Home

NaNorespiratioN system user maNual

1. 5 5 NANORESPIRATION SYSTEM USER MANUAL NANORESPIRATION SYSTEM USER MANUAL N AN ORESPI RATI ON SYSTEM Copyright 2010 Unisense A S USER MANUAL Version October 2010 UNISENSE A S TABLE CONTENTS CONGRATULATIONS WITH YOUR NEW PRODUCT SUPPORT ORDERING AND CONTACT INFORMATION OVERVIEW s ya sa ek eee LKM S Sn SYSTEM 5 MEASURING COMPONENTS OF THE SYSTEM NANORESPIRATION UNIT MICRO MANIPULATORS MICROSENSOR AMPLIFIER SOFTWARE SETTING UP an n An ache aches GETTING 5 BEFORE STARTING MEASUREMENTS LOADING THE ROSETTE POSITIONING THE MICROSENSOR MEASUREMENTS CALCULATIONS t54 ai eee wan bende ADVANCED 05 STORAGE AND REFERENCES n ale ne W n WARRANTY AND LIABILITY REPAIR OR ADJUSTMENT CONGRATULATIONS WITH YOUR NEW PRODUCT SUPPORT ORDERING AND CONTACT INFORMATION If you wish to order additional products or if you encounter any problems and need scientific technical assistance please do not hesitate to contact our sales and support team We will respond to your inquiry within
2. Step size 200 um Start position 400 um End point 2000 um Resting time 3 s Standby position 20000 um Motor travel speed 1000 um s Make a profile After the profile the sensor is positioned in the standby position 2 cm above the Rosette Rotate the disc gently so that the next capillary is in position for profiling and repeat profiling with the same SensorTrace PRO settings Continue until all the capillary profiles have been measured Another round of measurements can be made to evaluate the reproducibility of the measurements check the viability and well being of the organisms or to obtain a time series of respiration rates during incubation WARNING The depth of the capillary is approx 3mm but can vary at least 200 um It is therefore not recommended to measure deeper than 2 4 mm into the capillary 15 CALCULATIONS Concentration gradient profiles in the capillaries are converted to respiration rates by using the Excel spreadsheet Unisense Flux Analysis which is downloadable from our website The spreadsheet contains macros for opening and analyzing SensorTrace PRO datafiles For these macros to work the security settings of Excel must be set to low or medium before the analysis template spreadsheet is opened The spreadsheet contains buttons for selecting and importing SensorTrace PRO datafiles Once a datafile has been imported the spreadsheet will show which profiles are contained i
3. for the transfer of the organisms to the respirometer 1 Place the Rosette disc on flat clean surface under a dissection microscope 2 Fill the Rosette capillaries with a sterile incubation solution and transfer the respiring organisms to the capillaries using sterile micropipettes The central capillary cannot be used as it cannot be accessed by rotating the disc Leave one of the peripheral wells empty to serve as an empty reference measurement 3 Fill the depression of the Rosette disc holder with the incubation solution and place the Rosette disc in the Rosette disc holder Before proceeding make sure that the Rosette disc is completely inserted and firmly positioned in the disc holder Air bubbles may accidentally lift the Rosette disc slightly which could cause misalignment of sensor capillary opening IMPORTANT As the incubation container cannot be completely closed it may be difficult to obtain stable low oxygen conditions due to irregular influx of atmospheric oxygen or increase the risk of sensor breakage 4 Rotate the disc by gently pushing the disc appendage with a suitable tool e g tweezers so that the markings on the disc holder are aligned with the lines on the disc Make sure that the Rosette disk is still firmly positioned in the disk holder 5 Submerge the Rosette disc holder into the incubation solution by raising the elevator table with the incubation container Ensure that t
4. 8 mm By measuring the oxygen concentration gradient and knowing the capillary dimensions the oxygen flux towards the respiring organism can be calculated In steady state conditions The flux will be equal to the respiration rate of the organism in the capillary The oxygen gradient is measured with a Unisense oxygen microsensor The oxygen consumption of this sensor is so small that it does not affect the oxygen gradient in the well The respiration rates are calculated by exporting the measured oxygen gradient data into a Unisense Flow Analysis spreadsheet available from our website www unisense com Close up of rosette Oxygen conc Schematic drawing of Rosette capillary with organism and the measured oxygen concentration profile COMPONENTS OF THE SYSTEM The system consists on the following parts Nanorespiration unit Micromanipulator motorized mounted ona stable laboratory stand Oxygen microsensor Amplifier e g Microsensor Multimeter Monometer Data acquisition software Besides the supplied elements users would need the following Dissection microscope Elevator table Container for incubation solution Incubation chamber capable of maintaining desired temperature and gas composition The following sections describe the different elements briefly Please see separate manuals for more detailed descriptions and specification NANORESPIRATION UNIT The uni
