PSMI Patch Slice Micro-Incubator User`s Manual
Contents
1. Yol 7 Yoleasa Es Yn 100 Figure 2 Top View of PSMI 6 1 0 Cable Chamber Optical Window 7 Ch 1 Perfusion Lines Salt Bridge Well 8 Ch 2 Perfusion Lines Gas Inlet 9 Plate Ground Socket green Surface to Hold Magnets 10 Salt Bridge Ground Socket white 11 Thermal Fuse Cover Peltier Ring Seal Upper Plate 4 Cover Slip Dish Lower Plate Figure 3 Top Heat Transfer Plate of PSMI Expanded View HHHH Figure 4 Top Heat Transfer Plate of PSMI Assembled View Publication 5403 009 REV A 4 Harvard Apparatus PSMI Patch Slice Microincubator Description temperature to as close as 0 2 C These Peltier devices are sandwiched between the annular assemblies through which a central hole see Figure 3 on page 5 has been milled to provide support for that chamber and an optical window for the microscope This configuration maintains a controlled temperature at the level of the chamber by reducing heat loss through the base and maximizing heat transfer to the chamber The primary heat transfer to the controlled medium during perfusion occurs through the plastic tubes coiled inside a circling slot groove machined in the top heat exchange plate The extracellular medium first passes through these tubes and is warmed or cooled to the set temperature before reaching the chamber There are also two seconda2. b Blocked heat exchange tubing First flush tubing with 7096 alcohol and then perfuse distilled water for one hour If it is still a problem then change plastic tubing Very hot fins plate too hot to touch feed forward failure TURN OFF THE POWER IMMEDIATELY In this condition too much heat is generated at the Peltier junction nominally at ambient temperature Attempts to change the control temperature by chang ing the current have no effect Resume operation after the device has cooled off preferably after taking actions suggested under one of the following a You have not placed the device in contact with an adequate metallic ther mal mass or are trying to cool to too low a temperature Check that micro scope stage is thermally conducting Solutions Apply heat sink compound or silicone oil to the supporting metallic surface of the microscope and or install water perfusion APPENDIX 2 b Attempted operation outside the recommended range 10 50 C without water perfusion SEE APPENDIX 2 Controller is not supplying current See the manual for the controller in gen eral Problems specific to the PSMI are listed below If feed forward failure see 2 occurs with the TC 202A it automatically stops delivering current until the Peltier plate temperature cools off Use one of the solutions given in 2 to avoid repeated lapses in current Excessive 60 Hz pickup or baseline drift if electrically recording a Check
3. connector Thermistor AWG 26 Green Thermistor AWG 26 White Peltier 3 Peltier Plate Thermistor 4 Peltier 6 Peltier Plate Thermistor Ground Shield Publication 5403 009 REV A
4. PSMI Connecting this to a local ground can sometimes reduce 50 60 Hz 3 Chambers The Patch Slice Cover Slip Dish must be assembled before placing it in the central opening See Figure 1 on page 4 NOTE 1 22 mm glass cover slips are used for the bottom of the slice com partment of the Cover Slip Dish To prevent leakage of media yet allow easy replacement of the top one use stopcock or vacuum grease sample supplied both are composed of non toxic silicone oil wax Apply the grease to the inner ledge of the dish on the side where this ledge is closer to the external rim A glass cover slip is also needed for the other side see Figure 1 on page 4 to prevent condensation that would impair illumination from the con denser This one can be attached more permanently with a glue Sylgard used to coat patch pipettes would be a conveniently available choice NOTE 2 The temperature difference between the plate thermistor and the chamber as well as across the chamber itself can be reduced by coating the inside wall on the top surface of the chamber support ring of the PSMI with a good heat conductor heat compound messy silicone oil stopcock grease If using perfusion make sure the plastic tubes are inside the dish 4 Suggestions A superfusion micro incubator system consisting of a PSMI a matching con troller chamber and fluid control system has three modes of operation see Description section pages 4 6 each with its own routin
