Home

Mithras - Phenix Research Products

1. Figure 5 12 Connecting reagents to the injectors O Then the selected injector tubings are primed with 12 shots The Injector Sequence dialog box shows the respective in formation Wait for the prime process to finish Figure 5 13 BERTHOL CHNOLOGIES LB 940 Mithras 5 Mithras Software Functions Injector Sequence x Injector Prime Sequence Injecting Reagent Please wait for cycle to complete Injectors Injector 1 M Injector 2 M Injector 3 Cancel Next gt gt Figure 5 13 Prime injector tubings by 12 shots O The end of the prime cycle is displayed on the screen and the lt Close gt button is enabled Click lt Close gt to complete the process and to exit the Injector Sequence dialog box O If you are working with a prime plate this plate is automati cally moved out as soon as you click lt Close gt Remove the prime plate Figure 5 14 x Injector Prime Sequence Injector Prime Sequence Finished Please remove plate prior to running a new measurement Press Close to Exit Injectors M Injector 1 M Injector 2 M Injector 3 Cancel Figure 5 14 Finishing the prime process BERTHOLD 5 Mithras Software Functions LB 940 Mithras 5 4 4 Instrument Wash Injector tubings have to be washed whenever you are changing reagents as well as before starting and after completing your work Use the solutions recommended by the kit manufacturer Use a
2. OORROL r Operation Mode Batch Plate Repeats fi r Temperature Control Je Temperature 25 Celsius Abbrechen ibemehmen Figure 5 29 Measurement tab Shows the available operations Steps that can be selected are identified by a checkmark You can select only those steps that are supported by the instrument and the pre selected meas urement mode For example if you did not select any wells for injection on the Samples tab the Dispense step does not have a checkmark This ensure that the measurement sequence does not include any operations that cannot be carried out Operations required for the measurement sequence are copied from the left column into the center column and arranged in the desired order Individual operations can be copied for the measurement sequence and can be moved to any position 5 Mithras Software Functions LB 940 Mithras Click on an operation to show the respective parameters in the Operation column Double click on an operation to open the associated parameter dialog box to edit the parameters Copy To copy an operation to the center column double click on the respective operation in the left column This opens the parame ter window Enter the required parameters and confirm the en tries with lt OK gt The window is closed and the operation is en tered in the center column behind the last one chapter 5 5 5 Select order In the Operations col
3. Injectors M Injector 1 M Injector 2 M Injector 3 Figure 5 16 Loading the wash solution O Then the selected injector tubings are washed using the number of wash cycles defined in this dialog box The Injec tor Sequence dialog box displays a corresponding mes sage Wait until the process is finished Figure 5 17 Injector Sequence E Injector Wash Sequence Injecting Wash Solution Please wait for cycle to complete Number of Wash cycles 10 Injectors WK Injector 1 M Injector 2 M Injector 3 Cancel Next gt gt Figure 5 17 Washing the tubings BERTHOLD 5 Mithras Software Functions LB 940 Mithras O The end of the wash cycle is displayed on the screen and the lt Close gt button is enabled Click lt Close gt to complete the process and to exit the Injector Sequence dialog box O If you are working with a prime plate this plate is automati cally moved out as soon as you click lt Close gt Remove the prime plate Injector Sequence d x Injector Wash Sequence Injector Wash Sequence Finished Please remove plate prior to running a new measurement Press Close to Exit Number of Wash cycles 10 Injectors W Injector 1 M Injector 2 M Injector 3 Figure 5 18 Completion of wash process BERTHOLD ECHNOLOGIES LB 940 Mithras 5 Mithras Software Functions 5 4 5 Instrument Refresh The function Instrument Refresh may be used to refill re agent
4. MESPO01 f Sample 2 Firefly 3 Renilla Ratio Figure 5 50 Matrices in Calculation child window Note The substrate for the firefly reaction is added manually outside the instrument BERTHOLD LB 940 Mithras 5 Mithras Software Functions Readit measurements READIT PAR Injectors Injector 3 reading position Well selection All wells for measurement and injection Measurement sequence Injection 100 ul high speed by well Measurement 1 second counting time Measurement mode by well Note The measurement is carried out sequentially using 3 different microplates After measurement of a plate this plate is moved out unload it and load the next microplate In addition the measurement data is automatically entered into the EXCEL spreadsheet tier2master xls Calculation child window Sample POS position of cavity Value 1 raw data of measurement 1 Value 2 raw data of measurement 2 Value 3 raw data of measurement 3 MESP002 MESP002 MESP002 MESP002 MESP002 J H f Sample 2 Value1 3 Value2 Value3 Figure 5 51 Matrices in Calculation child window BERTHOLD as 5 Mithras Software Functions LB 940 Mithras 5 6 Measurement and Evaluation 1 Prepare instrument O If necessary connect wash solution and wash tubings In strument Wash see chapter 5 4 4 O Connect reagent bottle and prime tubings Instrument Pri
5. i Eluh teal jonexp Pesme feo SSS F es of lype Mikov Para Fie t pa i Cancel 4 Click on the Read button A bar with input fields and but tons appears below the displayed matrix Q The name of the parameter file used is displayed in the Read bottom row Flensme s Swn Gove Click to open the parameter file and you can edit aE the default settings etc File name enter a file name for the data Click lt Start gt to start the measurement after you have entered a file name for the data Name of data file Name of parameter file 5 Enter file name for the data 6 Click lt Start gt BERTHOLD 2 Quick Reference Guide LB 940 Mithras Oe r Coenin toe aed 7 The plate tray opens On the screen appears the prompt a to insert the microplate to be read i wie 4 Insert plate A1 at the rear left and click lt OK gt The plate is moved into the instrument and the pre defined measurement sequence is started 10 During measurement the data already available and the calculations can be viewed on the matrices of the menu Results 11 Upon completion of the measurement the plate stays in the instrument to protect it from external influences To unload it chose the command Unload Plate on the In strument menu 12 If Batch has been pre selected the system expects the ES stent en BO next microplate which is being read using the same pa rameters Insert plate and click lt OK gt 13
6. Excitation Filter F355 Umbelliferone Slot 41 X Excitation Aperture Small Normal Emission Filter F460 Umbelliferone Slot 41 X Counter position Top C Bottom Horizontal steps fi 1 10 Vertical steps fi 1 10 Paint displacement fo 1 0 1 50 mm Scanning mode Rectangular Round Figure 5 40 Scan properties dialog box Name of scan Define individual counting time Enter a value between 0 and 65 535 This range corresponds to 0 to 75 Watts and is divided in digital steps Select the excitation filter The pre defined filter with name and position in the filter slide is displayed Select the width of the excitation beam small 1 2 mm normal 2 0 mm Select the emission filter to be used The pre defined filter with name and position in the filter wheel is displayed Select if a fluorescence measurement should take place at the top or bottom Please keep in mind injections in reading posi tion for measurement at top is possible only with injector 4 Number of horizontal steps direction 1 12 Number of vertical steps direction A H Step width distance between data points Selectable dis tances 0 1 to 50 mm 5 Mithras Software Functions LB 940 Mithras Scanning mode Rectangular or round can be selected We recommend to se lect round for microplates with round wells since then the data points in the corner are automatically skipped During sc
7. Injector tubings have to be primed O prior to each measurement using the respective reagents A prime plate has to be used for the functions Prime tub ings Wash tubings and Refresh if no waste pump is in stalled For injector 4 please keep in mind This tubing can be primed washed or refreshed in single steps A prime plate always has to be used since a possibly installed aspiration pump cannot reach this position for technical rea sons 5 Mithras Software Functions LB 940 Mithras 5 4 1 Instrument Load Plate Select Instrument Load Plate to transport the plate slide into the instrument and to close the plate tray The instrument is closed light tight Open the plate tray only to load microplates so that as little light as possible may penetrate the counting chamber CAUTION If necessary allow for a delay between plate loading and start of measurement programmed in the measurement sequence since white microplates in particular may be phosphorescent after having been exposed to daylight 5 4 2 Instrument Unload Plate Select Instrument Unload Plate to move the plate slide out of the instrument and to unload a microplate Leave plate tray open only for loading microplates BERTH LD LB 940 Mithras 5 Mithras Software Functions 5 4 3 Instrument Prime NOTE CBERTHOLD Injector tubings have to be washed whenever changing re agents see chapter 5 4 4 and then primed with the new re
8. KTMA MES KTMA MES KTMA MES KTMA ME CT saroe 2 mega 3 Pea 2 TS Figure 5 45 Matrices in the Calculation child window BERTHOLD LB 940 Mithras 5 Mithras Software Functions Fluorescence repeated measurement Fluorescein Rep10 par Injectors Injector no 4 Well selection All wells for measurement and injection Measurement sequence Injection 100 ul low speed by well Measurement 0 1 second by well Lamp energy 7000 Excitation filter no 2 Emission filter no 2 Counter position Top Cycle time 30 seconds Total time 300 seconds Luminescence measurement Lumi1s PAR Injectors Injector 3 reading position Well selection All wells for measurement and injection Measurement sequence Injection 100 ul middle speed by well Measurement 1 second by well Emission filter no 8 Calculation child window Sample POS position of cavity Reader raw data Result average of matrix 2 Reader H AVE MA2 AVE MA2 AVE MA2 AVE MA2 Tsang 2 Fess Figure 5 46 Matrices in Calculation child window BERTHOLD 111 5 Mithras Software Functions LB 940 Mithras Luminescence measurement without injection Lumi_01s PAR Well selection All wells for measurement and injection Measurement sequence Measurement 1 second by well Emission filter no 8 Calculation child window Sample POS position the cavity Reader raw data Result average of ma
9. Results are presented in the Graphics window BERTHOLD LB 940 Mithras 3 System Description 3 System Description 3 1 Overview Fluorescence measurements Luminescence measurements FRET BRET The Mithras LB 940 is a Multilabel Reader for various fluores cence and luminescence measurements on microplates Up to 4 injectors may be used The strength of Mithras is its versatil ity as well as the fact that this instrument has been optimized for every measurement mode and therefore supplies excel lent results Clear device design simple handling as well as in strument control and data evaluation by the MikroWin2000 software make Mithras an indispensable measuring system in the research laboratory Fields of application high sensitivity due to improved optics highest sensitivity due to optimized optics measurement of the energy transfer between 2 fluorescent molecules measurement of the energy transfers from a luminescence re action to a fluorescent molecule An extremely low noise photomultiplier is used for these meas urements Highest sensitivities can be obtained due to using of single photon counting technology The reading accuracy is fur ther enhanced by using special methods to reduce crosstalk light transfer between neighboring samples The kinetics of fast luminescence and fluorescence reactions can be traced accurately by using ultra fast amplifiers Optimization for various measurement modes
10. buttons Select Operation Mode if needed Batch In this mode several plates can be read in succession using this proto col Plate Repeats The same plate can be read several times in succession Enter the num ber of repeats in this box Define temperature if your instrument includes a tem perature control input option 15 45 C Please note You can enter a target temperature in the instrument which is at least 5 C above room temperature i e if the room temperature is 20 C the minimum instrument temperature is 25 C Confirm entries with lt OK gt Options Samples Measurement r Measurement Sequence J 3 Shake Operations Dispense Dispense Luminometry Label inescence K Luminometry Kinetic S Luminometry Scannit R Luminometry Repeat Fluorometry Label K Fluorometry Kinetic Fluorometry Scannin R Fluorometry Repeate READIT TM OKERE KEK KK Bea Operation Mode I Batch Plate Repeats f1 r Temperature Control Celsius Options Samples Measurement Measurement Sequence MI SS Shake M Dispense M Luminometry Label M K Luminometry Kinetic OOO Operations Name Luminescence Counting Time 0 10 fak Delay Dispense O Luminometry Scannit O R Luminometryy Repeat m E3 Fluorometry Label M K Fluorometry Kinetic O S Fluorometry Scannin O
11. 19 73 Positioning Emission filter wheel Evaluation Evaluation parameters Excel file Excitation filter Clean Replace Excitation filter slide Excitation halogen lamp Export Export driver Export parameters Export Parameters Definition Export setup F Fan File Export File Export Setup File Open File Save Filter name input Fluorescein kin10 par Fluorescein Rep10 par Fluorescein par Fluorescence measurement Parameters Fluorescence measurements FRET Fuse Fuse replacement G Getting started H Hardlock Height adjustment of optics High voltage generation I Injection Injector configuration Injector positions 75 24 27 34 73 17 116 106 13 52 10 19 72 72 23 34 30 56 13 51 13 108 33 30 33 56 57 56 58 78 79 70 74 110 111 110 98 19 19 32 119 37 24 24 29 26 8 Appendix LB 940 Mithras Injector tubings Injectors Installation Installation Driver Instrument Boot Instrument Instrument Emission Filter Wheel Instrument Excitation Filter Slide Instrument Load Plate Instrument Prime Instrument Refresh Instrument Unload Instrument Unload Plate Instrument Wash Instrument control K Kinetics Kinetics measurement Parameters Kinetics measurement Fluor Parameters Kinetics measurement Lumi Parameters L Label LED Line Ascii Export Driver Load plate Lumi 1s Kinl0 PAR Lumi 1s Rep10
12. 42 C Operation modes Integral measurement 0 1 600 second Kinetics measurement total length up to 24 h Repeated measurement total length up to 24 h Plate repeats up to 999 Scanning up to 100 single data points Dispensing with 4 independent variable injectors Shaking 3 modes variable amplitude and speed BRET and BRET assays Dual luciferase reporter gene assay Readit protocol Delay up to 600 second CGBERTHOLD 7 Technical Data LB 940 Mithras Data evaluation Sample parameters Free plate loading samples standards controls Allocation of sample identifiers GLP documentation e g lot number Test kit validation formulas Evaluation Qualitative cut off determination with up to 3 categories Kinetics Linking of different types of evaluation Free input of calculation formulas Standard curve calculation only complete version Result display Presentation in multi matrix format or list format Graphical presentation of measured values Integrated statistics functions Export Output of all raw data and calculations manual or automatic in matrix or list format Output as a file network connection possible or to printer Robotic Integration Optional Ports RS232 9 pole Operating system Win98 Win2000 WinNT Win XP PC requirements Pentium processor 500 MHz or better CD ROM drive display 1024x768 or better serial port parallel p
13. Ber tholdTech Mithras with version number BertholdT ech Mithras Version 0 14 Corgon Instrument Configuration P Temperature Control Installed Gee I Dispenser Unit 1 installed sik eal IM Dispenser Unit 2 installed V Dispenser Unit 3 installed I Dispenser Unit 4 installed IV Fluorometry installed lV Luminometry installed P Z Mover installed Figure 5 2 Setup of device driver ComPort Define the PC port to which the Mithras instrument is con nected You may choose COM1 COM2 COM3 and COM4 Instrument Configuration Define the configuration of your Mithras instrument A selected item is identified by a checkmark v BERTHOL CHNOLOGIES 5 Mithras Software Functions LB 940 Mithras Temperature Control Installed Select this item if your instrument includes a temperature con trol Dispenser Unit installed Here you define which injector s is are installed This setting specifies how many injectors are controlled and are available in the software for definition of the measurement sequence Up to max 4 injectors can be installed at the same time Please note the position and function of the individual injectors Injector 1 Injection before reading position lumines cence Injector 2 Reading position luminescence Injector 3 Reading position luminescence Injector 4 Reading position fluorescence at top reading Fluorometry installed Select this item if t
14. Define the number of repeats per well Enter a value between 0 and 65 535 This range corresponds to 0 to 75 Watts and is divided in digital steps Select the excitation filter The pre defined filter with name and position in the filter slide is displayed Select the width of the excitation beam small 1 2 mm normal 2 0 mm Select the emission filter to be used The pre defined filter with name and position in the filter wheel is displayed 5 Mithras Software Functions LB 940 Mithras Counter position Select if a fluorescence measurement should take place at the top or bottom Please keep in mind injections in reading posi tion for measurement at top is possible only with injector 4 Use Injector 4 Enable injector 4 to perform an injection in counting position during the ongoing kinetics Injector 4 Cycle Define the cycle in which the injection should take place within the ongoing kinetics Volume Define the volume to be injected Inj 4 Speed Define the injection speed low middle high Operation Mode This function can be used in the plate or in the well mode Repeated operation allows optimized execution of kinetics measurements over longer periods of time Both operations Dispense and Repeated have to be used in the By wells mode if during a repeated measurement an injec tion triggering the reaction and immediately thereafter the first measurement should take place However if all selected wells are first to
