it may be necessary to remove the sup
48. side Move the myograph to the confocal microscope and attach bubbling to the bath Connect the myograph to the Myo Interface and turn the heating on Zeroing of the micrometer X Can now be done wires moved together until they are just touching and a normalization performed as described in the next section After the artery is normalized contractility tested and loading with the fluorescent dye completed move the heads downwards with the vertical micrometer to position the artery just above the cover slip Once you can see the jaws are close to the cover slip follow the force reading on the Myo Interface It is important to now advance slowly and carefully too much downwards force and you will put unnecessary strain on the transducer and risk cracking the cover slip When the artery is touching the bottom of the chamber the force reading with increase At this point move the artery up slightly The artery is now positioned for imaging and the objective should be able to focus on the vessel Note that movement artifacts can occur when the artery is stimulated with agonists or depolarized with potassium This factor can be somewhat avoided by the use of ratiometric dyes Contraction can be inhibited by wortmannin which covalently binds to the myosin light chain kinase Alternatively the artery can be hyperpolarized with potassium channel openers thereby preventing the opening of voltage sensitive calcium channels and contraction
49. ssel a little more difficult than in the normal wire myograph Another difference from the normal wire myograph is that the mounting supports for the jaws are longer and thus less rigid Slight pressure on the jaw can result in a significant stress upon the mounting supports On the transducer side this results in stress upon the transducer pin which could damage the transducer Furthermore there is a risk you will push the jaws out of alignment For these reasons the demands upon your carefulness and awareness are increased in these proce dures These instructions hopefully will help you to succeed with the mounting procedure Experience with mounting in a normal myograph is an advantage and will ease the process Procedure Prior to mounting 1 Avessel size 150 500 um is dissected as described in 5 1 3 Before mounting it is important that the cover slip diameter 19 mm the thick ness may need to be optimized to your optics at the bottom of the myograph is clean It is recommended that the glass be changed every day To ease this proc ess the top part of the myograph with the heads can be tilted up and out of the way this also permits easy access for cleaning To remove the old glass press up gently from below Remove the old grease and fix the new glass with a little grease high vacuum grease It is important to remove any air bubbles between the glass and the chamber to ensure a good water tight seal 4 Itis also important th
50. tem optional Myo Interface front panel optional Myo Interface rear panel pH meter Vacuum pum optional Myograph unit uum pump optional O Electronic valve optional Power supply Suction bottle Figure 2 1 The complete confocal 120CW system PAR Optional 2 2 Setting up step by step The chapter contains a complete step by step description of how to set up a complete myograph system as illustrated in fig 2 1 1 Myograph unit Myo Interface connection Connect the myograph 120CW unit to the Myo Interface using the grey 25 pin connection cable The end of the cable with the temperature probe is placed into the myograph chamber to monitor actual temperature 2 Myo Interface PC connection Data acquisition is possible either by connecting the Myo Interface directly to a PC or through a PowerLab data acquisition and analysis system optional I Direct PC connection Connect the Myo Interface to one of the COM ports on the PC using the serial cable delivered with the Confocal wire myograph 120CW system 14 DMT Confocal wire myograph system Model 120CW Il PowerLab optional Connect the Myo Interface to the PowerLab unit using BNC cables Connect Rec 1 on the Myo Interface to Input 1 on the PowerLab Rec 2 to Input 2 and so forth Connect the PowerLab unit to one of the USB ports on the PC using the USB cable delivered with the PowerLab system 3 Oxygen supply Connect the small pi
51. the way until it passes the lower right peg and enough wire is through to reach the lower right screw The jaws are screwed closely together The top end of the wire with the 90 angle should point upwards between the jaws and the wire should run underneath the right side pegs To ease the coming steps which maybe the most critical ones for a viable prepara tion the myograph is turned 90 degrees so the vessel runs from right to left Y User manual 120CW Chapter 5 33 34 20 The top end of the second wire is bent to wards the top right screw the screws are still named as if the myograph was not turned 90 and fixed below the screw head Take care that the vessel is not stretched or moved in the longitudinal direction You might pre vent this by holding the other end of the wire with your second pair of forceps 21 The lower end of the wire now needs to be fixed to the lower right screw Take care that the second wire is mounted parallel to the first wire and of course on the outside of it Remember again that the wire should be straight and tight 22 By looking in the microscope and at the same time watching the force signal the height of the left wire is adjusted to allow it to move freely when the wires are moved together or away from each other The height of the wire is adjusted by adjusting the Allen keys on the micrometer
52. the following list to check that the system is complete 1 Myo Interface unit e Myograph connection cable with a temperature probe e Power cord The shape of the AC plug varies by country be sure that the plug has the right shape for your location e Automatic voltage selector 2 Confocal wire myograph unit e Calibration kit including bridge balance and 2 g weight e Chamber cover e Vacuum valve manual 3 Accessories e 1 roll of 40 um stainless steel wire e 1 tube of high vacuum grease e 1tube of grease for linear slides e 4spare screws for mounting of jaws e 10 extra myograph window glasses e 2 Allen keys e 1 small screwdriver e 40 mm funnel e Serial cable 4 Manuals e User manual for Confocal wire myograph system model 120CW e Procedures for investigation of small vessels using small vessel myograph by Professor M J Mulvany Department of Pharmacology Aarhus University Denmark User manual 120CW Unpacking the myograph system 11 12 Chapter 1 System overview cc 1 1 Confocal wire myograph unit eee e 25 pin serial cable for commu nication with Myo Interface Vertical micrometer screw e Horizontal micrometer ceccoocooococooocoocooooo Allen screws for fine alignment of the myograph jaws Myograph jaw connected e Myograph jaw connected to to force transducer e ER horizontal micrometer Force transducer pin rain SEE