5. need for water cooling Ince C et al A teflon culture dish for high magnification microscopy and measurements in single cells Pflugers Arch 403 240 244 1985 Earthing and Interference pages 55 65 in Microelectrode Methods for Intracellular Recording and Iontophoresis Academic Press London ed R D Purves 1981 DeHaan R L et al J Gen Physiol 65 207 1975 Ince C et al Micro CO2 Incubator for use on a micro scope J Immuno Meth 60 269 275 1983 This paper describes the use of gas flow to reduce vertical and horizontal temperature gradients and to control pH in an opti cally accessible chamber Forsythe I An environmental chamber regulating temperature and super fusion of tissue cultured neurons during electrophysiological or optical stud ies Electrophysiology and MicroInjection 301 320 volume 4 of Methods in Neuro science Academic Press NY Ed Conn PM 1991 This article provides a complete description of the final version of the PDMI 2 and its operation Publication 5403 009 REV A 2 Harvard Apparatus PSMI Patch Slice Microincubator Appendices APPENDIX 1 INCLUDED ACCESSORIES l Aspirator PS ASD see Section 4 Perfusion page 11 figure 4 2 PS CSD Patch Slice Cover Slip Dish Includes Teflon frame sample 22 mm diameter glass cover slips and sample of vacuum grease to hold slice chamber cover slip 3 Teflon perfusant tubing Teflon may be more appropriate f
6. A matching temperature controller which in summary will 1 Allow the PSMI to either cool or heat the bath preparation as well as control temperatures near ambient equally well 2 Automatically switch the current direction when the sensed temperature is higher or lower than the set point temperature Allow fast changes in temperature Allow 0 2 C regulation Allow low noise electrical recording OM INE MO Automatically power shut off when excessive temperatures are reached so that possible system damage during feed forward failure is avoided See Troublesbooting on page 12 Publication 5403 009 REV A s Harvard Apparatus PSMI Patch Slice Microincubator Setup amp Operation Orientation Any references to right and lefi assume the unit is viewed from above with the electrical cable to the lefi CAUTION THIS UNIT MUST ONLY BE OPERATED WHILE IN GOOD CON TACT WITH A LARGE THERMAL MASS such as the metal stage of a microscope or by perfusion of water additionally through the lower plate For temperatures much more than 5 C below ambient the stage of a microscope may be insufficient by itself without such additional water perfusion See APPENDIX 2 1 Mounting on the Microscope The PSMI bottom plate has a flat surface for unrestrained mounting on various inverted microscope stages Accessories are available to lock the PSMI to the stages of the microscopes from several manufacturers The locking devic
7. PSMI Patch Slice Micro Incubator User s Manual PSMI Patch Slice Micro Incubator MA1 65 0044 MRE HARVARD APPARATUS Publication 5403 009 REV A WEEE RoHS Compliance Statement EU Directives WEEE and RoHS To Our Valued Customers We are committed to being a good corporate citizen As part of that commitment we strive to maintain an environmentally conscious manufacturing operation The European Union EU has enacted two Directives the first on product recycling Waste Electrical and Electronic Equipment WEEE and the second limiting the use of certain substances Restriction on the use of Hazardous Substances RoHS Over time these Directives will be implemented in the national laws of each EU Member State Once the final national regulations have been put into place recycling will be offered for our products which are within the scope of the WEEE Directive Products falling under the scope of the WEEE Directive available for sale after August 13 2005 will be identified with a wheelie bin symbol Two Categories of products covered by the WEEE Directive are currently exempt from the RoHS Directive Category 8 medical devices with the exception of implanted or infected products and Category 9 monitoring and control instruments Most of our products fall into either Category 8 or 9 and are currently exempt from the RoHS Directive We will continue to monitor the application of the RoHS Directive to its products an
8. atus warranties this instrument for a period of one year from date of purchase At its option Harvard Apparatus will repair or replace the unit if it is found to be defective as to workmanship or material This warranty does not extend to damage resulting from misuse neglect or abuse normal wear and tear or accident This warranty extends only to the original customer purchaser IN NO EVENT SHALL HARVARD APPARATUS BE LIABLE FOR INCI DENTAL OR CONSEQUENTIAL DAMAGES Some states do not allow exclu sion or limitation of incidental or consequential damages so the above limitation or exclusion may not apply to you THERE ARE NO IMPLIED WARRANTIES OF MERCHANTABILITY OR FITNESS FOR A PARTICULAR USE OR OF ANY OTHER NATURE Some states do not allow this limitation on an implied warranty so the above limitation may not apply to you If a defect arises within the one year warranty period promptly contact Harvard Apparatus Inc 84 October Hill Road Building 7 Holliston Massachusetts 01746 1388 using our toll free number 1 800 272 2775 Goods will not be accepted for return unless an RMA returned materials authorization number has been issued by our customer service department The customer is responsible for shipping charges Please allow a reasonable period of time for completion of repairs replacement and return If the unit is replaced the replacement unit is covered only for the remainder of the original warranty period dating from the pu