15. Measurement Mode CBERTHOLD Name of repeated measurement Define calculated total length of measurement Define interval counting time of a repeated measurement per well Define the time of a reading cycle Define the number of repeats per well automatically calculated from the previous data and the selected number of wells Select the filter to be used Usually no filter is used for lumi nescence measurements therefore filter slot 8 No Filter is defaulted Enable injector 2 or 3 to perform an injection in reading position during repeated measurement Define the repeated cycle at which the injection is to take place within the ongoing kinetics Define volume to be dispensed inj 2 or 3 Define injection speed low middle high This function can be used in the plate or well mode Repeated operation allows optimized execution of kinetics measurements over longer periods of time Both operations Dispense and Repeated have to be used in the By wells mode if during a repeated measurement an injec tion triggering the reaction and immediately thereafter the first measurement should take place However if all selected wells are first to be primed with reagent and the measurement series is to start thereafter you have to set both operations Dispense and Repeated to the By plate mode 5 Mithras Software Functions LB 940 Mithras Fluorometry Label Fluorescence measurement Doubl
16. PAR Lumi_01s PAR Lumils PAR Luminescence measurement Parameters Luminescence measurements M Main window Mains plug Mains switch Maintenance Matrix Ascii Export Driver Matrix system Matrix XLS Export Driver Measurement Measurement sequence Definition Measurement tab Measurement with one injection 59 7 29 38 8 47 68 73 69 60 61 67 68 60 64 59 94 99 99 99 94 93 98 54 34 112 113 112 111 93 19 44 31 31 118 54 45 52 17 116 12 85 85 41 Measurement without injections Menu File Installation Instrument Options Read View Menu bar Menu overview MikroWin2000 Multilabel Reader N Navigation bar Noise O Operating voltage Optics Options Read P Parameter file Contents Open Save Save PC port PC requirements Photomultiplier Photon counter Plate matrix Plate tray Plate type Positioning filters Power supply Prime Prime injector tubings Prime plate Prime tubings Program start Promega readit measurements Q Quick Reference Guide R Raw data Reading parameters 40 43 33 34 34 34 33 33 44 33 46 33 44 24 6 36 19 11 80 11 15 45 79 78 79 108 49 32 19 24 24 82 18 22 81 71 6 36 34 17 59 17 22 61 64 67 61 44 45 77 44 77 93 98 BERTHOLD LB 940 Mithras 8 Appendix Reading positions of PM Readit measurements READIT PAR READIT measurement Reage
17. R Fluorometry Repeate O READIT TM Operation Mode Ubenenmen Abbrechen Operation Meas operation Emission Filter Dispense by Well 8 Tl Batch Temperature Control Temperature Plate Repeats 1 Celsius Abbrechen Ber BERTHOLD TECHNOLOGIES LB 940 Mithras 2 Quick Reference Guide Define evaluation parameters see chapter 5 5 6 Evaluation parameters are selected using the options on the navigation bar or via the tool buttons and defined on the respective matrices Generally these pre defined parameters hardly have to be modified for simple lumi nescence and fluorescence measurements Check the following matrices anyway and make the required changes You can add matrices to each child window or deselect these matrices Options Matrix and change the pa rameters for measurement and evaluation on each matrix by well by row or column or for the entire microplate The exact procedure is described in the MikroWin2000 User Guide 12 Select Data and enter results sample identifier error aa information and dilution factors on the matrices Data 13 Select Template to change the partition of the sample matrix and define Template controls and standards The other matrices are parti tioned accordingly 14 Select Calculation to define calculation formulas for the individual matrices Calculation 15 Select Results AF Results and calculated values are
18. Reference Guide is structured as follows e The Quick Reference Guide is divided into separate sections so you can choose those chapters that are impor tant to you getting started software installation new definition of counting parameters creating counting pa rameters on the basis of existing parameter files measurement and evaluation e In each section you are guided through the various procedures step by step These steps are numbered con secutively in each section Explanations on the individual steps are added in small type font e Explanations on the various types of measurement are highlighted specifically e For your convenience illustrations are placed directly next to the respective text 2 1 Getting Started see chapters 4 1 4 4 Unpack and set up Mithras Unscrew instrument cover and open it Unscrew transport safety screw fixing the photomultiplier and the plate slide and keep transport safety screw in a safe place see chapter 4 4 Do not take instrument into operation before you have removed the transport safety devices as otherwise severe damage may occur 4 Close instrument cover again and fix it with screws 5 Check if the power supply is within the permissible range of the operating voltage of the Mithras Connect instrument only if this is the case Install hardlock supplied on parallel port of PC Connect Mithras to serial port of PC Connect instrument to power using the power cord sup
19. The photomultiplier can be raised in order to remove the emis sion filter wheel To do this pull off the locking button on the right side of the photomultiplier see Figure 3 3 and 3 4a and b Three reading positions of the PM The photomultiplier with counting head is mounted on a guide rail 1 4 circular arc so it can be moved to the optimum posi tion depending on the measurement to be performed M1 For luminescence measurements and BRET M2 For fluorescence measurements from below clear plates M3 For fluorescence from above J3 J2 J1 M N aa J4 M3 M1 M2 M3 measurement pos 1 2 and 3 J1 J2 J3 J4 injector 1 2 3 and 4 Figure 3 4a Reading positions of PM and injector positions GERTHOLD 25 3 System Description LB 940 Mithras Locking button Toothed belt for height adjustment of optics Figure 3 4b Photomultiplier in bottom reading position Four injector positions see also chapter 3 2 6 Injectors J1 pre position luminescence J2 and J3 reading position luminescence J4 reading position fluorescence GBERTHOLD LB 940 Mithras 3 System Description 3 2 5 Emission Filter Wheel Photomultiplier raised cover plate accessible Cover plate removed filter wheel accessible CGBERTHOLD The emission filter wheel located directly below the photomulti plier can accommodate up to eight different emission filters The desired filter is moved to the respective
20. after the other to the Operations column in the left column double click on the desired opera tion and enter the parameters in the respective window O Confirm your entries with lt OK gt The respective operation is entered in the Operations column behind the operation de fined last O In the Operations column the selected operations are first displayed in the order of their selection Click the buttons t and to change the order click x to delete an operation O The parameters of the operation selected in the Operations column are displayed in the Operation column O Select the operation mode O Enter the temperature if the instrument includes tempera ture control and temperature control is needed for the meas urement sequence O Confirm entries with lt OK gt CBERTHOLD LB 940 Mithras 5 Mithras Software Functions 5 5 5 Operations and their Parameters By plate By wells Perform on first plate repeat only CBERTHOLD Double click on an operation in the left column to open the re spective properties dialog box Click lt OK gt to accept the entries and append the respective operation to the last operation de fined in the Operations column Double click on an operation in the Operations column to open the properties dialog box and edit the entries Click lt OK gt to accept the entries The measurement mode By plate or By wells has to be se lected for nearly all operations By plate means that
21. agent to ensure that the desired volume is already injected with the first shot Use a prime plate whenever you are working with the prime function if no waste pump is installed A prime plate always has to be used for injector 4 Proceed as follows O Select Instrument Prime to open the Injector Sequence dialog box Figure 5 10 O Select the injectors to be primed All 3 injectors may be se lected at the same time Injector Sequence x Injector Prime Sequence Select Injectors When finished press Next Injectors M Injector 1 M Injector 2 M Injector 3 Cancel Figure 5 10 Select injectors O If no waste pump is installed you are prompted to load a prime plate The plate slide moves out Load the prime plate and click lt Next gt The plate tray is closed and the prime plate is placed below the injectors Figure 5 11 5 Mithras Software Functions LB 940 Mithras Injector Sequence x Injector Prime Sequence Load Prime Plate When finished press Next M Injector 1 M Injector 2 M Injector 3 Injectors Figure 5 11 Load prime plate O You are prompted to load reagents in the selected reagent positions Figure 5 12 0 As soon as you have loaded the reagents correctly click lt Next gt Injector Sequence x Injector Prime Sequence pe Reagent in the Reagent Positions Selected hen finished press Next Injectors M Injector 1 W Injector 2 W Injector 3
22. displayed after meas Beads urement EE Define export parameters see chapter 5 3 16 Prerequisite Install and set up export driver see also chapter 5 2 3 17 Select Export The following functions are file specific R i e they are valid only for the active parameter file Export 18 In the File Export dialog box select the item you want x e g Matrix XLS Export Driver if you want to create an Aive Expor iver Excel file MatixxLSExportDriver3 x Cancel 19 Confirm your selection with lt OK gt Weta x5 Export Driver 2 P Help 20 To export the data automatically after measurement se hie to a ad lect File Export Setup in the main window The File File Name Browse Export Setup dialog box is displayed Directory nn ee Il 21 Select desired export driver It has to be the same as the Fie Export setup x one selected in the File Export dialog box List ot Active Export Diver List of export driver BERTHOLD 13 2 Quick Reference Guide LB 940 Mithras Save parameter file see chapter 5 5 2 As soon as the measurement sequence has been generated and all parameters mentioned above have been defined as needed save the data to a parameter file 22 Select File Save As 23 Enter file name extension PAR and confirm with lt OK gt BERTHOLD LB 940 Mithras 2 Quick Reference Guide 2 3 2 Editing Parameter Files In this case an existing parameter file is m
23. its name If the matrix is identified by its name you have to enter the exact matrix name e g Results Click on the matrix name and then on lt Edit gt The edit mode is enabled and you can enter the matrix name via the keyboard Click lt Delete gt to delete the selected matrix from the matrix list Footer Text box for entering a footer Click the lt Add gt button to open a context menu and select a placeholder for the footer You may select several options one after another and separate the placeholders either by a tab character TB or by a keyboard entry comma space etc This context menu includes the same options as the header context menu Export Directory Target Define the target directory for the file The program proposes the subdirectory Transfer in the MikroWin2000 program direc tory Click the lt gt button to select another directory The Look in dialog box is displayed and you can select the desired direc tory GERTHOLD ss 5 Mithras Software Functions LB 940 Mithras Matrix Ascii Export Driver Select this export driver to define an Ascii matrix file Parame ters header matrix and footer as well as the target directory for data storage are entered in the same manner as for an Excel file see previous section In addition you have to specify the Operation Mode settings Operation Mode Export only valid test If this item is selected data export takes place only if a test is valid Includ
24. layout in these three boxes Header Text box for entering a header Click on the lt Add gt button to open a context menu and select a placeholder for the header You may select several options one after another and separate the placeholders either by a tab character TB or by a key board entry comma space etc Matrix Name HMX Date DT Time TM Plate Identifier HPI Template Identifier T Tabulator TB Figure 5 5 Context menu for entering header placeholders Example Header with date time and plate identification separated by tab characters DT TB TM TB PI 52 BERTHOLD LB 940 Mithras 5 Mithras Software Functions Matrix In this text box you enter the matrices whose data you wish to export In general one exports only data from the result matri ces But you may also select any other matrices with provisional results Make sure that the matrix specified here is identical with the number of the result matrix in the parameter file You should always use the same matrix number for the results in all parameter files With respect to the supplied basic parameter files the results are typically found on Matrix 3 Click lt Add gt to open the context menu and select the matrix number 1 15 or define the matrix name Several matrices can be selected one after the other They are entered in the matrix list the Selection mode column shows if the respective matrix is identified by its number or by
25. located in the rear left corner Click lt OK gt to confirm the prompt Then the plate moves into the instrument below the photomultiplier and the se lected wells are read Select the Results dialog box to view the results on the ma trices Results may be displayed graphically printed out or ex ported e g as an Excel file 4 Getting Started and First Measurement LB 940 Mithras CBERTHOLD LB 940 Mithras 4 Getting Started and First Measurement 4 6 3 Fluorescence Measurement without Injections CBERTHOLD Qudauauda da Q Start MikroWin2000 software Select File Open Select file type par in the Open dialog box Open the basic parameter file Fluoreszein par Click the lt Read gt button in the main window Enter a file name for the data in the status bar The file name of the parameter file with the extension dat is defaulted Click the lt Start gt button The plate slide moves out and on the screen you see the prompt to load a microplate Place a prepared microplate onto the plate slide make sure A1 is located in the rear left corner Click lt OK gt to confirm the prompt The plate moves into the instrument below the photomultiplier and the selected wells are read Select the Results dialog box to view the results on the ma trices Results may be displayed graphically printed out or ex ported e g as an Excel file 5 Mithras Software Functions LB 940 Mithras 5 Mithras Softw
26. solution with one shot if the tubings have already been primed This function is used for example when the last meas urement took place some time ago and you are not sure if the tubing is completely primed Use a prime plate whenever you are working with the refresh function if no waste pump is installed NOTE A prime plate always has to be used for injector 4 O Select Instrument Refresh to open the Injector Se quence dialog box O Select the injectors for the refresh function O Click lt Next gt O The refresh function is carried out Injector Sequence e x Injector Refresh Sequence poeci Injectors When finished press Next r Injectors M Injector 1 M Injector 2 M Injector 3 Figure 5 19 Refresh function O If no waste pump is installed you are prompted to place a prime plate into the plate tray The plate slide moves out Load the prime plate and click lt Next gt The plate tray is closed and the prime plate is placed below the injectors CGBERTHOLD 5 Mithras Software Functions LB 940 Mithras 5 4 6 Instrument Unload Injector Select Instrument Unload Injector to pump reagents back into the bottle Make sure that bottles are connected to the respective in fectors 5 4 7 Instrument Boot Instrument If the Mithras instrument has been turned off and on again without having shut down the MikroWin2000 program you have to select the item Instrument Boot Instrument
27. the cover to have access to the inside of the instrument The cover is held in place by a gas pressure spring Figure 3 3 LB 940 Mithras with raised cover Inside the instrument you find the photomultiplier the emission filter wheel the electronics with the high voltage generation the injector tubings and tips the toothed belt for height adjustment of the entire optics and the microplate slide which is moved to the reading and injection positions during measurement The photomultiplier measures light with high sensitivity and low noise The spectral sensitivity is within a range that is suitable for all bio and chemiluminescence applications The photomultiplier operates as an ultra fast photon counter The photo electrons released from the photo cathode by the light quanta are multiplied via the dynode chain at the anode these photons trigger a fast pulse with a rise time of a few nanoseconds These counts are amplified by a very fast ampli fier Low energy single counts created by the noise of the pho tomultiplier are suppressed by a threshold discriminator The single counts are counted digitally their total number is directly proportional to the emitted quantity of light Gas pressure spring Photomultiplier in reading position 1 Guide rail for photomultiplier Injection tip of injector 4 GBERTHOLD LB 940 Mithras 3 System Description So called counts cts are used as unit of measure for the raw data
28. 1 Add program icon to the MikroWin2000 program folder gae oa xl Setup will add program icons to the Program Folder listed below You may type a new folder name or select one from the existing Folders list Click Next to continue 12 Click lt Next gt Installation is carried out and successful completion is indicated 13 Click lt Finish gt Program Folders Mikro J00 Existing Folders Brother HL 1650_1670N Series Caere Anwendungen Creative Hewlett Packard HP PhotoSmart MGI SoftDVD MAX Microsoft Office Tools xl as owes BERTHOLD o TECHNOLOGIES 2 Quick Reference Guide LB 940 Mithras 2 2 2 Driver Software Installation 1 Insert CD containing the Berthold Technologies driver software The installation routine starts automatically 2 Select language and confirm with lt OK gt The setup as sistant is started 3 Click lt Next gt in the Welcome dialog box The driver is sur automatically installed to the MikroWin2000 folder and the appropriate subdirectory Setup has finished installing Centro on your computer Setup can launch the Read Me file and Centro Choose the options you want below 4 Upon successful driver installation the Setup Complete dialog box appears To be able to work with the new software you have to restart your computer 5 Choose Yes restart computer and click on lt Finish gt Your computer is restarted and all program components are
