Model # HSSE-2/3 INSTRUCTION MANUAL

Transcription

1 Model # HSSE-2/3 INSTRUCTION MANUAL ALA Scientific Instruments Inc. 60 Marine street Farmingdale, NY Tel. # Fax: # support@alascience.com Table of Contents: Page# Page 1 of 28

2 INTRODUCTION... 4 HSSE COMPONENTS... 5 Reservoirs & bracket...5 Valve Box...5 HSSE front end (microbore Teflon tubing holder)...5 VC 3-4 Valve Controller...5 SETTING UP THE HSSE... 6 CLEANING AND MAINTENANCE... 8 VC 3 CONTROLLER... 9 Control Box Front Membrane Panel...9 Control Box Rear panel VC 3 4 CONTROLLER INSTRUCTIONS Power Buttons Valve Control Modes Manual ON/OFF Control: Manual MOMENTARY Control: TTL Mode Control: ANALOG Voltage Control: Latching Valves Control: Rear Panel Connections TTL SYNC OUT: ANALOG Voltage Control: ANALOG OUT: AUX OUT: USB: DIGITAL IN: DB-9M to BNC Breakout cable: TO VALVES: DC POWER: POWER ON/OFF: GND: FUSE: SPILL SENSOR INPUT: To reset the system after a spill RUNNING EXPERIMENTS Preliminary Considerations Setup Testing visually and with an open electrode Real experiments Theoretical and practical aspects of rapid solution exchange Estimating the speed of solution exchange Table 1. The most commonly used valve combinations DIAGRAM 1. - GENERAL SYSTEM DIAGRAM 2. - HSSE FRONT END IN PETRI DISH DIAGRAM 3. - EQUIVALENT CIRCUIT OF HSSE Page 2 of 28

3 DIAGRAM 4. - DETAIL OF FRONT END OF HSSE PHOTOS FIGURE 1 - SLOW PATCH / FAST PATCH REFERENCES LIMITED WARRANTY Page 3 of 28

4 Introduction The HSSE-2/3 is a fairly simple instrument designed to exchange solutions at the recording pipette tip in under 1msec. Data by Dilger et al, have shown this type of instrument to be able to exchange solutions in the µsec range. The basic principle behind the HSSE is that of hydraulics rather than mechanics. Other systems that can deliver this high performance are usually based on Piezo micropositioners that move a series, usually two, output tubes in a single dimensional plane. In those types of systems, tubing must be precisely aligned and continuous flow of both solutions into the cell bath (a source of contamination) is necessary. Furthermore, these systems require sophisticated theta tube applicators and complex software with D/A driving capability in order to steer the Piezoelectric device. The HSSE requires only one TTL line, and only one solution need flow into the cell bath at a time, making driving simple and reducing contamination in the cell bath. Even though the solenoid of the pinch valve that drives the HSSE is not as fast as a Piezoelectric device, hydraulic pressure and reliable synchronization allow this system to rival and even exceed the performance of Piezoelectric systems. The use of a four way pinch valve means that there are no internally wetted parts to be cleaned or flushed. Soiled tubes that cannot be flushed clean can be replaced. A single armature attached to the solenoid of the valve is responsible for solution switching. This ensures that the stopping of one solution and the starting of the other will occur in the most rapid possible time course. In addition, the HSSE includes two additional pinch valves to control the flow of solution to waste. Since the HSSE is gravity driven, the flow of solution out of the tip of the Teflon tubings can be increased significantly if the solution that is about to be released to the patch is already flowing. The secondary pinch valves allow such flow to be started moments before application to preserve valuable solutions. Page 4 of 28

5 HSSE Components Reservoirs & bracket Consisting of: 9 x 60ml reservoirs with luer stopcocks and tubing 1 bracket with 8 reservoir mounting clips and rod mount. 2 x 18 length aluminum rod magnetic base Valve Box Consisting of: Bracket with one NR 4-way pinch valve and three Sirai 3-way pinch valves Hamilton Valve Panel with two Hamilton rotational valves (4 position) Contains 9-pin connector to attach to valve controller Tubing assembly HSSE front end (microbore Teflon tubing holder) Small polycarbonate holder (This item has three orthogonal opposed channels for holding 300µm o.d. Teflon tubing). See photo #2. VC 3-4 Valve Controller Four channel valve control interface. Membrane panel control featuring manual on-off and momentary-on control for all valves, TTL input for data acquisition system command of valve function and analog voltage control of valves (optional cable required). A wall transformer power supply is included. Page 5 of 28

