MitoViewer MV-2 User Manual
Contents
1. The back panel features a USB connector with a small LED indicator DC voltage power input jack Ain analog input BNC connector to enable display of additional parameter side by side with NADH on the MitoViewer software DIO connector enables triggering or synchronizing of the MitoViewer with external devices System ON OFF y Probe connector Probe connector pushbutton with LED j RI detection channel Light source channel indicator MitoViewer front panel USB connector with Analog in DIO 12 VDC LED indicator BNC connector BNC connector Power jack MitoViewer MV 2 back panel 9 Page MitoViewer User Manual Prizmatix Standard MitoViewer MV 2 fiber optic probe The standard fiber optic probe features a needle type distal end designed for measurements on exposed tissue by means of a micromanipulator or within a cannula The probe contains seven fibers which are arranged in a honeycomb structure in the distal end The central fiber is the excitation fiber which delivers the UV LED light to the tissue The six satellite fibers collect the light from the tissue and deliver it to the detection unit in the MitoViewer The proximal end of the probe features two branches connectorized with optical SMA connectors The thin branch contains the single excitation fiber and should be connected to the Light Source Unit port on the front panel of the MitoViewer The thick branch contains the six collection fibers and should be con2. low S N conditions the auto ranging feature may be engaged 11 During Auto Ranging the LED Power will automatically change to bring the PMT detectors within reasonable operation gain 12 The Smoothing constant may be changed by clicking on the current value and choosing different value Pegs PMT1 Refi 3 25 PMT2 Fluor Ihz LED PD 13 At the end of the measurement press the stop button to end the UV LED excitation and to stop data acquisition Note Always stop the software by only using the Stop button 28 Page MitoViewer User Manual Prizmatix 14 Data storage The data is stored in an ASCII Tab delimited text file TXT file The file can be reviewed by simple editor exe Sys a5 5 TextFile txt Hotepad He Gat Format Yew Help 3 3 2010 11 50 55 AM 79 000 100 074 99 966 99 942 5737 27010 11 50 56 AM 70 000 100 036 99 890 99 855 5 3 2010 11 50 57 AM 31 000 100 045 99 816 99 771 5 3 2010 11 50 58 AM 37 000 100 034 99 766 99 732 5 3 2010 11 50 59 AM 33 000 100 009 99 772 99 764 57372010 11 51 00 AM 34 000 99 978 99 727 99 749 5 3 2010 11 51 01 AM 35 000 99 975 99 746 99 771 5 3 2010 11 51 02 AM 36 000 99 997 99 741 99 744 3 3 2010 11 51 03 AM F 000 99 981 99 753 99 773 5 3 2010 11 51 04 AM 38 000 99 968 99 806 99 838 3 3 2010 11 51 05 AM 39 000 99 935 99 762 99 826 5 3 2010 11 51 06 AM 40 000 99 888 99 795 99 907 3 2010 11 51 07 AM 41 000 99 869 99 816 99 948 5 3 2010 11 51 08 AM 47 000 99 867 99
3. 8271 99 954 3 2010 11 51 09 AM 43 000 99 867 99 844 99 978 3 201L0 11 51 10 AM 44 000 99 884 99 877 99 993 3 2010 11 51 11 AM 45 000 99 881 99 816 99 935 57 3 2010 11 51 12 AM 46 000 99 853 99 777 99 923 3 2010 11 51 13 AM 47 000 99 857 99 746 99 890 Following is the description of the file columns 1 Time 2 Point number from start of the file 3 Refl Value not normalized to PD 4 Fluor value not normalized to PD 5 NADH 6 LED Photodiode 7 Marker The Red arrow shows the Marker signature 29 Page ui g et DA 4 ecoooce au 0 99 907 0 000 99 911 0 000 99 911 0 000 99 899 0 000 99 900 0 000 99 897 06 000 99 694 0 000 99 880 0 000 MitoViewer User Manual Prizmatix The file can be imported and analyzed in Microsoft Excel In order to import the data into Excel choose Text Files prn txt crv option at Files of type selection at File Open dialog box as shown in following figure i a xazm Ste Type Date Modfied 11 KB Text Document 2 21 2010 12 19 AM Tex Files s pen mbt i The file will be imported into excel in correct order as shown here r TextFile txt Microsoft Excel lt ba oe Opa EON i Ta ta ayi ao oo x fa ei aj ay fh Calibri Genera DO Formatting 3 Insert E ar J 4a B Fon ala a as _ Seem al E ete 3 Delete a J
4. Due to the dual dependence of the NADH redox state on O2 availability and ATP turnover the cause for a change in fluorescent signal is not unambiguous However combined with the information provided by other technologies 31 Page MitoViewer User Manual Prizmatix and parameters such as microcirculatory blood flow a more accurate interpretation of the NADH fluorescence changes can be accomplished There is general agreement on the characteristics of the NADH spectra and their biochemical significance when measured in vivo and in vitro The absorption and fluorescence spectra of NADH the reduced form have been well characterized at different levels of organizational complexity in solution cell suspension tissue slices perfused organs and under in vivo monitoring conditions The conceptual foundations for tissue mitochondrial NADH fluorometry were established in the early 50 s by Britton Chance He defined various metabolic states of activity or rest for in vitro mitochondria preparation These metabolic states depend on the availability of oxygen substrate and phosphate acceptor ADP and are associated with different redox states of NAD NADH In the current paper the brain is given as an in vivo example due to the relatively large amount of published material on this organ yet the same technology and data interpretation could be presented for other organs in vivo and in vitro As in most optical monitoring systems the main critical
