Radnoti Modular Wire Myograph System Assembly and Basic Principles

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Pierce Dickerson
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2 List of Components for the M1000 The Radnoti M1000 is the start of the Wire Myograph product line. The chamber is inclusive of Isometric force transducer and amplifier. The minimal recomended configuration would be one M1000 Wire Myograph Chamber, one RadTemp temperature controller and one M1901 Horizontal Calibration Adapter. The M1000 Wire Myograph chamber with Transducer and Amplifier includes the following: Description Part # Myograph Chamber w/ Transducer & Amp M1000 The M1000 includes the following: * Replacement Glass Tube Set M In-Flow glass tube M Out-Flow glass tube M Oxygenating glass tube M Silicone-Luer adapter M9254 +Micro vessell support bracket M9750 Tissue strip support M9700 *Thermister M9603 Bnc Cable *Lock down screws (includes spare set) M9601 *Lock down washers (includes spare set) M9602 M Field stimulation Electrode M9260 Hex tool M Micron wire (pack) M Micron wire (pack) M Micron wire (pack) M9125 * One set is installed in the chamber and the other set is a spare + Chamber is shipped with Jaws installed unless specified differently 6

support bracket (fixed) Micro vessel support bracket (adjustable) Thermocouple 5ml chamber

3 Myograph Chamber Heater Controller Connection Buffer inlet tube Buffer outlet tube Gas inlet tube (sintered glass tipped) Transducer Amplifier Connection Transducer housing Micro vessel support bracket (fixed) Micro vessel support bracket (adjustable) Thermocouple 5ml chamber volume X axis adjustment Tissue Strip Holder 7 Z axis adjustment (minor) Y axis adjustment micrometer

4 Myograph Chamber Overview The Radnoti Wire Myograph systems are designed for researchers performing contractile force studies on small ring samples ranging from mouse aortic rings and small intestinal ring samples down to micro-vessel preparation, such as mesenteric arteries. Precision Alignment Using precision fabricated micro vessel support brackets, the system accommodates lumen diameters down to 60μm and segment lengths up to 3mm. For samples with lumen diameters of 1mm or more, the standard Radnoti Tissue Organ Bath systems are recommended. To mount the vessel segment, two individual parallel wires are passed through the sample lumen and attached to the micro vessel support brackets. One bracket is connected to a precision 0 0.5gm capacitance type isometric force transducer (switch able to 0-5gm, and also available in 0-2gm/0-20gm configurations) that provides a true isometric measurement, i.e. less than 12.5μm deflection at 0.5gm force, which is critical when working with small vessel samples. The opposing support bracket is connected to a precision micrometer controlling the X axis movement, with Y and minor Z axis positioning controls conveniently positioned on the myochamber stage, allowing for precise alignment of the micro vessel support brackets. The wires are then anchored via a locking screw on each of the brackets and pre-load tension applied via adjustment of the X axis micrometer. An added feature is a safety lock for the ultra sensitive force transducer. The researcher is able to disable the movement of the micro vessel support bracket in a manner that protects the transducer. The researcher is able to focus on mounting the sample with less worry about damaging the mechanics of the system. 8

5 Options for the Myograph System Description Part # Myograph Calibration Weight Set (Recomended) M1904 Myograph Horizontal Calibration Adapter (Recomended) M1901 RADTEMP heater controller (Recomended) RADTEMP Setup Options Myograph Lighted Mounting Station M1950-MS Base for Single Myograph Chamber M1950-B1 Base for Four Myograph Chambers M1950-B4 Stainless Steel Support Rods 6 M1950-TR6 Stainless Steel Support Rods 4 M1950-TR4 Stainless Steel Support Rods 3 M1950-TR3 Stainless Steel Support Rods 2 M1950-TR2 Options for Buffer Delivery (Dry Block Heater) Description Part # Dry Block Temperature Control Unit MDBH Aluminum Insert with 4 wells MDBH-BL Reservoir for Dry Block Heater 120ml Oxygenating Bubbler for Tubing Kit for Dry Block / Single Chamber MDB-100 Automated Fluidic Control System recommended for use with Dry Block Temperature Control Unit and Reservoirs Description Part # Micro Fluidic Control Pump Module MFCPM USB Fluidic Pump Controller MFCPM-BT Micro Fluidic Control Software MFCPM-S 9