29. 2 Driver Installation 47 5 2 3 Driver Setup 47 5 3 Export 56 GERTHOLD Contents LB 940 Mithras 5 3 1 Manual Data Export 57 5 3 2 Automatic Data Export 58 5 4 Instrument Control and Operation 59 5 4 1 Instrument Load Plate 60 5 4 2 Instrument Unload Plate 60 5 4 3 Instrument Prime 61 5 4 4 Instrument Wash 64 5 4 5 Instrument Refresh 67 5 4 6 Instrument Unload Injector 68 5 4 7 Instrument Boot Instrument 68 5 4 8 Excitation Filter Excitation Filter Slide 69 5 4 9 Emission Filter Emission Filter Wheel 73 5 5 Reading Parameters 77 5 5 1 Overview 77 5 5 2 Open Save Parameter File 78 5 5 3 Well Selection 80 5 5 4 Definition of Measurement Sequence 85 5 5 5 Operations and their Parameters 89 5 5 6 Definition of Evaluation Parameters in Child Windows 106 5 5 7 Definition of Export Parameters 108 5 5 8 Saving Parameter Files 108 5 5 9 Basic Parameter Files 109 5 6 Measurement and Evaluation 116 6 MAINTENANCE 118 6 1 Cleaning the Instrument 118 6 2 Cleaning Tubings 118 6 3 Fuse Replacement 119 6 4 Preparations for Transport 120 7 TECHNICAL DATA 121 8 APPENDIX 123 8 1 Index 123 Po CGBERTHOLD LB 940 Mithras Explanation of LED s and Beeps LED Instrument status lights up green Instrument OK and connection to PC OK lights up yellow Instrument OK no connection to PC flashes yellow 1 short beep New CAN is installed after power on of instrument lights up yellow 1 l
30. 40 Mithras has been manufac tured in accordance with the safety requirements for electronic and medical measuring systems If the law lays down regula tions on the installation and or operation of sample measuring system then it is the operator s responsibility to adhere to them NOTE NOTE The manufacturer has done everything possible to guarantee that the equipment functions safely both electrically and me chanically The user has to make sure that the instrument will be set up and installed properly to guarantee safe operation The instruments have been tested by the manufacturer and are supplied in a condition that allows safe and reliable operation O The instruments may only be operated by personnel who have been trained on the use of the system It is strongly recommended that all users read this manual prior to use O Remove the transport safety devices before you turn on the instrument O Use the instrument only for the designated application O BERTHOLD TECHNOLOGIES assumes no liability for any dam ages including those to third parties caused by improper use or handling of the instrument O The user is responsible for connecting the instrument in ac cordance with the valid regulations for electrical instruments O The instrument is provided with a 3 pole grounded plug If your wall outlet does not allow connection of a 3 pole plug have a suitable wall outlet installed by qualified personnel or use an adapt
31. CBERTHOLD O Depending on the selected measurement mode the pho tomultiplier may move to three different positions to get to the optimum reading position O The entire optics is height adjustable to compensate for dif ferent heights of the microplates In this manner the mi croplate is automatically moved up to the photomultiplier ensuring that there is no gap between photomultiplier and microplate O The instrument includes separate devices for excitation and emission filter with replaceable single filters 3 System Description LB 940 Mithras Moreover universal application of the Mithras is ensured by the following features O Free choice of plate formats 96 and 384 wells with auto matic setting of the optimum light path 6 12 24 48 and 1536 well plate formats are also freely selectable if the op tion automatic plate height adjustment is available O Use of up to 4 injectors with freely adjustable volume and adjustable injection force JET injection technology O Shaking functions O Temperature control optional O Barcode reader optional O Connection to lab automation systems optional O Comprehensive software for instrument control and data evaluation MikroWin2000 O In addition to the software basic parameter files are sup plied for various measurement modes kinetics measure ment and repeated measurement for luminescence and fluo rescence standardized luminescence and fluorescence measurement
32. ECHNOLOGIES 6 Maintenance LB 940 Mithras 6 Maintenance 6 1 Cleaning the Instrument Cleaning the instrument surface The surface of the instrument is protected by a washable finish Dirty or dusty surfaces should be cleaned using a damp cloth If necessary use a mild detergent Do not use a scouring agent Cleaning the inside of the instrument Before opening the instrument turn it off and disconnect it from power supply Open the screw on the instrument cover to clean the instrument inside Then raise the cover If necessary the photomultiplier can be raised as well Always keep the plate slide as well as the inside of the instru ment e g below the plate slide clean Wipe off any dirt using a damp cloth Use cotton buds for corners Remove dirt fairly quickly so it does not get dry and may not have any adverse ef fect on the movement of the plate slide 6 2 Cleaning Tubings Injector tubings have to be washed O before starting work O before changing reagents O at the end of each work session before turning off the in strument O after longer periods of inactivity Use solutions recommended by the kit manufacturer e g distilled water diluted alcohol hypochlorite solution Injector tubings have to be primed O prior to each measurement using the respective reagents Please refer to chapter 5 4 3 and 5 4 4 for more information on washing or priming the tubings CBERTHOLD LB 940 Mit
33. MikroWin Version 4 18 Mithras Software Version 0 05 Mithras Driver Version 0 16 July 2002 LB 940 Multilabel Reader Mithras Id No 38099BA2 Rev No 00 LB 940 Mithras Contents Contents 1 SAFETY INSTRUCTIONS 3 2 QUICK REFERENCE GUIDE 6 2 1 Getting Started 6 2 2 Installation of MikroWin and Driver Software 8 2 2 1 MikroWin2000 Installation 8 2 2 2 Driver Software Installation 10 2 3 Definition of Measurement Sequence and Parameters 11 2 3 1 Creation of New Measurement Sequence and Definition of Parameters 11 2 3 2 Editing Parameter Files 15 2 4 Measurement and Evaluation 17 3 SYSTEM DESCRIPTION 19 3 1 Overview 19 3 2 Reader Unit 21 3 2 1 Plate Tray 22 3 2 2 Excitation Filter Slide 23 3 2 3 Components Inside the Instrument 24 3 2 4 Photomultiplier 24 3 2 5 Emission Filter Wheel 27 3 2 6 Injectors 29 3 2 7 Excitation Halogen Lamp with Fan 30 3 2 8 Connections 31 3 2 9 PC 32 3 3 Software 33 3 3 1 Structure 33 3 3 2 Brief Explanation of Menus and Functions 33 4 GETTING STARTED AND FIRST MEASUREMENT 35 4 1 Setup Site 35 4 2 Space Required 35 4 3 Unpacking 35 4 4 Connecting 36 4 5 Software Installation 39 4 6 First Measurement 40 4 6 1 Luminescence Measurement without Injections 40 4 6 2 Luminescence Measurement with one Injection 41 4 6 3 Fluorescence Measurement without Injections 43 5 MITHRAS SOFTWARE FUNCTIONS 44 5 1 Software Structure and Operation 44 5 2 Installation 47 5 2 1 MikroWin2000 Installation 47 5 2
34. Name text box O Define usage of filter for fluorescence or luminescence measurement O Click lt OK gt in the Add Filter dialog box The dialog box is closed and the new filter name is listed in the Unused Filter text box in the Excitation Filter Slide dialog box O Delete a filter name highlight the respective filter name and then click lt Remove gt GERTHOLD LB 940 Mithras 5 Mithras Software Functions Positioning filters Depending on the position of the filter on the excitation filter slide allocate the respective filter names to positions 1 to 5 see Figure 5 22 In the parameter files only those filter posi tions are displayed for selection which are occupied here Only those filters can be used for measurements which have been defined in the software Therefore make sure that the allocation in the software matches the actual filter positions Proceed as follows O Prerequisite is that filter names are displayed in the Unused Filters text box see previous page o Drag the filter name from the Unused Filters text box to one of the positions 1 to 5 of the schematic illustration of the excitation filter slide O You may also drag a filter name from one position on the slide to another or from a slide position back to the Unused Filters text box Excitation Filter Slide xj Unused Filters F485 FITC Fluorescein All filters can be dragged from the Unused Filters list to an empty vd position i
35. Quick Reference Guide 2 3 Definition of Measurement Sequence and Parameters There are two alternative ways of defining the measurement sequence and parameters a b Measurement and evaluation parameters are defined completely new see section 2 3 1 An existing parameter file is used and the parameters are modified as needed Either you use the parameter files supplied by Berthold Technologies each includes a typical run for each type of measurement see sec tion 2 3 2 Or you use parameter files you have created yourself and edit this file accordingly Please note A saved parameter file includes selected wells individual operations and evaluation parameters e g calculation formulas 2 3 1 Creation of New Measurement Sequence and Definition of Parameters In the MikroWin2000 main window select the menu item Edit Reset Untitled par is displayed as tempo rary file name in the status bar Select Options Read The Options Read dialog box is displayed Select BertholdTech Mithras from the Device drop down list Click on lt Options gt The Options dialog box appears showing two tabs Samples and Measurement Well selection see chapter 5 5 3 5 On the Samples tab select plate type desired wells and measurement order by rows or by columns Select plate type 6 12 24 48 96 384 or 1536 wells In the row Plate Mode enable the item Measurement and then select the wells for the measurement Select d
36. Started and First Measurement 4 1 Setup Site The Multilabel Reader Mithras LB 940 has to be set up in dry fairly dust free rooms and protected from exposure to direct sunlight and significant temperature fluctuations It should not be set up next to a radiator or an air conditioning 4 2 Space Required Dimensions of LB 940 330 x 430 x 270 mm W x D x H Set the instrument up such that the rear panel with the connec tion ports is easily accessible so it can be turned on and off easily any time Allow for sufficient space on the side for the waste and liquid bottles reagents wash solution Do not set up the Mithras instrument close to the PC 4 3 Unpacking The cardboard box is reusable and should be used again when ever the instrument has to be transported When unpacking the instrument make sure the shipment is complete and shows no sign of damage The careful packing usually rules out transport damages Should the instrument or instrument parts be damaged anyway please inform the ship ping agent or the BERTHOLD TECHNOLOGIES service department immediately Unpack instrument and remove foamed inserts with care CAUTION GERTHOLD ss 4 Getting Started and First Measurement LB 940 Mithras 4 4 Connecting Remove transport safety screw Check if power supply is within the permissible range of the Mithras s operating voltage Connect instrument only if this is the case O Carefully take instru
37. The software initializes the instrument again and establishes correct communication between instrument and PC Otherwise instru ment control is not possible CBERTHOLD LB 940 Mithras 5 Mithras Software Functions 5 4 8 Excitation Filter Excitation Filter Slide BERTHOLD Definition and maintenance of the excitation filter used takes place via the menu item Instrument Excitation Filter Slide The following options are available O Enter the name and position of the filter used which are then available for selection when defining the reading pa rameters in the parameter files O Move the excitation filter slide in and out to change or clean the filter Select the menu item Instrument Excitation Filter Slide to open the Excitation Filter Slide dialog box Excitation Filter Slide x Unused Filters F485 FITC Fluorescein All filters can be dragged from the Unused Filters list to an empty position in the slide or from one position to an empty one or back to the list Excitation Filter Slide Positions Add Remove Eject Slide Cancel Add Remove filter Eject filter slide Figure 5 20 Control of excitation filter 5 Mithras Software Functions LB 940 Mithras Entering filter names Click lt Add gt to open the Add Filter dialog box Add Filter X Name I ag r Usage I Fluorometry T Luminometry Figure 5 21 Entering a filter name O Enter name of filter in the
38. a defined operation is first performed for all selected wells of the plate before the next step starts By wells means that all consecutive operations are first performed for the 1 well then for the 2 well etc If you choose Perform on first plate repeat only the respec tive operation is performed only once at the start of a series of repeat measurements see also Repeat 5 Mithras Software Functions LB 940 Mithras Delay before an operation Double click on to open the Delay properties dia log box and define the delay between two operations Delay properties x m Parameters Duration fl 0 1 3600 sec er ance m Measurement Mode C By plate By wells I Perform on first plate repeat onl Figure 5 31 Delay properties dialog box Duration Define delay between 1 and 3600 seconds Measurement Mode This function can be used in the plate or well mode BERTHOLD IES LB 940 Mithras 5 Mithras Software Functions Shake Shake Duration Speed Diameter Type Measurement Mode BERTHOLD Double click on Shake to open the Shake properties dia log box and define the parameters for microplate shaking Shake properties x r Parameters Duration 0 1 600 sec Speed Slow Nomal C Fast Diameter joi 0 1 5mm Type Linear Orbital Double orbit Cancel r Measurement Mode C By plate By wells J Perform
39. ach menu item via Options Matrix and define parameters for each matrix for measure ment and evaluation by well by row or column or for the entire microplate To edit matrices double click on the respective tab For more information please refer to the MikroWin2000 User Guide Child windows Data child window The data supplied by the Reader Unit are entered on matrix 1 z You may enter sample ID error information and dilution factor ed on the defaulted matrices Data Template child window Here you may change the partition of the sample matrix and de j fine controls and standards The other matrices are partitioned A accordingly Template Calculation child window Three matrices show the well positions Sample_ID the meas urement data Reader_Values and the calculated results Re al sults Any formula may be used for measurement data and Calculation results You can type a formula directly into the calculation for mula text box or select it from the Add Formula dialog box A formula can be defined for individual wells columns or rows or for the entire plate BERTHOLD LB 940 Mithras 5 Mithras Software Functions Select formula O Select the desired tab in the Calculation child window and click on _ Add Formula The Add Formula dialog box opens H Common program variables Common statistic functions using plate partition Statistic functions using normal partition Statistic
40. anning one well with up to 100 data points can be measured To measure the well of a 96 well microplate 6 mm with 100 data points evenly and completely you have to define the fol lowing parameters horizontal and vertical steps each 10 point displacement 0 6 mm However you may also focus on the center of one well and se lect smaller distances BERTHOLD LB 940 Mithras 5 Mithras Software Functions R Fluoromety Repeat Repeated measurement Fluorescence Name TotalTime Counting Time Cycle Time Repeats Lamp Energy Excitation Filter Excitation Aperture Emission Filter BERTHOLD Double click on R Fluorometry Repeat to open the Repeated dia log box Parameters N Re eated ee Cancel 1 86400 s TotalTime 300 Counting Time 01 005 6008 Cycle Time fo 24 85 6000 s Repeats 10 1 999 Lamp Energy 7000 0 65535 Excitation Filter F485 FITC Fluorescein Slot 42 v Excitation C Small Normal Emission Filter F535 FITC Fluorescein Slot 42 aa Counter position Top gt Bottom Injector 4 Cycle fo 0 10 Volume 10 X Speed low bi r Operation Mode By plate By wells Figure 5 41 Repeated dialog box Name of repeated measurement Define the calculated total length of the measurement Define the individual counting time of a repeated measurement per well Define the cycle time
41. are Functions 5 1 Software Structure and Operation In the following section you find information on the structure of the software For more detailed information please consult the MikroWin2000 software online help Program start Double click on the program icon to start the software The main window is displayed Main window Upon program start this main window includes six child win dows i e Data Template Calculation Results Statistics and Graphics You can work with these windows in accor dance with the Windows Multi Document Port MDI defini tion In addition the navigation bar can be displayed from this navigation bar you can select the child windows with the re spective views Menu bar All program functions can be selected from the menu bar Tool bar The tool bar of the main window is located directly below the menu bar providing quick access to various windows and to the print edit and file operations simply by clicking on the re spective tool button Status bar The program status bar is located at the bottom of the main window It contains information on the currently loaded data and parameter file Context sensitive help information is available in the left section of the status bar The name of the active pa rameter file par is displayed in the right hand section Result file The raw data of the measurement is saved to a result file ex tension dat Furthermore this file may contain the respective
42. ause damage or lead to false results CAUTION Select Instrument Load Plate to move the plate slide into the instrument and to close the plate tray light tight a GERTHOLD LB 940 Mithras 3 System Description 3 2 2 Excitation Filter Slide To the right of the plate tray there is a compartment containing the excitation filter slide instrument front panel Figure 3 1 To replace or clean the filter you have to eject the slide via soft ware Proceed as follows O Select Instrument Excitation Filter Slide O In the Excitation Filter Slide dialog box click on the button lt Eject Slide gt The door opens slightly and the slide moves out a bit O Open the door all the way by hand and pull the filter holder out at both metal pins bottom and top at filter holder O Clean or replace filter O If you replace the filter drag the respective filter into the Ex citation Filter Slide dialog box to the appropriate positions See the explanations in chapter 5 4 8 O Push in filter holder all the way into the slide O Click lt OK gt in the Excitation Filter Slide dialog box The slide moves all the way into the instrument and the door is closed again GERTHOLD Ee 3 System Description LB 940 Mithras 3 2 3 Components Inside the Instrument Locking button for photo multiplier Electronics Filter wheel with cover plate 3 2 4 Photomultiplier Open the screw on the instrument cover and raise