5. bling witha gas mixture with a fixed oxygen partial pressure 13 Secure the precalibrated microsensor in the micromanipulator so the tip is approx 1 cm above the Rosette Rotate the Rosette disc so the lines of the Rosette disc are aligned with the markings on the Rosette disc holder By manually turning the x and y screws of the micromanipulators align the sensor tip so that it is slightly above the top of a capillary and positioned as close to the capillary center as possible It is necessary to study the electrode tip carefully with a dissection microscope during this operation to ensure that it is not damaged Set actual depth in SensorTrace PRO software to 0 From this point do NOT change the x y position of the micromanipulator Retract the sensor tip approx 20 000 um using the SensorTrace PRO motor control of the micromanipulator so it is possible to remove the Rosette disc from the disc holder without damaging the sensor MEASUREMENTS After 1 hour of incubation steady state oxygen gradients will have been established in the glass capillaries with a slope proportional to the respiration rate of the organisms Make sure that the disc lines are always aligned with the markings on the disc holder Keep track of which capillary is in position for profiling Measure the oxygen profile in the capillary according to the manuals for sensors and software We recommend the following SensorTrace PRO settings
6. cifications Unisense will replace the sensor s free of charge Unisense will only replace dysfunctional sensors if they have been tested according with the instructions in the manual upon receipt of the sensor s The warranty does not include repair or replacement necessitated by accident neglect misuse unauthorized repair or modification of the product Physical damage to the tip of the sensor is not covered by the warranty Liability In no event will Unisense be liable for any direct indirect consequential or incidental damages including lost profits or for any claim by any third party arising out of the use the results of use or the inability to use this product REPAIR OR ADJUSTMENT Sensors and electrodes cannot be repaired Other equipment that is not covered by the warranty will if possible be repaired by Unisense with appropriate charges paid by the customer In case of return of equipment please contact us for a return authorization For further information please see the documents Conditions for Sale and Delivery for Unisense and Warranty and Shipping Information 20 TROUBLESHOOTING Problem Possible cause 1 Solution Possible cause 2 Solution Possible cause 3 Solution Non linear profiles The profiles are not in steady state Extend the pre incubation period Well or incubation solution contaminated with oxygen consuming microorganisms Autoclave Rosette disc and incubatio
7. e PC with the RS 232 cable If you do not have an RC 232 port on your computer use the provided adapter cable for the USB port The adapter may need to be installed Place the microsensor amplifier s near the stand 0 5m 1 5ft and connect it to a power source if not battery or USB powered Connect the amplifier to the PC using the USB cable Install the Unisense SensorTrace PRO software GETTING STARTED The Rosette must be submerged in the type of liquid medium freshwater saltwater physiological media etc in which the organism s respiration is being studied The aqueous incubation solution needs to be collected prepared before the measurements are initiated It is recommended to autoclave the solutions and the Rosette before use in order to avoid contamination with undesired oxygen consuming microorganisms The novel measurement principle employed in the Nanorespi rometer is based on the measurements of steady state i e stable oxygen profiles in the Rosette capillaries generated by the respiration of the enclosed organism Therefore it is very important that the aqueous incubation solution has a stable and defined oxygen concentration This is most readily achieved by letting the solution equilibrate with an atmospheric oxygen partial pressure by aeration If a lower oxygen condition or a special gas mixture is required e g high CO as part of a carbonate buffering system special precautions must be taken in order to ens