6 Setting up the HSSE When you receive the HSSE-2/3 it may be in several parts. There will be one magnetic base and two aluminum posts. Screw one of the posts to the magnetic base. Place the magnetic base on a steel table or plate to secure it from falling over. Mount the valve box to the post using the rod holder and securing screw located on the rear of the valve box. The second aluminum post should now be screwed to the first post. The eight plus one optional (3 rd solution flow) reservoirs (60ml syringes) should be placed in the eight clips on the reservoir bracket. At this stage, the plungers can be removed from the syringes, but they should be saved to be used for priming if necessary. Each reservoir should have a luer stopcock connected at its base, and then a short length of tubing with two male luer-lock fittings on the ends. This length of tubing is used to connect the reservoir with the Hamilton rotational valve. The reservoir bracket should be mounted on the top rod using the rod holder located on the bracket and secured with the thumb screw. Prior to mounting, make sure the magnetic base is secure to a steel table or plate for safety and security (THE MAGNETIC BASE SHOULD ALWAYS BE ON SO THAT THE STAND CANNOT FALL OVER). Each reservoir is connected to the manual (Hamilton) valve via the side ports on the valves. The order of connection is based on the labeling of the front panel of the valve panel. Looking at the back of the valve, it would be counterclockwise 1-4. The eight reservoirs should be thought of as two groups of four, the right four and the left four. This right and left designation will follow through to the front end which also has a right and left side tube. The single output of the valve is located on the rear. A tube leads from here to the 4-way NR pinch valve. The right hand manual (Hamilton) valve is connected to the right side of the pinch valve, and the left manual (Hamilton) valve to the left side. In alternative configurations you may wish to connect the output of one Hamilton valve to the #4 valve so that one side of valve #1 can be a permanent wash. PLEASE NOTE: Tubing lengths may be shortened as necessary just by cutting tubing and replacing fittings. Page 6 of 28

7 The valve box has four valves attached to it with a 9-pin connector to attach to the VC 3 controller. This assembly comes with tubing attached and connected. Should it come off, please follow the diagram at the back of this manual for re-connection. The valve bracket assembly is placed on the stand and adjusted so that the height of the valves is just a few centimeters above the microscope stage. The cable should be plugged into the VC 3 (See photo 1 for recommended positioning). The VC 3-4 controller can be placed outside the Faraday cage. The valves must be located right next to the microscope stage. The VC 3-4 should be controlled via TTL to valve one, and any other valve you wish to control via computer. A BNC cable is required for connection to a D/O from a data acquisition system. The VC 3-4 has a ground plug on the rear for safe grounding for electrical noise. It is advisable to make use of it. The VC 3-4 power supply can be plugged in to any mains supply. It is then connected to the VC 3-4 at the rear. The front end of the HSSE-2/3 is designed to hold the two pieces of Teflon tubing at right angles to each other in the same plane (Diagram 4). A third middle position is available for an additional stream associated with valve #4, or any other combination you wish to make. It must be on the microscope stage and the Teflon tubing can be cut to the shortest possible length for connection from the HSSE front end and the 4-way pinch valve. The front end is designed to mount on a small manipulator and reach into a small Petri dish. This design can be used to do rapid solution exchange on cells attached to the bottom of a dish, or even a slice. The preferred configuration is a cell attached to the tip of a patch pipette. In any event, the tubes must sit at least 2-3 mm below the surface of the liquid (Diagram 2). If you need to thread the tubing into the holder, cut the ends first with a razor blade at a precise right angle. Then thread through the holes and bring out to a point where the edges of each tube touch each other in the even right angle configuration. Remember that the pipette tip will have to be brought to this area, so treat it as valuable real estate and be sure it is in view in the microscope. It should be noted that special long working distance objectives may need to be used with this equipment. Page 7 of 28

8 Cleaning and Maintenance The HSSE-2/3 requires very little maintenance. No parts need to be oiled or tested regularly. Parts of the system can be wiped with a damp cloth. Never submerge electrical components. Valves are very well sealed so that if they get splashed they can be wiped off and returned to service immediately. The controller is splash proof and should be wiped off if it becomes wet. NEVER SUBMERGE THE CONTROLLER. Always unplug system before performing maintenance. It is a good idea to filter solutions, but not essential for HSSE-2/3 performance since the smallest tube is 300µm. ID. The HSSE-2/3 should be drained and flushed out after each day s usage. Distilled water is the best thing to use, but detergents and anything compatible with silicone can be used for cleaning. All tubing as well as reservoirs should be washed out. This includes the Teflon tubes at the front end. Simply add the cleaning fluid to all the reservoirs and open each valve to allow flow through every tube. This is important because fungus and bacteria can grow in any unclean tube. BE CAREFUL WHEN REMOVING OR REPLACING FULL RESERVOIRS IN THEIR CLIPS; SOLUTION CAN SPLASH OUT. KEEP RESERVOIRS COVERED WITH YOUR HAND OR WITH THE SYRINGE PLUNGER IF NECESSARY. Silicone tubes that are pinched by the valves will wear out after a time and require replacement. Typically, they lose their elasticity and no longer open properly when the valve opens. To prolong the life of a tube you can change the crush point on the tube by pulling it slightly to a new position. This may work several times before the tube has to be replaced. Please note that slightly different tubing can be used for the NR valve and the pinch valves, a slightly softer one for the pinch, but it is not absolutely necessary. The same tubing is supplied for both valves from the factory. Please order more from your representative if you should run out. Other silicone tubes that match the size and density of those provided can be used as well. Please check for functionality because the NR valve is dependent on the tubing having a certain amount of spring in order to function correctly. The tubing should be.8mm ID and 2.3mm OD of medium density Pt cured silicone or Silastic. Please note: The pinch valves may not function when silicone tubes are not present in the pincher. The valves may heat up after prolonged usage but this should not raise the temperature of the fluid. The controller operates the valve with the corresponding number. The three, 3-way valves can be opened by pushing in the button on their front. Page 8 of 28