5. new The system will automatically recognize the new hardware and will report that the new hardware is ready for use 5 The following dialog box will appear 22 Mew Data Acquisition Device MI DAtime has detected a new data acquisition device USB 6009 What would you like to do x Do Begin 4 Measurement with This Device T5 Run Test Panels Configure and Test This Device Using NI Measurement amp Sutomation Explore O Take No Action Always Take This Action Cancel 6 Click on Configure and Test This Device 17 Page MitoViewer User Manual Prizmatix 7 The following screen titled NI USB 6009 Dev1 Measurement amp Automation Explorer will appear E m USG e009 Tevi MA UnMeEn t AUTOMatiOn Exshorer Doet ten erronei Qyvesetvevce E Create lask Value Geax mal Jp Devies and Interfaces thd 40AIFC ey MI DtOrroe Devine Ni DAGms Device Basics 8 The National Instruments USB 6009 device will be listed in the side menu Make sure that the NI USB 6009 OEM or NI USB 6009 is marked as Dev1 9 Ifthe NI USB 6009 OEM is named as Dev2 or by other name rename it to Dev1 10 In order to rename the NI USB 6009 OEM as Dev1 e Click on the NI USB 6009 OEM with left button of the mouse and select option Rename from the list e Change the name of the NI USB 6009 OEM to Dev1 and click anywhere outside The name will change to NI USB 6009 OEM Dev1 18 Page Mit
6. performed with excitation light provided by Mercury lamps at 365nm and not at the NADH absorption peak at 340nm The MitoViewer was designed according to this tradition The MitoViewer device uses 365nm UV LED for excitation of the NADH fluorescence Fluor signal This excitation light is also utilized for the correction of hemodynamic artifacts by measuring the reflection light intensity at same excitation wavelength of 365nm the Refl signal The light is transmitted from and to the device by means of a flexible fiber optic probe The software displays the Refl Fluor NADH corrected fluorescence signal and the photodiode PD signal 3 Page MitoViewer User Manual Prizmatix Main Features of the Mito Viewer Suitable for various experimental animal models and variety of organs under in vitro in vivo conditions LabVIEW user friendly interface Calculates corrected NADH independent of hemodynamic changes in the tissue Measurements taken via flexible fiberoptic probe Custom made probes can be supplied to suit experimental set ups Possible integration of the MitoViewer probe with other probes in existing set ups The MitoViewer is suitable for the testing of Ischemia hypoxia anoxia heperoxia or hypercapnia in various organs 1 e brain kidney liver or muscle tissue Activation of the brain by cortical spreading depression or seizures Changing of heart metabolism by isotropic and chronotropic drugs Drug safety and e
7. MitoViewer MV 2 User Manual Software version 1 1 Main Office European Sales Office North America Sales Office Phone 972 8 929 7844 Phone 44 0 77 9172 9592 Phone 1 248 436 8085 Fax 972 8 929 8772 Fax 44 0 20 7681 2977 Fax 1 248 281 5236 Sales prizmatix com Sales europe prizmatix com Sales uSa prizmatix com P O B 4234 Modtiin Illite 71919 Israel Prizmatix Table of Contents MitoViewer MV 2 System Description ccccccssccescccsscceeccessccsecesecesscceeeseseeeeceseseeceseeesessensesees 3 MitoViewer Principle Of NADH Monitoring ccsscccccssssceecceeseceeceeesseeeeeeeeeceeseaaeeceesueeeeeeseaegeeeees 3 Main Features Of the MitO ViWEL cccccsssssseccccccseesssecccecsaueeeseececeeaeasseeeeeesssaeseeeeeeessaaaseseeeesaaenees 4 The MitoViewer is suitable for the testing Of cccccccssccccseeceeeseceeeeeeceeuscseeesceeeueceeseuecetseeeetsners 4 TEENY VG SPO UY sejiccctcn crcactasrhancinnessmeicae suena acnaseaincext oe eu enone uatoce zeae cee saeasacececuscectioucuaet ses A 5 Set Up orhe DEVICE corn i ge gaisi E N 6 FAC Ve MY aE A E EE 6 SV SES IO CUI serrerirnri iieri an E O E ENERE EEEE IEEE E E EERS 8 Additional equipment not provided with the MitoViewer aessessessssesesersesreresrererrererreess 11 SOTEW ae IS Cal ION aaar E A 12 Installation of the National Instruments software esssssssesesssssserrssssssrerressssereressssseene 12 Installation of the Prizmatix Mito