43. be primed with reagent and the measurement series is to start thereafter you have to set both operations Dispense and Repeated to the By plate mode CBERTHOLD LB 940 Mithras 5 Mithras Software Functions H READIT TM READIT measurement Name Counting Emission Filter BERTHOLD The Readit operation allows you to run the Readit tests by Promega for SNP determination This operation comprises se quential measurement of 3 microplates and automatic entry of the measured data into the EXCEL spreadsheet tier2master xls Double click on X READIT TM to open the Readit dialog box m Parameters Name c i ance Counting Time fo 1 0 05 600 s Emission Filter No Filter Slot A8 x Figure 5 42 Readit dialog box Name of Readit measurement Duration of measurement in seconds Select the emission filter to be used The pre defined filter with name and position in the filter wheel is displayed here 5 Mithras Software Functions LB 940 Mithras 5 5 6 Definition of Evaluation Parameters in Child Windows Evaluation parameters are selected using items on the naviga tion bar or via tool buttons and defined on the respective ma trices In general default settings are defined such that they hardly need to be changed for simple luminescence measure ments Check the following matrices anyway and if necessary edit the settings You may add more matrixes in e
44. chnical Data T Technical Data Operating voltage 100 230 V Frequency 50 60 Hz 180 VA Power consumption 70 VA Safety standards EN 61010 1 EN 61326 1 EN 61000 3 2 EN 61000 3 3 EN 61326 Temperature range Storage 0 40 C Operation 15 35 C Humidity 10 85 no condensation Dimensions 472 x 494 x 374 mm W x D x H including injectors Weight 52 kg Detection unit Low noise photomultiplier with single photon counting technology 9107 SA Reading technologies Luminescence TOP reading fluorescence Bottom reading fluorescence BRET FRET Excitation source Halogen lamp 75 W 340 to 700 nm Excitation filter 355 nm 485 nm Emission filter 460 nm 535 nm 400 nm 515 nm 480 nm 530 nm with BRET option Sensitivity lt 20 amol ATP lt 10 fmol FITC Dynamic range gt 6 decades Crosstalk Low crosstalk due to crosstalk reduction methods 5 x lt 10 Injector Up to 4 injectors variable volumes each 10 100 ul JET injection tech nology injections in plates with up to 384 wells Plate formats All microplates with 6 to 1536 wells external dimensions 86 0 x 128 2 x 17 0 mm W x L x H Petri dish Terasaki plates filter Plate height adjustment Optional automatic detection and adjustment to different plate heights 14 0 mm to 25 0 mm Temperature control Optional 5 C above room temperature to
45. ct the injectors and the num ber of shots to wash the tubings Don t forget to load a prime plate if no waste pump is installed in the instrument you always have to load a prime plate for injector 4 The instrument detects this and prompts you accordingly Refresh To refresh tubings with one shot Please keep in mind that you have to load a prime plate if no waste pump is installed in the instrument you always have to load a prime plate for injector 4 The instrument detects this and prompts you accordingly Unload Injector To pump any solution remaining in the tubings back to the bot tle Excitation Filter Slide To move the excitation filter slide out in instrument front panel and to define the filter used Emission Filter Wheel To change and to define the emission filter Boot Instrument To establish communication between instrument and PC if the Mithras instrument has been turned on an off again while work ing with MikroWin2000 Options menu To define the reading parameters Select Read to open the Options Read dialog box to define the wells to be read and the reading sequence including individual operations Installation menu To define the settings directories password etc in the Instal lation Settings dialog box To select and define the drivers for the Mithras instrument and for the export function in the Installation Driver dialog CBERTHOLD LB 940 Mithras 4 Getting Started and First Measurement 4 Getting
46. cted well area With the mouse button held down drag the mouse pointer di agonally across the desired well area and then release the mouse button The wells inside this rectangle are highlighted You can define several independent rectangular well areas on one plate d Single wells Click on the desired well e Clear selection Clear all Click right mouse button and select Clear from the context menu Undo function selected last Select last function once more i e enable the respective Plate Mode and click once more on the wells you want to deselect or click on the empty button in the top left corner between A and 1 BERTHOLD LB 940 Mithras 5 Mithras Software Functions 5 5 4 Definition of Measurement Sequence Left column Operations column BERTHOLD The measurement sequence is defined in the Options dialog box on the Measurement tab First select the wells for measurement and injection on the Samples tab then define the measurement sequence with the individual operations To do this select the Measurement tab Options X Samples Measurement r Measurement Sequence Operations Operation Shake Dispense oe Luminometry Label K Luminometry Kinetic C Dispense GI 8 Delay gei K KKK K S Luminometry Scannit R Luminometry Repeat S Fluorometry Label K Fluorometry Kinetic S Fluorometry Scannin R Fluorometry Repeate O READIT TM
47. ction Measurement sequence Calculation child window CBERTHOLD No injector All wells for measurement Measurement 1 Renilla Luciferase with emission filter no 5 by well 1 second Measurement 2 GFP with emission filter no 6 by well 1 second Sample POS position of cavity DeepBlueC raw data of measurement 1 GFP2 raw data of measurement 2 Ratio measurement 2 measurement 1 5 Mithras Software Functions LB 940 Mithras Fluorescence measurement Fluorescein par Injectors None Well selection All wells for measurement and injection Measurement sequence Measurement 0 1 second by plate Lamp energy 7000 Excitation filter no 2 Emission filter no 2 Counter position Top Calculation child window Sample POS position of cavity Result average value Plate limit value calculation Fluorescence kinetics measurement Fluorescein kin10 par injectors Injector no 4 Well selection All wells for measurement and injection Measurement sequence Injection 100 ul middle speed by well Measurement 10 seconds total time 1 second interval time 11 data points No delay Lamp energy 7000 Excitation filter no 2 Emission filter no 2 Counter position Top Calculation child window Sample POS Position of cavity Peak Maximum of kinetics measurement Integral Integral of kinetics measurement T Slope Time at maximum slope T Max Time at maximum Time at maximum of kinetics H KTMA MES
48. driver you want to use for data export Click on the arrow button to open the list showing the available driv ers and select the driver you want Please keep in mind that you have set up the driver you have selected here in the menu Installation Driver Export Otherwise no data will be trans ferred Export Target Information File Name Shows the file name of the active parameter file An extension identifying the selected driver is appended XLS for Excel files and TXT for Ascii files The file name can be edited Directory The target directory has been defined by the selected export driver during installation Click the lt Browse gt button to select another target directory Click lt OK gt to accept your selection BERTHOLD 5 Mithras Software Functions LB 940 Mithras 5 3 2 Automatic Data Export Choose the menu item File Export Setup to select the export driver that is to be loaded automatically upon successful com pletion of a reader run If a driver has been selected for the ac tive parameter file data evaluation is performed after comple tion of the respective measurement and data export is carried out in accordance with the selected driver Please keep in mind This function is only valid for the active parameter file Prerequisite for automatic data export is that the respective ex port driver has been installed and set up in the menu item In stallation Driver see chapter 5 1 and the export driver
49. e click on Fluorametry Label to open the Fluorometry dialog box for entry of the parameters for the fluorescence measurement m Parameters Name Fluorescence Cancel Counting Time o 1 0 05 600 s ey Lamp Energy fo 0 65535 Excitation Filter F355 Umbelliferone Slot 41 7 Excitation Aperture Small Normal Emission Filter F460 Umbelliferone Slot 41 v Counter position Top Bottom Operation Mode C By plate By wells Figure 5 38 Fluorometry dialog box Name Name of measurement Counting Time Counting time in seconds Lamp Energy Enter a value between 0 and 65 535 This range corresponds to 0 to 75 Watts and is divided in digital steps Excitation Filter Select the desired excitation filter The pre defined filter with name and position in the filter slide is displayed Excitation Aperture Select the desired width of the excitation beam small 1 2 mm normal 2 0 mm Emission Filter Select the emission filter to be used The pre defined filter with name and position in the filter wheel is displayed Counter position Select if a fluorescence measurement should take place at the top or bottom Please keep in mind injections in reading posi tion for measurement at top is possible only with injector 4 Operation Mode This function can be used in the plate or in the well mode BERTH LD LB 940 Mithras 5 Mithras Software Functions K F
50. e matrix name title If this item is selected the matrix name is exported as well Line Ascii Export Driver Select this export driver to define an Ascii file with row wise data presentation Header and footer are defined by clicking the lt Add gt button The context menu includes the same options as the matrix ex port drivers Line In this text box you describe the structure of the line Click on the respective lt Add gt button to open the context menu showing the available options see illustration to the left ine ui Position A01 Position 401 PS Well position numbers below 10 are preceded by 0 Position 41 PO0 Error HER Position A1 Plate Identifier PI Well position without 0 Template Identifier T Test Name TS Error ableton TB Shows the error messages of the respective well Matrix 1 01 A Matrix 2 02 Matrix 1 9 Matrix 3 03 Selection of matrices whose data will be accepted Matrix 4 04 Matrix 5 05 Matrix Name Matrix 6 06 If this option is selected you have to enter the exact matrix Matrix 7 0 name e g lt Results gt instead of lt Name gt Matrix 8 08 Matrix 9 09 Matrix Name lt Name gt Figure 5 6 Context menu Line BERTHOLD LB 940 Mithras 5 Mithras Software Functions Data Selection In this group box you can select the data to be exported Consider Plate Partition If this item is selected only the main well is exported if wells have been combined S
51. e saved with this file There are two different export functions a Manual data export File Export you select the export driver for the respective measurement If you wish to export data after a measurement click the lt Export gt button on the tool bar b Automatic data export after each measurement Prereq uisite is that the export driver has been defined in the menu item File Export Setup After storing the parameter file this setting is specific to this parameter file CBERTHOLD LB 940 Mithras 5 Mithras Software Functions 5 3 1 Manual Data Export The following dialog supports manual export of program data The data to be exported the format as well as the export desti nation depend on the selected driver and its configuration The actual data export is carried out by an export driver if you click on the lt Export gt button after a measurement O Open the parameter file you need O Select File Export to open the File Export dialog box Datei Export x Abbrechen Hilfe Neues Bild mit englischer Software m ktiver Export Treiber Matix Ascii Export Driver Matrix Ascii Export Driver Line Ascii Export Driver Matrix XLS Export Driver m Export Ziel Informationen Dateiname Untitled TxT Suchen Verzeichnis C Programme MikroWwin 2000 Transfer Figure 5 8 File Export dialog box with open driver list Active Export Driver Select the export
52. e to the op erator The tubings from the solution bottles are connected to the injector ports using screw type caps The instrument rear panel includes a device for installation of the supply bottles reagent bottle backpack Injector ports 4 3 2 1 Figure 3 6 Mithras rear panel with reagent bottles back pack Four injector positions inside the reader unit J1 pre position luminescence J2 and J3 reading position luminescence J4 reading position fluorescence Injector configuration Luminescence injection into the well located 1 position before the reading position Luminescence injection into the well in reading position Fluorescence top reading position Injector 4 will be discussed in a separate section for technical reasons J4 may only be washed and primed if a prime plate has been inserted as there is no aspiration pump installed at this point 3 System Description LB 940 Mithras Injector parameters The parameters for control of the injector are entered into the software O For measurement in menu item Options Read O For washing and priming on the Instrument menu 3 2 7 Excitation Halogen Lamp with Fan The fan is located on the right instrument side and behind it in side the instrument the excitation halogen lamp 0 75 W The fan works in continuous service when the instrument is turned on Mithras works correctly only if the fan is working properly If the fan is faulty the i
53. ee cloth or better a micro fiber cloth as used for cleaning eye glasses O Insert filter wheel again It can only be inserted in a certain position The metal pin of the filter wheel has to be fitted into the hole in the base plate without twisting this hole to keep the original change position The filter wheel is inserted cor rectly when it rests flat on the base plate O Put cover plate on filter wheel and make sure the round countersinks are flush with the dowel pins Then turn the cover plate slightly clockwise so that the countersink in the center of the cover plate is exactly facing the back O Hold photomultiplier pull locking button left and carefully tilt photomultiplier down again It has to be inserted directly into in the respective opening Check as follows it must be possible to push the PMT with the cover plate easily to the mobile reading positions For a certain time after filter re placement the photomultiplier may show increased back ground values O Close instrument cover again and fix it with screws BERTHOLD LB 940 Mithras 5 Mithras Software Functions 5 5 Reading Parameters 5 5 1 Overview The test specific configuration required to perform and evaluate tests is saved to parameter files extension par The Mithras Multilabel Reader can perform different types of luminescence and fluorescence measurements Each type of measurement has its own typical sequence Berthold Tech nologies sup
54. el Kinetics Print Setup Boot Instrument Scanning 3 3 2 Brief Explanation of Menus and Functions File menu To open save export or print files and enter the respective parameters Select the desired export driver for the active parameter file via the Export item Enable an automatic export function for the active parameter file via Export Setup To do this select the desired export driver It must be the same as the driver selected in the menu item File Export If this function is enabled data is exported automatically upon completion of a measurement See also chapter 5 3 View menu To select special views For example you can show or hide the navigation bar or the tool bar Read menu To start a measurement using the active parameter file GERTHOLD s Es 3 System Description LB 940 Mithras Instrument menu Instrument functions are presented on this menu Load Plate To move the plate slide into the instrument below the photomul tiplier Unload Plate To move the plate slide out of the instrument to unload a mi croplate and load a new one Prime To prime the injector tubings Select the injectors and the num ber of shots and then start the prime function Please don t for get to load a prime plate if no waste pump is installed in the in strument you always have to load a prime plate for injector 4 The instrument detects this and prompts you accordingly Wash To wash the injector tubings Sele
55. ells are depicted differently so that you can see at one glance for which operation s the wells have been selected BERTHOLD LB 940 Mithras 5 Mithras Software Functions Presentation of selected wells Wells which have been selected for measurement are divided into 4 segments against a gray background AN AN A 7A AA A 9A 7A AA LLLE For wells selected for injector 1 the right segment or the seg ment in the bottom right corner is green For wells selected for injector 2 the left segment or the seg ment in the top left corner is blue For wells selected for injector 3 the bottom segment or the segment in the bottom left corner is red For wells selected for injector 4 the segment in the top right corner is yellow GERTHOLD 5 Mithras Software Functions LB 940 Mithras Well selection For well selection you first have to select the mode first select Measurement then select the wells with the cursor Jaee eeel EaP Figure 5 28 Well selection a All wells on a plate Click on the empty button in the top left corner between A and 1 All wells on the microplate are selected in accordance with the pre defined mode b Injections into all wells selected for measurement Select the injector and click on the empty button in the top left corner between A and 1 All wells selected for measurement are in addition selected for the pre selected injector c Sele