9 VC 3 Controller Control Box Front Membrane Panel VC 3 4 Control Front Panel 1. Soft Power Button with LED - Green LED indicates when system is powered. It is also used to reset the spill sensor alarm. 2. Channel LED - Above each Valve button is a green LED that indicates when the power to that particular valve is ON. 3. Valve Switches - membrane switch (channels 1-8) for valve activation. Default valve switch setting is ON-OFF. 4. MOMENTARY - Sets all Valve switches to MOMENTARY ON mode. 5. SPILL DETECTED indicator- LED will light when a spill is detected by the spill sensor cable connected to the rear of the controller. (requires optional spill sensor) 6. LATCHING - Sets the controller to operate in Latching mode. When active the valve that is ON will be switched OFF when any other valve is switched ON. 7. TTL ON - Sets the controller to accept TTL pulses to control valves via the Digital I/O port on the controller rear panel. Page 9 of 28

10 Control Box Rear panel VC 3 Control Rear Panel AUX OUT 12V DC- Port for additional 12V dc valve connection. Positive center pin. 9. DC Power Input - 3.3A. 10. Power Switch - Main power switch to turns VC 3 controller ON/OFF. 11. FUSE - Controller fuse. 3.15A 250 V Fast Blow 5x20mm fuse. 12. SPILL SENSOR INPUT Connector for the spill sensor cable. (Cable is optional) 13. GND - Port (banana jack) to connect controller to ground. 14. TO VALVES - DB-9 FEMALE connector to connect controller to valve manifold. 15. DIGITAL INPUT - DB-9 MALE connector for TTL input control of valve channel 1-8 via DB-9 to BNC breakout cable. (cable is optional) 16. USB port to connect controller to PC via USB cable. 17. ANALOG IN - BNC connector to control valves via an analog voltage input in 0.5 V steps. 18. ANALOG OUT - BNC connector that outputs an analog voltage in 0.5 V/ valve steps with a +/-100mv threshold. 19. TTL SYNC OUT - BNC connector that outputs a TTL high signal whenever a valve is ON. Page 10 of 28

11 VC 3 4 Controller Instructions Power Buttons The VC 3 valve controller has two power switches. The main power is located on the rear of the unit. This rocker switch will turn input power ON or OFF to the system. The other power switch is a soft power button located on the membrane panel. This switch serves a dual purpose. First it is used to turn power ON or OFF to the membrane panel controls. Secondly it is used as a reset button for the spill sensor. When the spill alarm is activated, press the soft power button to reset it. Activating the soft power button will also calibrate the spill sensor to its new capacitive value. ( Sensor should be cleaned and dried before calibration) Valve Control Modes The VC 3 4 is a four channel 12V DC valve controller. The control box has a membrane panel with pushbutton switches to control the valves and other functions. The controller is designed to control in one of three ways, Manual switch, TTL input or Analog input. The controller can also be enabled to control latching valves. The VC 3 is designed with Low voltage circuitry that will automatically drop the valve voltage after opening the valve. This option is for researchers who are concerned about the transmission of heat to solutions from the warming of valves. Manual ON/OFF Control: Manually each valve is controlled by an individual membrane panel switch. Each switch is numbered, 1 through 4, corresponding to each valve. Press the membrane switch once to energize turn ON the corresponding valve. The valve will remain on until the membrane switch is pressed again. Press the membrane switch again to de-energize, turn OFF, the valve. A green LED will indicate when a switch is ON. The controller is able to activate all eight valves at the same time. This aids in flushing out the reservoirs during a cleaning procedure. It is not recommended to activate more than one valve at a time during an experiment since solution mixing cannot be measured accurately. Manual MOMENTARY Control: To enable the membrane switches to act as momentary switches press the MOMENTARY button on the controller. A green LED will indicate the momentary setting is enabled. In the momentary mode you must press and hold down the membrane switch to energize, turn ON, a valve. As long as you hold down the switch the valve remains ON. Release the switch and the valve turns OFF. Page 11 of 28