9. d will comply with any changes as they apply Do Not Dispose Product with Municipal Waste Special Collection Disposal Required E Harvard Apparatus PSMI Patch Slice Microincubator Table of Contents SUBJECT PAGE NO General Information sseeeeeeeseeceeeeeeeeeeeeeeeeeees 2 Introduction tee adichatesntendeeccdeastentspstaussaeabnccdse 3 D tTeg i D 4 6 Controling the Micro Incubator 7 8 Set Up and Operation 9 10 POrfuSl OM e 11 12 Maintenance e 13 Trouble Shooting ceeeeeee 14 FOOMOLES epe t E TUNE 15 Appendices Appendix 1 Included Accessories 16 Appendix 2 Useful Accessories 16 Appendix 3 Theory of Device Operation 16 Appendix 4 Specifications 17 Appendix 5 Thermistor Calibration Table 17 Appendix 6 PSMI Wiring Codes 18 Publication 5403 009 REV A Harvard Apparatus PSMI Patch Slice Microincubator General Information Serial Numbers All inquires concerning our product should refer to the serial number of the unit Serial numbers are located on the bottom plate Calibrations All electrical apparatus is calibrated at rated voltage and frequency While the flow will stay calibrated the peak will vary Warranty Harvard Appar
10. during flow 5 Small chamber 22 mm x 5 5 mm and upstream volume allow rapid media change 6 Electrical isolation of heat exchange plates from chamber electrical ground This allows reduction of electrical noise by using a single external connection between signal ground and power ground see Figure 2 9 on page 5 Publication 5403 009 REV A Harvard Apparatus PSMI Patch Slice Microincubator Description FIGURE 1 PSMI ASSEMBLY The Open Perfusion Micro Incubator PSMI is an annular shaped assembly sur rounding the central chamber the Patch Slice Cover Slip Dish PS CSD The PSMI contains two metal annual plate assemblies to effect the heat transfer to the chosen chamber The lower plate assembly black anodized aluminum plate bas a flat bottom sur face to which the appropriate securing ring is attached to form a mating surface with the microscope This lower assembly serves also as a radiator of peltier waste heat via radial cooling fins see Figure 1 above on its outside diameter The top plate see Figure 1 above is also made from black anodized aluminum It forms one of the heat exchanges and also serves to support the inserted chamber Heat transfer to the chosen chamber is driven by two Peltier thermoelectric devices which when powered by a suitable feedback controller can regulate the chamber Publication 5403 009 REV A Harvard Apparatus PSMI Patch Slice Microincubator Description
11. e Gradients Gas Perfusion This is intended primarily to reduce the heat loss to or gain from the local atmos phere thereby reducing temperature gradients across the dish It is possible to con trol the gas mixture above the perfusion chamber The gas passes over the same heat exchange as the fluids Any desirable gas mixture may be introduced into the micro incubator e g 5 CO air mixture can be used with a bicarbonate buffer medi um An inlet for CO has been incorporated into the PSMI s Publication 5403 009 REV A E Harvard Apparatus PSMI Patch Slice Microincubator Maintenance The PSMI requires only a minimum of maintenance Periodically replace the tub ing to avoid clogging by dust particles or growth of micro organisms with daily use every two months The period between changes can be increased by flushing with distilled water after using followed perhaps by modest heating to dry out the tubing An occasional perfusion of 70 alcohol helps Disassembly for replacement of tubing Remove the screws outside rim of the plastic lid Lift off lid observing carefully how the perfusion tubes are fed through and their entry and exit points from the outer circular bobbin of the exposed heat exchange plate Remove the tubing If better heat transfer is needed use heat sink compound white paste zinc oxide contact an electronics repair shop to obtain some or silicone oil in the bobbin s groove Wind new
