Home

FLUOstar* OPTIMA Software User Manual

1. 232 m Meas stmt time 0 05 2 oL Inj time 2 0 5 4 Vel gr inj tima 7 0 2 ii 12 14 5 4 2 D E Start nj 1 Start injection 2 Start measwemert EM Note Only the input elements for volume groups where a pump is defined in the corresponding layout definition are available see chapter 4 6 Injection Cycles Volume group 1 injection cycle The cycle in which the injection of volume group 1 will be performed The defined cycle number cannot be greater than the number of cycles defined Volume group 2 injection cycle Volume group 4 injection cycle Same for volume group 2 4 Equidistant kinetic cycles If you use this option the cycle time for ail cycles is as long as the cycle with the latest longest injection or measurement requires otherwise the time for all cycles without injection can be smaller Start Times Using the Start Times box it is also possible to meet special requirements regarding timing on one well in a slow kinetic The start of the injection s or of the measurement in Basic Parameters or Kinetic Windows sheet can be delayed up to 1200 seconds on each well after the time when the well reaches the measurement position and the positioning delay is over 2002 11 15 0413F0003A 61 123 FLUOstar OPTIMA Software User Manual BMG Labtechnologies Volume group X injection start time Defines the time when the injection of volume g
2. So TOP aa Lm t 07 s EL HIM b E l5 _ et s Uu uM atr moe EL WT pea A oue ec CU v PU eI 2 loce c 0255 2 4 MIC at veles ase n Ype 1 QI 2 IU LES 1 E wn wae PE LN MIB e 2257 5 gt 5 r m 5 1 EA vo i i gt M 4 EN T 1 i t VR To E at LI k M 1 T A 1 UM m EM Display Mode The measurement results can be displayed as Curve The measurement results can be displayed as a curve of points or lines If there is more than one chromatic used multichromatic more than one filter pair you will see the results of all chromatics displayed together Measurement values of last measured cycle interval You will see the last measurement values in numeric style Due to space limitations only the results for up to two chromatics filter pairs will be shown the first two To see values from an earlier cycle interval you can change the number in the Cycles Intervals input box of the Current State window Note When you use this option in plate mode with an update display setting see below of more often than only after a cycle is completed for all wells which are not yet measured in the current cycle will be displayed to avoid that measurement values of different cycles can b
3. cU Ware ch ae ep Din Hn 1 ige etne JR Doo Tb aPC iv asw DIALS EN P mE ax ver E MES ees EE et em 1 on ee PE S P Dames DU E un DLE SU we Ll MENS MELT ons QE X Ru 8 T b A 1 M we e x ET Fut ls dmg c Sau a qes3 e t E L X o 1 1 M oium dart 5 Tee SAO Me Jira a eta ae Ni aum UM aT Sb har D GOD QT SII Jd MORN tuc emm OP gd UL amv VT oe Ie cx At ta xx Do Eye CIR X ieiti UA aero quem e ons qe injection start tities 30 1299 ELLE A vat roc Srl Moo ons soon Tal Uu eflc 1S we ott Pease iXas e c awe Da do We FEMA Ey Ua nc vus PSUR UE 04 3 crm 8 tont r eter M 1 2 4 16 12 14 Post MEN dey Startin 2 Stert infection 1 Start measurement 1 bs M t oo _ V tt 1 DU SS aes E i t siege a ELI t att LM I QUIT Qo o l2 cux ee m E E LIL 1 vemm oy zu u s oo 2t Lm Moers E ETET T T IER aa a Tix rare rar a en v x f tege 27 EM I aice a ag t ms vun Wat Volume group X injection start time Defines the time
4. c The data path where the ASCII files will be stored This can be a network directory too Filename The file name can be the test name or one of the following options given in the drop down menu e protocol Name of the used test protocol e lt D1 gt lt ID3 gt The information written in the plate identification window before the e lt 1 gt 2002 11 15 measurement begins Tests will be assigned consecutive numbers When the FLUOstar OPTIMA software is restarted the numbers begin again with 1 You can change the start number for example enter the number 5 and the test numbers will increase consecutively starting at 5 You can add a character in order to increase the number of digits used lt 1 gt will produce file names 001 002 003 etc If you want a number to be changed oniy after e g every tenths plate you should add this value in parenthesis before the gt character e g lt 1 10 gt To use the Total no of executed runs instead of the number of executed runs after program start add T before the e g lt 1 gt 0413F0003A 29 123 FLUOstar OPTIMA Software User Manual BMG Labtechnologies AMI e lt date gt e lt time gt 30 123 To use the No of executed runs for the used protocol used test definition add a P before the e g lt 1 gt You can see a Run Statistics after pressing the 224 button
5. ob eset Dey mae Mae gem on OT tuat aos iS Ta unes un sp Og c 2 Es m QBINT n A Y E wem co luo SS GB un EM z LO E E a Eate T e L Oi o 7 A a rc uta ee Tnm oie 07 1 E Ml ago RT Uu eoe VENIR il uei I oos a TOTe Tt s eR vert Sj S d he mi E TM fon nU B leo DIL DIEN t tof Eel ALT 217 TW T rit r lon uu sun QSE UW 7 SO VEM Gv Roh es t t MEM TP aoa um Di 2 AE we m SUC c i SO rye QOEMVEL ah or wo ie a ek aur E E a ial PEE ee ee FAP aU D IE HEEESHT a JI ds gt gel e Time 0 iib GM c lois 00 7 4 MUTO LEUR UE ae Bs E Yngo VA oan Wins vt E zi n RE T M A yar E t tt wit b nr TERIS reba ie rer a ries ir e DC L ot tte me vce Ye uti poses o DONTI Ine oio CO fui DM is tn o77 Vor BIS e ul m LAG oet cott te JR o ili ere o 3 ME for 57 6 u D LE to ee E Beta 5L 6 LOU vno Y ot EYOU to T 0 Ta Ca Mei t 07 tos QU F etat eE OLR MIU
6. Data Definit Definit The test protocols are stored in this directory Data The measurement data s stored in this directory You will find here lt number gt dbf raw data files and the file Measure dbf test runs overview file It is possible at any time to redirect the path of the data and to store the information in another directory You can do this in the FLUOstar OPTIMA control software by selecting Setup and then Path or Login see also Path and Login section 3 4 2002 11 15 0413F0003A 13 123 FLUOstar OPTIMA Software User Manual BMG Labtechnologies 2 2 Main Screen After logging on the main screen for the control software opens An information screen appears in the middle containing details of the software and firmware versions Note Write down the software version and firmware version so it is easily accessible if you need technical support You can also retrieve this information by selecting Help and About FLUOstar OPTIMA in the main menu FLUOstar OPTIMA Reade Serial No 413 0001 Software Versio 1 20 0 Finmmeare Version 1 10 D The status bar at the bottom of the main screen shows the currently selected reading mode the user logged in and the path used for storing the measurement results You can change these settings by clicking the respective field of the status bar The little icon in the user field shows whether the user is allowed to change test protoc
7. 00 00 00 00 00 00 00 00 00 0 so ej ETE EY dug rds BMG Labtechnologies It is possible to stop the process of adding new entries to the run log by checking the Stop checkbox You can print the entire run log or a marked part of the run log The run log will automatically be saved into a the file FLUOstar OPTIMA log in the FLUOstar OPTIMA main directory usually Program Files BMG FLUOoptima You can switch off the generation of the run log file by changing the line RunLog true in the configuration file Program FilesXBMGXFLUOoptimaXVFLUOstar OPTIMA ini section Debug into RunLog false Note 122 123 0413F0003A At the next program start the existing content of this file will be erased Whenever you need support from BMG due to a software firmware malfunction you should send us the log file together with a description of the nature of the problem 2002 11 15 BMG Labtechnologies FLUOstar OPTIMA Software User Manual 9 Support If you have any problems questions regarding the software the instruments you should visit our web page http www bmgiabtech com and read the Frequently Asked Questions FAQ on the Support page f you can not find an answer there please contact BMG using the following emai addresses e Problems questions regarding software suppornt bmglabtech com e Problems questions regarding the instruments tech se