12 TTL Mode Control: The VC 3 4 controller has a digital I/O port to enable valve control via TTL signals. This port allows you to control each valve using a single TTL high signal. To enable this feature press the TTL ON button on the front panel. A green LED will indicate the TTL mode is enabled. The digital I/O port is a DB-9 Male connector on the rear of the controller. Using an optional DB-9M to BNC breakout cable allows the VC 3 to be controlled by most acquisitions systems digital outputs. Multiple valve control is possible by using this port. This is useful when priming the system reservoirs and when running a cleaning procedure. ANALOG Voltage Control: A BNC connector is used to input an analog voltage to control the valves. An analog voltage from 0.5V to 4.0 V dc in 0.5V steps will control valves 1-8, respectively. There is a +/-100mV threshold. To operate in this mode, the MOMENTARY mode switch must first be enabled. Connect the ANALOG IN port to your acquisition system analog out port via a BNC male cable to control all valves. Latching Valves Control: The VC 3 4 controller can be set to latching mode. By pressing the LATCHING button on the controller the green led will indicate that the controller is in latching valve mode. When active the valve that is switched ON will be switched OFF when any other valve is switched ON. Only one valve can be ON at a time in this mode. This feature allows for fast manual solution switching by eliminating the need to switch a valve OFF before switching another ON. Page 12 of 28

13 Rear Panel Connections TTL SYNC OUT: The sync out BNC port can be used as either a TTL marker or to sync (trigger) the controller to another device. A TTL high (+5V) signal is output whenever a valve is turned ON (energized). ANALOG Voltage Control: As stated above (Control Modes section), this feature allows for the control of valves via an analog input. A BNC connector is used to input an analog voltage to control the valves. An analog voltage from 0.5V to 2.0 V dc in 0.5V steps will control valves 1-4, respectively. There is a +/-100mV threshold. To operate in this mode, the MOMENTARY mode switch must first be enabled. Connect the ANALOG IN port to your acquisition system analog out port via a BNC male cable to control all valves. ANALOG OUT: BNC connector is used to output an analog voltage to representing a valve opening. An analog voltage from 0.5V to 2.0 V in 0.5V steps represents valves 1-4, respectively. AUX OUT: The AUX OUT port outputs 12V dc whenever any valve is turned ON. This port can be used to connect an additional 12V dc valve. Center pin on the connector is positive (+). USB: The USB connector is used to connect the VC 3 controller to a computer. This port is used for programming and maintenance at the factory only. Page 13 of 28

14 DIGITAL IN: The digital input is used to control the valves via a TTL signal. The DB-9 male connector pins 1-4 is used to input TTL signals to valves 1-4, respectively, with pin 9 being common ground. The DB-9F to 4BNC breakout cable is used to connect the VC 3 to an acquisition systems digital output. DB-9M to BNC Breakout cable: The TTL valve control feature uses a DB-9Female connector as its input. To facilitate connection to the digital outputs of an acquisition system the DB9BNC4 cable can be used. (Cable is optional) Cable is color coded according to above image. Red corresponds to valve1, green to valve 2 and so forth. Connect the DB-9Female connector to the rear of the VC 3 4 controller. Connect the BNC s to the digital outputs of your acquisition system. TO VALVES: This DB-9 Female port is used to connect the controller to the valve manifold (VM-4). A DB-9M/M cable is supplied with the VC 3 perfusion system. Pins 1-4 correspond to valves 1-4, respectively. Pin 9 is the common +12 V DC. DC POWER: The DC power jack is the main power input to connect the universal 15V DC power supply supplied with the VC 3 system. POWER ON/OFF: Power ON/OFF switch will turn the main power to the controller ON or OFF. Page 14 of 28

15 GND: The VC 3 controller can be connected to ground via a banana jack on the rear panel. FUSE: The VC 3 fuse holder is located on the rear panel and uses 5x20mm fuses. The rated value for the VC 3 controller is a 250V 3.15A fuse. Replace fuse by using a straight edge screw driver to twist off fuse holder cap. Use fuse of stated rated value only. SPILL SENSOR INPUT: The VC 3 has been designed with a built in spill sensor. This feature will allow for the protection against overflows from chamber or dishes onto expensive optics or electronics. To use this feature simply connect the spill sensor cable supplied with the VC 3 system to this port. Place the sensor wire around the area you wish to protect from liquid spills and turn ON the controller The VC 3 controller auto calibrates the spill sensor every time it is turned ON. Therefore, it is important that the spill sensor cable be connected before the controller is turned ON. If a spill occurs the sensor will detect it and two things will happen. 1) There will be a soft power shut down. This means power to the valves will be cut off. The main power will still be ON. 2) An audible and visual alarm will be activated. The audible alarm will be a chirping sound. The visual alarm is a red LED on the controller will blink repeatedly. To reset the system after a spill After a spill the alarm will sound and power will be cut to the valves. Follow the procedure below to reset the controller: 1) Press the Soft power switch on the controller panel. This will turn the alarm off. 2) Turn OFF main power on rear of controller. 3) Remove spill sensor cable from controller. 4) It is important to carefully clean and dry the sensor wire after a spill. Depending on what was spilled on the sensor, use distilled water to wash off any solution on the sensor. Carefully dry the sensor with an absorbent towel (paper towel). 5) Once dry replace spill sensor cable on the controller. Page 15 of 28