8. he walls of the container do NOT touch the Rosette disc holder If the Rosette disc holder is touched by the container the position of the Rosette capillary may be shifted relative to the microsensor position which may cause microsensor breakage when profiling is initiated 6 Let the Rosette with the organisms equilibrate in the solution for 1 hour to obtain steady state profiles before the measurements are initiated POSITIONING THE MICROSENSOR Successful respiration measurements require a precise positioning of the sensor relative to the Rosette capillaries The sensor position only needs to be adjusted in one of the outer Rosette capillaries The revolver principle then ensures that all the other capillaries in the outer Rosette ring can be brought in position for profiling by rotating the Rosette disc The sensor x y position can only be adjusted manually and correct positioning is a challenging pre measurement routine It is recommended to practise this with a dummy sensor before the experiments are started in order to minimize the risk of breaking the sensor during measurements IMPORTANT It is important that the incubation solution is in equilibrium with the gas mixture before the measurements are initiated If the oxygen partial pressure in the incubation solution changes during measurements steady state profiles cannot be obtained A stable partial pressure may be obtained by constant bub
9. ignal is directly proportional to the oxygen concentration at the sensor tip provided that the medium composition and temperature are kept constant AMPLIFIER A Unisense microsensor delivers a very small signal current and it takes a specialized high quality amplifier such as the Unisense Multimeter to amplify the signal to a measurable level The microsensor is connected to the Multimeter which maintains the polarization of the sensor tip cathode as well as measures the current produced in picoampere A similar polarization of the guard cathode is necessary to prevent oxygen from diffusing though the sensor shaft to the electrode tip puen6 slqeD 6nid ona dij 10suos Schematic drawing of oxygen sensor All Unisense amplifiers have A D converters built in and they connected directly to the PC SOFTWARE SensorTrace PRO is a PC Windows program for automated data acquisition and motor control It measures vertical concentration profiles which can be used to calculate analyte flux such as respiration rates SensorTrace PRO can read data from the microsensor amplifier via the A D converter integrated or stand alone ADC 216 416 816USB that is connected to a PC and the program can also control the motorized micromanipulator via a motor controller The program allows recording calibration plotting and storing of multiple automatically or manually measured microprofiles of the oxygen concentrat
10. ion gradients in the Rosette SETTING UP The following section will describe how to set up a NanoRespirometer System as shown in the diagram above Please consult individual component manuals for further details Micromanipulator mounted on MMS Microsensor Thermometer amp One channel Meter Multi channel Meter Calibration Chamber Nanorespirometer Unit Motorcontroller Data Acquisition Lab stand Schemativ view of the NanoRespiration System 1 Place the laboratory stand 1518 ona smooth surface Adjust the feet to prevent the stand from rocking If the sample is not to be placed on the stand plate the stand should be placed at an appropriate distance from the sample to allow space for the micromanipulator 2 Assemble the nanorespiration unit and mount it on the LS18 stand Leave enough space below the unit for the elevator table with media and enough space above it for the micromanipulator 3 Mount the motorized micromanipulator MMS on the stand with the fixation screw at a suitable height so that the clamp for holding the oxygen microsensor is positioned about 12 5 cm 5 above the rosette in the nanorespiration unit when the micromanipulator z axis is completely retracted If motorized connect the motor port of the motor controller to the motor stage using the motor cable If motorized connect the PC port of the motor controller to the RS 232 serial port of th
11. ld be designed where gas control is possible This will be the case for most mammalian embryo studies Please contact Unisense for advice CORROSION STUDIES Metal particle corrosion due to exposure to different solutions can be examined using the Unisense NanoRespiration System 17 STORAGE AND MAINTENANCE Unisense recommends that the micromanipulator and the lab WARNING stand NOT the stage are protected with silicone spray before use Do not spray the This is especially important if salty water is used as an incubation MMS with silicone solution This will prolong the lifetime of the unit and will help to keep the surfaces clean Rinse the micromanipulator and stands with distilled water if salt crystals or other corrosive material have been deposited on them Keep the micromanipulator and motor free of sand and dust The Rosette capillaries should be cleaned after use This is achieved by flushing a suitable cleaning solution into to the capillary followed by a thorough flush with distilled water A syringe equipped with a 0 4 mm needle is a useful tool for this To evaluate the result of the cleaning we recommend examination with a dissection microscope or an ordinary upright microscope The Rosette disc should be autoclaves or acid washed to ensure that substances with unwanted oxygen consumption are removed Sensor Depending on which substance is present on the sensor tip or membrane the sensor can be cleaned with different