8. 245 272 AAG 25 2 Mb 230 297 5 Description of the chart Order by rows The order of the wells appears sorted by rows on the microplate defaut Order by The order of the wells appears sorted by columns on the microplate columns The coordinates of the wel in the microplate A01 TOW A column m Cont Content of the well as labeled in the layout defi nition you can select a specific well or a group of wells to be displayed in the Signal Curve sheet by highlighting the well name s in the Cont Column if do not select anything here the signal curve for all wells will be displayed although with a maximum of 253 wells 2002 11 15 0413F0003A 97 123 FLUOstar OPTIMA Software User Manual BMG Labtechnologies CH Channel Number of the used filter setting The numbering will always start with 1 which corresponds to the first defined filter in the multichromatic sheet see section 4 8 Multichromatics t In the channel column the t stands for time For more than one cycle or interval the t row shows the time in seconds in which the cycle interval occurred the channel column the C stands for Celsius If the incubator was used during the measurement or if the Temperature Monitoring Feature is switched on see chapter 5 2 this row displays the temperature of the instrument at the particular kinetic cycle only plate mode Cycles
9. Calculates the standard deviation of the raw data for each well over the selected range of cycles intervals divided by the average of the raw data for this well range expressed in percent Use average of blanks from different groups average of blanks of all groups Here you can collect the blank values from different groups as average instead of using an individual blank value for each group This box appears only if there is more than one group defined Range Pull Down Menu You can select the range you are interested in for each table Rangel Rare 2002 11 15 0413F0003A 105 123 FLUOstar OPTIMA Software User Manual BMG Labtechnologies Range 7 The measurement values of range 1 Calc Range start 1 Stop 1 on the raw data worksheet will be used in the corresponding table Range 2 The measurement values of range 2 Calc Range start 2 Stop 2 on the raw data worksheet will be used in the corresponding table 7 5 2 Table Content Pull Down Menu of replicates Layout Standard concentration Volumes 1 Volumes 2 Yolumes Volumes 4 Sample IDs Dilution Factors Raw Data The raw data with no calculations will be displayed Raw Data blank The average of the blanks background is subtracted from the raw data and displayed Averages The average of the replicates will be displayed Averages blank The average of the blanks subtracted from the average of replicate
10. Note All settings from the program configuration window besides the Auto Login option are user specific therefore each user can select his preferences independently 28 123 0413F0003A 2002 11 15 BMG Labtechnologies FLUOSstar OPTIMA Software User Manual 3 3 3 ASCII Export Function If you click the Define format button in the Program Configuration window see chapter 3 3 1 the Define Format for ASCII Export window will appear In this window you can choose the name and format of the file and where the data is saved File Directory I TE Ner Xm Mn DERE eot PM ut SR ie o S TUUS TET ut cM t ust VUE A M DELLI 7 n QM o Filename and Pah d amah i 0 305 OS RS 0 i uoc Let mn t t tM t tan tt Sata DS AD nana nt EN i EG l 1 T 27 t 2 75 1 Ta ME Doni aot oci 1 2 woe SMS 2 que syle ee 5 o 21 TO ard Me T 1 i elne Format for ASTI E xport Wimggt et SedehASClidat y desiname Plate Topit dat rename old file by adding date time een uo CUT RM o e I a8 ns s Itti uM to tae DODO te x n DIN d Brio teeth cedes Ear nle sani 1 e a L O ATUM TD m MT ee Yep ie M
11. Note Even if you choose this option it is possible to define different kinetic cycle times for different kinetic windows but the software will ensure that the timing of all wells is equal by adding waiting times after processing each well Minimum cycle time The minimum cycle times can only be calculated by the reader If you do not use the Equidistant kinetic cycles option the Minimum cycle time can be different in different kinetic windows depending on the Measurement start time and the Number of flashes After defining all necessary settings do not forget the filters click the button Check timing The shortest possible cycle times will be displayed If you have defined a cycle time in any of the kinetic windows which is shorter than the corresponding minimum time it will automatically be corrected At the bottom of the test protocol definition window the calculated Total measurement time for the entire plate will be displayed Note The Check timing function is only available if the reader is switched on 2002 11 15 0413F0003A 47 123 FLUOstar OPTIMA Software User Manua BMG Labtechnologies 4 4 Well Mode Fluorescence Tests Well mode tests should be selected for fast kinetics i e if the reaction usually initiated by an injection occurs over a very finite amount of time Measurements can then be performed in small intervals when the reaction occurs In well mode each well as defined in the layout will be measu
12. p EMG LABTECHNOLOGIES 86 Wel Fluorescence BMG LABTECHNOLOGIES 95 Fluorescence TF SK ZINJAETTITIN I AB TEULHNIIE Hb Hate BMG LABTECHNOLOGIES 95 Well o Fluwescenee 2 EBC TEST BMG LABTECHNOLOGIES 96 Plate Fluorescence RE PLATE MODE TST BMG LABTECHNOLOGIES 96 Plate Fluorescence x TOM S WELL MODE TST GREINER 384 Well Fluorescence Double click on the desired protocol or select the protocol and click OK Click Cancel to return the main menu Note To select the first protocol beginning with e g T simply press the key T 76 123 0413F0003A 2002 11 15 BMG Labtechnologies FLUOstar OPTIMA Software User Manual 6 1 Plate Identification After selecting the test protocol the next window gives you the option to add identifiers to the test to perform a gain adjustment or to start the measurement This is an optional feature that allows you to give the plate specific identifying features such as a title a description or a number Three identifiers with up to 20 alphanumeric characters are possible The drop down menu for each identifier allows you to choose several options for the IDs or you can manually type in a description fart lest Hun A UITAE a us itn Betray i S Plate Na 1 FEE TEM QUIM n TUM e TTD Sie ea ar hae rn OU RC m uisum DEN Sein cora te rn e CVM
13. pull down menu or the 2X button 3 1 1 Reader Configuration The FLUOstar OPTIMA can measure fluorescence intensity and time resolved fluorescence and optional absorbance and luminescence In addition the POLARstar OPTIMA can read in fluorescence polarization mode Using the Setup Reader Configuration function you can designate the measurement procedure He ader L Onu ager z E Bs sty TT S Permi rari rrp 2002 11 15 0413F0003A 17 123 FLUOStar OPTIMA Software User Manual BMG Labtechnologies 3 1 2 Connection You must select the corresponding communication port on the PC and check the communication status between the instrument and the You can only select com ports which are available on the PC If there is no communication between the PC and the instrument check that the power to the instrument is switched on If there is still no communication try a different com port emm Connection check allows you to validate communication between the the instrument If there is no communication a message box wil appear 18 123 0413F0003A 2002 11 15 BMG Labtechnologies FLUOstar OPTIMA Software User Manual 3 1 3 Filters The FLUOstar OPTIMA can be configured with up to 8 excitation filters and up to 8 emission filters The position in the table should correspond to the filter position in the filter
14. 1 ee cUm ami 1 lI ee 1 if 1 1 A vu Geita i DU Lu HIME eee Bo beds bt Eb For logging in as the standard user USER by default no password is necessary The initial administrator password is bmg When the password is entered the Administrator buttons will become available and new users can be created or changes for existing user entries can be made see next page Run Only If this field is checked the respective user is only able to start pre defined test protocols The user can perform a gain adjustment and enter plate sample IDs but can not change or delete an existing test protocol or copy import protocols or create new protocols Changing offset or filter values or microplate definitions is also not possible for this type of user The Run Only property can only be set unset by the administrator When using this option it is strongly recommended to select Run Only also for the defauit user USER or to define a password for this account and to change the administrator password to something other than bmg Note Instead of setting unsetting this option every time a protocol change is necessary you can also create two user entries using the same directory one of these entries with Run Only option and the other one without this option 12 123 0413F0003A 2002 11 15 BMG Labtechnologies FLUOstar OPTIMA Sof