8. RENT RETURN POLICY 1 Definitions As used in this Agreement the following terms have the following mearings SS eee eee 14 Page MitoViewer User Manual Prizmatix 8 Accept the license agreement and click Next Following dialog box with end user agreement will appear Mi DAGQm 8 7 1 License Agreement Vou mutt ecoem the Eeence et citplaed balma be proces MILHOSUF AML CURE SERVICES MS2ML 4 0 IMPORTANT READ CAREFULLY This Microsoft End User Leerese Ageement EULA legal agreement between you either an indrideal of a single entity and Micmeoft Comoration for the Tehicrosef software identified shove which may inchade compoter softwar aepociated mecha printed feriak and online or electronic documentation SOFTWARE By downlosding installing pying or otherwise using the SOFTWARE you apee to be bound by the terma of thas EULA If ado nol agree io the tama of thia EULA do not wstal oruse the SOFTWARE The SOFTWARE i pamected by copyright law and iniemational copynghi treaties as well m other Decta Ti A ti s M fi or its the tithe ght and othe Netettorteet Peat iti tothe SUETUIDE The CAETWABE t Nesesad ene cele xs 9 Accept the license and click Next Following Dialog will appear Click Next to Start the installation F X sE NI DAQmx 8 7 1 Overall Progress Me Currently installing NI Uninstaller Part 1 of 96 Copying new files Cancel 15 Page MitoView
9. Viewer software ccccsssseeccccecseesseeeceeesueaeeeeeeessaeees 20 MitoViewer Software Usage ievcccdesscudseastadvocexetancvaanrsnsettawammedesiuennbonsianeniansthenedianastencedstncdoasvaunnerenbat 24 Appendix A Real time evaluation of mitochondrial function by monitoring NADH redox state anene N OV W T er EA E 31 Appendix B Typical Results while monitoring the Brain in VIVO essesssssssessrseesrrrsrrrresrererrererreres 34 2 Page MitoViewer User Manual Prizmatix MitoViewer MV 2 System Description The MitoViewer MV 2 is a unique optical monitoring device enabling real time evaluation of mitochondrial function under in vitro and in vivo conditions The MitoViewer MV 2 provides significant and critical information originating from the intracellular Nano organelles the mitochondria which provide energy for cellular functions The measurement is based on the auto fluorescence of the intramitochondrial NADH which serves as a control marker in energy generation processes by the respiratory chain in the mitochondria NADH redox state is sensitive to the intracellular oxygen level and to energy consumption by the tissue MitoViewer Principle of NADH Monitoring MitoViewer measurement is based on the auto fluorescence of the intramitochondrial NADH The Mitochondrial NADH absorption band ranges from 300nm to 400nm with the NADH fluorescence emission peaking at around 450nm Due to historical reasons most NADH measurements were
10. Viewer_ 1 017 Installation Wizard It is strongly recommended that you exit all Windows programs before running this setup program Click Cancel to quit the setup program then close any programs you have running Click Next to continue the installation WARNING This program is protected by copyright law and international treaties Unauthorized reproduction or distribution of this program or any portion of it may result in severe civil and criminal penalties and will be prosecuted to the maximum extent possible under law 20 Page MitoViewer User Manual Prizmatix 4 Press Next the following dialog box will appear jie Mito iewer_1 017 Setup Destination Folder Select a folder where the application will be installed The installation wizard will install the files for MitoViewer_1 017 in the following folder To install into a different folder click the Browse button and select another folder You can choose not to install MitoViewer_1 017 by clicking Cancel to exit the installation wizard Destination Folder C Program Files MitoViewer Browse 5 If you would like to install the Mito Viewer software into different location use Browse button to change the installation location Press Next to continue the setup The following dialog box will appear jie Mito iewer_1 017 Setup x Ready to Install the Application JA Click Next to begin installation iB ec Click
11. al be invclaiied on ihe local hand drive fe Maacunennent Stoke fot Waua E NET Fremewedk 1 1 Languages NET Fremework 20 Languages Microrot Vieus C Support The festure end ts selected aubco Microtot Virusi Basic 6 0 Suppe regure up to 223 MB of disk space ORCA aisble Suppest z E Hi Laii Signe gees 2 511 w gt Diinechory for NI DAQmo 8 7 1 C Program Fleri atonal Inetruments WM DAGS _ 7 Press Next to approve the installation of the additional features of the driver The following dialog box with license agreement will appear 20 HIOAOms 8 7 1 Licente Agreement Tou mad ace tee boerne dirie ieee bin peed NATIONAL INSTRUMENTS SOFTWARE LICENSE AGREEMENT INSTALLATION NOTICE THIS IS A CONTRACT BEFORE YOU DOWNLOAD THE SOFTWARE OR COMPLETE THE INSTALLATION PROCESS CAREFULLY READ THIS AGREEMENT BY DOWNLOADING THE SOFTWARE ANDOR CLICHMNG THE APPLICABLE BUTTON To COMPLETE THE INSTALLATION PROCESS YOU CONSENT TO THE TERMS OF THIS AGREEMENT AND YOU AGREE TO BE BOUND EY THIS AGREEMENT IF YOU DO NOTWISH TO RARFCOMF A PARTY TO THIS AGREEMENT AMD AP ROUND AY ALL OF ITS TERMA AND CONDITIONS CLICK THE APPROPRIATE BUTTON TO CANCEL THE INSTALLATION PROCESS DO NOT INSTALL OR USE THE SOFTWARE AND RETURN THE SOFTWARE WITHIN THIRTY 30 DAYS OF RECEIPT OF THE SOFTWARE NCLUDING ALL ACCOMPANYING WRITTEN MATERIALS ALONG WITH THEIR CONTAINERS TO THE PLACE YOU OBTAINED THEM ALL RETURNS SHALL BE SUBJECT TO NTS THEN CUR