56. er for safe grounding Please observe the safety specifications of the grounded plug GERTHOLD s 1 Safety Instructions LB 940 Mithras O The instruments are designed according to the IEC 1010 1 _ or EN 610 10 1 regulations for electrical measuring systems O Do not open any instrument doors as long as the instrument is in operation O Service and repair work may be carried out by qualified per sonnel only O The operator may only perform the maintenance work de scribed in this user guide O Use only parts described in this manual for servicing O Disconnect power supply before opening the instrument O Pull the power cord to disconnect instrument from power supply O Turn instrument off before pulling the power cord O If the hood is removed the safety provisions are no longer complied with Watch out for moving parts Inside the in strument temperatures may be present which may cause burns Some parts of the instrument may still be hot after power off although this is not immediately obvious O The electronic unit of the detector generates high voltage Do not touch it during operation O Caution Risk of explosion if battery is inserted incor rectly The battery may be replaced only using the same battery type or a type recommended by BERTHOLD TECHNOLOGIES by a person authorized by BERTHOLD TECHNOLOGIES Spent batteries have to be disposed off in accordance with the manufacturer s instructions O The ins
57. ering for export of the file see chapter 5 2 5 5 7 Definition of Export Parameters You have to define export parameters if you want to export data to an Ascii or Excel file Prerequisite is that the export driver s has have been installed and set up accordingly see chapter 5 2 Please refer to chapter 5 3 2 to define automatic data export after each measurement Please see chapter 5 3 1 to perform manual data export 5 5 8 Saving Parameter Files Select File Save to save the active parameter file Select File Save As to save the file under a new name or to create a new parameter file Untitled par See also chapter 5 5 2 BERTHOLD LB 940 Mithras 5 Mithras Software Functions 5 5 9 Basic Parameter Files BRET Assay Bret par Fifteen different basic parameter files are supplied for the most common types of measurement Different print formats are as sociated with the individual parameter files The following basic configurations are included Injectors Well selection Measurement sequence Calculation child window BRET Assay Bret2 par No injector All wells for measurement Measurement 1 Renilla Luciferase with emission filter no 3 by well 1 second Measurement 2 eYFP with emission filter no 4 by well 1 second Sample POS position of cavity RLuc raw data of measurement 1 eYFP raw data of measurement 2 Ratio measurement 2 measurement 1 Injectors Well sele
58. esired injector in the row Plate Mode and then select the wells into which the respective injector is to dispense Presentation Wells selected for reading Wells selected for injector 1 Wells selected for injector 2 Wells selected for injector 3 HERBE Wells selected for injector 4 BERTHOLD Neues Bild mit englischer Software Optionen Read r Ger teauswahl Abbrechen Hilfe Optionen Readertreiber tigre auliges Treiberversion far 4 a Reader device driver for Berthold Beschreibung Technologies Multilabel Reader Mithras Options x Samples Measurement Plate parameter Plate type 8x12 Berthold plat Br jate x Plate Mode Measurement is e e Cis Measurement Order By Rows By Columns T Meander S 2 Quick Reference Guide LB 940 Mithras Define measurement sequence see chapter 5 5 4 6 10 11 After you have selected the wells click on the Meas urement tab to define the measurement sequence with the individual operations Copy the respective operations one after the other into the Operations column e double click the desired operation in the left column e enter the desired parameters in the respective pa rameter box and click lt OK gt to confirm you entries In the Operations column you may change the se Tt quence of selected operations by clicking on the and
59. functions using free partition Statistic functions using control overlay Threshold calculation functions Curvefit calculation functions Kinetic calculation functions Scanning calculation functions Pattern calculation functions Basic mathematical functions Text output functions Control variables Curvefit variables Program operators Figure 5 43 Add Formula dialog box O Click on the plus sign of the desired formula type to dis play the list of available formulas O Select the formula you want from the formula list and click lt OK gt The formula is entered into the Calculation formula of Position text box Please consult the respective chap ter in the MikroWin2000 User Guide Calculation Formula of Position A02 MEA Figure 5 44 Calculation formula text box O Define the plate area to which the formula is to be applied For the entire plate Click on button For a column or row Click on the respective column or row button For a single well First select the well then select the for mula and place the cursor into the formula text box Then push the lt Return gt key BERTHOLD bis ECHNOLOGIES 5 Mithras Software Functions LB 940 Mithras Results child window The displayed matrices are identical with those defined in the Calculation child window Here the data and calculated values E are displayed These matrices are used depending on their Results numb
60. has been selected in the menu item File Export see chapter 5 2 1 O Open parameter file O Select File Export Setup to open the File Export Setup dialog box Datei Exporteinrichtung X m Liste der aktiven Exporttreibe List of export driver Abbrechen LS Export Driver __ihilte Entfernen Figure 5 9 File Export Setup dialog box List of Active Export Driver Select the export driver you want to use for automatic data ex port upon successful completion of a reader run Click on the arrow button to open the list showing the available drivers and select the driver you want The selected drivers appear in the text box directly below the drop down list box To delete a driver from the list select this driver and then click lt Remove gt Click lt OK gt to accept your selection BERTHOLD LB 940 Mithras 5 Mithras Software Functions 5 4 Instrument Control and Operation GBERTHOLD The Mithras Multilabel Reader is controlled and operated via the MikroWin 2000 software This includes opening and clos ing of the plate tray as well as priming and washing the injector tubings Injector tubings have to be washed O before starting work O before changing reagents O at the end of each work session before turning off the in strument O after longer periods of inactivity Use solutions recommended by the kit manufacturer e g distilled water diluted alcohol hypochlorite solution
61. he Mithras unit is equipped for fluorescence measurements A selected item is identified by a checkmark v Luminometry installed Select this item if the Mithras unit is equipped for luminescence measurements A selected item is identified by a checkmark v Z Mover installed Select this item if the Mithras unit includes an automatic plate height adjustment mechanism for the microplates in Z direction vertical A selected item is identified by a checkmark v lt Get Configuration gt Download configuration from instrument lt OK gt Save configuration lt Cancel gt Discard new entries CBERTHOLD LB 940 Mithras 5 Mithras Software Functions Export Driver Setup Neues Bild mit englischer Software Matrix Ascii Export Driver Line Ascii Export Driver Matrix XLS Export Driver BERTHOLD Export drivers have to be installed if you want to export data In addition you have to set up the export driver and you have to specify data structure data matrices as well as header and footer Data is exported depending on the driver selected and configured in this dialog box To use another data format you can select another driver before running a measurement or set up the selected driver new O In the Installation Driver dialog box select the Export tab to view the available drivers You may choose Installation Treiber x Reader CurveFit Export Sampler m Liste der installierten Ex
62. hras 6 Maintenance 6 3 Fuse Replacement The fuse is located on the instrument rear panel in a black fuse holder to the left of the mains switch Figure 6 1 Instrument rear panel Proceed as follows O Turn instrument off and pull power cord O Insert screwdriver or similar tool into fuse holder recess and lift it out by applying slight pressure O Take faulty fuses out O Insert new fuses Use only the fuse type specified O Insert fuse holder again CGBERTHOLD 6 Maintenance LB 940 Mithras 6 4 Preparations for Transport The following safety provisions have to be taken to trans port or ship the instrument Preparation for transport of instrument O Turn instrument off and disconnect it from mains O Open instrument cover O Move plate tray manually to the position where transport safety screw 2 can be inserted and tightened between the hole in the transport plate If the screw does not catch move the height adjustment of the optics down by hand by turning the toothed belt clockwise O Once transport safety screw 2 has been fixed move the height adjustment of the optics carefully up by hand by turn ing the toothed belt counter clockwise O Tighten transport safety screw 2 ensure through slight sideways motion by a few millimeters that the detector is in the correct position Transport safety screw no 1 no 2 Figure 6 2 Instrument rear panel CBERTHOLD I E s LB 940 Mithras 7 Te
63. inetic S Fluorometry Scannin R Fluorometry Repeate OOONWNOORN BBS READIT TM m Operation Mode T Batch Plate Repeats fi m Temperature Control fe Tempercure j Celsius Abbrechen Figure 5 30 Defined measurement sequence with display of reading parameters UEemenmen Temperature Control Define the desired temperature if your instrument includes a temperature control input option 15 45 C Please note You can enter a target temperature in the instru ment which is at least 5 C above room temperature BERTHOLD ECHNOLOGIES 5 Mithras Software Functions LB 940 Mithras Proceed as follows to define a new measurement sequence O Select Edit Reset Untitled par is displayed as temporary file name in the bottom right corner of the status bar O Select Options Read to open the Options Read dialog box O Select BertholdTech Mithras from the device drive list O Click lt Options gt to open the Options dialog box with the Samples and Measurement tab O Select the plate type on the Samples tab O In the row Plate Mode enable the item Measurement and then select the wells for measurement O Select injector in the Plate Mode row and then select the wells into which the selected injector is to dispense O After selection of the wells select the Measurement tab and define the measurement sequence with the individual opera tions O Copy these operations one
64. injec tion Select Instrument Prime The Injector Sequence dialog box is displayed Select injector 3 If no waste pump is connected you have to place a prime plate onto the plate tray In this case the plate tray opens Insert the prime plate and click lt Next gt The plate tray is closed and the prime plate is placed below the injectors Connect the reagents to the respective injectors Click lt Next gt as soon as you have connected the reagents correctly Then the selected injector tubing is filled with 12 shots A corresponding message is displayed in the Injector Se quence dialog box Wait until the prime process is finished As soon as the prime process is finished a corresponding message is displayed and the lt Close gt button is enabled Click on lt Close gt to complete the process and to close the Injector Sequence dialog box If you are working with a prime plate this plate is automati cally moved out as soon as you click lt Close gt Remove the prime plate and close the plate tray again by selecting In strument Load Plate As soon as the tubing is primed click the lt Read gt button in the main window Enter a file name for the data in the status bar The file name of the parameter file with the extension dat is defaulted Click the lt Start gt button The plate slide moves out and you are prompted to load a microplate Insert a prepared microplate onto the plate slide such that A1 is
65. installed in the Windows operating system Click Finish to complete Setup 6 Click on the program icon Eo to start MikroWin2000 7 The main window of MikroWin2000 is displayed The main window with menu bar tool bar and navigation bar is displayed It includes all menus and menu items In addition all child windows data template calculation result statistics graphics are displayed these windows can be minimized or maximized as needed The navigation bar can be deselected in the View menu See MikroWin2000 software User Guide 8 Set up driver Select menu item Installation Driver and verify or define parameters see chapter 5 2 3 9 Select menu item Options Read The Options Read x i i i por i Type of measurement dialog box is displayed Driver Selection j Device fBerholdTechCENTRO x Endpoint C Kinetics 10 Open the Device drop down list and select the item Ber v ia tholdTech Mithras Bet oe i ascription echnologies Microplate Luminometer The respective drivers and pre defined parameter files are loaded The other text boxes in this dialog box are filled in p Common Adjustments automatically Measurement filter Shake time z p second s Reference filter Shake mode 11 Define excitation and emission filter on the Instrument menu and install filters into the instrument accordingly see chapters 5 4 8 and 5 4 9 GERTHOLD LB 940 Mithras 2
66. ith name and position in the filter slide is displayed 5 Mithras Software Functions LB 940 Mithras Excitation Aperture Select the width of the excitation beam small 1 2 mm normal 2 0 mm Emission Filter Select the emission filter to be used The pre defined filter with name and position in the filter wheel is displayed Counter position Select if a fluorescence measurement should take place at the top or bottom Please keep in mind injections in reading posi tion for measurement at top is possible only with injector 4 Use Injector 4 Enable injector 4 to perform an injection in counting position during the ongoing kinetics Injector Delay Time counted from start of kinetics measurement after which the injection should take place within the ongoing kinetics Volume Define the volume to be injected Inj 4 Speed Define the injection speed low middle high BERTHOLD LB 940 Mithras 5 Mithras Software Functions S Fluorometry Scannin Scanning Fluorescence Name Counting Time Lamp Energy Excitation Filter Excitation Aperture Emission Filter Counter position Horizontal steps Vertical steps Point displacement BERTHOLD Double click on S Fluorometry Scannin to open the Scan proper ties dialog box Scanning always takes place in the By wells mode m Parameters oK OK Name Scannin Cancel Counting Time fo 1 0 05 600 s Eon Lamp Energy jo 0 65535
67. jections Coelenterazin amp eYFP BRET 2 measurements without injections Deep Blue C amp GFP Dual Luciferase Reporter Gene Assays with 2 injections Dual Luciferase Reporter Gene Assays without injections Dual Luciferase Reporter Gene Assays in 384 well plate Fluorescence kinetics measurement with injector 4 Fluorescence repeated measurement with injector 4 Fluorescence measurement without injection Luminescence kinetics measurement with injector 3 Luminescence repeated measurement with injector 3 Luminescence measurement with injector 3 Luminescence measurement without injection Promega Readit measurements Fluorescence measurement without injection Save the changes to these files under a new name to keep the basic parameter files Matrices Operation CGBERTHOLD The matrix system of the program is largely comparable to a spreadsheet software However the number of input fields is limited to 8 x 12 positions representing the typical microplate format Plates with less or more cavities may also be emulated Using this matrix system all calculations which are required to evaluate a test can be carried out Operation and handling of the program follow the usual Win dows conventions Special user instructions will be described whenever required 5 Mithras Software Functions LB 940 Mithras The following sections of chapter 5 describe only those func tions in detail which are relevant for control of the Mi
68. kip Control Positions If this item is selected only wells which are not used as controls will be exported Only used Sample ID s Only the data of samples having an ID will be transferred Only numeric Sample ID s Only the data of samples having a numeric ID will be trans ferred Line Ascii Export Driver Version 4 08 r Export Layout Header Add Line Add Footer Add m Data Selection m Data Sequence IV Consider Plate Partition BIE Gee T Skip Control Positions I Only used Sample ID s I Only numeric Sample ID s Export only valid test Operation Mode r Export Directory Cancel Target E ProgrammeMikrowin 20005 Transfer 7 Hel elp Figure 5 7 Parameters for a Line Ascii file Data Sequence In this group box you define the order of the data to be ex ported A B C D means that the data in row A on the mi croplate will be exported then those in row B etc 1 2 3 4 means that the data in column 1 of the microplate will be exported then those in column 2 etc Operation Mode Export only valid test If this item is checked data export takes place only if a test is valid BERTH LQ ss 5 Mithras Software Functions LB 940 Mithras 5 3 Export Prerequisite for exporting data files is the correct installation and setup of the export driver see chapter 5 2 The following export functions apply only to the active parame ter file and have to b
69. l counting time Emission Filter Select the filter to be used Usually no filter is used for lumi nescence measurements therefore filter slot 8 No Filter is defaulted Horizontal steps Number of horizontal steps direction 1 12 Vertical steps Number of vertical steps direction A H Point displacement Step width distance between data points selectable dis tances 0 1 to 50 mm Scanning mode Rectangular or round can be selected We recommend to se lect round for microplates with round wells since then the data points in the corner are automatically skipped During scanning one well with up to 100 data points can be measured To measure the well of a 96 well microplate 6 mm with 100 data points evenly and completely you have to define the fol lowing parameters horizontal and vertical steps 10 each point displacement 0 6 mm However you may also focus on the center of one well and se lect smaller distances BERTHOLD 5 Mithras Software Functions LB 940 Mithras R Luminometry Repeat Repeated measurement Luminescence Double click on R Luminametry Repeat to open the Repeated dia log box Repeated MECO ZASIGIE po SAE Speed M Figure 5 37 Repeated dialog box LB 940 Mithras 5 Mithras Software Functions Name TotalTime Counting Time Cycle Time Repeats Emission Filter Use Inj 2 Use Inj 3 Injector Cycle Volume Speed