12. e procedures and specialized tricks of the trade This manual supplies a starting point for your specialized application allow time to perfect your own procedures See Perfusion section pages 11 12 for perfusion suggestions The articles pub lished by the developer 1 2 7 and others 3 4 5 6 for the closely related Open Perfusion Micro Incubator may also be useful Foot notes on page 15 Publication 5403 009 REV A Harvard Apparatus PSMI Patch Slice Microincubator Perfusion 7 Figure 4 LU ASP Aspirator Outlet from the chamber is by direct suction The included aspirator LU ASP was originally developed by Dr Can Ince University of Leiden for patch clamp applica tions The design of this aspirator cleverly avoids fluid level variations a source of elec trical noise in electrical recording experiments The aspirator has a magnetic base to grip a matching magnetic surface see 5 Figure 2 page 5 on the top of the top plas tic plate of the PSMI Connect up a source of suction with a liquid trap to this aspira tor The level of fluid in the chamber is determined by the height of this aspirator and can be changed with its thumbscrew The oscillations inherent in any peristaltic perfu sion are easily damped with bubble traps Such traps also allow the independent cham ber grounding required for low noise electrical recording Fluid Perfusion For reliability at low flow rates a peristaltic pump is preferable to gra
13. es Ranges between 4 mm between 15 30 C and 20 microns between 30 40 C One pair of Silastic one pair of Teflon tubing See Figure 1 on page 4 APPENDIX 5 THERMISTOR CALIBRATION TABLE RESISTANCE DATA PSMI INTERNAL THERMISTOR Temperature Resistance Temperature Resistance o C 271 6 Kilohms 250 C 100 0 Kilohms 258 3 26 95 3 6 245 7 27 91 3 7 233 8 28 87 4 8 222 5 29 83 6 9 211 9 30 80 0 10 201 7 31 76 6 11 192 2 32 73 3 12 183 1 33 70 2 13 174 5 34 67 3 14 166 3 35 64 4 15 158 6 36 61 75 16 151 3 37 59 2 17 144 3 38 56 75 18 137 7 39 54 5 19 131 4 40 52 2 20 125 5 41 50 1 21 119 8 42 48 0 22 114 5 43 46 1 23 109 4 44 44 3 24 104 5 45 42 5 Publication 5403 009 REV A E Harvard Apparatus PSMI Patch Slice Microincubator Appendices From the first release to the market of the PSMI Micro Incubators to the current design there have been various design changes A wiring color code is provided below to aid the wiring of the PSMI cable to the chosen temperature controller s power con trol output The PSMI is provided with a connector which matches the TC 202A out put connector For other options the table below may be used In any case we suggest that you contact Harvard Apparatus Inc for instructions or more details APPENDIX 6 PSMI Wiring Codes Wire Size Final Peltier AWG 20 Red Peltier AWG 20 Black Signal Ground salt bridge AWG 26 1 mm White connector AC Ground 60 Hz noise AWG 26 1 mm Green
14. es come in two forms a As alignment rings that assemble to the bottom of the PSMI and in turn fit on opening in the microscope stage b As fixing platforms that fit a locking mechanism of Zeis or Leica attachable mechanical stages Using a stage attachment accessory has the advantage of improved mechanical stabil ity and gives the user the ability to use the microscope stage built in X Y manipula tors to position the chamber in the field of view The following microscope stage locking accessories are available a PDMI ARN Alignment Ring for Nikon Diaphot old or 300 200 TMD stage 107 75 mm x 1 78 mm H 4 242 x 0 071 H b PDMI ARZ Alignment Ring for Zeiss Axiovert gliding or rotary stages 102 75 mm x 1 78 mm H 4 046 x 0 071 H c PDMIARO Alignment Ring for Olympus IX50 70 or IMT2 fixed stage 109 73 mm x 1 78 mm H 4 320 x 0 071 H d PDMI FPZ Fixing Platform for Zeiss Axiovert with attachable mechanical stage e PDMI FPL Fixing Platform for Leica DAS Microscope DMIL and DMIRB E with attachable mechanical stage f PDMI ARL Alignment Ring for Leica Microscope 88 mm x 1 8 mm H 3 52 x 0 072 H These rings or platforms are easily attached to the bottom plate with 3 small screws supplied with each ring or platform 2 Electrical Connections The PSMI main cable has a multi pin connector at its end This connector matches the TC 202A front panel I O jack This cable provides the electrical connections f
15. for ground path between the chamber ground connection and the metal plates Keep the top plastic surface of the PSMI clean especially of dried saline salt solution Silicone oil may be helpful b Drift in baseline when electrically recording is most likely due to one of the Ag AgCl junctions in the user s complete recording circuit c Ground loops causing noise are avoided by using only one grounding point preferably near the chamber Connect the patch clamp ground the bath ground and most likely the heat exchange plate ground green pin here The source of ground loops can be subtle If problems remain see Appendix 4 Static discharge during perfusion a Check earthing system and fluid level in bubble traps The simplest way to solve this problem is to use stainless steel needles in the bubble traps and earth them Publication 5403 009 REV A E Harvard Apparatus PSMI Patch Slice Microincubator Footnotes Forsythe I D et al A chamber for electrophysiological recording from cul tured neurons allowing perfusion and temperature control J Neurosci Meth 25 19 27 1988 This article describes a prototype of the Open Perfusion Micro Incubator PDMI 2 without provision for gas flow Forsythe I D et al An open perfusion micro incubator for electrophysio logical recording in vitro J Physiol Lond 410 5P 1989 This article describes the addition of gas flow to the PDMI 2 and the elimination of the