15. Dij i UM 1 E HN 7 IIoc un t 2a 74 IE tov HL R 1 ee Boltomqight nF 7113 60 Bolom ight Yin 7439 be Tor Doe Dip PM ee QT c Me 5 tto Made a UI doe NT MIT nu A LU ioc US a lU Su n i BRAD DORIA LEB c mV TN MEER e Peet o t ot wee 7 a n n sde t t a mi t E 1 Le Ue AR afsew Sys mT ott S Sak Sar ors Uoc QU woe MY ov 7 7 UY DET D DEMO 070571 X nie Qu Nott 6001 EHE 3 ai Name of the new microplate Select the total number of wells in the plate 1536 384 96 48 24 12 or 6 wells The outer length of the entire microplate from border to border Outer width of entire microplate from top to bottom X 1 Distance from the center of the upper left well to the left border of the microplate Y 1 Distance from the center of the upper left well to the top border of the microplate x n Distance from the center of the lower right well to the left border of the microplate Yin Distance from the center of the lower right well to the top border of the microplate Copy Select a microplate that you want to copy A window will appear allowing you to rename the microplate The dimensions are copied exactly This option allows you to edit a microplate s dimensions without changing the original Export Select a microplate definition that you want to expo
16. Ill EMEN ANI Wut lied Ius Lee Ue Po DEL fate PANE BU Wit up4 Duo ME Ts E I cu T BMG Labtechnologies FLUOSsta rt OPTIMA Software User Manual Version 1 20 0 FLUOstar OPTIMA Software User Manual BMG Labtechnologies This manual was designed to guide FLUOstar POLARstar OPTIMA users through the software features Although these instructions were carefully written and checked we cannot accept responsibility for problems encountered when using this manual Suggestions for improving this manual will be gratefully accepted BMG Labtechnologies reserves the right to change or update this manual at any time The Revision Number is stated at the bottom of every page r SU Dl 1 E mm ET n a r 1 a D of je E p a D dq 1 BMG Labtechnologies Ltd PO Box 73 Aylesbury HP 20 2 QJ United Kingdom Phone 44 1296 33 66 50 Fax 44 1296 33 66 51 uk irl gbmglabtech com BMG Labtechnologies S A R L 7 Rue Roland Martin 94500 Champigny s Marne France Phone 33 1 48 86 20 20 Fax 33148864707 france bmglabtech com 9l Jlogies GmbH BMG Labtechnologies inc 2415 Presidential Dr Bldg 204 118 Durham NC 27703 USA Phone 1 919 806 1735 Fax 1919 806 8526 usa bmglabtech com BMG Labtechnologies Pty Ltd P
17. 0 5 seconds Multichromatics Define up to 8 filter settings for multiple absorbance analysis Select 280 and 260 excitation absorbance filters for DNA absorbance 68 123 0413FQ003A 2002 11 15 BMG Labtechnologies FLUOstar OPTIMA Software User Manual 4 11 Luminescence Tests You must select the luminescence mode in the reader configuration window see section 3 1 1 before defining a luminescence protocol Luminescence can be measured in plate or well mode Most parameters are similar to fluorescence protocols see chapter 4 3 Plate Mode Fluorescence Tests or 4 4 Well Mode Fluorescence Tests Multichromatic measurements are possible Flate mode Il uminescenc ce Ur Up ah 24 fM i d D SUAE e EH t i 3h ot 2 03 A d pe wy mI 010 x Positioning delay At least 0 2 seconds are recommended for luminescence tests Measurement interval time Defines the measurement time defines how long the light is measured By increasing this time you will get higher and usually more accurate measurement values Interval time only in well mode You can define the duration of each interval from 20ms to 25 0 seconds When the instrument is active you can click the Check timing button and the interval time is automatically validated by the instrument if you do not use multichromatics the minimum interval time is equal to the defined measurement interval time If you u
18. 12 Rhxlimnted Methylumb Coumerin Mocitied Fluorescence plate made Kinetic window 1 02 3 4 Piate type Brits LABTECHNOLOGES No of cycles 9 Cycle time s 143 JE Meas stat time Ma of flashes 0 Chromatic No 1 2 3 4 Gain 118 48 118 3 Excitation titer 54 355 340 485 Emission filter 590 460 440 520 Last req value volume group 1 2 3 4 V olurme Lil 20 Used pump Positioning delay 02 Injection sped pis 50 injection cycle 1 E Injection start s 00 Shaking after inject s Pause cycle 0 Pause duration fs 0 Resding direction 1 _ Calculation Start 1 Stopt 2 Start2 3 Stop2 3 Comment 110 pl color reagent per vrei Software version control 1 20 8 Software version evaluation 1 20 8 0031 Serial number 413 0034 User USER All the settings are described in section 4 Defining Protocols Note for the last required value it is the last required value to be saved together with an automatic gain adjustment A gain can be typed in manually and hence the required value 96 is not taken into account 116 123 0413F0003A 2002 11 15 BMG Labtechnologies FLUOstar OPTIMA Software User Manual 8 Known Problems and Solutions There are at least five possible errors caused by Microsoft 1 4 6 1 Error Cannot open Data Path 2 Error 429 ActiveX component can t create object or retur
19. Averages of repiicates Starti 1 Stopi 8 X B 3 B ll 24000 22000 21000 Log 20000 MAMMA E RENE 19000 0 5 10 15 20 2h 30 vi Zo0m TIME sec 33 5 You can choose to show the curve in Linear or Logarithmic form You can change the scale for the units by clicking on the scale in the lower left corner If threshold is the evaluation type selected in the Evaluation Worksheet see chapter 7 5 the legend will show a threshold line as The and the desired threshold number Blank Corr Appears only if blanks are defined If checked the average of the blanks will be subtracted from each data point of the corresponding signal curves The blank curve itself will not be shown Averages of replicates If checked the averages of each selected content and channel will be calculated and displayed as signat curve In the legend the names of the contents appear instead of the names of the wells Y Zoom A scrollbar to zoom the signal curve in y direction Lin Log Buttons to switch between linear and logarithmic scaling of the y axis 2002 11 15 0413F0003A 99 123 FLUOstar OPTIMA Software User Manual BMG Labtechnologies Ratio calculation between filter pairs This feature is for e g FURA 2 applications and only available for the fluorescence measurement In this software version V1 20 0 it is only activated for the multichromatic fluorescence measurement type There appear three dropdown boxes You c
20. Counting down is also possible use a instead of the Identical to consecutive numbers except now the file name will be consecutive alphabetical letters The number of letters used can be modified i e A TEHE will result in AAC etc insert current date You can specify the date format if you add a format description after date using yy or yyyy for the year m or mm for the month and d or dd for the day yy year with two digits 1999 gt 99 2000 gt 00 year with four digits m one or two digits for the month January gt 1 December gt 12 mm month with two digits January 2 01 mmm abbreviated name of the month January gt Jan mmmm full name of the month d day with one or two digits dd day always with two digits ddd abbreviated name of the day Monday gt Mo dddd full name of the day ddddd date in the format defined as Short Date Format under windows Settings Control Panel Regional Settings dddddd date in the format defined as Long Date Format under windows Example date yyyy mm dd If you do not specify the format yyyymmdd will be used insert current time You can specify the time format if you add a format description after lt time h or hh for the hour one or always two digits m or mm for the minute Or SS for the second t time in the format defined as Short Time Format under windows Settings Control Panel
21. ID for this well should be in the same line beginning with position 12 If you want to include dilution factors these values should be entered beginning at position 44 Format for Sample ID Excel files jj H2 xi Probe 007 1 H3 Probe 007 2 E B4 X3 Probe 007 3 mel Bb 2 007 4 1 C2 S3 Standard 5096 C3 54 2 C4 B Blank Cb Blank The well order in the files does not matter If there are no dilution factors included the dilution factors entered so far will not be changed Export IDs Export current sample Ds and if the appropriate option described below is selected also the dilution factors into a text or XLS Excel format file Automatically enter sample IDs previously used with this test protocol if this option is checked then the last used sample Ds for the selected test protocol automatically will be reentered as default values Export Print also lines without sample ID entry If this option is checked all lines will be exported or printed otherwise only the lines where a sample ID was entered would be exported or printed When exporting into an Excel file all lines will always be exported include dilution factors when exporting printing If this option is checked the dilution factors will also be exported or printed Note Exporting the table into a HTML file in addition to the XLS or ASCII format is possible after right clicking the table The settin