12. n solution Fluctuating temperature or oxygen concentration in the incubation solution Improve temperature and oxygen concentration control If problems arise with a single component of the NanoRespiration System please consult the individual component manuals If you encounter other problems and need scientific technical assistance please contact sales unisense com for online support we will answer you within one workday 21 UNISENSE UNISENSE DENMARK www unisense com info unisense com
13. n the datafile and enable the user to select four profiles that are displayed and analysed together The user can then select which part of the concentration profiles should be used to calculate respiration rates The respiration rates of the chosen profiles are presented in different units For a more detailed description of the algorithms used by the Unisense Flux Analysis spread sheet consult the detailed manual TEMPERATURE Keep good track of the temperature as both the sensor signal and the oxygen solubility depend on temperature The temperature coefficient varies from sensor to sensor but is approximately 2 3 signal increase per More information is available at on our website www unisense com ADVANCED USE ADJUSTMENT OF NANORESPIRATION SYSTEM SENSITIVITY If the standard configuration of the Unisense NanoRespiration System does not provide sufficient sensitivity for your application it is possible to increase the sensitivity by changing the Rosette dimensions Narrowing the diameters of the Rosette capillaries or increasing the capillary length lowers the detection limit whereas wider and shorter capillaries will decrease the sensitivity of the system Unisense would be happy to provide special Rosettes for customers with special requirements LOW OXYGEN EXPERIMENTS If respiration measurement needs to be conducted under low oxygen conditions or using a special gas mixture a special incubation container shou
14. one working day E mail sales unisense com Unisense A S Tueager 1 DK 8200 Aarhus N Denmark Tel 45 8944 9500 Fax 45 8944 9549 Further documentation and support is available at our website www unisense com REPLACEMENT OF SENSORS Unisense will replace sensors that have been damaged during shipment provided that The sensors were tested immediately upon receipt in accordance with the delivery note and the manual The seal is still intact The sensors are returned to Unisense for inspection within two weeks The sensors are correctly packed for return to Unisense in accordance with the note included in the sensor box OVERVIEW Unisense has developed the Nano respiration system to measure respiration rates of very small immobile organisms like embryos eggs etc with exceptional precision and sensitivity This manual gives an overview of the components comprising the complete measuring system provided by Unisense It also includes detailed operating instructions to measure respiration rates with Unisense NanoRespiration System Detailed manuals describing the individual components of the NanoRespiration System can be obtained from Unisense website at www unisense com SYSTEM FEATURES Novel proprietary steady state measurement principle patented The oxygen concentration around the organism is constant facilitating prolonged and repeated measurements without stress Very low detection limit
15. s for oxygen consumption sensitivity below 0 06 nL Hr 0 7 fmol s High accuracy and reproducibility 0 03 nL h Fast measurement of the respiration rate 2 min Abroad range of respiration rates can be measured 0 1 to 5 nL hr Insignificant oxygen consumption by the sensor e Organisms placed in oxygen impermeable and chemically inert glass capillaries Easy sterilisation by autoclavation Spreadsheet available for automated calculation of respiration rates Small sensor diameter which does not disrupt oxygen flux to the respiring organism e Organism is placed a suitable liquid medium during measurement MEASURING PRINCIPLE The central part of the NanoRespiration System is a rosette of seven fused glass capillaries the rosette with a sealed glass bottom Each capillary illustration is filled with a suitable liquid medium and the respiring organism is placed on the glass bottom The glass is completely impermeable to oxygen but the oxygen supply to Oxygen microsensor the respiring organism is maintained au by molecular diffusion from the overlaying liguid media down through the glass capillary see illustration As the organism consumes oxygen by respiration a linear steady state Rosette glass Wel oxygen gradient will be established S in the capillary This is usually reached rganisi within one hour due to the small 9 dimensions of the capillary length 3 mm diameter 0 6