6 Options for systems using Water Jacketed Reservoirs Description Part # 2 Bar Lab Stand Base Only B2 Universal Stand Clamp Stainless Steel Rod Stainless Steel Rod Water Jacketed Reservoir 1 Liter Ring Clamp for 1 Liter Reservoir Oxygenating Bubbler for 1 Liter Reservoir Water Jacketed Reservoir.5 Liter Ring Clamp for.5 Liter Reservoir Oxygenating Bubbler for.5 Liter Reservoir Water Jacketed Reservoir.25 Liter Ring Clamp for.25 Liter Reservoir Oxygenating Bubbler for.25 Liter Reservoir In-Flow/Out-Flow Manifold for 2 Chambers In-Flow/Out-Flow Manifold for 4 Chambers Gas TNV Manifold for 2 Chambers Gas TNV Manifold for 4 Chambers Tubing Kit for Myograph Chamber/WJ M1900 Electronic Field Stimulation (EFS) Options RADSTIM Components: RADSTIM Module RADSTIM Power Supply RADSTIM ACS Junction Box (Includes ACS Software) RS1000 RS1000-PS RS1000-ACSJB *RADSTIM Power Supply able to power up to 4 RADSTIM modules at a time. *RADSTIM ACS Junction Box able to control up to 24 compatialbe devices. 10

7 Water Jacketed Reservoir Setup for Single Chamber (Option) Perfusate To Waste (Vacuum flask recommended) Gas 11

8 Water Jacketed Reservoir Setup for 2 Chambers (Option) Way Stop Cock 30 Perfusate 30 Gas In Way Stop Cock Gas 30 To Waste (Vacuum flask recommended) 12

9 4 Channel Myograph Setup (Option) G1 G2 G3 G4 G5 Gas G5 G4 G3 G2 G1 Flow Flow Flow Flow Flow Flow G Out-Flow V1 V2 V3 V4 Flow V4 V3 V2 V1 Flow Flow Flow Flow V VC VC VC VC 13

10 4 Channel Myograph Setup Cont. (Option) In-Flow P1 P2 P3 P4 P4 P3 P2 P1 Flow Flow Flow Flow P Flow PC PC PC PC 14

11 Dry Block Temperature Control (Option) Transducer Amplifier The Radnoti Dry Block Temperature Control is designed for up to 4 Radnoti ( ) 120ml non-water-jacketed Dry Block reservoirs. The reservoirs require oxygenating bubbler with fluid return port for non water jacketed reservoir. When used in combination with Radnoti Fluidic Control Pump Modules allow for buffer temperature control and automated buffer delivery. For a single sample chamber configuration users may populate with 1-4 reservoirs. For multiple reservoirs, up to four per dry block temperature controller. Pump #1 (In-Flow) Pump #2 (Out-Flow) Out-Flow Pump Return to reservoir or to waste via manifold 15

12 Placing Sample in Chmber The standard maximum sample opening on the brackets is 2.5mm (3mm and 3.5mm are also availalbe. Fixation of the wire The wire is attached to the bracket by use of the slotted head stainless steel screws with black washers. Loosen both screws then rap the wire clockwise around the screw between the washer and the bracket itself. Using the.006 wide slots on the front of the bracket place the wire in the slots then insert ring sample in bracket opening then attached to the second screw in the same way. Repeat on opposite bracket only insert wire through ring sample. Fixation of the wire Release of the transducer safety lock Once the vessel has been mounted, rotate clockwise to disengage. The transducer is now ready for use. Transducer lock Using the micrometer head located on the myograph unit, rotatate counterclockwise until the desired pre-load is achieved. Repeat as required during equilbration period. 16

Transducer Calibration This transducer operates on the principle of converting picofarad capacitance changes into an amplified DC output voltage by means of a patented circuit (U.S.