22. Lists the number of the particular interval well mode or cycle plate mode Intervals Calc Range Start 1 Stop 1 Calc Range Start 1 2 You can select the kinetic cycles intervals that you want included the data calculations by entering the first cycle interval in Calc Range Start 1 and the last cycle interval in Calc Stop 1 The cycles intervals you select will be highlighted in red The calculation start and stop values defined in the test protocol control part are used see chapter 4 3 1 and 4 4 1 as default values for this range Calc Range Start 2 Stop 2 Here you can select a second range of kinetic intervals cycles With this function it is possible to calculate the difference or the quotient of range 1 and range 2 The cycles intervals selected for range 2 will be highlighted in green Ali cycles intervals after the calculation stop cycle interval defined in the test protocol control part are selected as default for range 2 98 123 0413F0003A 2002 11 15 BMG Labtechnologies FLUOstar OPTIMA Software User Manual 7 4 Signal Curve Worksheet The signal curve worksheet graphically plots the data points for one well or a group of wells selected in the Raw Data Worksheet see chapter 7 3 Cont description This sheet only appears if you have more than one kinetic point FLUOmtar OPTIMA Test CA TEST 2001 A012 21 05 18 BMG Labtechnologes ID 1 2 5 UTP DF Blark Corr
23. Mode Fluorescence Tests and 4 4 Well Mode Fluorescence Tests wil explain the difference between the two modes ude Fluurescence Dam Uns Vn CUT PD D t S A After the mode is selected the next window allows you to enter the test parameters 2002 11 15 0413F0003A 39 123 FLUOstar OPTIMA Software User Manual BMG Labtechnologies 4 3 Plate Mode Fluorescence Tests Plate mode can be used for slow kinetics i e the reaction lasts for an extended period of time even after injection All wells defined in the layout are read once during a plate cycle it is possible to read up to 250 cycles injection can be defined for any cycle all defined wells will receive an injection followed by a measurement cycle Key words Cycles The number of times the entire plate will be measured Each well is read only once per cycle if no multichromatics are used otherwise it will be measured as many times as chromatics are used within each cycle Cycle time The amount of time it takes to measure the plate during one cycle You can use the Minimum cycle time as calculated by the instrument fastest possible time or enter a higher time if you want a delay between the cycles 4 3 1 Basic Parameters Plate Mode Made o luoresceneec E oe Example test using plate i de I Test Name Assign a test name as you would like it to appear on the list of test protocols Microplate Use the pull down menu
24. O Box 469 Mount Eliza Victoria 3930 Australia Phone 61 3 5973 4744 Fax 61 35973 4711 australia bmglabtech com Copyright 2001 2002 BMG Labtechnologies All rights reserved Ali BMG Labtechnologies brand and product names are trademarks of BMG Labtechnologies Other brand and product names are trademarks or registered trademarks of their respective holders 2 123 0413F0003A 2002 11 15 BMG Labtechnologies FLUOstar OPTIMA Software User Manual 1 1 1 1 2 1 3 1 3 1 1 3 2 1 4 2 2 4 2 1 1 2 2 2 3 3 3 1 3 1 3 1 3 1 3 1 3 1 ON a 3 2 3 3 3 3 1 3 3 2 3 3 3 3 9 4 3 4 3 5 4 1 42 4 3 4 3 1 4 3 2 4 3 3 44 4 4 1 44 2 4 5 4 5 1 4 6 4 7 4 7 1 4 7 2 4 8 4 8 1 4 9 4 10 4 11 4 12 2002 11 15 TABLE OF CONTENTS INSTALLATION SYSTEM REQUIREMENTS A VERSION OF FLUOSTAR OPTIMA SOFTWARE IS ALREADY INSTALLED SOFTWARE INSTALLATION EXCEL INSTALLATION FLUOSTAR OPTIMA SOFTWARE INSTALLATION UNINSTALLING THE FLUOSTAR OPTIMA SOFTWARE CONTROL SOFTWARE OVERVIEW LOGIN SCREEN USER DIRECTORIES MAIN SCREEN ICONS AND MENU COMMANDS SETUP INSTRUMENT SETUP READER CONFIGURATION CONNECTION FILTERS READER OFFSETS MICROPLATES FIRMWARE UPDATE PROGRAM CONFIGURATION PREFERENCES SHEET ViEW SHEET ASCII EXPORT FUNCTION RUN STATISTICS LOGIN DATA PATH DEFINING PROTOCOLS PROTOCOL SELECTION WINDOW CREATING A NEW TEST PROTOCOL PLATE MODE FLUORESCENCE TESTS BASIC PARAMETERS PLA
25. Regional Settings tt time in the format defined as Long Time Format under windows am pm or a p or AM PM or Am Pm use 12 hours format and show am or pm a or p Example lt time hh mm ss gt If you do not specify the format hhmmss will be used 0413F0003A 2002 11 15 BMG Labtechnologies FLUOstar OPTIMA Software User Manuai You can use more than one of these parameters at one time in the filename example protocol plate 1 477887 dat If a file extension is not specified then DAT will be added automatically If file exists If a file with the same name already exists then there are several options Rename the old file by adding date and time to distinguish it from the more recent file Overwrite old file Append the new data to the existing file it will list separate tests in the same file each test is separated by a dashed line Backup Here you can choose what to do if the data storage to the above defined directory fails no backup file Show an error message when creation writing into the defined ASCII file failed The measurement results are still available in the database files dBase format for usage with e g Excel create backup file only if main file writing fails If creation writing into the defined ASCII file failed the ASCII file will be stored in the specified backup directory same filename and behavior if file already exists as defined above If writing into the backup
26. as default values for calculation range 1 All cycles behind the entered stop cycle are used as presetting for range 2 Pause before cycle You can define a pause for plate mode tests The default setting is 0 which results in an uninterrupted measurement If you want a pause enter the cycle number before which the reader should pause The reader will pause the measurement before the defined cycle If you have defined 05 as pause time the Test Run Pause window will appear otherwise the measurement will automatically be continued after the defined pause time Test Run Pause mw ES ES dn how use c ife io the gt Ex Use ihe TERTE bion ux the in the caption bar of the window you can see the time elapsed since the beginning of the pause The pause window provides the possibility of bringing the plate out to make a manual injection to incubate or to change the plate or stopping the test run You can also pause after a measurement has started plate mode only with more than one cycle Go to Measure and select Pause after current Cycle Note itis possible to define a pause before the first cycle e g to incubate the plate for a certain time Comment Here you can enter a short description of the test protocol up to 255 characters The first 100 characters of this description will also be visible later in the comment field of the evaluation sheet of the FLUOstar OPTIMA evaluat
27. evaluation part so this error should occur after installation only if you install a new Microsoft program which may have deleted or changed this registry key 8 3 Error 1004 VBA initialization failed When you get this error message after starting the evaluation part some Excel add ins are missing or are not enabled e g the VBA macro language Please start the evaluation part directly from the windows start menu and not using the Excel button in the control part There will be a message telling you what parts are missing and asking you whether you want to install these Excel parts Please answer Yes Using Excel XP it is also necessary to set the security level to medium or low as the high level will not allow execution of our set of macros To do so please use the Excel menu command Extras Security 8 4 Error 40009 current row If this message appears when you open the evaluation worksheet you must update the Dao350 dll file installed on your computer Close Excel and open a Windows Explorer e Open Program Files Common Files Microsoft Shared Dao e Rename the existing Dao350 dll file in the folder to Daoold dll e From the FLUOstar OPTIMA installation CD ROM go to the directory FLUOstar OPTIMA Vx xx x Installation from CD ROM Evaluation Excel97_RunTimeError40009 Copy the file Dao350 dll from the CD and paste it inthe Microsoft Shared Dao folder Reopen the software There should b