12. ale aeea ere aoma 2 ae Clipboard 7 Font SJ Alignment EE Number al Styles JL eetts Editing 3 M41 G a Fa Fi A SS Ga Da Ea Eaa E aaa l J J 31 5 3 2010 11 50 30 100 036 99 89 99 855 99 971 o B2 5 3 2010 11 50 31 100 045 99 816 99 771 99 957 o 33 5 3 2010 11 50 32 100 034 99 766 99 732 99 946 o 34 5 3 2010 11 50 33 100 009 99 772 99 764 99 936 o 35 5 3 2010 11 51 34 99 978 99 727 99 749 99 924 o 36 5 3 2010 11 51 35 99 975 99 746 99 771 99 912 o EFA 5 3 2010 11 51 36 99 997 99 741 99 744 99 904 o 38 5 3 2010 11 51 37 99 981 99 753 99 773 99 905 100 39 5 3 2010 11 51 38 99 968 99 806 99 838 99 897 o 40 5 3 2010 11 51 39 99 935 99 762 99 826 99 906 o 41 5 3 2010 11 51 40 99 888 99 795 99 907 99 907 o 42 5 3 2010 11 51 41 99 869 99 816 99 948 99 911 o 43 5 3 2010 11 51 42 99 867 99 821 99 954 99 911 o 44 5 3 2010 11 51 43 99 867 99 844 99 978 99 899 o 5 3 2010 11 51 44 99 884 99 877 99 993 99 9 o 46 5 3 2010 11 51 45 99 881 99 816 99 935 99 897 o SZ 5 3 2010 11 51 46 99 853 99 777 99 923 99 894 o 48 5 3 2010 11 51 47 99 857 99 746 99 89 99 88 o 4 gt gt TextFile J 4 i Ready 30 Page MitoViewer User Manual Prizmatix Appendix A Real time evaluation of mitochondrial function by monitoring NADH redox state using the MitoViewer The MitoViewer MV 2 is based on principles and technologies that have emerged during the last five decades and the user is strongly advised to read a few publicat
13. cence Fluor from the tissue to the Detection Unit Detection Unit provides detection to transform the Refl and Fluor light into electrical signals which are transmitted to the electronics board for amplification Electronics Unit provides amplification for the Refl and Fluor signals as well as control of the detector gain and driver for the UV LED The amplified signals are transmitted to Analog to Digital convertor module USB 6009 module provides Analog to Digital A D conversion for the Refl and Fluor signals and Digital to Analog D A conversion for control signals sent from a PC to control the function of the Mito Viewer PC Personal Computer enables control of the MitoViewer operation through the MitoViewer software Power Adaptor provides the DC voltage for operation of the MitoViewer PMT1 Refl Signal PMT1 Gain Signal Detection National Fiber Unit Instruments Optic 7 PERE z USB 6009 Far I E Light UV LED Current D A A D l Source module I l l Unit PD Signal l l l Power l EEREN ENEE ee i i Adaptor Tissue MitoViewer subunits diagram 8 Page MitoViewer User Manual Prizmatix The front panel features SMA optical connectors for excitation and collection branches of the fiber optic probe and the main power ON OFF switch The yellow LED indicator above the Light Source Unit optical output connector is turned ON when the UV light is emitted during measurement
14. e due to the compensation of blood via the circle of Willis Only after reopening two of the carotid arteries the signals recovered to the baseline levels 35 Page MitoViewer User Manual Prizmatix iii 5 ATT 7 A ii 14 41 38 MAIDO M00 144500 144500 1447000 148800 i4300 145000 MSN BE E Fig B3 Effect of Ischemia Occlusion of 2 carotid arteries The effects of deep anesthesia are presented in Fig B4 compared to a ten minute control period shown in Fig B5 When the preparation of the monitored tissue is good it is possible to have very stable signals After injection of the Urethane the Fluor and the NADH signals was elevated slightly to a higher level probably due to decrease in oxygen consumption IP Injection of Urethane C i z C 3 MO IZ mt pe gt a E ee E IE p Fig B4 Effect of deep anaesthesia induced by IP injection of Urethane 36 Page MitoViewer User Manual Prizmatix Fig B5 Stability of signals during 10 minutes The effects of the increase in energy consumption shown in Fig B6 were induced by exposure of the brain to Cortical Spreading Depression by high level of potassium In the normoxic brain the oxygen supply is not limited and accordinly the Fluor blue and the NADH red decreased due to the oxidation of NADH Under this condition the ATP turnover was dramatically increased and the extra oxygen supply was provided by an increase in microcirculatory blo