16 6) Turn main power ON from rear of controller. 7) Turn soft power switch ON from controller membrane power. 8) Controller will calibrate itself to the spill sensor cable. 9) System is now ready to be used again. It is sometimes necessary to turn soft power OFF and ON after following above procedure for the system to calibrate. Running Experiments Preliminary Considerations 1. Bath solution level. The rate of solution flow into the bath is ml/min (depending on the height of the reservoirs), so you will need suction or a pump to remove excess solution from the bath (We recommend our Levelock (ALA LL-2) bath level regulator). 2. It is a good idea to have a way of perfusing normal saline into the bath to dilute the bath concentration of agonists and drugs, either continuously or when necessary. This will add to the solution removal requirements. 3. It is helpful to attach drip chambers to the solution reservoirs. This makes it easier to determine that a solution is flowing and keeps the height of the solution and, therefore, the driving force, constant. 4. The valve controller can control up to 4 valves; three (2 Sirai + 1 NR pinch valves) are used in normal ( 2 solution flow) operation. The fourth one could be used for a valve that controls the flow of normal saline to the bath as described above (A.2) or as a third solution flow to the front end. The controller can be used manually with the membrane switches or by 5V TTL signals from an acquisition system. It is recommended to use the TTL feature of the VC 3 to control the valves. One TTL signal is necessary to perform timed-rapid solution exchanges. Separate triggering of valves 2 and 3 allows for faster solution exchanges (see note at end of section B) and can also be used to automate the flushing of tubing when new solutions are to be introduced. NOTE: The TTL feature cannot be used simultaneously with the manual feature. 5. As solutions are used, reservoirs can be moved up and down in their clips to even out solution flow due to gravity. Be sure you have access to reservoirs during the experiment. Page 16 of 28

17 Setup 1. Fill solution reservoirs. Reservoir selection valves should be set between positions so that no solution will flow. The 4 reservoirs on the left will usually contain agonist-free solutions (normal); the 4 reservoirs on the right will usually contain solution + agonist (test). Two examples are: Example 1 - Agonist concentration-response experiment Left Reservoir 1 Left Reservoirs 2-4 Right Reservoir 1 Right Reservoir 2 Right Reservoir 3 Right Reservoir 4 Example 2 - Drug concentration-response experiment. Left Reservoir 1 Left Reservoir 2 Left Reservoir 3 Left Reservoir 4 Right Reservoir 1 Right Reservoir 2 Right Reservoir 3 Right Reservoir 4 Saline (S) only Empty S + agonist concentration 1 (A1) S + A2 S + A3 Empty Saline (S) only S + drug concentration 1 (D1) S + D2 Open Empty + A S + A + D1 S + A + D2 Empty Leaving the #4 reservoirs empty allows the user to introduce air into the perfusion tubing to provide a physical and visual gap when changing from one solution to another. This can also be accomplished by including a 3-way stopcock valve after the reservoir. 2. Fill tubing with solution. Place Front End Tubing Holder into culture dish (or something else to catch solution that flows). Refer to Diagram 1 and 2. Valve 1-4-tube (NR) pinch valve - makes rapid change between normal & test solutions (to both the front end and a waste collector). Valve 2 - pinch valve - controls flow of NORMAL solution to waste. Valve 3 - pinch valve - controls flow of TEST solution to waste. Valve 4 pinch valve adds option to have a third solution flow to Cell, has Cell/waste option built in with y connection. Left reservoir 1. When the manual valve (Left Hamilton) is open and all of the pinch valves are OFF, solution will flow from the left reservoirs to the normal outflow tube ( A in Diagram 1). However, air in the tubing may slow or stop the flow. To speed up removal of air, turn ON valves 1 and 2 to direct solution to waste. Turn Valves 1 and 2 OFF after solution flows freely. Removal of air from narrow tubing may require some pressure. For example, temporarily attach syringe plunger to reservoir. Page 17 of 28

18 Right reservoir 1. When the manual valve is open and all of the pinch valves are OFF, solution will NOT flow from the right reservoirs to the front end (This may seem unclear on Diagram 1, but it is the case.). Turn ON valve 3 to direct test solution to waste. Shut valve 3 OFF when solution flows freely. Remove air from narrow tubing as above, but first turn ON valve 1 to direct test solution to front end. Optional Reservoir 1. Valve 4 (3-way Sirai pinch valve) controls flow from this reservoir to waste or to front end manifold. This gives the option of having control of three solutions to Cell. This can be your wash channel, or you can switch it with the right or left side of valve one so that the test solution is always switched with a wash. Valve 4 can flow to waste or to the cell on its own, and you can select which way on/off directs the fluid by switching the tubing in the tracks on the valve front. The system is now primed for the experiment. Note: the somewhat complicated plumbing diagram of Valve 1 in which the valve controls the flow of solution both to the front end and to the waste, allows the user to start the flow of TEST solution to the drain ms before turning Valve 1 on. Then, when Valve 1 is turned on, the flow of test solution to the drain is stopped and the flow of test solution to the front end is started simultaneously. The fact that the test solution has already been flowing through the wide tubing allows faster changes of solution. Testing visually and with an open electrode Before using the system to perfuse a patch, it is a good idea to practice by visually observing flows and by using an open patch electrode. Visual inspection of flows is most easily done by using solutions with different salt concentrations. You can see the solutions by their different refractive indices. Many combinations are possible. For example, fill a tissue culture dish with deionized water and put normal saline solutions into two of the reservoirs (one left and one right). Prime the system as described above. Focus on the side edges of the front end tubing with the microscope. Select the solutions with the manual valve. When left reservoir 1 is selected, you should see a narrow stream of solution flowing from one of the tubes (note - sometimes phase contrast can obscure this, try adjusting the polarizer). Activate valve 1. You should see the stream flowing from the other tube. Note where the two streams intersect. This is where the patch electrode should be placed. (Diagram 1,2, and 4). An open patch electrode can be used to identify optimal positions for the patch and to estimate the speed of solution exchange. The idea is to change from one salt concentration to another and measure the current that flows due to the change in junction potential. Here is one combination of solutions that works well. Fill the electrode with 150 mm KCl solution, fill the dish with 150 mm NaCl, fill left reservoir 1 with 150 mm NaCl and fill right reservoir 1 with 75 mm NaCl. Prime the system with these solutions. Setup patch clamp amplifier to measure currents of about 1 na. Set up the stimulator to provide a 5 Volt TTL, 200 ms pulse to input 1 of the valve controller and to trigger data acquisition (pipette potential should be zero). Focus on the side edges of the front end tubing. Place the open electrode at the position determined above and move the electrode up or down Page 18 of 28