13 Transducer Calibration This transducer operates on the principle of converting picofarad capacitance changes into an amplified DC output voltage by means of a patented circuit (U.S. Patent Number ). The transducer consists of a stiff beam suspended between two capacitor plates. This forms a differential capacitor. Using this principal, the beam can be exceptionally stiff, approaching the ideal of measuring force without motion. As an example, for a force of 2 grams, the beam deflection is a maximum of only 5 microns in either the 0.2 or 2.0 gram range mode. The linearity is within +1% with a high DC voltage output and freedom from drift. GENERAL INFORMATION Amplifier / Power Supply a) The entire transducer is powered by a 12 volt AC wall transformer (see Fig. 2). b) There is no ON/OFF switch. The unit can remain powered, as shown on the digital meter, for the entire experiment. Front Panel Controls Range Selector The range selector switch has two positions: 0.5 gram and 5 gram 0.5 gram position: a 0.5 gram force produces a 5 VDC output. 5 gram position: a 5 gram force produces a 5 VDC output. 17

14 Transducer Calibration Cont. OFFSET The OFFSET control enables the user to set a zero output voltage for any pre-load value. It is essentially a Tare Control device since it balances out static loads such as the weight of the muscle preparation prior to contraction and the support wire or string. Internal Controls and Adjustments On the rear panel of the power supply are three small access holes to the trim pots (see Fig.3). The high and low gain trim pots are used to adjust the range. The right-hand trim pot is factory set to deliver 5.0 VDC for 0.5 gram. The center trim pot is factory set to deliver 5.0 VDC for 5.0 gram. Either can be adjusted to deliver more or less output as conditions dictate. The OFFSET adjustment trim pot is located on the extreme left and has been factory adjusted to determine the offset control. It also con be adjusted in situations where the front panel offset control does not have sufficient range. It can be used to correct for static loads equivalent to signals between +0.6v and -1.2v. Electrical Output A BNC jack on the rear of the amplifier is provided for output connections Output voltage is 0 to 5.0 VDC depending upon the position of the range selector switch. *Output impedance is 2 kilo-ohms for direct Connection to all recorders The transducer is linear to 100% overload (4 or 40 grams, depending on setting). An internal frequency response jumper is factory set to 1000Hz The jumper is located inside the chassis next to the trim pots. After unplugging the unit, the jumper can be moved to a second position forward towards the front of the amplifier. This will reduce the frequency response to 10 Hz for situations where signal dampening Maintenance and Storage The transducer heads are very fragile. Care should be taken to store the Individual Myography chambers in clean dry a safe place during non use. 18

housing for the z-axis adjustment. Slide the calibration block over the z-axis adjustment cover as shown. Once in place slide the adapter back to the screw stop in the horizontal calibration adapter.

15 Verifying Transducer Calibration Radnoti has developed a methodology for calibrating the myograph chamber in the horizontal position. This method requires the use of the provided calibration pulley block, 4-0 silk and calibration weights The horizontal calibration adapter mounts directly atop the Myograph chamber, sliding over the housing for the z-axis adjustment. Slide the calibration block over the z-axis adjustment cover as shown. Once in place slide the adapter back to the screw stop in the horizontal calibration adapter. This will insure repetitive placement of the horizontal calibration adapter from chamber to chamber and from use to use. You will then need to run the 4-0 silk (hook end first) from the upper corner of the Myograph mounting bracket support, under the lower pulley wheel, over the upper pulley wheel and leaving the ring end hanging down. Then calibrate first in the 5gm force range of the transducer. Position the range selection switch to 5gm. Briefly remove the 4-0 suture hook from the Myograph wire support arm to obtain a zero reading. 19

16 Verifying Transducer Calibration Cont. If the display does not read zero, adjust the zero offset on the transducer amplifier to read zero. Once zero has been established, replace the suture hook on the Myograph bracket support arm. Hang the provided 5gm weight on the ring side of the 4-0 suture that is hanging down off the upper pulley. Once the weight is in place, be sure that each of the pulley wheels is in the center of the bracket. Gently, using the provided forceps, locate the 4-0 suture onto each of the pulleys as close to center as possible. 20

Verifying Transducer Calibration Cont. Double check that the 4-0 suture line remains at the center of the pulleys and that the 4-0 suture line hook remains at the apex of the Myograph bracket support.