16. gs This problem is acute with patch slice recording in which a water immersion microscope objective is used The recording and cleaning pipettes need access at very shallow angles with respect to the horizontal axis and continuous perfusion is required to maintain oxygenation within the slice The PSMI permits control of temperature of both a perfused liquid and overlying gaseous medium for the submerged tissue slice Application of drugs or a change in the ionic composition is achieved without disturbing the set temperature or any electrodes The tissue chamber for the PSMI is the PS CSD with two replaceable 22 mm diameter cover slips one for the floor of the slice chamber and one below it to prevent condensation that would otherwise block distort condenser illumina tion from below The PSMI uses Peltier devices to drive the heat exchange APPENDIX 3 describes their operation and identifies their advantages in this application The design of the PSMI avoids the normal need for Peltier cooling water except at the lowest control temperatures Design Considerations 1 Mechanical access for water immersion objective for its superior optics 2 Mechanical access for patch pipette amp cleaning pipette with access angles as small as 15 20 with respect to the horizontal 3 Allows temperature regulation both above and below room temperature with perfusant media supplied at room temperature 4 Maintenance of constant low fluid level
17. he Peltier leads of about 4 volts Two methods of control are possible manual or feedback A manual system uses a DC power supply the direction and magnitude of the current through the micro incubator is adjusted by hand For better performance and more stable temperature control the feedback method is the best choice In this method the current sup plied is automatically adjusted in magnitude and with the TC 2024A direction depending on the difference between the actual and the desired temperature The advantage of feedback is that the calibration curve chamber temperature versus controller setting is unaffected by ambient temperature In the cooling direction however the lowest temperature reachable will still depend on the degree of con tact with the microscope stage and the ambient temperature MANUAL CONTROL The power supply should deliver constant current rather than voltage and be adjustable up to the maximum needed for the desired temperature range in any case 6 amperes At higher perfusion rates more current will be needed for a given temperature for static solutions less current This data will also be affected by the ambient the depth of media in the chamber and the degree of contact of the PSMI with a large metal surface such as a microscope stage For accurate calibration produce your own graph of plate temperature see APPENDIX 5 for converting measured resistance to temperature versus current measured eithe
18. or a Power to the PSMI Peltier heat pumps b The PSMI built in temperature feedback thermistor A table of its electrical resistance versus temperature is shown in Appendix 5 page 17 Note that this temperature is not exactly the same as that in the media in the dish E Harvard Apparatus PSMI Patch Slice Microincubator Setup amp Operation c System ground The micro incubator s ground scheme is designed to provide the best possible noise shielding for demanding electrical recordings The PSMI heat exchange plates are anodized and thus not electrically in contact with the microscope stage These plates are also electrically separated from the Peltier power leads One of those Peltier power wires is grounded with in the TC 202A The micro incubator body heat exchange plates is ground ed to the temperature controller chassis by the metal shell connector on the PSMI cable Two independent separate from the main cable ground pathways are also available when single channel or similar demanding electrical recordings are to be performed a Salt Bridge Ground The Ag AgCl disc in a salt bridge well see 3 Figure 2 page 5 is connected to a small 1 mm diameter white color socket see 10 Figure 2 page 5 This allows grounding of the recording chamber via an integral agar salt bridge b Shield Ground A small 1 mm diameter green color socket see 9 Figure 2 page 5 connected to the cable shield and aluminum components of the