15. er User Manual Prizmatix 10 During the Installation Process you will need to insert Disk 2 of 2 E NI DAQmx 8 7 1 Insert Source Please insert the disk labeled NI DAQms 8 7 1 Disk 2 If the required files may be found elsewhere please enter that location below EA iz The specified folder does not contain the correct volume Cone 11 At the end of the process following dialog box will appear Press Next to approve i Hi DAQm x 8 7 1 Inctatahon Complete The features m thie malalston have been succesiully updated You may be promoted bo rebel ater diirit this chads 12 The following dialog box appears Click Restart to reboot the computer M DAQmx 8 7 1 ray ou must restat pou computer io compbete thea operation wah aae ter acetal phan ar ara shat dove the computer If you ehoces bo restart Lote PEKA Golan AEA BAO warn ines axes cf Wile Aeee 16 Page MitoViewer User Manual Prizmatix Configuration of National Instruments USB 6009 device l hardware procedure The new hardware is National Instruments A D module called USB 6009 or USB After the software has been properly installed and the computer restarted connect the USB cable as follows plug the square Type B USB connector to the USB port on the back of the Mito Viewer MV 2 and the rectangular Type A USB connector to the USB port in your computer The computer will identify the new hardware and will start with Found
16. factor in achieving good NADH results is the stable contact between the fiber optic probe and the analyzed tissue This goal is more easily achieved in brain monitoring and therefore the typical results of this organ will be presented Total backscattered light reflectance signal Due to the scattering of excitation light by the tissue some small part of the excitation light is reflected back to the tissue surface collected by the collecting fibers and transmitted to the photodetector This light intensity is referred to as backscattered or reflectance signal A certain amount of light is absorbed by the tissue primarily due to hemoglobin The reflectance signals are inversely correlated to the level of light absorption by the tissue Since hemoglobin is the main absorption element in the tissue upon blood elimination from the tissue the Reflectance signal increases dramatically Conversely when the tissue becomes more perfused with blood Reflectance decreases Accordingly Reflectance 32 Page MitoViewer User Manual Prizmatix provides information regarding blood volume in the tissue This feature of the Reflectance signal also helps to correct artifacts in the NADH fluorescence signal due to changes in tissue blood volume When the blood volume increases the Reflectance and the Fluorescence signals decrease even when the oxygen levels remain the same and vice versa Various algorithms including F R and F R have been used for correc
17. fficacy monitored in vivo in various organs Organ transplants for on mitochondrial function during the postoperative period 4 Page MitoViewer User Manual Prizmatix Health and Safety Before applying power to the power adaptor of the system ensure that the protective conductor of the three conductor mains power cord is correctly connected to the protective earth contact of the socket outlet Improper grounding can cause electric shock with implications for your health or even death Prizmatix products are NOT authorized for use as components in life support devices or systems The MitoViewer MV 2 is for use as laboratory equipment only It is not cleared or authorized for clinical use The MitoViewer must not be operated in explosion endangered environments Any maintenance shall ONLY be performed by a technician authorized by Prizmatix Cellular phones or other radio transmitters should not be used within the vicinity of 3 4 meters of the unit The MitoViewer MV 2 emits UV and other intense light Intense UV light can be emitted by the system and from the probe tip during operation Precautions must be taken to prevent looking directly at the UV light with unprotected eyes or illuminating the UV light on skin Do not look directly into the UV light through the optical system or the probe tip during operation of the device This can cause permanent damage to the eyes even if only exposed for brief periods Keep the metal ca
18. ig B2 shows the responses to 2 events affecting the oxygen levels in the brain in opposite directions In the left side the rat was exposed to hypoxia mixture of 6 oxygen and 94 nitrogen while in the right side of the figure to hyperoxia 100 oxygen As expected under hypoxia the Fluor signal and NADH were moderately elevated compared to anoxia and the Refl signal shows the same direction as in anoxia When the rat was 34 Page MitoViewer User Manual Prizmatix exposed to hyperoxia the NADH became more oxidized decrease signal and the Refl signal was elevated due to decrease in blood volume in the tissue 6 Oxygen ar 100 Oxygen ar 200 m rr E a ee ee ee ee 0 ee A ee an a v F o E ee 2 ee ee E ee ee gs ae ee es es se aO O N Tenn ee T T ee ee S PoE A A d eee ee w r d ee a E ee eee a hore TTT ee ee _ EE ee ee ee ee ee ee E E E E E E eee S ee ee Li I iidol en 1312000 izio 131400 15900 13 1600 131700 131000 119000 1k Tite Fig B2 Responses to Anoxia 100 nitrogen In Fig B3 the effects of partial ischemia occlusion of right and left carotid arteries are presented As expected the blood volume decreased momentarily due to the occlusion as seen in the increase of the Refl signal The Fluor and the NADH signals were initially elevated to high levels followed by a spontaneous partial recovery Under this situation the ischemia is not complet