28. file fails too then there will be an error message This setting is recommended if the main directory is on a network drive always create backup file The ASCII file will be stored in the specified main directory A second copy will be stored in the backup directory same filename and behavior if file already exists as defined above There will be no error message when writing into the main file failed But there will be an error message if writing into the backup file fails always create backup file no error message when this fails The ASCII file will be stored in the specified main directory A second copy will be stored in the backup directory same filename and behavior if file already exists as defined above There will be no error message when writing into the main and or backup file failed Directory Specify the directory for storing the backup copy of the ASCII file 2002 11 15 0413F0003A 31 123 FLUOstar OPTIMA Software User Manual BMG Labtechnologies Data Format On the second sheet Format you can specify the data format Style The data can appear as a table raw data side by side in a matrix or a column list You can select the following options from the pull down menu e List the data with the well number included e Include or exclude non measured wells Tests that have more than one cycle will have data blocks representing the results from each cycle Multichromatic measurements will appear with t
29. fluorophore to be analyzed Excitation and Emission filter If you only want to analyze one fluorophore you can select the filter combinations directly in this window When you use the pull down menu the list of filters as defined under Setup Filters see chapter 3 1 3 Filters is displayed Gain This value will be used to adjust the sensitivity of the photo multiplier tube PMT A higher gain factor will increase the signal Choose a gain value which will keep the measurement result of higher concentrations within the range of the instrument i e O to 65000 relative fluorescence units You can enter the gain settings here or perform an automatic gain adjustment before the measurement Integration Start and Time This is for time resolved assays For integration start enter the time in which the measurement should begin after the flash see also chapter 4 3 2 This time should provide the maximum signal from the fluorophore after the flash has dissipated The integration time is the length of the measurement i e how long the PMT reads the emission light To find the optimal times for the fluorophore see section 4 8 1 42 123 0413F0003A 2002 11 15 BMG Labtechnologies FLUOstar OPTIMA Software User Manual Calculation Range Start Stop You can enter the cycles which you want to use for calculations here This can also be done later in the Evaluation part of the software Note The values specified here are used
30. for the directory The default directory is Program Files Microsoft Office If an older version of Excel already exists a new directory should be created to prevent a conflict between the two Excel versions Click on OK to install e f Excel 97 already exists on the PC Start the Office or Excel installation program Choose add remove components n the section Microsoft Excel Program files select Change option Add ins Change option select Solver and analysis tool pack press OK Than choose Data Access Change Options select Data Access Objects for Visual Basic select Data Base Driver Change Option select All press OK A standard installation of Excel 2000 XP should contain all necessary parts This might not be true for small business editions Note Excel installation should be prior to the FLUOstar OPTIMA software installation or run time errors can occur If it is ever necessary to reinstall Excel it can be necessary to reinstall the FLUOstar OPTIMA software Therefore important data and definitions should be saved on a diskette or another directory prior to reinstallation 6 123 0413F0003A 2002 11 15 BMG Labtechnologies FLUOstar OPTIMA Software User Manual 1 3 2 FLUOstar OPTIMA Software Installation There are two parts to the FLUOstar OPTIMA software the Control part and the Evaluation part Control setup ins
31. format defined as Long Time Format under windows will be used Clear IDs Delete the plate IDs that are entered Get last IDs Recall the ID settings used for the last test run performed by the user currently logged in Automatically enter the previous plate IDs when using same test protocol If this box is checked then the same plate identifiers will be entered automatically when using the same protocol You can still edit the identifiers or use the Clear IDs button 78 123 0413F0003A 2002 11 15 x Ee A ANN EY mE EEREEEEERLENGN D Y Y Y EHLLLLELL es BMG Labtechnologies FLUOstar OPTIMA Software User Manual 6 2 Gain Adjustment By clicking the tab for Gain Adjustment the sheet with the gain settings and the layout defined will appear The purpose of a gain adjustment is to optimize the signal amplification so that the results have the maximum sensitivity and dynamic range The gain is usually performed on the well containing the standard with the highest concentration of fluorophore highest intensity This sets the gain so there is no overflow in the higher intensity wells an overflow means the relative fluorescent units of a well exceeds the maximum range i e 65 000 rfu Zoom Feature For 384 and 1536 well formats the gain adjustment screen has a zoom function allowing you to zoom in a certain section of the layout To zoom in and out of the screen use the ae mm I5 icons The gain adjustm
32. ie DE M VO tns IBXE Ceo GARIS US ToS piv tt uvis Wort COEM 1 n E Mer NS a 1 x eus Dr egi E re re Tah ect H B e A M z Ive 7l eun le LN 1 5 soy eatem n n scl M ex 1 Filters For polarization measurement two identical emission filters should be installed in the filter wheel positioned 180 from each other When you select one filter the other filter is automatically selected Gain and K Factor You can specify a gain value for both channels The necessary gain may differ slightly between the channels The K factor is a fine adjustment for the two channels It is best to perform an automatic Gain and K factor adjustment right before test start see chapter 6 2 3 Gain Adjustment Fluorescence Polarization No of flashes BMG Labtechnologies recommends 50 for the number of flashes for the most accurate results All flashes are averaged together for one intensity value Positioning delay The positioning delay in fluorescence polarization tests is recommended to be 1 second so that the surface of the liquid is stable before measurement 2002 11 15 0413F0003A 71 123 FLUOstar OPTIMA Software User Manual BMG Labtechnologies 5 Priming Incubation 5 1 Priming The FLUOstar OPTIMA may have up to 2 pumps Before starting a test run it is necessary that all used injector pumps are initialized This can be achi
33. index Notes When switching on the group mode all existing layout entries will be converted to group When switching off the group mode all layout entries not belonging to group will be erased 2002 11 15 0413F0003A 57 123 FLUOstar OPTIMA Software User Manuai BMG Labtechnologies 4 6 Concentrations Volumes Shaking To define the standard concentrations and the injection volumes go to the Concentrations Volumes Shaking sheet In this sheet you will see a table that lists contents and their reference numbers Concentration refers to the known concentration of the standards Volume 1 4 refers to the injection volumes of volume groups 1 4 respectively The concentrations of standards and volumes for injection can be entered manually into each space or by using the auto function well kode Luminescence T m T 0 F Iz 1 E Fh 3 B T cid tou TER LE 1 __ m a n n ut RE VII E EH DEA Dy CIAO DA rem 1 TRA rh DADA II a D ence iiri qua X i EN I 2 r ul p Min 1 ariar NEM x AE FRETI ar wrote dussssssssssss The Auto function can be used to define the concentrations and volumes without entering them manually Define the start value and select Factor Increment or Decrement and type in a factor Then click with the right mouse bu