70. le under a new name to keep the basic pa rameters e Select File Save As e Enter file name extension par and confirm with lt OK gt BERTHOLD LB 940 Mithras 2 Quick Reference Guide 2 4 Measurement and Evaluation 1 The Mithras instrument has to be connected completely and powered on 2 Prime injector tubings used see chapter 5 4 3 e Select Instrument Prime Injector Prime Sequence e In the Injector Sequence dialog box select injectors Eeee pene to be primed and click lt Next gt When finished press Next e f no waste pump is connected you are prompted to place a prime plate in the plate loading area A prime plate always has to be inserted for injector 4 Injector Injectors 4 always has to be processed separately from injec Aee ee pipers tors 1 to 3 Then click lt Next gt e Connect the reagents to the respective injectors aa Click lt Next gt Then 13 shots per injector are carried out e The end of the prime process is displayed Remove prime plate if you have used one and click lt Close gt The Injector Sequence dialog box is closed 3 Open parameter file par for measurement see chap ee 7 ter 5 5 2 The matrices associated with this parameter Lookin a Paa J amp A e mel file are displayed depending on your choice of the iif dero EKn menus Data Template Calculation Results DLR Consistency c D Hom EA DLR Quenching c E DLR Tubing c iiep
71. luation parameters b Using an existing parameter file and modifying the parame ters as needed Either you use the basic parameter files supplied by Berthold Technologies each includes a typi cal measurement sequence for each type of measurement Or you use parameter files you have created yourself and edit them 5 Mithras Software Functions LB 940 Mithras 5 5 2 Open Save Parameter File Create new parameter file O Select Edit Reset to create a new parameter file The file name Untitled par is displayed in the status bar Open parameter file O Select File Open or click the lt Open gt button on the tool bar to open an existing parameter file The Open dialog box is displayed Neues Bild mit englischer Software Suchenin Pao A Bret par Eg Dir par Ej Lumi 1s Dir Kin10 par Bret1 par Ea DIr384 par EJ Lumi 1s Kin10 par Bret2 par Ej Fluorescein Kin10 par Z Lumi 1s Rep10 par Dir par E Fluorescein Rep10 par Lumi par Dir_0 par E Fluorescein par Ej Lumi_O 1s par Dir_384 par Ekin par EJ Nom par gt Dateiname Dateityp Mikrowin Para File par x Abbrechen Figure 5 25 Open dialog box O From the Files of type drop down list box select the type MikroWin Para File par The contents of the directory Para is displayed showing the available parameter files O Select the file you want and click lt Open gt O If the parameter file is located in another directory ope
72. luorometry Kinetic Kinetics measurement Fluorescence Name TotalTime Counting Time Repeats Delay Lamp Energy Excitation Filter BERTHOLD Double click on K Fluorometry Kinetic to open the Kinetics prop erties dialog box A kinetics measurement always takes place in the By wells mode m Parameters Name Kinetic TotalT ime fi 1 864005 Counting Time 01 0 05 600s Repeats fv a9 Delay jo 0 6005 Lamp Energy 0 0 65535 Excitation Filter F355 Umbeliferone Slot Al f Excitation Aperture Small Normal Emission Filter F460 Umbelliferone Slot 41 bed Counter position Top Bottom Injector 4 Delay fo 0 600 Volume 10 b Speed low aE Figure 5 39 Kinetics parameters Name of kinetics measurement Specify the total counting time for each selected well Define the individual counting time of a kinetics sequence of one well The number of data points per well is automatically calculated from the entered parameters including the Delay Calculation takes place as soon as you click lt OK gt The calculated value is then displayed on the table in the Operation column Delay time between individual measurements of a kinetics se quence of one well presently not implemented Enter a value between 0 and 65 535 This range corresponds to 0 to 75 Watts and is divided in digital steps Select the excitation filter The pre defined filter w
73. m Description LB 940 Mithras Reagent outlet If a waste pump is installed connect a waste tubing to it and put this tubing into a waste bottle Observe all statutory requirements for handling patient samples and reagents Fuse The instrument fuse is located next to the mains switch in a black fuse holder For fuse replacement please see chapter 6 Maintenance 3 2 9 PC Requirements Pentium processor 500 MHz or better 128 MB RAM Graphics card 1024x768 or better CD ROM drive Operating system Win98 Win2000 Win NT Win XP Serial port RS232 Parallel port 32 CBERTHOLD LB 940 Mithras 3 System Description 3 3 Software 3 3 1 Structure The software for control of the Multilabel Reader Mithras is an extension of the Windows application MikroWin2000 The illustration below shows the menus and functions of the program Only the system functions printed in boldface type are described in this user guide For information on all other func tions please refer to the user guide and the MikroWin2000 online help Instrument Options Installation New Undo Tool Bar Load Plate Read Settings Open Copy Status Bar Unload Plate Threshold Automation Save Paste Navigation Bar Prime Controls Driver Save As Reset Help Card Bar Wash Definitions Export Refresh Matrix Export Setup Unload Injector Result Print Excit Filter Slide Curve Fit Print Preview Emiss Filter Whe
74. me see chapter 5 4 3 2 Open and edit parameter file O Open parameter file par for measurement File Open see chapter 5 5 2 O Select Options Read to edit well selection and measure ment sequence O You may edit the matrices Data Template Calculation Results 3 Start measurement Q To start the measurement sequence click on the Read button in Read the main window A status bar is displayed below the matrix with text boxes and buttons for the measurement E Fiepame J sus t S Ston Unititied dat Norm par Name of data file Name of parameter file Figure 5 52 Status bar after start of a measurement The name of the parameter file used is displayed in the bottom row right Click to open the parameter file used and edit the default settings if necessary The Options Read dialog box opens Click on lt Options gt to open the Options dialog box to view and edit the well selection and the measurement sequence File name Enter a name for the data file It consists of the name of the parameter file and has the extension dat The file name may be overwritten but the extension cannot be changed Click lt Start gt to start the measurement provided you have en tered a file name for the data BERTHOLD LB 940 Mithras 5 Mithras Software Functions As soon as you have clicked on lt Start gt the plate slide moves out of the instrument On the screen you see the prompt to load a micro
75. me of a kinetics sequence of one well The number of data points per well is automatically calculated from the parameters defined including delay Click lt OK gt to start calculation The calculated value is displayed on the tab in the Operation column Delay between single measurements of a kinetics sequence of one well this function is not yet available Select the filter to be used Usually no filter is used for lumi nescence measurements therefore filter slot 8 No Filter is defaulted Enable injector 3 to perform an injection in reading position dur ing on going kinetics Time counted from the start of the kinetics measurement after which the injection should take place within the on going kinet ics Define volume to be dispensed inj 3 Define injection speed low middle high BERTHOLD LB 940 Mithras 5 Mithras Software Functions S Scanning Luminescence measurement Double click on Scanning to open the Scan properties dia log box Scanning always takes place in the by wells mode Parameters J Name Scanning Cancel jo 0 05 600 s ma Counting Time Emission Filter No Filter Slot A8 Horizontal steps fi 1 10 Vertical steps fi 1 10 Point displacement fot 0 1 50 mm Scanning mode Rectangular Round Figure 5 36 Scan properties dialog box Name Name of scan Counting Time Define individua
76. ment out of cardboard box and put it onto your workplace Remove transport safety screw O Two red tapes remind you to remove the transport safety screw a O Unscrew instrument cover and open it The signal tapes in dicate the position of both screws a O Unscrew transport safety screw 1 using an Allan key It fixes the photomultiplier to the guide rail so it cannot move during transport and is not damaged b O Keep transport safety screw in a safe place It has to be re used when transporting the instrument O Remove second transport safety screw and also keep it ina safe place c O Close instrument cover and fix it again with screws Figure 4 1 LB 940 inside view Install both transport safety screws again before transport ing the instrument Please read chapter 6 4 Preparations for Transport BERTHOL CHNOLOGIES LB 940 Mithras 4 Getting Started and First Measurement Electrical connections O Connect hardlock supplied with the instrument to the paral lel port of your PC O Connect Mithras instrument PC port to serial port of PC or laptop O Connect instrument to the wall outlet using the power cord supplied with the instrument O If you have not already done so connect the computer to the wall outlet Figure 4 2 Mithras rear panel with connections BERTHOLD CHNOLOGIES 4 Getting Started and First Measurement LB 940 Mithras Waste and reagent tubings O If the ins
77. n used Filters text box Proceed as follows to replace the filter wheel O Open instrument open screw and raise cover O With the instrument turned on select the menu item Instru ment Emission Filter Wheel The Emission Filter Wheel dialog box appears showing a schematic presentation of the filter wheel Click on the lt Position Wheel gt button The filter wheel is turned to the replacement position This position is ex tremely important and should not be twisted when taking out the wheel The photomultiplier is located in reading position 1 all the way to the back O Pull off locking button to the right of the photomultiplier and raise the photomultiplier O Turn cover plate such that the 3 round countersinks are flush with the dowel pins then take it off O Take out filter wheel and make sure that the base plate is not twisted 5 Mithras Software Functions LB 940 Mithras Replacing filters O Using a fine pair of tweezers pull out the lock washer and take the filter out O Place new filter into the filter wheel such that the colored side is facing up and the metallized side is facing down If the side of the filter contains an arrow mark the arrow has to point up towards the light O Push in lock washer to fix the filter O In the Emission Filter Wheel dialog box drag the filter onto the respective positions of the filter wheel name Cleaning filters O Filters should be cleaned using a lint fr
78. n order to control the Multilabel Reader Mithras via the soft ware you have to set up the respective drivers To work with Mithras you have to set up the Reader and Export drivers cor rectly Select the menu item Installation Driver to open the Installation Driver dialog box with a separate tab for each driver type Installation Treiber x Reader CurveFit Export Sampler m Liste der installierten Reader Treiber Berthold ech Mithras Bibliothek Hinzuf gen Null Device 4 12 NULLDEV1 X BertholdTech CENTRO 0 21 CENTRO XDL Entfernen 0 14 MITHRAS XDL Einrichten Abbrechen Figure 5 1 Installation driver dialog box UBemenmen Hilfe 5 Mithras Software Functions LB 940 Mithras Installation Driver dialog box List of installed drivers This list includes all currently installed drivers of a special type Reader CurveFit Export and Sampler Each driver type ap pears on a separate tab The left column shows the names of the installed drivers the center column shows the version number of the driver and the right column includes the names of the library file used for exe cution of the driver functions lt Add Driver gt Click this button to add a new driver or to update an existing driver You need a program library i e a file with the extension xdl e g Nulldev1 xdl Such a file may be found on a special driver disk a program CD the local hard drive or on the ne
79. n the Look in drop down list box and select the directory you want The file name of the opened parameter appears on the status bar in the bottom right corner of the screen Please keep in mind All changes in the reading parameters refer to the open pa rameter file Changes are taken over for good when the pa rameter file is saved BERTHOLD LB 940 Mithras 5 Mithras Software Functions Save parameter file Select File Save so save the active parameter file Select File Save As if you want to save the file under a new name or if you have created a new parameter file Unti tled par Enter the file name in the Save As dialog box and select the file type MikroWin Para File extension par The directory pre defined in the Installation settings dialog box is defaulted You can change the directory Click lt OK gt and the new parameter file is saved to the pre selected directory What does a parameter file contain CBERTHOLD A parameter file includes Well selection Options Read Measurement sequence Options Read Evaluation parameters Child windows Data Template Calculation Sample parameters Options menu Parameter for controls limit values Export parameters Manual export File Export Automatic export File Export Setup 5 Mithras Software Functions LB 940 Mithras 5 5 3 Well Selection Before defining the measurement sequence you have to select the wells of the microplate you want t
80. n the slide or from one position to an empty one or Pa back to the list Excitation Fier Slide Positions Add jemove Eject Slide Cancel Figure 5 22 Placing filter names with drag amp drop 1 Drag amp Drop Place mouse pointer over filter name push left mouse button and drag mouse pointer with mouse button held down to the de sired position The filter name is dragged along Release left mouse button as soon as the filter name has reached the desired position You can drop the filter name only in the Unused Filters box or on one of the 5 filter positions BERTHOLD ECHNOLOGIES 5 Mithras Software Functions LB 940 Mithras Replacing Cleaning excitation filters The compartment with the excitation filter slide is located to the right of the plate tray instrument front panel Proceed as follows O Click lt Eject Slide gt in the Excitation Filter Slide dialog box The door opens slightly and the slide moves out a bit O Open the door all the way by hand and pull out the filter holder at both metal pins bottom and top on filter holder O Clean filter or replace it and insert it again O If you replace the filter and insert it again you have to drag the respective filter in the Excitation Filter Slide dialog box to the respective positions see previous page O Push filter holder into the slide again all the way O Click lt OK gt in the Excitation Filter Slide dialog box The slide moves into the ins
81. ng transport the plate slide has to be secured by a safety screw O For instrument cleaning please refer to the respective sec tions in this manual O Reliable instrument function can be guaranteed only when original spare parts are used The tests and service work recommended by the manufacturer has to be performed to make sure that the operator remains safe and that the instrument continues to work correctly Any service and maintenance work not described in this user guide has to be performed by authorized service personnel Special Spare Parts These spare parts are essential for safe operation of the instrument always use original spare parts supplied by the manufacturer or an authorized distributor Fuse 3A T 5x20 type 179500 ELU ID No 12259 or 3A T 5x20 type 19198 Wickmann ID No 12259 Power supply 100 240VAC 24V 6 5A type JWS 150 24 Lambda ID No 34846 Lithium battery 3V 220mAh type CR2032 Varta ID No 17391 GERTHOLD 5 2 Quick Reference Guide LB 940 Mithras 2 Quick Reference Guide This Quick Reference Guide will lead you step by step through the various work processes i e getting started running measurements and performing evaluations It provides a quick overview on how to work with the software and the instrument Mithras is a very versatile instrument for different types of applications in the field of luminescence and fluores cence measurement To meet the needs of different users this Quick
82. nstrument has to be turned off to rule out overheating and the fan has to be replaced The excitation halogen lamp can be set via software in 65 535 digital steps between 0 and 75 Watt see also chapter 5 5 5 Figure 3 7 Right instrument side fan and excitation halogen lamp If the excitation halogen lamp is faulty first screw off the fan and take it off Then pull the halogen lamp carefully out and re move it BERTHOLD CHNOLOGIES LB 940 Mithras 3 System Description 3 2 8 Connections Connections PC power supply mains switch and instrument fuse are located on the instrument rear panel to the left next to the reagent bottles backpack The reagent outlet is located on the right instrument side Connections Mains switch Reagent outlet Figure 3 8 Connections and mains switch on instrument rear panel Serial port Connect a computer PC laptop for control of the LB 940 Mithras to the 9 pole serial port Mains plug Connect the instrument to mains using the cable supplied with the instrument with grounded conductor Mains switch Push the mains switch to turn the instrument on and off Turn the instrument on before starting the software so the program can establish communication with the instrument If the instru ment is turned on after you have started the software you have to select Instrument Boot Instrument to establish communi cation with the instrument BERTHOLD 31 ECHNOLOGIES 3 Syste
83. nt change Reagent container Reagent outlet Reagent tubing Refresh Repeat mode Repeated measurement Repeated measurement Fluor Parameters Repeated measurement Lumi Parameters Result file S Safety instructions Samples tab Scan parameters Scanning Parameters Scanning Fluor Parameters Sensitivity Serial port Setup site Shake Software BERTHOLD 25 45 77 115 105 64 38 6 32 38 7 38 34 86 45 77 103 96 44 11 81 95 101 101 24 31 35 91 Functions Installation Structure and operation Space required Special spare parts Status bar System description T Technical data Temperature control Tool bar Top reading position Transport safety screw Typographical conventions U Unload injector Unload plate Unpacking Ww Wash Wash cycles Wash injector tubings Wash tubings Waste pump Well presentation Well selection 44 39 44 35 44 121 12 50 87 44 38 6 36 3 34 34 35 34 64 59 64 6 17 32 38 61 83 11 80 84