19 so that the tip is in focus. Apply gentle pressure to the electrode to avoid diffusion of solution and/or debris into the tip of the electrode. Trigger the stimulator to give a single pulse. You should see a rectangular outward current when the 75 mm NaCl solution hits the patch. If there are fluctuations in the current response, make small adjustments in the position of the electrode. It should be easy to find the sweet area in which the response is rectangular. If you do not see any response, check that the solutions are flowing; you will be able to see the 75 mm NaCl solution because of the refractive index difference. If the solutions are flowing and there is still no response, turn on Valve 1 with the membrane switch and move the electrode until you see a change in current. At that point, the electrode should have a good approximate position. Return to previous step to fine tune the position. If the response has a slow onset and offset, you can try several things. Apply some more pressure to the pipette. Check for debris on the tip of the pipette. Check that there are no air bubbles in the front end tubing. Check that the reservoirs still have solution in them. Check that there are no obstructions or kinks in the tubing. If you have Shock Wave Problems they are created by the #1 pinch valve as it compresses the silicone tubing. To minimize the shock wave the HSSE-2/3 incorporates silicone tubing with minimal ID (inside diameter). The smaller the ID, the less solution there is in the part of the tubing that is pinched. By reducing this volume, the shock wave can be reduced. However, if the tubing ID becomes too small, flow is restricted. Therefore, there must be a compromise between flow and shock wave. Your HSSE comes with a few sizes of silicone tubing for replacement in valve #1 so that the optimum operation can be achieved. Real experiments 1. Start with solutions that should give a maximal response (e.g. saturating agonist concentrations). This will make it easier to locate the optimal position for the patch. The type of current response you get will depend on the kinetics of the current being studied. The onset time will depend on agonist binding and channel gating kinetics. 2. The peak response may be flat but it will show a decay if there is desensitization. The offset time will also depend on gating kinetics. 3. The optimal position will be the one in which the onset is fast and there are no sudden fluctuations in the current (assuming that there are many channels being activated - if single channels are seen, it may be necessary to calculate the ensemble average in order to identify irregularities). 4. After data are obtained for one experimental condition, the solution from another reservoir will have to be introduced into the tubing. This can be done with the patch in place provided care is taken to avoid air getting into the narrow tubing. The procedure is slightly different for the left and right reservoirs, just as it was for the initial filling. Page 19 of 28