17 Verifying Transducer Calibration Cont. Double check that the 4-0 suture line remains at the center of the pulleys and that the 4-0 suture line hook remains at the apex of the Myograph bracket support. Notice that the reading may not be at 5v at this time. This is do to resistance or stickage between the pulleys and the suture line. To obtain an accurate reading, gently lift and reset the weight using the forceps. This is best done by clasping the suture line approximately 5mm bellow the upper pulley between the weight and the pulley. Lift 1 2mm and then gently bring back down. Do this several times until the reading is accurate at 5v. 21

Verifying Transducer Calibration Cont. To verify linearity you will want to hang a mid range weight (2gm weight provided). Follow the same steps as given for the first 5gm weight.

18 Verifying Transducer Calibration Cont. To verify linearity you will want to hang a mid range weight (2gm weight provided). Follow the same steps as given for the first 5gm weight. Once the weight is in place and the resistance or stickage has been removed by repeated the method described, the reading should now be 2v. For use in the 0.5gm range Position the range selection switch to 0.5gm. Briefly remove the 4-0 suture hook from the Myograph wire support arm to obtain a zero reading. If the display does not read zero, adjust the zero offset on the transducer amplifier to read zero Once zero has been established replace the suture hook on the Myograph bracket support arm. 22

19 Verify Transducer Calibration Cont. Hang the provided 500mg weight on the ring side of the 4-0 suture that is hanging down off the upper pulley. Once the weight is in place, be sure that each of the pulley wheels is in the center of the bracket. Double check that the 4-0 suture line remains at the center of the pulleys and that the 4-0 suture line hook remains at the apex of the Myograph bracket support. Notice that the reading may not be at 5v at this time. This is do to resistance or stickage between the pulleys and the suture line. 23

20 Verify Transducer Calibration Cont. To obtain an accurate reading, gently lift and reset the weight using the forceps. This is best done by clasping the suture line approximately 5mm bellow the upper pulley between the weight and the pulley. Lift 1 2mm and then gently bring back down. Do this several times until the reading is accurate at 5v. To verify linearity you will want to hang a mid range weight (200mg weight provided). Follow the same steps as given for the first 500mg weight. Once the weight is in place and the resistance or stickage has been removed by repeating the method described, the reading should now be 2v. The chamber is now calibrated and ready for use. 24

RADTEMP Heating Controller RADTEMP Set Point Adjustment Pushing the Set point button briefly will show the current setting. Pushing it again returns to the temperature display.

Quickly release the button when the desired setting is displayed. Now depress the set point button again for greater than 3 seconds to save the setting.

21 RADTEMP Heating Controller RADTEMP Set Point Adjustment Pushing the Set point button briefly will show the current setting. Pushing it again returns to the temperature display. Pushing the Set point button once briefly and then holding the button again for more than 3 seconds starts the display incriminating in 0.1C increments. The Set point has a range of 33-38C. Quickly release the button when the desired setting is displayed. Now depress the set point button again for greater than 3 seconds to save the setting. The display then returns to displaying the temperature. If a connection to the temperature sensor is open the the display will show OPE. If the temperature sensor is below 24C then the display will show LLL. The temperature is 0.1C accurate only in a range from 33 to 38C. Maximum temperature should not exceed 50C. 25

Radnoti Isometric Force Transducer A

Radnoti 2006 Radnoti Isometric Force Transducer 159901A Use of Radnoti 159901A Isometric Force Transducer This transducer operates on the principle of converting picofarad capacitance changes into an amplified