19. or use of some perfusants 4 Two 1 mm plugs Leads for plates ground connections 5 Thermistor holder 6 Nylon mesh 1pcs of 4in x4in APPENDIX 2 USEFUL ACCESSORIES 1 A small waterproof temperature probe to measure the actual temperature in various parts of the chamber Only thermocouple type probes will be small enough 2 Metal tube loop for water perfusion necessary for very low temperature to prevent feed forward condition place in circular groove 45 cm wide on top of air fins Available from Harvard Apparatus APPENDIX 3 PELTIER DEVICE OPERATION A thermoelectric module is a solid state device consisting of a series of semiconductors Most commercial devices are made from p and n doped bismuth or lead telluride The passage of electric current normally generates only heat The contradictory concept of the Peltier effect is due to Jean Peltier who in the nineteenth century discovered that the passage of current through two dissimilar conductors results in the junction either heating up or cooling down Each device consists of many cou ples mounted in series and connected by copper strips sometimes overlying this is a thin piece of ceramic an electrical insulator but thermal conductor In principle they work as heat pumps that is when current is passed through the junction the device pumps heat in one direction reversing the current reverses the direction of the heat flow Thus depending on the capacities of
20. r with the current meter of the power supply or with a separate current meter con nected in series with the PSMI and the scope The plate s thermistor will be 1 2 C further from ambient than the chambers temperature Due to the low electrical resistance of the PSMI 0 7 ohm it may be useful to put a power resistor in series with it to spread the operating range out over the control dial of the power supply Power supplies with sufficient current capacity tend to have voltages larger than the maximum of the PSMI See Setup and Operation pages 9 10 for identification of leads in the control cable of the PSMI Publication 5403 009 REV A Harvard Apparatus PSMI Patch Slice Microincubator Controlling Micro Incubator FEEDBACK CONTROL The control for temperature can be from the thermistor mounted on the top heat exchange plate built in thermistor compatible with the TC 202A temperature controller The TC 202A also has a so called bath thermistor optionally used with the Open Perfusion Micro Incubator for measuring or controlling directly in the chamber For most patch slice perfusion geometries it is too large Due to the fast perfusion rate relative to volume in the chamber the temperature of the slice relative to that of the plate thermistor will be only 1 2 C closer to ambient To achieve the feedback control and obtain maximum benefits from your PSMI Micro Incubator we strongly recommend that you use the TC 202
21. rchase of the original device This warranty gives you specific rights and you may also have other rights which vary from state to state Repair Facilities and Parts Harvard Apparatus stocks replacement and repair parts When ordering please describe parts as completely as possible preferably using our part numbers If prac tical enclose a sample or drawing We offer a complete reconditioning service CAUTION This unit is not registered with the C A U T i O N FDA and is not for clinical use on human patients NOT FOR CLINICAL USE ON HUMAN PATIENTS Publication 5403 009 REV A E Harvard Apparatus PSMI Patch Slice Microincubator Introduction The PSMI Patch Slice Micro Incubator is a unique environmental control unit that with its matching temperature controller TC 202A enables patch clamp record ings to be made from a brain slice using an upright microscope with water immer sion objective Tissue slices principally from the brain are increasingly being used for electrophysi ological and optical studies Although this preparation offers an environment in which the majority of synaptic connections are maintained less consideration has been given to the microscope s environment where experimental examination takes place One reason for this is the design problem associated with providing environ mental conditioning without limiting optical or electrode access or reducing the quality of any electrical recordin
22. ry heat transfers The first of these is by direct conduction from the support surface and collar to the chamber while the other is through convec tion from air or gas flowing over the same controlled plate below a covering annu lar black acrylic cover Several different modes of operation are permissible 1 Continuous perfusion at a constant set temperature 2 Switching perfusion of extracellular medium at a set temperature 3 Rapid temperature changes with perfusion Why perfuse the recording chamber The ability to change the extracellular environment by perfusing the experimental chamber is important for two reasons first excised tissue both requires continued oxygenation and the washing away of substances produced by the excision second perfusion is essential for quantitative ionic and pharmacological studies or for studies of distributed synaptic inputs or network activity in which drugs need to be applied to a large area Perfusion is especially useful for the application and sub sequent washout of an antagonist at a precise concentration Publication 5403 009 REV A 2 Harvard Apparatus PSMI Patch Slice Microincubator Controlling Micro Incubator The PSMI has self contained Peltier heating cooling elements Current passed in one direction will heat the enclosed dish reversed current will cool it WARNING The maximum sustained current is about 6 amperes This corresponds to a maxi mum voltage applied across t