20 5. Left reservoir: Turn on Valves 1 and 2. Open the manual valve to an empty reservoir to introduce some air into the wide tubing, then open the manual valve to reservoir #2. Follow the air bubble as it passes through the tubing to the drain. After the bubble is safely out of the way, turn off Valves 1 & 2. Left Solution 2 will now start on its way to the patch (Remember to use the syringe plunger if fluid flow from the reservoir is sluggish). 6. Right reservoir: Turn on Valve 3. Open the manual valve to an empty reservoir to introduce some air into the wide tubing then open the manual valve to reservoir #2. Follow the air bubble as it passes through the tubing to the drain. After the bubble is safely out of the way, turn off Valve 3. Turn on Valve 1. Right Solution 2 will start on its way to the patch. 7. The time needed for the new solutions to traverse the narrow tubing should be determined experimentally. It will depend on the differences between solutions 1 and 2. For example, if you are going from a low (drug or agonist concentration e.g. 0) to a high concentration (100 µm), waiting until the exchange is 99% complete will give a concentration of 99 µm instead of 100 µm. On the other hand, if you are going from 100 to 0, 99% exchange means you have 1 µm instead of 0. Even a 99.9% exchange to 0.1 µm might leave enough drug or agonist to be significantly different from 0. A guideline is to allow the solutions to flow for 30 seconds, make some test applications, flow for another 30 seconds and repeat the tests. Continue until the test applications produce stable results. 8. Experiments with drugs can be done in different modes to determine different aspects of the drug s action. The best first step is to include the drug in both the NORMAL and TEST solutions so that the experiments are done under equilibrium drug concentration conditions. This experiment may also reveal whether the drug acts on both closed and open states of the channel. The other experimental modes may provide useful information, but if the speed of solution exchange is not fast enough, these experiments are difficult to interpret. Including the drug in the TEST solution only, may reveal the speed with which the drug acts, but if solution exchange is slow, it may reveal only the time course of the solution exchange. Including the drug in the NORMAL solution only may reveal the speed with which the drug s action reverses, but if solution exchange is slow, it will produce a confusing story. Theoretical and practical aspects of rapid solution exchange 1. Three factors determine the speed of solution exchange: the amount of mixed solution (intermediate between NORMAL and TEST) produced by the system, the speed with which the solution flows (this determines the time needed for mixed solution to pass the patch), and the time it takes the TEST solution to diffuse to the channels in the patch. 2. The HSSE-2/3 has no internal volume in which solutions get mixed. However, bath solution will diffuse into the tip of the TEST solution tube when the TEST solution is not flowing. You may find that the first response after a long period of NORMAL solution flow is slower than subsequent ones separated by only a few seconds. You may choose to disregard the first response of an ensemble because of this. If long intervals are unavoidable (e.g. recovery from desensitization is slow), you can adjust Page 20 of 28

21 Valve 1 so that some TEST solution bleeds out of the tubing, thus preventing bath solution from entering the tip. There are 2 tapped holes for #4-40 screws on the top of Valve 1. Inset a screw into one of the holes and tighten until solution can be seen oozing out of the TEST tube (test this with a solution of different refractive index). If you tighten too much, the valve will lock in the ON position. Ideally, bleeding should be done transiently just before making the solution change. 3. Mixed solution can also arise from having both NORMAL and TEST solutions flowing simultaneously. With the HSSE-2/3, this is determined by two factors: the movement of the valve pincher and the relaxation of the pinched tubing. The valve can be made to move more quickly by using a larger voltage to the solenoid. However, DO NOT APPLY MORE THAN 12 VOLTS TO THE CONTROLLER as this will damage the electronics. A specially designed controller would be needed for this approach. The improvements in solution exchange time (~20 µs for an open electrode) are probably unnecessary for most experiments. (Please contact the manufacturer or your dealer for information on higher voltage controllers.) 4. Given that some amount of mixed solution is unavoidable, the idea is to move it past the patch as quickly as possible. Faster solution flows can be achieved by placing the reservoirs higher. The risk is that the patch may not tolerate the faster flow. 5. The thickness of the unstirred layer around a patch is not known. Given that solution exchange times of ~50 µs have been achieved for ACh applied to outside-out patches, we can use the Einstein diffusion relationship to calculate 0.2 µm as an upper limit for the diffusion distance (r 2 =2Dt= cm 2 /s s). Longer solution exchange times for inside-out patches can be expected. We have found that some outside-out patches show a considerably slower response than others under the same conditions. We hypothesize that not all patches have membranes resembling smooth hemispheres (grapes) but may be wrinkled like raisins. If channels are buried deep within a wrinkle, agonists and drugs must overcome a large diffusion barrier to reach them. We have found that slight positive pressure sometimes turns a slow patch into a fast patch (it is more likely that a broken patch will be the result). We now use an electrode solution with a higher osmolarity than the perfusion solution (by adding 10 mm glucose) and it appears that slow patches are found less frequently. 6. Debris attached to the patch or near the tip of the electrode will increase the solution exchange time. Debris may arise from inadequate filtering of perfusion solutions or from cells detaching from the culture dish. In either case, try to get rid of it! A thick coating of Sylgard near the tip of the pipette may also increase the solution exchange time; avoid this if possible. Page 21 of 28

22 Estimating the speed of solution exchange 1. Determining the time needed for a complete solution exchange is not trivial. The rise time of the response of an open electrode to a change in electrolyte concentration provides a lower limit on the solution exchange time. The unstirred layer around a patch will make the times longer. The onset time of a current response to rapid agonist application depends on many kinetic factors as well as the solution exchange time. If supersaturating concentrations of agonist are used to activate a channel with a rapid opening rate (e.g. the muscle ACh receptor), the onset time might be faster than the solution exchange time. Rapid channel block might also affect the onset time. 2. Very good estimates of solution exchange times can come from experiments in which the permeant ion concentration is changed rapidly. This assumes that the permeant ion concentration does not affect channel kinetics. For a ligand-gated channel, the following experiment can be used. 3. Left reservoir #1: S 4. Right reservoir #1: S + super-saturated (e.g. 20 EC 50 ) agonist concentration 5. Right reservoir #2: 50% diluted S + saturated agonist concentration (simultaneous change in saline concentration and agonist concentration) 6. Application of solution 1 (control) should produce a fast response (but note that the peak response to super-saturated agonist might be achieved before the solution exchange is complete). 7. If solution exchange is slow, application of solution 2 will produce a peak current close to control, followed by a decay to about 50% of control (Diagram 3, left). The decay represents the time course of the saline concentration at the channels (and the agonist concentration as well). A faster solution exchange will result in less of an overshoot decay. The best situation would be the absence of an overshoot (Diagram 3, right). Then, one can say that by the time the current has reached its peak, the solution exchange is complete. Table 1. The most commonly used valve combinations. Normal Solution to Patch Test Solution to Patch Normal Solution to Drain Test Solution to Drain Valve 1 Valve 2 Valve 3 Purpose 1 OFF OFF OFF Perfuse Patch w/normal 2 OFF ON ON Prepare for Rapid Exchange 3 ON ON ON Perfuse Patch w/test 4 ON ON OFF Flush Normal to Drain 5 OFF OFF ON Flush Test to Drain Page 22 of 28