34. me or Eri ar mm c A Em T o Bert arabes a p 3 nic Ua lo I Tm kl Erat 5 T hos Eur a he Fra page 3 T3 T Lal un D SUA Ev AT m E I 1 nite Peay 4 SET E 88 123 0Q413F0003A 2002 11 15 BMG Labtechnologies FLUOstar OPTIMA Software User Manual You can select a start and an end color It is also possible to use colors from the rainbow spectrum If your measurement values use only a part of the total measurement range i e 0 30000 it might be useful to change the start and or end value to enlarge the used range of the color gradient Similar to the Two and Three colors option it is only possible to display the results from one chromatic Note For this option it is important to use a graphic mode with more than 256 colors windows control panel Layout Shows which wells are used for samples standards and blanks Curve Display The settings in this group will be used for the Curve display in the Current State window and for the Current State Zooming window Auto scale The limits for the graph will bo selected automatically according to the measurement results Manual scale This options allows you to personally specify the graph limits You can use this function to enlarge parts of the graph of special interest Max Maximum value displayed Min Minimum value displayed Update
35. new FLUOstar OPTIMA software version over an existing version you should export your test protocols and microplate definitions if any changes are made and export your measurement data It is possible to re import these files into the new software version after installation Write down the filter settings You will find the export function in the test selection window Test Setup Test Protocol for test protocols Select all the definitions you would like to export and click the Export button To export measurement data of already performed test runs start the evaluation part of the FLUOstar OPTIMA software The export function is in the FLUOstar OPTIMA pull down menu at the top of the Excel window Export Test Runs After the installation of the new software use the Import function to bring the test protocols and test runs inio the new software Notes Do not try to copy the data or definition folder in Windows Explorer and attempt to paste it into the new user folder This could result in the loss of your previous data due to possible changes in the data format For complete removal of an old software version see chapter 1 4 A de installation of a former version is in general not necessary you can install the new version direct over an existing version The database format for microplate definitions and the filter database has not been changed since version 1 10 0 therefore it is possible to perform a custom installa
36. pump this will be marked by an exclamation mark and a description will inform you about the origin of the timing problem In addition you will see tables containing the timing for measurements and the injection times If you use the Shaking after injection option definable in the Concentrations Volumes Shaking sheet you will see these times as well Note The pump action times listed include time for shaking An injection action can contain different steps ER VS Injection Shaking Injections are not possible during shaking 2002 11 15 0413F0003A 67 123 FLUOstar OPTIMA Software User Manual BMG Labtechnologies 4 10 Absorbance Tests You must select the absorbance mode in the reader configuration window see section 3 1 1 before defining an absorbance measurement protocol Most test parameters are similar to those listed in fluorescence mode see chapter 4 3 Plate Mode Fluorescence Tests or 4 4 Well Mode Fluorescence Tests You can still choose Well mode or Plate mode i 3 i Filter settings Important parameter for absorbance mode is the filter setting The absorbance filter should be in a position on the excitation side The emission side should have a position defined as empty No of flashes In absorbance mode the number of flashes should be defined as at least 20 to minimize the deviation from flash to flash Positioning delay The positioning delay should be defined as at least
37. the left between the second and third table You can select optional calculations between the tables here Description Explanation All 3 tables are independent Tablet Table2 The content of table 1 is divided by the content of table 2 results are shown in table 3 Table2 Tablei The content of table 2 is divided by the content of table 1 results are shown in table 3 Table1 Table2 Values from table 2 are subtracted from the values from table 1 the results are shown in table 3 Table Table1 Values from table 1 are subtracted from the values from table 2 and the results are shown i in table 3 Polarization The polarization values are calculated using table1 and table 2 results are shown i in table 3 Anisotropy The anisotropy values are calculated using table1 and table 2 results are shown in table 3 Intensity The intensity values are calculated using table and table 2 results are shown in table 3 Note The items marked with an asterisk appear only in polarization mode When a calculation is chosen then all three tables will convert to the same type of data i e Raw Data Averages etc If no calculation is possible the third table will be gray 108 123 0413F0003A 2002 11 15 BMG Labtechnologies FLUOstar OPTIMA Software User Manuai Note f there is a division by zero the respective value will be set to empty in the evaluation sheet Re
38. the test run You can specify the date format if you add a format description after lt date using yy or yyyy for the year m or mm for the month and d or dd for the day yy year with two digits 1999 99 2000 gt 00 yyyy year with four digits m one or two digits for the month January gt 1 December gt 12 mm month with two digits January 01 mmm abbreviated name of the month January Jan mmmm fuli name of the month d day with one or two digits dd day with two digits ddd abbreviated name of the day Monday gt Mo dddd full name of the day ddddd date in the format defined as Short Date Format under windows Settings Control Panel Regiona Settings dddddd date in the format defined as Long Date Format under windows Example lt date yyyy_mm_dd gt If you do not specify the format the date format defined as Short Date Format under windows will be used lt time gt Time of the test run You can specify the time format if you add a format description after lt time h or hh for the hour mormm forthe minute S or 85 for the second t time in the format defined as Short Time Format under windows Settings Control Panel Regional Settings tt time in the format defined as Long Time Format under windows am pm or a p or AM PM or Am Pm use 12 hours format and show am or pm a or p Example lt time hh mm ss gt If you do not specify the format the time
39. timing button and the interval times are automatically validated by the instrument If you want to increase an interval time if you want a delay between the intervals you can manually enter a time For example the instrument gives a time of 0 4 seconds but you can change this to 5 seconds so that there will be a delay of 4 6 seconds between intervals End time This value is automatically calculated Start time plus Number of intervals multiplied by Interval time The start time for the next kinetic window needs to be higher than this end time Minimum interval time The minimum interval times this time value might be different for different kinetic windows can only be calculated by the reader After defining all necessary settings press the button Check timing The shortest possible interval times will be displayed If you have defined an interval time in any of the kinetic windows which is shorter than the respective minimum time it will automatically be corrected f necessary the start times for all following kinetic windows will also be corrected At the bottom of the test protocol definition window the calculated Total measurement time for the entire plate will be displayed Note The Check timing function is only available if the reader is switched on 94 123 0413F0003A 2002 11 15 BMG Labtechnologies FLUOstar OPTIMA Software User Manual 4 5 Layout Definition This sheet contains a grid representing the
40. total ection Tinig Trang Overton n a RE Di c nd Newt 29 Start time Defines the time when the measurement for the selected kinetic window will start relative to the time when the measurement position is reached plus position delay The start time for a kinetic window needs to be higher than the end time of the kinetic window before 2002 11 15 0413F0003A 53 123 FLUOstar OPTIMA Software User Manual BMG Labtechnologies Number of intervals This is the amount of times a measurement will be successively taken on each well You can define up to a total of 250 intervals Number of flashes all modes except luminescence You can define up to 200 flashes per measurement interval All measurement values obtained for all flashes defined for one interval are averaged to produce one intensity value per well The greater the number of flashes the greater the accuracy For fluorescence measurements 10 flashes are usually adequate Note Increasing the number of flashes also increases the reading time Measurement interval time only luminescence mode Defines the measurement time defines how long the light is measured By increasing this time you will get higher and usually more accurate measurement values Interval time You can define the duration of each interval from 0 02 to 25 0 seconds When the instrument is switched on you can click the Check