16. solutes The standard method is to rinse with 96 ethanol then rinse with 0 01 M HCl and rinse with water This will remove most substances Alternatively it is possible to rinse with 0 1M NaOH isopropanol or different detergent Store the oxygen sensor according to the sensor manual REFERENCES Abe A Non Invasive and Sensitive Method for Measuring Cellular Respiration With a Scanning Electrochemical Microscopy to Evaluate Embryo Quality Journal of Mammalian Ova Research 2007 24 70 78 Lopes A S Larsen H Ramsing Lovendahl Raty M Peippo J Greve Callesen H Respiration Rates of Individual Bovine in Vitro Produced Embryos Measured With a Novel Non Invasive and Highly Sensitive Microsensor System Reproduction 2005 130 669 679 Nielsen P Larsen L H Ramlov H and Hansen B W 2006 Respiration rates of subitaneous eggs from a marine calanoid copepod monitored by nanorespirometry Journal of Comparative Physiology B Biochemical Systemic and Environmental Physiology 28 Nov 2006 19 WARRANTY AND LIABILITY Microsensors are consumable items The sensors are tested thoroughly before packaging and shipment Warranty for sensors N O sensors 60 days from shipment Standard Oxygen sensors 180 days from shipment All other sensors excluding special sensors 90 days from shipment If within the above specified period the sensor s fail to perform according to the spe
17. t consists of a Rosette of 7 fused glass capillaries the Rosette a Rosette disc made of UDEL polysulfone a Rosette disc holder made of UDEL polysulfone Holder and Rosette disc astable metal frame Micro MANIPULATORS The oxygen microsensor tip needs to be lowered in very precise steps into the liquid filled glass capillary in order to be able to resolve the oxygen gradient within the capillary created by the respiring organism To accomplish this the microsensor must be mounted micro manipulator Unisense MM33 The micro manipulator is firmly attached to a stable laboratory stand Motorized sensor positioning is superior to manual positioning due to a higher level of precision faster positioning and general ease of operation therefore it is recommended to use a motorized micromanipulator system for the NanoRespiration System MICROSENSOR The Unisense oxygen microsensor is a miniaturized Clark type oxygen sensor with an internal reference and a guard cathode Due to the minute tip size excellent response time and insignificant stirring sensitivity the Unisense oxygen sensor facilitates reliable and fast measurements of high spatial resolution Driven by the external partial pressure oxygen from the environment will diffuse through the sensor tip membrane and be reduced at the gold cathode surface The picoammeter see below converts the resulting reduction current to a detectable signal This sensor s
18. ure a stable defined oxygen concentration and gas mixture Temperature will have a strong influence on a the oxygen solubility for any aqueous incubation solution b the obtained microsensor signal at a given concentration and c the activity level and respiration rate of the organism being studied Therefore care should be taken to ensure stable temperature conditions during respiration measurements It is also very important to carry out sensor calibration at exactly the same temperature and salinity as experienced by the organism in the NanoRespiration System BEFORE STARTING MEASUREMENTS 1 Prepare the incubation solution and find design a suitable container for the incubation where stable oxygen and temperature conditions can be maintained WARNING Vigorous bubbling of aqueous solutions with a dry gas mixture may cause the water to cool considerably Monitor temperature to find a suitable bubbling rate which does not cool the water 11 2 Transfer the organisms of interest to sterile Petri dish containing the incubation solution Individual organisms can then be selected and transferred to the Rosette with a sterile micropipette 3 Calibrate the oxygen microsensor according to the sensor manual LOADING THE ROSETTE To make sure that no oxygen consuming microorganisms are contaminating the glass capillaries it is recommended to autoclave boil the Rosette disc before experiments and to use sterile pipettes

NaNorespiratioN system user maNual

Contents

Download Pdf Manuals

Related Search

Related Contents