23 Diagram 1. - General System Page 23 of 28

24 Diagram 2. - HSSE Front End in Petri Dish Diagram of HSSE front-end in Petri Dish Teflon tubes lead to corresponding right and left side of valve No. 1. Teflon tubes are inserted into silicone tubing of valve No. 1. Tubes are held in front-end piece by friction. Super glue can be used if necessary to hold tubes firmly. Mounting rod can be held in Position pipette tip with cell attached at apex of the three tubes. Tubes are held at 90 by the holder. The edges of the tubes can touch. For cells on bottom of dish, position output tubes as close as possible with target cell in between. Check angle to insure correct fluid flow path over cell. The HSSE-2/3 front-end is suspended from a micropositioner so that the front ends of the two Teflon HSSE front-end is designed to be used with a low wall petri dish or other chamber. Mounting rod has slight camber to help in positioning. Mounting rod can be bent to accommodate a particular situation,, material is 316 stainless steel. tubes can reach into a shallow petri dish. The ends of the tubes must be submerged and should be several mm below the surface. The system works with both inverted and up-right microscopes. The output tubes can either be pointed at a cell on the bottom of the dishr cell membrane attached to a pipette. Please note that whole-cell results are likely to have slightly larger time scale than cell attached or membrane patch recordings. Page 24 of 28

25 Diagram 3. - Equivalent circuit of HSSE-2 V2 to Drain V3 Test Normal V1 to Front End Diagram 4. - Detail of front end of HSSE-2 Place the tip of the patch electrode at the intersection between the two flows. Page 25 of 28

26 Photos Photo 1: Valve box with three 3-way Pinch valves, 4 way NR pinch valve and two 4-way manual valves. Photo 2: Close-up of HSSE front end on microscope stage. Page 26 of 28

27 Figure 1 - Slow Patch / Fast Patch 300 µm ACh 300 µm ACh 1/2 NaCl 1/2 NaCl Control τ =1ms Control 50 pa 10 ms Slow Patch 50 pa 10 ms Fast Patch FIGURE 1: Testing the speed of rapid perfusion using co-application of agonist and low permeant ion concentration (1/2 NaCl). A slow patch is characterized by an onset overshoot in the response. The fast patch has no onset overshoot. Both patches show an offset overshoot occurring at the removal of agonist. This arises because the NaCl returns to normal before the channels close. The insets show higher resolution views of the 1/2 NaCl responses (50 µs per point). For the fast patch, the solution exchange is complete well within 5 data points (<250 µs). References Brett RS, Dilger JP, Adams PR, Lancaster B (1986) A method for the rapid exchange of solutions bathing excised membrane patches. Biophys J 50: Dilger JP, Brett RS, (1990) Direct measurement of the concentration and time-dependent open probability of the nicotinic acetylcholine receptor channel. Biophys J 57: Dilger JP, Brett RS, Mody HI, (1993) The effects of isoflurane on acetylcholine receptor channels: 2 Currents elicited by rapid perfusion of acetylcholine. Molecular Pharmacology 44: Liu Y, Dilger JP (1991) Opening rate of acetylcholine receptor channels. Biophys J 60: Page 27 of 28

28 Limited Warranty ALA Scientific Instruments, Inc. agrees to warranty this product for a period of 1 year from the date of shipment. Repair or replacement, at our option, of parts and or materials shall be limited to those which prove to be defective under normal usage. This warranty is void if this product is used in a manner inconsistent with that set forth by this manual. User is responsible for return shipment to dealer or factory. Goods must be opened upon receipt or within three days thereof to claim shipping damage. Neither ALA Scientific nor our consultants or employees make any claim to accuracy of this product nor to that of the data obtained therewith. This product has been manufactured with the latest scientific information available to our knowledge; it is up to the user to verify results using proper scientific methods. This product has no clinical application; it is intended strictly for biological research. Usage on human subjects constitutes a violation of law. This instrument meets the safety and EMC requirements of the European Union. Your warranty rights may vary from state to state. Teflon is a trademark of the Dupont Co., Wilmington, DE Page 28 of 28