41. wells of the microplate that you selected You can define wells containing samples blanks standards and controls Mi Mode scence KEEPS vex en eget Leda se wc yep TIT E 2 43 B 82 83 84 85 8 87 98 89 90 91 92 93 94 95 99 97 W 99 10 101 102 103 104 105 106 107 108 103 1107111 11124413 114 115 116 117 118 119 120 OB 121 122 123 124 125 128127128 129 130 131 132 123 134 135 136 137 138 1390 1 5 141 142 143 144 145 145 147 148 149 150 151 152 153 154 155 156 157 158 159 180 7 161 162 183 164 165 186 187 168 169 170 171 172 173 174 175 176 177 178 179180 181 1821183184 165 166 197 180 189 190 191 192 193 194 195 196 197 198 199 200 1 201 202 203 204 205 206 207 208 209 210 211 212 213 214 215 216 217 218 219220 2 221 222 223 224 225 226 227 228 228 230231 232 233 234 235 238 237 238 239 240 3 241 242 243 244 245 246 247 248 249 250 251 252 253 254 295 255 257 258 269260 4 5 15 B B 7 5 6 5 45 46 65 66 251 282 203 254 255 256 257 258 268 270 271 272 273 274 275 276 277 278 279 280 281 282 203 204 285 286 287 288 289 290 291 292 293 294 295 296 297 298 299 300 m 302 303 30 305 306 307 308 309 lt ED 3t E 313 15 6 3t7 3 38 238 0 Bit If you edit a 384 or 1536 weil layout you can use the zoom buttons AI to enlarge or reduce the layout display Content In the content pull down menu you can select the type of sample that the w
42. you to copy an entire test run with all the raw data 105 layout etc It is then possible in a safe way to modify the data and save the changes without loosing the original data Select the test run you wish to copy by adding the test name than choose Copy Test run The copied test run appears the test run list with an C to the left of the name For example if you want to delete raw data from the calculations you can copy the original test run and then in the copied test run delete the data you do not want to use and save these modifications Export Test Run With the export function you can copy the test run onto a diskette a new drive or directory Select a test run or a group of test runs and then choose Export Test Run A new window will ask for the destination drive and directory as well as the file name The extension for the test run export file is In addition to this file a directory will be created lt name gt RU containing one file for each exported test run Do not forget this directory and its content when you copy the exported file For example you may select a test run export and choose the A drive as the destination You can give the file a name e g Test1 run 2002 11 15 0413F0003A 95 123 FLUOstar OPTIMA Software User Manual BMG Labtechnologies Import Test Run With the import function you can copy a test run from a disk or another drive Click on Import Test
43. 02 409 782 ES B tes ees 0978 1958 770 294 0976 19558 2934 4 1958 3912 e UEM ERE T4824 BBB 3912 3912 489 2994 EE 3912 2934 we THE e MIE US Tes MM 5068 08758 5868 860 0378 18956 489 1 2 3 4 5 6 ri 9 10 11 12 mom MO O DD 299 9002 2024010 217 0920 259 0555 tabe deem eee 177 480 154 8757 S664 Tablet Table2 EES _ 976916 8492004 9247055 6099754 9560232 8 973 9123 1142027 323 41 741 8844 6728003 875 6358 803 9687 829587 9040 o 789 9334 955 5232 854 8746 726 2294 8456334 1000 1226 862 3640 271 1 2 3 4 6 f 8 9 10 Gray fields contain deleted values 2002 11 15 0413F0003A 103 123 FLUOstar OPTIMA Software User Manual BMG Labtechnologies Calculation Pull Down Menu for Kinetic Evaluations The menu on the left side has additional calculation options and evaluation types These calculations are for kinetic assays in which there are more than one cycle interval They are related to the selected intervals cycles defined by the calculation start 1 stop 1 resp start 2 stop 2 controls on the Raw Data sheet Sum You can add all the kinetic points together for each well Select Sum in the pull down menu The sum of the selected range will be listed in the tables of the evaluation sheet Maximum Finds the maximum value for a set of intervals for each well Select Maximum in the pull down menu The maximum of the selected ra
44. 12 123 0413F0003A 2002 11 15 BMG Labtechnologies FLUOstar OPTIMA Software User Manual Calculation of the Unknowns there are two tables below the graph Standard concentrations ran oo 1 2 j 4 5 6 Fi 9 10 11 12 Calculated concentrations Table calculation None Ranges Channel 1 Rav data blark A 8SE D7 Z B9E 07 142 07 9 37E 08 E 286E 07 128E 07 9 67 08 3 5608 5 1 3 3 14 5 G 7 8 10 1 The signal of the respective sample is lower or higher than the asymptote min or max values Used for calculation First Table Standard Concentrations This table contains the standard concentrations as defined in the layout Concentration and Volumes Second Table Galculated Concentrations This table contains the calculated concentrations based on the graph of the standard concentrations They are calculated by using the formula for linear regression segmental regression point to point 2 and 3 polynomial or the 4 parameter fit Used for Calculation With these radio buttons at the bottom of the sheet you can select the standard of the group which is used for calculating the sample values With the button Group selected which is activated by default every group uses its own standards for calculation Note In the case of 2 and 3 degree polynomial equations it is possible that there are problems calculating the unknowns The po
45. 3F0003A 111 123 FLUOstar OPTIMA Software User Manual BMG Labtechnologies Curve Fits There is a pull down menu for selecting one of six curve fits below the graph You can choose the one that best fits the data and provides the best results for calculating the unknowns Note a The Segmental Regression curve fit is useful especially for solubility applications It tries to split the data range into two regions with optimal regression fit displaying also the coordinates of the intersection point If the parameter r for the whole data range is bigger than 0 98 only one regression line will be drawn b The 4 Parameter Fit option is not available if the data is not suitable for this type of curve fit For this option parameter a means the minimum b the maximum c the tuming point and d the slope of the asymptote Parameter c is also known as the CSO Values which are greater than the maximum or lower than the minimum of the 4 Parameter Fit asymptote parameters a resp b are not shown Edit start parameters for curve fit If you choose the 4 parameter fit calculation you have the option to edit the start parameters for the curve optimization The graph is now plotted with the default start parameters You can edit the start parameters by changing the values in the parameter table Each time you press the Optimize fit button an optimization routine is done With the Reset button you can reset the values to the original ones 1
46. 7 2 Test Runs Worksheet The Test Runs worksheet is automatically displayed when Excel is opened It lists all the test runs that have been saved or imported At the bottom of the worksheet the user path is shown only the test runs stored in this directory are displayed on the worksheet E Miciesedt E zrel als m EE C0413 SF DARK VALUEW 5 8 384 413 0006 2001 1018 16 32 45 384 9 1 PLATE 2001 10 17 10 45 48 96 7 gt CABS WELL 2001 10 17 10 01 12 96 C LUMI TIMEOPT 20011017 0233242 9 5 4C LUMI PLATE 200 110 18 171144 A MULTI uu 2001 10 15 17 01 8 9 3 CATEST _ VTP DR ae EE 2001 02 16 22 43 96 2 _ TEST 700 0014 2001 02 15 15 36 9 1 ISTE Test tuns TUDSER H el UU E x lr jpg iy z S T Tug AM LN n AM Ic Testname The testname is listed first and appears as it is defined in the protocol definition There is a small column to the left of the testname If a test has been modified in any way a marker will appear in this column If the test run has only been copied or imported a C will be used as marker This distinguishes the test from the original If the test run has been modified e g wells have been taken out see Save under 7 3 Raw Data Worksheet or Sample IDs see chapter 7 6 have been changed this test run will be marked using 101 7102 ID3 These are t