84. o measure and those which are to receive an injection O To edit an existing parameter file open this file File Open O To create a new parameter file select Edit Reset in the main window Untitled par is displayed as temporary file name in the bottom right corner of the screen O Select Options Read to open the Options Read dialog box Neues Bild mit englischer Software Optionen Read Gerateauswahl Readertreiber a et ae ligne rs Abbrechen Treiberversion for 4 ___ Hite B See Reader device driver for Berthold eschreibung T echnologies Multilabel Reader Mithras Optionen Figure 5 26 Options Read dialog box Driver Selection Device From the Device drop down list select BertholdTech Mithras to define the measurement sequence for Mithras Version Shows the driver version This entry cannot be edited Description Describes the selected driver As soon as you have set up the Mithras driver click the lt Options gt button to open the Options dialog box with the Samples and Measurement tabs Figure 5 27 On the Samples tab select the plate type and the wells to be measured and to receive an injection The measurement sequence is defined on the Measurement tab BERTHOLD LB 940 Mithras 5 Mithras Software Functions Options x Samples Measurement Plate parameter Plate type 8x12 Berthold plate X Plate Mode Measurement cma Oi Oa Cis Measurement Order B
85. odified Basically all MikroWin2000 parameter files can be edited The basic parameter files supplied by Berthold Technologies include typical measurement sequences of some major measurement types Using this data you just have to make a few changes to define the measurement se quence for your own requirements A parameter file includes the selected wells the pre defined operations and the evaluation parameters e g calculations result presentation and export The type of measurement is indicated in the file name Depending on your instrument setup it may happen that not all defaulted parameter files can be util ized to the full extent see chapter 5 1 Save the changes to these files under a new name in order to keep the basic parameter files Select File Open in the main window P wnae Gl SA il fen Select the file type par in the Open dialog box pem an Select the desired parameter file and confirm with paeh lt OK gt GRIDLR Tubing c Eep fad DLR Tubng2 c Ilte A parameter file is active until a new one is opened or Dir Eluh until you exit the program The name of the open file ap ik pears in the bottom right corner of the screen Fleneme feon 4 Select Options Read and in the Options Read dia log box click on the lt Options gt button The Options dia log box appears The wells selected for measurement and injections are displayed on the Samples tab 5 Change well selection as needed see page 11 f
86. on first plate repeat onl Figure 5 32 Shake properties dialog box Define shake duration in seconds Define shake speed You may choose Slow Normal or Fast Define amplitude of shake motion Select shake mode linear orbital or double orbital This function can be used in the plate or well mode 5 Mithras Software Functions LB 940 Mithras Dispense Dispense Injector Volume Speed Measurement Mode Double click on Dispense to open the Dispense properties dialog box This operation is available only if wells have been selected for injections on the Sample tab Please keep in mind that you have to define this operation separately for each injector Dispense properties X m Parameters Injector Cancel Volume 110 ka Speed flow w m Measurement Mode C By plate By wells T Perform on first plate repeat only Figure 5 33 Dispense properties dialog box Select injector no 1 2 3 or 4 Only those injectors are dis played for which wells have been selected on the Samples tab Define volume to be dispensed 10 to 100 ul Define injection speed low middle high This function can be used in the plate or well mode BERTHOLD LB 940 Mithras 5 Mithras Software Functions Luminometry Label Luminescence measurement Double click on L Luminametry Label to open the Luminometry dialog box and enter the parameter
87. onger beep CAN correctly installed lights up red Shortly after power on of the instrument during initialization flashes red 2 short beeps Error after power on of instrument CAN module not correctly installed GERTHOLD a LB 940 Mithras Note The MikroWin Lite software is supplied as standard version with the Mithras instrument This software version does not include all functions described in this user guide and in the MikroWin user guide The versions Advanced I and Ad vanced Il which include additional functions are also available Typographical Conventions lt Add formula gt Buttons are printed inside angular brackets in lt OK gt lt Close gt boldface type Menu File Open dialog box Menu titles and dialog boxes are printed in bold face type File Open Menu items are also printed in boldface type Options Read menu and submenu item are separated by a verti cal line E BERTH LD LB 940 Mithras 1 Safety Instructions 1 Safety Instructions This User Guide includes information and warnings that have to be observed by the user to ensure safe instrument operation NOTE Please do always act according to the following safety in structions before as well as during operation of the sys tem Before taking the instrument into operation it is abso lutely essential to read the user manual as otherwise the safety of instrument and operator cannot be ensured The Multilabel Reader LB 9
88. or more details see chapter 5 5 3 6 Select Measurement tab The pre defined measure ment sequence is displayed 7 Change measurement sequence as needed see page 12 for more details see 5 5 4 For differences in the individual types of measurement see chapter 5 5 9 BERTH LQ ss 2 Quick Reference Guide LB 940 Mithras Verify evaluation parameters Evaluation parameters are selected using the options on the navigation bar or via tool buttons and set on the matrices see page 13 The name of the open parameter file appears Untitled dat Norm par in the bottom right corner of the screen For more information please refer to the MikroWin2000 Name of data file Name of parameter file user guide 8 Select Data and edit matrices if necessary E Data 9 Select Template and edit matrices if necessary Ei Template 10 Select Calculation and edit matrices if necessary t Calculation For differences between the individual types of measurements see chapter 5 5 9 11 Select Results Matrices are displayed depending on the pre selected calculation matrices Calculated results are displayed Results depending on the pre defined formulas A 12 Define export parameters see page 13 13 Save parameter file see page 14 As soon as the measurement sequence has been created and all parameters have been defined as needed save the parameter fi
89. ort Software Windows BERTHOLD TECHNOLOGIES MikroWin2000 Order Information Order Number Mithras LB 940 940 38099 10 incl Mikrowin 2000 Lite Injector 1 pre position L 10 100 ul 940 37772 11 Injector 2 reading position L 10 100 ul 940 37772 12 Injector 3 reading position L 10 100 ul 940 37772 13 Injector 4 reading position F 10 100 ul 940 37772 14 Waste pump for injectors 1 to 3 940 Plate height adjustment 940 Robot integration module 940 Temperature control 940 Bottom reading fluorescence 940 BRET package 940 MikroWin 2000 full version 37854 025 MikroWin 2000 full version German language 37854 020 122 CBERTHOLD LB 940 Mithras 8 Appendix 8 Appendix 8 1 Index A Add formula Ascii file Aspiration pump B Basic parameter files Contents Batch mode Battery Beeps Boot instrument BRET Bret par Bret2 par C Child window Calculation Data Results Template Child windows Cleaning filters Cleaning the instrument Cleaning tubings ComPort Connecting Connections D Data export Automatic Manual Delay Dimensions Dispense parameters DLR PAR DLR384 PAR Drag amp Drop Driver setup Driver software E Emission filter BERTHOLD 107 54 22 15 45 109 86 34 19 109 109 106 106 108 106 44 106 28 76 118 118 49 36 31 58 57 90 35 92 113 114 71 47 10 10
90. ox drag the filter onto the respective positions of the filter wheel name Cleaning filters O Filters should be cleaned using a lint free cloth or better a micro fiber cloth as used for cleaning eye glasses O Insert filter wheel again It can only be inserted in a certain position the metal pin of the filter wheel has to be fitted into the hole in the base plate without twisting this hole to keep the original change position The filter wheel is inserted cor rectly when it rests flat on the base plate O Put cover plate on filter wheel and make sure the round countersinks are flush with the dowel pins Then turn the cover plate slightly clockwise so that the countersink in the center of the cover plate is exactly facing the back see Fig ure 3 5 O Hold photomultiplier pull locking button left and carefully tilt photomultiplier down again It has to be inserted directly into in the respective opening Check as follows it must be possible to push the PMT with the cover plate easily to the mobile reading positions For a certain time after filter re placement the photomultiplier may show increased back ground values O Close instrument cover again and fix it with screws BERTHOLD LB 940 Mithras 3 System Description 3 2 6 Injectors Injector 1 Injector 2 3 Injector 4 CGBERTHOLD The injectors optional are located inside the instrument on the rear panel The injector ports are directly accessibl
91. plate Load plate to continue Figure 5 53 Loading a microplate for measurement Load microplate with A1 at rear left and click lt OK gt The plate is moved into the instrument and the pre defined measurement sequence is carried out During measurement you may view the already available data and calculations on the matrices of the Results menu Figure 5 54 Upon completion of the measurement the plate is ejected If you have pre selected the item Batch the system expects the next microplate which will be measured using the same pa rameters Measurement States Measuremert ect periormed Calculation Stems No enor detected 1 2 3 4 5 amp 7 8 g 10 n 12 6 000 0 000 000 0 000 0 900 0 000 0 000 0 000 0 000 0 000 0 000 0 000 0 000 0 080 0 000 0 600 0 000 0 009 0 000 9 000 0 000 0 900 0 000 0 000 0 000 0 090 9 000 9 900 0 900 9 009 0 000 0 090 9 000 9 900 0 900 9 009 0 000 0 080 0 000 0 000 0 000 0 009 0 000 0 020 6 000 0 000 0 000 0 009 0 000 0 080 0 000 0 600 0 000 0 009 0 000 0 020 0 000 0 000 0 000 0 000 0 000 0 000 9 000 0 900 0 900 0 009 0 000 0 020 9 000 0 900 0 900 0 009 0 000 0 080 6 000 0 600 0 900 0 009 0 000 0 000 6 000 0 000 0 000 0 009 0 000 0 080 6 000 0 900 0 000 0 009 0 000 0 080 0 000 0 600 0 000 6 000 f Sample O 2 Reader Values 3 Resuts nied dat Unetied par Figure 5 54 On line presentation of measured data Results are presented in the Graphics menu BERTHOLD as
92. plied 9 If a waste pump is installed in the instrument connect tubing to the reagent outlet and pass it into a suitable bottle to collect the wash solution EE CBERTHOLD LB 940 Mithras 2 Quick Reference Guide 10 Connect reagent tubing to injectors using a screw cap Rear panel The injectors are located on the right side of the instru ment and are numbered as follows from left to right 14 Injector 4 3 2 1 injection into the well located 1 position before lumi nescence reading position 2 3 injection in lumines cence reading position 4 injection in top reading posi tion 11 Turn instrument on GERTHOLD 2 Quick Reference Guide LB 940 Mithras 2 2 Installation of MikroWin and Driver Software 2 2 1 MikroWin2000 Installation 1 Close all Windows applications before you start install ing the software 2 Insert MikroWin2000 CD into CD tray The installation routine starts automatically 3 Select language and confirm with lt OK gt The setup as sistant is started Enter name and company and click lt Next gt Choose destination location see screen shot to the right The following path is defaulted C Program Files MikroWin2000 If you wish to install the program to another folder click lt Browse gt and select another folder Click lt Next gt Select the setup type We recommend that you choose Typical for your first installation to ensure that all program components are ins
93. plies special basic parameter files including typi cal measurement sequences which can easily be adapted to each user s specific needs You do not have to create a com pletely new measurement sequence but you may utilize and modify an existing structure The following basic parameter files are supplied Bret1 par Bret2 par Dir par Dir_0 par Dir_384 par Fluorescein Kin1 par Fluorescein Rep10 par Fluorescein par Lumi 1s Kin10 par Lumi 1s Rep10 par Lumi 1s par Lumi_0 1s par Readit par Umbelliferone par BRET 2 measurements without injections Coelenterazin amp eYFP BRET 2 measurements without injections Deep Blue C amp GFP Dual Luciferase Reporter Gene Assays with 2 injections Dual Luciferase Reporter Gene Assays without injections Dual Luciferase Reporter Gene Assays in 384 well plate Fluorescence kinetics measurement with injector 4 Fluorescence repeated measurement with injector 4 Fluorescence measurement without injection Luminescence kinetics measurement with injector 3 Luminescence repeated measurement with injector 3 Luminescence measurement with injector 3 Luminescence measurement without injection Promega Readit measurements Fluorescence measurement without injection NOTE CGBERTHOLD Save the changes to these files under a new name to keep the basic parameter files Reading parameters can be defined in two different ways a Completely new definition of measurement and eva
94. port Treiber Bibliothek Hinzufiigen Matrix Ascii Export Driver ASCIIS XDL Line Ascii Export Driver 4 08 ASCIIZ DL Entemen Matris XLS Export Driver 4 03 LS1 XDL Matrix XLS Export Driver 2 4 03 xLS1 xDL Matris XLS Export Driver 3 4 03 xLS1 xDL Hilfe UBemenmen Abbrechen Figure 5 3 Selection of export drivers Driver for data export to an Ascii file with matrix structure The data is presented in the form of a matrix Driver for data export to an Ascii file with line structure The data is presented row wise Driver for data export to an Excel file with matrix structure Three XLS drivers are available they can be set up for different purposes e g different matrix numbers or header and footer O Select driver by clicking on the respective line O Click the lt Driver Setup gt button to open the respective dia log box and enter the parameters for data export 5 Mithras Software Functions LB 940 Mithras Matrix XLS Export Driver If you select the Excel export driver you have to define the fol lowing configuration Matrix XLS Export Driver Version 4 03 Export Layout Header pa Add C Help Matrix Selection mode Matrix No 3 Matrix identified by Number Add Matrix Edit Delete sae poem Add m Export Directory Target C Programme MikroWin 2000 T ranster zd Figure 5 4 Excel export driver setup Export Layout Define the Excel file
95. prime plate whenever you are working with the wash function if no waste pump is installed Nee A prime plate always has to be used for injector 4 Proceed as follows O Select Instrument Wash to open the Injector Sequence dialog box O Select the injectors you want to wash All 3 injectors can be selected at the same time Figure 5 15 O Enter the number of wash cycles One wash cycle consists of one shot 10 cycles ensure that the injection system is washed completely Injector Sequence x Injector Wash Sequence Select Injectors When finished press Next Number of Wash cycles 10 Injectors W Injector 1 M Injector 2 M Injector 3 Figure 5 15 Selection of injectors and definition of the number of wash cycles O If no waste pump is installed you are prompted to place a prime plate into the plate tray The plate slide moves out Load the prime plate and click lt Next gt The plate tray is closed and the prime plate is placed below the injector tips Never carry out a wash cycle without prime plate BERTHOLD LB 940 Mithras 5 Mithras Software Functions O Then you are prompted to load the wash solution in the se lected reagent positions Figure 5 16 O Click lt Next gt as soon as you have connected the wash solu tions correctly Injector Sequence x Injector Wash Sequence Load Wash Solution in the Reagent Positions Selected When finished press Next Number of Wash cycles 10
96. reading position 1 2 or 3 under control of the software See also chapter 5 4 9 Countersink Figure 3 5 Filter positions on emission filter wheel Proceed as follows to replace the filter wheel o o Open instrument open screw and raise cover With the instrument turned on select the menu item Instru ment Emission Filter Wheel The Emission Filter Wheel dialog box appears showing a schematic presentation of the filter wheel Click on the lt Position Wheel gt button Then the filter wheel is turned into the replacement position This position is ex tremely important and should not be twisted when taking out the wheel The photomultiplier is located in reading position 1 all the way at the back Pull off locking button to the right of the photomultiplier and raise the photomultiplier Turn cover plate such that the 3 round countersinks are flush with the dowel pins then take it off from above Take out filter wheel and make sure that the base plate is not twisted 3 System Description LB 940 Mithras Replacing filters O Using a fine pair of tweezers pull out the lock washer and take the filter out O Place new filter into the filter wheel such that the colored side is facing up and the metallized side is facing down If the side of the filter contains an arrow mark the arrow has to point up towards the light O Push in lock washer to fix the filter O In the Emission Filter Wheel dialog b
97. s Dual Luciferase Reporter Gene Assay Readit etc BERTHOLD LB 940 Mithras 3 System Description 3 2 Reader Unit The Mithras LB 940 is a desktop instrument with small foot print It can be set up on any lab workplace Plate tray Excitation filter slide Figure 3 1 LB 940 Mithras To obtain good and consistent results please keep the follow ing in mind NOTE O Do not expose instrument to direct sunlight O Set up instrument in dry rooms O Open plate tray only for loading or cleaning to keep light and dust out O Open instrument cover only for cleaning Prior to a meas urement the instrument cover has to be closed light tight for a longer period of time O Keep plate tray free from dirt O Absorb reagents spilled inside instrument immediately using a clean and dry household tissue and then clean the respec tive location 3 System Description LB 940 Mithras 3 2 1 Plate Tray The instrument front panel includes the plate tray It can be opened and closed under control of the MikroWin2000 soft ware Figure 3 2 Open plate tray Select Instrument Unload Plate to open the plate tray then place the microplate on the tray during priming or washing a prime plate if no aspiration pump is installed Position A1 of the microplate has to be in the rear left corner Load the mi croplate such that it rests completely on the plate tray and is fixed by holders Incorrectly loaded microplates may c