19. ions before starting to use device routinely The broad spectrum of publications on NADH monitoring was reviewed most recently by Mayevsky and Rogatsky 2007 Several other articles appear in the list of publications attached to this document Accordingly only few significant points will be discuss in the following paragraphs Introduction Only the reduced form of NAD 1 e NADH absorbs UV light at nearly the entire UVA spectrum area 315 400 nm and fluoresces at 420 480 nm A small part of the excitation light delivered to the tissue by the excitation optical fiber will be absorbed and re emitted as fluorescence by NADH Any change in the redox status of NAD NADH will be reflected in the intensity of the emitted fluorescence while the more reduced state NADH shows grater intensity of fluorescence than the oxidized state NAD The redox state of NADH and hence the fluorescence depends on the availability of oxygen as well as on the rate of ATP turnover ADP availability When ATP consumption is constant a decrease in intracellular O2 shifts the redox potential to a more reduced state thereby increasing NADH and fluorescence intensity When the O2 level is 0 NADH accumulates to its maximal level On the other hand when O2 is not a limiting factor any increase in ATP turnover ADP availability induces a shift in the mitochondrial NADH redox potential to a more oxidized state thus decreasing the fluorescence signal intensity
20. nected to the Detection Unit port in the front panel of the Mito Viewer General Specifications Total length 100 cm 10cm 0 Common length 80 cm 5cm 0 Fibers Plastic optical fibers 750um NA 0 5 Number of excitation fibers 1 Number of detection fibers 6 Connectors SMA 905 x2 Tip Stainless steel Tube 12G 0 109 OD length 20mm Maximum temperature 70 C Sterilization method Ethylene Oxide gas Do not apply Autoclave sterilization Biocompatibility Materials in contact with the tissue are known to be inert to the tissues However ISO 10993 or USP VI tests were not performed on the probe to establish this claim 10 Page MitoViewer User Manual Prizmatix Additional equipment not provided with the MitoViewer 1 PC with Windows XP Vista 7 operating system 2 Methods and means for stabilizing the probe on the monitored tissue may require special mechanisms or procedures Consult Prizmatix regarding your specific application 11 Page MitoViewer User Manual Prizmatix Software Installation The software installation is performed in two steps 1 Installation of the National Instruments software e NI DAQmx Driver e Measurement and Automation Explorer 2 Setup of Prizmatix MitoViewer software Installation of the National Instruments software 1 Insert the National Instruments installation CD 1 of 2 of NI DAQmx into the CD drive 2 The auto run will start the setup software please follo
21. oViewer User Manual Prizmatix 11 Click on Self test icon to check the functionality of the NI USB 6009 OEM X HUSB 6009 Dew Moasurement amp Automation Explorer Tet Panels Qi Peret Dete i Creste Tack My System T aaua Gex eal 5 Ery fee oe bimi eg M DAGms Devies NI Bi NDUS8 6009 Devi DAQm x Device E FAI Px Sytem ureden i E Er Basics JG Software SQ i Drivers bs o Remote Systems What do you wani lo do e Byn fre HHA noe I eat Fon ole 12 A report message should appear The device has passed the self test 13 Close the Measurement amp Automation Explorer 14 Tip another option to access the Measurement amp Automation Explorer is by clicking the following icon on the desktop ww gt Measurement amp AUTOMAton 19 Page MitoViewer User Manual Prizmatix Installation of the Prizmatix MitoViewer software 1 Insert the Prizmatix MitoViewer software installation CD into the CD drive 2 Run the setup exe to initialize the installation process data 27 085 KB install l IA Inisthsi Windows Installer Package Installer For the Windows Inst 2 108 KE Microsoft Corporation EA Insthsiv setup Installer For the Windows Inst SI Installer Bootstrapper Microsoft Corporation Yational Instruments setup Configuration Settings 1 KE 3 The following dialog box will appear ie Mito iewer_1 017 Setup 15 x Welcome to the Mito
22. od flow 50 E I l F E 1 E I a 15 37 19 1 3900 154000 184100 15400 I54 00 1500 154500 154600 15047518 Fig B6 Effect of Cortical Spreading Depression SD induced by topical application 0 5 M KCl 37 Page MitoViewer User Manual Prizmatix References Chance B et al Science 137 499 508 1962 Mayevsky A and Chance B Brain Res 98 149 165 1975 Mayevsky A and Chance B Science 217 537 540 1982 Mayevsky A Brain Res Rev 7 49 68 1984 Mayevsky A Mitochondrion 9 165 179 2009 Mayevsky A and Chance B Mitochondrion 7 330 339 2007 Mayevsky A and Rogatsky G G Am J Physiol Cell Physiol 292 C615 C640 2007 38 Page MitoViewer User Manual