53. time User manual 120CW Chapter 3 17 18 move the line to be programmed hour Minute or second to the top of the display Use the numeric keys to enter the time value and press Enter to confirm To control the timer function press the Info key and the display shows the F key options for the Timer menu Choose one of the F keys to proceed with the following options or press Info again to return to the Main menu F1 Proceed to the Setup menu F2 Start the countdown the time values are entered in the Timer menu F3 Stop or pause the countdown F4 Reset the entered time values or to turn off the alarm Setup menu Calibrate Displays the signal to be calibrated either force or pH Valve dly The time in seconds that the electronic valve optional to the system remains open when activated Contrast The actual contrast setting in the display Baudrate Data transmission rate from the Myo Interface to a PC via the RS 232 Option The option line allows access to submenus using specific access codes Use the numeric keys to change the valve delay or display contrast settings and press Enter to confirm Press the Info key and the display shows the F key options available for the Setup menu Press Info again to return to the Setup menu or one of the F keys to proceed with one of these options F1 Press F1 to proceed to the Main menu F4 Calibrate Force or pH Having chosen in the Setup menu which signal to calibrate press F4 to i
54. tion of how to adjust calibrate and maintain the Confocal wire myograph unit to ensure the equipment is always performing to its highest standard 4 1 Adjustment of supports A successful mounting of any kind of tubular tissue segment in the confocal wire myo graph is very dependent on perfectly matching supports The supports are matched prior to the shipment but daily use of the myograph system and greasing of the trans ducer pinhole will over time create a need for an adjustment of the supports Adjustment of the supports is performed using the following step by step procedure Please note that the amount of force on the screws should be very little to avoid breaking the threads The procedure is illustrated in fig 4 1 1 Carefully loosen screw A on the top of the support connected to the force trans ducer Align the horizontal support and carefully tighten the screw again 2 Loosen screw B on the top of the support connected to the linear slide Align the horizontal support matching the force transducer connected support as carefully as possible and gently tighten the screw again 3 Loosen screw C on the linear slide to roughly match the linear slide support to the force transducer support in the horizontal plane as illustrated in fig 4 1 Tighten the screw before proceeding with step 4 4 The plate on which the linear slide is mounted is balanced on top of a small stain less steel ball making it possible to finely adjust the
55. ures of the Confocal wire myograph system Chapter 4 contains procedures describing general as well as daily maintenance of the myograph unit e g adjustment of supports weight calibration of the force trans ducer and cleaning instructions Chapter 5 describes how to get started using the wire myograph system This in cludes a complete dissection and mounting procedure for small mesenteric arteries and a few basic pharmacological experiments Appendices contain additional information about normalization theory ocular calibra tion myograph service Shipping instructions system specifications equipment lists accessories and spare parts and fuse replacement DMI Confocal wire myograph system Model 120CW Unpacking the myograph system Please take a few minutes to carefully inspect your new Confocal Wire Myograph System for damage which may have occurred during handling and shipping If you suspect any kind of damage please contact us immediately and we will take care of the problems as soon as possible If the packing material appears damaged please retain it until a possible claim has been settled We recommend that you store the packing material for any possible future transport of the Confocal wire myograph system In case of transport and the original packing material is unavailable please contact DMT Sales Department for advice and packing Instructions After unpacking your new Confocal wire myograph system please use
56. wire myograph system Model 120CW 5 5 Endothelium function The reasons for checking endothelium function may include 1 To check whether the relaxing function of the endothelium is intact The procedure is performed to make sure that the endothelium is not damaged during the dissec tion or mounting procedure 2 If an experiment requires removal of the endothelium this procedure is useful to check whether the endothelial cells were successfully removed The procedure can be performed after the vessel segment has been heated equili brated and normalized Preferably the procedure should be done after performing a standard start to make sure that the vessel segment is viable The present procedure is for use with rat mesenteric arteries Another procedure may be needed for other animal species and tissue or vessel types Principles of checking endothelium function Stimulating a vessel segment with acetylcholine causes a release of nitric oxide NO also known as EDRF from the endothelium cells and subsequent relaxation of the vascular smooth muscle cells If the endothelium is undamaged by the dissection and mounting procedures then a substantial relaxation will occur With complete removal or damaged endothelium a partial relaxation or no relaxation to acetylcholine is observed It is important to note that the amount of NO or EDRF in a vessel is often dependent upon its size In certain vessels endothelium derived hyperpolarizing f
57. yograph chamber 12 Holding the vessel close to the proximal end with the forceps the vessel is mounted onto the wire 13 The vessel can now be cut off to give it the right length to fit the gap between the short est pegs 2 mm Add a little bit more the width of one peg Explanation follows in step 15 DMT Confocal wire myograph system Model 120CW 14 45 The jaws are now moved widely apart and the wire is released so the vessel can pass the pegs without touching them and thereby be damaged The vessel is pulled gently up the wire and the wire is positioned underneath the top left peg Note When the proximal end of the vessel is fixed between the wire and the peg this pre vents the vessel slipping down and becoming compressed when the second wire is inserted The vessel should not be fixed at the distal end A fixation at the distal end would hinder the vessel to move freely in the longitudinal direction 16 17 18 19 The wire is fixed with the lower left screw and at the same time is pulled to run tight from the screws along the head and un derneath the left pegs The vessel should run parallel to the front of the head A long wire 25 mm is bent one third from the top in a 90 angle This wire is guided into the vessel along the first wire from the top end of the vessel When the wire is through the whole length of the vessel it is cautiously pulled through the rest of
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