98. s for measurement r Parameters Name Luminescence z i ance Counting Time fo 1 0 05 600 Emission Filter No Filter Slot 48 m Operation Mode By plate By wells Figure 5 34 Definition of reading parameters Name Name of measurement Counting Time Duration of measurement in seconds Emission Filter Select the filter to be used Usually no filter is used for lumi nescence measurements therefore filter slot 8 No Filter is defaulted Measurement Mode This function can be used in the plate or well mode BERTHOLD TECHNOLOGIES 5 Mithras Software Functions LB 940 Mithras K Luminometry Kinetic Kinetics measurement Luminescence Name TotalTime Interval Repeats Delay Emission Filter Use injector 3 Injector delay Volume Speed Double click on K Luminometry Kinetic to open the Kinetics prop erties dialog box A kinetics measurement always takes place in the by wells mode Kinetics properties m Parameters Name Kinetic Cancel TotalT ime fi 1 86400 s Counting Time 01 005 600 Repeats mM 1 999 Delay jo 0 6005 No Filter Slot A8 z Injector 3 Delay fo 0 600 s Volume 10 X Emission Filter Speed Figure 5 35 Kinetics properties dialog box Name of kinetics measurement Define total counting time for each selected well Define interval counting ti
99. sample identifier for each cavity Any errors that may occur dur ing measurement are also stored In addition the result file in cludes the data from the child windows Data Results Statis tics and Graphics as well as further information such as date and time of measurement and the parameter file used CBERTHOLD LB 940 Mithras 5 Mithras Software Functions Parameter file The parameter file extension par includes all test specific settings which are required to run and evaluate tests including all measurement parameters well selection measurement se quence filter selection etc all calculation parameters data from the child windows Template and Calculation as well as all print and export settings Basic parameter files Bret1 par Bret2 par Dir par Dir_0 par Dir_384 par Fluorescein Kin1 par Fluorescein Rep10 par Fluorescein par Lumi 1s Kin10 par Lumi 1s Rep10 par Lumi 1s par Lumi_0 1s par Readit par Umbelliferone par The basic parameter files supplied by Berthold Technologies include typical measurement sequences of some major types of measurement Using this data you just have to make a few changes to define the measurement sequence for your own re quirements A parameter file includes selected wells pre defined operations and evaluation parameters e g calcula tions result presentation and export The file name indicates the respective type of measurement BRET 2 measurements without in
100. t work Click the lt Add gt button to open a dialog box from which you can select the drive and directory of the new library file Click the lt Browse gt button to search for a special folder If the directory of the library file has been defined click lt OK gt to open another dialog listing all drivers library files available in the selected directory Select one or several entries from the list and click lt OK gt to install the selected drivers lt Delete Driver gt To delete an installed driver you first have to select one from the list of installed drivers Click the lt Delete Driver gt button to remove the selected driver lt Driver Setup gt Click the lt Driver Setup gt button to configure an installed driver Each new driver should be set up prior to its first use A driver specific dialog opens in which you can define the required con figuration The Reader driver dialog typically includes the com port settings used as well as information on the injectors in stalled in the instrument etc This information should be verified after first installation A general description of these dialogs is not possible since each driver has its own configuration dialog CBERTHOLD LB 940 Mithras 5 Mithras Software Functions Mithras Driver Setup O In the Installation Driver dialog box click on the Reader tab and select the instrument driver BertholdTech Mithras O Then click lt Driver Setup gt to open the dialog box
101. talled If you are familiar with the system you may choose Custom to select the components you need for your application 8 Click lt Next gt 9 Select the desired components or deselect those com ponents you don t want to install The option Curvefit is not really needed for working with Mithras and may be deselected 10 Click lt Next gt Choose Destination Location S x Setup will install MikroWin 2000 in the following folder To install to this folder click Next To install to a different folder click Browse and select another folder You can choose notto install MikroWin 2000 by clicking Cancel to exit Setup Destination Folder ee Files MikroWin 2000 lt Back Next gt Cancel Setup Type x e Typical Program will be installed with the most common ical options Recommended for most users Compact Program will be installed with minimum required C Custom You may choose the options you want to install Recommended for advanced users Destination Folder Pogam Fest Browse aoa care Select Components J x Selectthe components you wantto install clear the components you do notwantto install Components Description This component includes all available curvefit drivers Space Required 340K Space Available 3057568 K lt Back Next gt Cancel BERTHOLD LB 940 Mithras 2 Quick Reference Guide 1
102. ter name in the Name text box O Define usage of filter for fluorescence or luminescence measurement O Click lt OK gt in the Add Filter dialog box The dialog box is closed and the new filter name is listed in the Unused Filter text box O Remove filter name highlight the respective filter name and click lt Remove gt Add Filter x l None Cancel r Usage D Fluorometry T Luminometry Figure 5 24 Entering a filter name GERTHOLD LB 940 Mithras 5 Mithras Software Functions Photomultiplier raised cover plate accessible Cover plate removed filter wheel accessible CBERTHOLD Positioning filters Depending on the position of the filter in the filter wheel allo cate the respective filter names to positions 1 to 8 see Figure 5 22 In the parameter files only those filter positions are dis played for selection which are occupied here Only those filters can be used for measurements which have been defined in the software Therefore make sure that the allocation in the soft ware matches the actual filter positions Proceed as follows O Prerequisite is that filter names are displayed in the Unused Filters text box O Drag the desired filter name from the Unused Filters text box to one of the positions 1 to 8 of the schematic illustration of the filter wheel O You may also drag a filter name from one position on the slide to another or from a wheel position back to the U
103. thras in strument In addition we will discuss some menu items which are essential for creating parameter files and result output These functions are printed in boldface type in the menu over view below Menu overview Instrument Options Installation New Undo Tool Bar Load Plate Read Settings Open Copy Status Bar Unload Plate Threshold Automation Save Paste Navigation Bar Prime Controls Driver Save As Reset Help Card Bar Wash Definitions Export Refresh Matrix Export Setup Unload Injector Result Print Excit Filter Slide Curve Fit Print Preview Emiss Filter Wheel Kinetics Print Setup Boot Instrument Scanning Menu items printed in boldface type are described in the follow ing chapters Chapter File Open Open parameter files 5 5 2 Open data files 5 6 File Save As Save parameter files 5 5 2 Save data files 5 6 File Export Setup Export data files 5 3 Read Measurement 5 6 Instrument Instrument control functions 5 4 Options Read Definition of measurement sequence 5 5 3 5 5 4 and 5 5 5 Installation Driver Driver setup 5 2 2 BERTHOLD HNOLOGIES LB 940 Mithras 5 Mithras Software Functions 5 2 Installation 5 2 1 MikroWin2000 Installation 5 2 2 Driver Installation 5 2 3 Driver Setup Neues Bild mit englischer Software BERTHOLD see Quick Reference Guide page 8 see Quick Reference Guide page 10 I
104. trix 2 Reader H AVE MA2 AVE MA2 AVE MA2 AVE MA2 Figure 5 47 Matrices in Calculation child window Kinetics measurement Lumi 1s Kin10 PAR Injectors Injector 3 reading position Well selection All wells for measurement and injection Measurement sequence Injector 3 100 ul middle speed by well Delay 0 second Measurement 10 seconds total time 1 second interval time 11 data points no delay Measurement mode by well Emission filter no 8 Calculation child window Sample POS position of cavity Peak Maximum of kinetics measurement Integral Integral of kinetics measurement T Slope Time at maximum of slope T Max Time at maximum of kinetics 112 BERTHOLD LB 940 Mithras 5 Mithras Software Functions Luminescence Repeated measurement Lumi 1s Rep10 PAR Injectors Injector 3 Well selection All wells for measurement and injection Measurement sequence Injector 3 100 ul low speed by well Measurement Total Time 300s Counting time 0 1 seconds Cycle time 30s Emission filter no 8 Calculation child window Sample POS position of cavity Peak Maximum of repeated measurement Integral Integral of repeated measurement T Slope Time at maximum of slope T Max Time at maximum of kinetics H KMAdx MES KMAX MES KMAX MES KMAX MES KMAx MES f Sample 2 Peak 3 Integral T Slope 5 T Max Figure 5 48 Matrices in Calculation child window D
105. trument and the door is closed again CBERTHOLD LB 940 Mithras 5 Mithras Software Functions 5 4 9 Emission Filter Emission Filter Wheel BERTHOLD The emission filter used is defined and serviced managed using the menu item Instrument Emission Filter Wheel The fol lowing options are available O Enter the name and position of the filter placed in the filter wheel which is then available for selection when defining the reading parameters in the parameter files O Position the emission filter wheel to change or clean the fil ter Select the menu item Instrument Emission Filter Wheel to open the Emission Filter Wheel dialog box Drag the filters used to the desired filter wheel position Emission Filter Wheel xi Unused Filters Emission Filter Wheel Positions All fiters can be dragged from the Unused Filters list to an empty position in the wheel or from one position to an empty one or back to the list Add Position wheel Cancel Add Remove filter Position filter wheel Figure 5 23 Emission Filter Wheel dialog box Up to eight different emission filters can be placed into the emission filter wheel located directly below the photomultiplier The desired filter is moved to the respective reading position 1 2 or 3 under control of the software 5 Mithras Software Functions LB 940 Mithras Entering filter names O Click lt Add gt to open the Add Filter dialog box O Enter fil
106. trument has to be set up such that the mains switch is easily accessible O If you can see that the instrument has become unsafe to use switch it off and disconnect it from power supply O If liquid gets inside the instruments pull the power cord Clean the unit or have it cleaned by an authorized service center O Protect yourself from electrostatic charge as discharge could damage sensitive instrument parts especially sensi tive parts of the computer and electronics cards O The system always has to be primed with solutions recom mended by the kit manufacturer O Use only reagents recommended by the kit manufacturer O Use reagents only in accordance with the kit manufacturer s instructions a BERTH LD LB 940 Mithras 1 Safety Instructions O Do not use any flammable or explosive solutions or liquids whose mixture is flammable or explosive O Waste when priming washing the tubings always has to be disposed off properly if a waste pump is installed a bottle has to be connected If no waste pump is present a suitable prime plate has to be placed below the injectors during prim ing washing O Injector solutions may be pumped back only if the appropri ate reagent bottle is connected O Observe all statutory requirements for handling biological waste reagents and patient samples O The operator is responsible for the use of reagents O The Mithras instrument should be shipped in its own case Duri
107. trument includes a waste pump connect tubing supplied to the reagent outlet O Pass waste tubing into a suitable bottle to collect the wash solution O Connect reagent bottle Fix reagent tubing to the injectors with screws The injectors are located on the instrument rear panel Reagent bottles backpack and are numbered as follows from right to left Injector 1 Luminescence injection into the well located on position before the reading position Injector 2 3 Luminescence injection into the well in reading position Injector 4 Fluorescence Top reading position For technical reasons injector 4 will be discussed in a separate section J4 may only be washed and primed if a prime plate has been inserted as there is no aspiration pump installed at this point Injector ports 4 3 2 1 Reagent outlet Figure 4 3 Injector ports on rear panel Power on O Push mains switch on instrument rear panel to turn instru ment on GERTHOLD LB 940 Mithras 4 Getting Started and First Measurement 4 5 Software Installation O Install MikroWin2000 software on your computer see chap ter 2 2 1 O Install driver software see chapter 2 2 2 and select the driver for the Mithras instrument O Define excitation and emission filters on the Instrument menu and install them in the instrument accordingly see chapter 5 4 8 and 5 4 9 GERTHOLD 4 Getting Started and First Measurement LB 940 Mithras 4 6 First Meas
108. ual Luciferase Reporter Gene Assay DLR PAR Injectors Injector 2 and injector 3 Well selection All wells for measurement and two injections Measurement sequence Injection 1 100 ul low speed by well Delay 2 seconds Measurement 1 Firefly 10 seconds by well Injection 2 100 ul middle speed by well Delay 2 seconds Measurement 2 Renilla 10 seconds by well Calculation child window Sample POS position of cavity Firefly data of measurement 1 after injection 1 Renilla data of measurement 2 after injection 2 Ratio data of measurement 1 data of measurement 2 MESPOO1 MESPOO1 MESPO01 H MESPOO01 MESPO01 f Sample 2 Firefly 3 Renilla Ratio Figure 5 49 Matrices in Calculation child window BERTHOLD s 5 Mithras Software Functions LB 940 Mithras Dual Luciferase Reporter Gene Assay in 384 well microplate DLR384 PAR Injectors Injector 2 and injector 3 Well selection All wells for measurement and 1 injection Measurement sequence Measurement 1 Firefly 10 seconds by well Injection 1 Delay 1 Injection 2 100 ul low speed by well Delay 2 seconds Measurement 2 Renilla 10 seconds by well Calculation child window Sample POS position of cavity Firefly data of measurement 1 Renilla data of measurement 2 after injection 2 Ratio data of measurement 1 data of measurement 2 H MESPOO1 MESPOO1 MESPOO1 MESPOO1
109. umn the selected operations are first listed in the order of their selection To change the order click on the buttons s and Select the operation which you want to move to another position and click on the respective button until you have reached the desired position The arrows on both buttons indicate whether the operation is moved up or down Clear operation To delete an operation from the measurement sequence select this operation and then click on the x button Operation column This column shows the parameters of an individual operation selected in the center column see Figure 5 26 Operation Mode Select the desired mode Batch In this mode several plates can be measured one after the other using the same protocol Measurement sequence After it has been processed the first plate is moved out of the instrument Load the next plate and start measurement with the same parameters Repeat The same plate can be measured several times in succession Define the number of repeats here BERTHOLD LB 940 Mithras 5 Mithras Software Functions Samples Measurement m Measurement Sequence Operations Operation Delay AAE Name Luminescence Shake x ieee Counting Time 0 10 Di C Dispense Meas operation by Well eer pense G Dela Emission Filter 8 Luminometry Label p y K Luminometry Kinetic sponse S Luminometry Scannit R Luminometry Repeat Fluorometry Label K Fluorometry K
110. urement 4 6 1 Luminescence Measurement without Injections Start MikroWin2000 software Select File Open Select file type par in the Open dialog box Open basic parameter file Lumi_01s par Click lt Read gt button in the main window Quodauda da Enter a file name for the data in the status bar The file name of the parameter file with the extension dat is defaulted Q Click the lt Start gt button The plate slide moves out and on the screen you see the prompt to load a microplate O Place a prepared microplate onto the plate slide make sure A1 is located in the rear left corner O Click lt OK gt to confirm the prompt The plate moves into the instrument below the photomultiplier and the selected wells are read O Select the Results dialog box to view the results on the ma trices O Results may be displayed graphically printed out or ex ported e g as an Excel file CBERTHOLD LB 940 Mithras 4 Getting Started and First Measurement 4 6 2 Luminescence Measurement with one Injection Example Luminescence measurement injection in reading position CBERTHOLD o o o o o Connect respective reagent bottle to injector 3 Start MikroWin2000 software Select File Open Select file type par in the Open dialog box Open basic parameter file Lumi1s par for a luminescence measurement with one injection Inj 3 in reading position All 96 wells have been pre selected for reading and
111. y Rows C By Columns I Meander Ubemenmen Abbrechen Figure 5 27 Options dialog box Samples tab Samples tab Plate parameter Plate type Select the desired plate type with 12 24 48 96 384 or 1536 wells The plate matrix is displayed showing the respec tive number of wells only with option automatic plate height adjustment Plate Mode Select the wells to be measured with the cursor The active Plate Mode indicates for which operation the wells are to be selected First activate the Measurement mode and with the cursor select the wells to be measured Then enable the injec tor Inj 1 Inj 2 Inj 3 or Inj 4 and select the wells to receive an injection by the enabled injector Up to four injections can be defined for each well Injections can be selected only for those wells which have already been selected for measurement Only injectors installed in the software can be selected see In stallation Driver chapter 5 2 3 BERTHOL CHNOLOGIES 5 Mithras Software Functions LB 940 Mithras Measurement Order By Row By columns Choose if the measurement sequence is to be processed by rows or by columns and within this selection meandering Plate matrix A schematic presentation of a microplate is displayed showing the selected microplate type Select the wells to be measured and to receive an injection Depending on the defined opera tion the selected w

Mithras - Phenix Research Products

Contents

Download Pdf Manuals

Related Search

Related Contents