23. owing screen will appear Main screen of MitoViewer software The main screen comprises the following controls and indicators l Se ee ee E Main graph area displaying the Refl Fluor NADH and PD parameters as function of time Current numerical values of Refl Fluor NADH and PD Control buttons Marker Normalization Clear Chart and Stop LED power level control panel Refl PMT detector control panel Fluor PMT detector control panel Detectors Values indicator and smoothing control panel Auto Ranging feature control panel 24 Page MitoViewer User Manual Prizmatix In order to start measurement the following steps shall be performed l At LED Power panel press ON button to enable the UV excitation light and start data acquisition Increase the LED power level control slider close to the level indicated by the yellow arrow Watch the LED power level at the Detectors Values indicator panel LED PD At the start of the measurements the LED PD value will be within the range of 3 5 to 4 5 Gradually increase the Refl PMT1 gain control slider while watching the PMTI1 Refl indicator at Detectors Values indicator panel At this initial stage of the measurements the PMT1 Refl value will be about 4 If the PMT1 Refl signal is too low or too high the LED indicator will change to red If the signal level is within acceptable limits the LED indicator will change to green Gradually increase the Fluor PMT2 gain con
24. ps of optical units closed unless the fiberoptic probe is connected 5 Page MitoViewer User Manual Prizmatix Set Up of the Device Remove the device from packaging and inspect the device for lose components or any sign of damage Notify Prizmatix if the device appears damaged in any way do not install damaged device Package List MitoViewer MV 2 Fiberoptic Probe D Software Installation CDs x3 USB A to USB B Cable 6 Page MitoViewer User Manual Prizmatix ltem_ Description Y SCQTTY Mitochondrial Function 1 Monitoring System with stainless steel straight tip Rien eens oo Installation CDs software and drivers Cable to connect the MitoViewer MV 2 to Ot the computer mains voltage 6 Power Adaptor Universal power adaptor Fiber Optic MitoViewer Probe MIV 2 Power Adaptor a ee USB Cable Tissue The MitoViewer setup 7 Page MitoViewer User Manual Prizmatix System overview The MitoViewer system comprises the following main subunits Light Source Unit provides UV light at 365nm for NADH fluorescence excitation and tissue reflection measurements Also included is a reference photodiode that enables correction of signals in cases of intensity changes of the LED during measurement Fiber Optic Probe transmits the UV light from the Light Source Unit to the measured tissue site and transmits the collected light the reflection Refl and the fluores
25. the Back button to reenter the installation information or click Cancel to exit the wizard Cancel 21 Page MitoViewer User Manual Prizmatix 6 Press Next to approve the installation of the software The following dialog box will appear showing the process of the installation fe Mito iewer_1 017 Setup Updating System The features you selected are currently being installed pmanmimmnini 7 At the end of the process the following dialog box will appear Press Finish to approve ie Mito iewer_1 017 Setup Pm x MitoViewer_1 017 has been successfully installed Click the Finish button to exit this installation Lead ittSh i Parcel 22 Page MitoViewer User Manual Prizmatix 8 In order to run the MitoViewer software go to Start gt Programs gt MitoViwer gt MitoViwer as in following figure Poo fev Windows Explorer fh Programs b fan Games r Microsoft Excel A Documents gt I CCD Profiler gt mA National Instruments gt e Settings a IF Birdvision gt 22 Search fH Mitoviewer B gt Mitoviewer_1 017 Help and Support Run of P Log Off user Windows XP Professional Qj Turn OFF Computer start 23 Page MitoViewer User Manual Prizmatix MitoViewer Software Usage Connect all cables as described in the Set Up of the Device section Run the MitoViewer software as described at the end of previous section The foll