23. the heat source and sink a tem perature gradient is built up across the device The thermoelectric device is thus sandwiched between the object of interest and a suitable thermal mass or ground Use of Peltier devices Peltier devices are essential for rapid temperature changes or when the desired tem perature is close to ambient temperature because they can actively correct the actu al temperature whether too high or too low instead of relying on the slow passive return of a monopolar system These advantages are only available when a bipo lar controller is used see Description section pages 4 6 Peltier devices also offer the flexibility to cool as well as heat in the same chamber Such a system further Publication 5403 009 REV A E Harvard Apparatus PSMI Patch Slice Microincubator Appendices allows examination of the temperature dependence Q10 of biological properties by changing the command temperature rapidly during an experiment Finally low ering the temperature eases the study of ion channels with rapid kinetics APPENDIX 4 SPECIFICATIONS Electrical maximum Temperature range Perfusion rate Thermal Expansion Plastic Perfusant Tubes 6 amperes continuous approximately 3 volts From 10 C below ambient to 50 C on the con ducting stage of a microscope but without supple mentary water cooling of the heat fins Up to 3 ml per minute See OPTIONAL REASSEMBLY page 13 for higher rat
24. tubing APPENDIX 4 through the same slots from the bobbin itself Cut the tubes to the desired lengths and thread them through those slots A dab of silicone cement bathroom caulk clear smells like vinegar to hold the tubing as it goes into the exit slots may be useful Reseat the plastic ring that circles the outside of the exposed heat exchange plate first rubbing a little silicone oil on its top and bottom surfaces and position the plastic plate Reattach the plastic lid with the screws Be careful in tightening the screws to avoid stripping OPTIONAL REASSEMBLY For faster perfusion rates lengthen the tubing on the bobbin by selecting another exit point or else reduce its wall thickness see APPENDIX 4 Either allows the faster moving fluid to still get enough heat transfer before reaching the chamber SUGGESTION Use only one tube and wind it one extra turn one and three quarters total for the same exit point This should allow perfusions of as much as o 6 ml min or connect ch 1 out to ch 2 in WARNING DO NOT FURTHER DISASSEMBLE THE PSMI ONCE THE TOP HEAT PLATE IS REMOVED REASSEMBIY FOR PERFORMANCE THAT MEETS SPECIFICATIONS IS DIFFICULT AND IT MAY NEED TO BE SENT TO OUR PREMISES FOR ADJUSTMENTS Publication 5403 009 REV A Harvard Apparatus PSMI Patch Slice Microincubator Troubleshooting Slow perfusion rate or excessive fluid build up in the bubble trap a Due to excessive perfusion rate
25. vity feed This also makes it easier to maintain fluid level on changing solutions A four channel pump is ideal since this allows for perfusion of several different solutions Two small 90 elbow white inputs CH1 IN or CH2 IN are provided in the micro incubator In addition two small 90 elbow white outputs CH1 OUT or CH2 OUT are provided for tubes that can be user directed to the desired location in the chamber Changing the perfusate can be achieved by switching between the two corresponding pump channels attached via a valve to one of the PSMI s perfusion inlets Complete Layout of Liquid Perfusion For one possible complete layout of liquid perfusion see Figure 5 on next page Publication 5403 009 REV A Harvard Apparatus PSMI Patch Slice Microincubator Perfusion gt B A Bubble o Traps Reservoirs Figure 5 Perfusion System Temperature Gradients The temperature at any particular point in the recording chamber is dependent on the mode of operation and the rate of perfusion The horizontal temperature gradient depends on media perfusion rate gas flow rate depth of media and the presence or absence of a cover lid For absolute determination during a particular experiment it is essential to measure the temperature directly OUT e IN PSMI PSMI Perfusion Rate 1 0 ml min Static with Gas Flow and Lid with Gas Flow and Lid Figure 6 Isotberm Maps Temperatur
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