26. tion It is recommended to study the best correction technique for the specific monitored tissue although the two approaches can be applied in most cases For details see Mayevsky and Rogatsky 2007 33 Page MitoViewer User Manual Prizmatix Appendix B Typical Results while monitoring the Brain in vivo The fiber optic probe of the MitoViewer was attached to the surface of the brain via an appropriate holder cemented to the skull with dental acrylic cement We used rats 200 250 grams anesthetized and operated as discussed in the various papers appears in the attached list Fig B1 presents typical responses of the brain to complete oxygen depletion by exposing the rat to 100 nitrogen As can be seen the Fluor signal blue is elevated due to inhibition of the respiratory chain activity The Refl signal green is decreasing as expected under this condition due to the elevation in blood volume in the monitored tissue The corrected NADH signal black shows a symmetrical increase and decrease during the anoxic cycle _ EE ee ee ee ee eee eal E A rl a nee OR a A o o ae a ee ee ral A ia eS ee Ee eS See i O O A eee eee S S S S O W o 8 S y y y ED EE Ee ee ee eee d eee M eS RN ERS a ea OOO To WMO T ee T T ee eee eee oe 13 50 00 13 51 00 13 52 00 13 53 00 13 54 00 13 55 00 13 56 00 13 57 00 15 58 26 Fig B1 Responses to Anoxia 100 nitrogen F
27. trol slider while watching the PMT2 Fluor indicator at Detectors Values indicator panel At this initial stage of measurement the PMT2 Fluor value will be around 4 If the PMT2 Fluor signal is too low or too high the LED indicator will change to red If the signal level is within limits the LED indicator will change to green Detectors Values Ass emiren d 0 976 PHT2 Fle d 3 78 LED Po srw a i 25 Page MitoViewer User Manual Prizmatix 6 Press the Normalization button to normalize all signals to 100 The Normalization event will be marked by the marker ie E E Prizmatix MitoViewer 7 In order to clear unwanted traces from the chart use the Clear Chart button This will clear the chart area and the chart drawing will start from the left side of the graph The data in the file will not be influenced from this operation 26 Page MitoViewer User Manual Prizmatix 8 In order to mark important events during the measurements use the Marker button on the right side of the main screen The marker will appear at the chart screen and on the stored data file 9 Scaling of the chart can be performed by simply clicking on the upper or lower scale and changing the maximum or minimum value of the scale 27 Page MitoViewer User Manual Prizmatix 10 In order to enable continuous measurements without detector saturations or too
28. uage wersions than previous releases of NI DAQ aang Wen vinan rA DAQ device support excludes V gt DAO Remete 5O41 1200 Seres DAG the DAQCard S00 700 fariu parallel pot DAD Pads and seve al other older devicss Do not install HI DAG 7 0 or laterd your icing applications include unsupporled components Peler to the HI DAO Readme he on the ditribution 5 Press Next and the following dialog box will appear amp NI DAQmx 8 7 14 Destination Drectory Select the pimay inatalston dracio Browse button and peleci another folder ou can peleci To change the default folder for incialing Natonal Incinuments software cick the indradual component locators in the need dag Derman Daectoy C Program Files National Instruments 13 Page MitoViewer User Manual Prizmatix 6 If you would like to install the National Instruments software into different location use the Browse button to change the installation location Press Next to continue the setup Next the following dialog box will appear EE Hi NAQm s f_7 1 Features Celect Heap joshet bo rriei gt NI DAQems 6 7 1 NI DAQme the nent generation HIDAQ diver wit LabiEW 6 5 Support Teve Y E E OaE ay yll x LabVIEW 8 2 Suppo Programa pout sb ea Labview 805 feature and advantage over the previous vernon LabVIEW 7 1 Suppo LabviEW Riesl Time Support eS vg Lawson tt Sco B gt S Labi Real Time Su Measurement Studes for Voua C Tha feature v
29. w the instructions that appear on the screen 3 Click Install Software in the following dialog box Install Software Minstruments View ReadMe File Browse CD Exit NI DAQ Data Acquisition Driver Software ni com dataacquisition 12 Page MitoViewer User Manual Prizmatix 4 Following dialog box will appear a H1 DAOm x 8 7 1 Product Information Pieace road the following infomation about lhe products lo ba misled HI DAQ 8 7 Device Supporl ADAG me 8 7 supports the latas DAO devices including C Senas H Sanes E Senes 5 Senes B Senas USE Sene Analog Output avefom Counte Timer Digital 10 NI 653x DSA SOI and SCC dewees The Last version of NI LAQme to suppor the PCI 704 1 8006 device is 8 1 F you need support lor this dewice donot instal the version of NI DAGm Rieter lo the NICAL Readme tor supporled device models Trodtiona NI DAQ Legacy is an older diwar wath an outdated API tha support tome addtional oder DAU devices but not ai of the latest DAJ deviens developed with earler versions of NI DAQ will tun under Traditional MI DAQ Legacy You can us both NI DaOma and Tneditional NI D40 Legacy in the seme computer but musi rset dences before changing from Traditional NI DAO Legacy to NI D4Qimo Refer to the NI DAQ Readme lor guidelines Support Removed ham M DAD 7 0 amd later NI DAQ 7 0 and latet supports diferent devices operating systems aophcabon software and programming lang
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