47. CULATIONS ON THE THREE TABLES IN THE EVALUATION SHEET 103 7 5 2 TABLE CONTENT PULL DOWN MENU 106 1 5 3 DATA FOR STANDARD CURVE 108 7 6 SAMPLE IDS WORKSHEET 110 STANDARD CURVE WORKSHEET 111 7 8 RESULT LIST WORKSHEET 114 7 9 PROTOCOL SETTINGS WORKSHEET 116 3 KNOWN PROBLEMS AND SOLUTIONS 117 8 1 ERROR CANNOT OPEN DATA PATH 117 8 2 ERROR 429 ACTIVEX COMPONENT CAN T CREATE OBJECT OR RETURN REFERENCE TO THIS OBJECT 117 8 3 ERROR 1004 VBA INITIALIZATION FAILED 118 8 4 ERROR 40009 NO CURRENT ROW 118 8 5 DISABLED PLATE IN OUT BUTTONS 118 8 6 ACCESS RIGHTS UNDER WINDOWS NT 2000 XP 119 8 6 1 FILE ACCESS PERMISSIONS 119 8 6 2 REGISTRY ACCESS 121 8 7 RUN LOG WINDOW 122 9 SUPPORT 123 4 123 0413F0003A 2002 11 15 BMG Labtechnologies FLUOSstar OPTIMA Software User Manual 1 Installation 1 1 System Requirements o Computer with Pentium CPU we recommend Intel Pentium III or higher with at least 500 MHz clock rate e Minimum 128 MB RAM we recommend 256 MB or higher especially when you are using Windows 2000 or XP One free serial communication port e 20 MB free hard disk memory for software installation e Microsoft Windows 98 ME or Windows NT 2000 XP e Excel 97 2000 XP for data reduction 1 2 Aversion of FLUOstar OPTIMA Software is already installed Please go to section 1 3 Software Installation if there is not a previously installed version of the FLUOstar OPTIMA software Before installing a
48. FLUOstar OPTIMA Software User Manual Back flush lf the pumps have been in use it is important to flush out any solutions that could be considered a contamination reagent non contaminating reagent could be i e distilled water or a water alcohol solution The back flush feature also allows you to conserve expensive reagents since the entire syringe can be emptied after use Invert dispensing function The default position of the plunger is at the bottom of the syringe barrel In this position the injection starts with the liquid in the syringe barrel being pumped through the tubing and then the syringe is refilled as the plunger comes back down It is possible to change the order of the plungers movements by clicking on Invert dispensing function The plunger will begin at the top of the syringe barrel and the syringe barrel will in this case first fill and then dispense through the tubing The invert function is helpful in cases where the solutions have particles or cells that may settle at the bottom of the syringe barrel and therefore will not be dispensed uniformly 2002 11 15 0413F 00034 73 123 FLUOstar OPTIMA Software User Manual BMG Labtechnologies 5 2 Incubation 5 2 1 Temperature Control F geri EL The incubation can be activated through the temperature button or by selecting Temperature under the Measure menu The temperature range of the incubator is 25 C to 45 C optio
49. If an overflow occurs the measurement value will be 65 000 The measurement range for luminescence depends on the measurement interval time max 200 000 000 for 100 s To avoid an overflow it is necessary to set that the gain so the values stay within the range of the instrument In the top left comer of the gain adjustment screen is a Required Value number This value refers to the percentage of the maximum value of the dynamic range The default value is 90 Therefore when you perform a gain adjustment on the well with the highest concentration of fluorophore the result will be 9096 of the maximum fluorescence value i e 65 000 x 0 90 This prevents an overflow in case of deviation of the intensities over the plate If the measurement is an endpoint test then the required value can remain at the default value of 9096 For a kinetic assay an injection can increase the intensity values over the entire plate If you do not know the final results you should lower the required value to 5096 or less of maximum value in order to have enough space for higher intensity values and to prevent an overflow 82 123 0413F0003A 2002 11 15 BMG Labtechnologies FLUOStar OPTIMA Software User Manual 6 3 Sample IDs Dilution Factors You may enter identification values for each well in this sheet These values will be shown in the Sample IDs sheet and in the Evaluation sheet when Sample IDs is selected in the pull down box of the FLUOst
50. P you can choose whether you want to get the start menu entry in the section for all users or in the section for the current user only You may also decide whether the program should be usable also for non power users If you do not check this box the program might not be usable for non power users depending on the operating system or the current setting of the system policies on your computer beginning with Windows 2000 Microsoft changed the default access permissions for some registry keys and newly installed program files in a way that a non power user is by default not able to use nearly any newly installed program either than some Microsoft certified programs When you use this option the access permission is changed for the BMG Labtechnologies registry keys and files to enable program usage for all users For more information about access rights see chapter 8 6 8 123 0413F0003A 2002 11 15 BMG Labtechnologies FLUOstar OPTIMA Software User Manual Custom Installation If you use the custom installation mode the next window displays a list of the components that can be installed during the setup Alanis i omponients t d 0 2 MB T 161 By selecting an item with a single mouse clicK you can obtain a description of the individual software component You can then decide if this feature needs to be installed Backup files The installation program will create a bac
51. Run and the next window will ask for the file name and the directory and drive where it is located Highlight the test run and select OK The test run is added to the list on the Test runs worksheet Delete Test Run If you want to delete a test run select it and choose Delete Test Run You can select and delete several test runs at a time Switch to Control Part With this function you can switch back to the FLUOstar OPTIMA control software or use the button in the toolbar The evaluation software will stay open in the background To access the Raw data Signal curve and Standard curve worksheets for a test run double click on the desired test run 96 123 0413F0003A 2002 11 15 BMG Labtechnologies FLUOstar OPTIMA Software User Manual 7 3 Raw Data Worksheet This worksheet displays all the raw data for each interval or cycle from the measurement Each row represents the data from a specific well Each column represents the data at a particular interval well mode or cycle plate mode Test Name A STANDARD OE Rhoads 0 2 3 ORIN 1 42 17 S Order by row i ph Order by Calc Range Btart 1 s Calc Range Start 2 ell 3 4 5 f 4 10 T E E n th mn me Be i AM pu E FM 48119 T i Be 902 Ae S QOL 44 Ae s 963 As T 8 1 Aas 4 553 peal 2 E 3 KOS ud 0 27 _ AM 15 40 43 36 38 AM 32 34
52. TE MODE TIME RESOLVED TEST PROTOCOLS KINETIC WINDOWS PLATE MODE WELL MODE FLUORESCENCE TESTS BASIC PARAMETERS WELL MODE KINETIC WINDOWS WELL MODE LAYOUT DEFINITION USING LAYOUT GROUPS CONCENTRATIONS VOLUMES SHAKING INJECTION TIMING INJECTION TIMING PLATE MODE INJECTION TIMING WELL MODE MULTICHROMATICS MULTICHROMATICS IN TIME RESOLVED FLUORESCENCE TIMING OVERVIEW ABSORBANCE TESTS LUMINESCENCE TESTS FLUORESCENCE POLARIZATION TESTS 0413F0003A 3 123 FLUOstar OPTIMA Software User Manual BMG Labtechnologies 5 PRIMING INCUBATION 72 5 1 PRIMING 72 5 2 INCUBATION 74 5 2 1 TEMPERATURE CONTROL 74 5 2 2 TEMPERATURE MONITORING FEATURE 74 5 2 3 AUTO POWER ON INCUBATION 75 6 PERFORMING A MEASUREMENT 76 6 1 PLATE IDENTIFICATION 77 6 2 GAIN ADJUSTMENT 79 6 2 1 GAIN ADJUSTMENT FLUORESCENCE AND LUMINESCENCE MODE 79 6 2 2 GAIN ADJUSTMENT ABSORBANCE MODE 80 6 2 3 GAIN ADJUSTMENT FLUORESCENCE POLARIZATION 81 6 2 4 REQUIRED VALUE 82 6 3 SAMPLE IDS DILUTION FACTORS 83 6 4 MEASUREMENT 85 6 4 1 TIME DURATION 85 6 4 2 STOPPING THE TEST RUN 85 6 5 CURRENT STATE GRAPHICS 86 6 5 1 CURRENT STATE OVERVIEW 86 6 5 2 CURRENT STATE OPTIONS 87 6 5 3 ZOOM FEATURE 91 6 5 4 TIMING OVERVIEW 92 7 EVALUATION SOFTWARE DATA REDUCTION 93 7 1 THE WORKSHEETS 93 7 2 TEST RUNS WORKSHEET 94 7 2 1 FLUOSTAR OPTIMA PULL DOWN MENU 95 7 3 RAW DATA WORKSHEET 97 SIGNAL CURVE WORKSHEET 99 T 5 EVALUATION WORKSHEET 101 7 5 1 CAL
53. UOstar OPTIMA Software User Manual BMG Labtechnologies In addition to this file extension TST there will be an additional file with the extension TSM and a subdirectory same name but ending with TS containing the layout information and the sample IDs for the exported test s Do not forget the second file and the complete subdirectory when sending the exported test protocol s to someone else Import Import a test protocol from another drive and directory by clicking on Import Choose the directory A list of files with the extension TST will appear under file name select the desired file and click on Open The imported test protocols will be added to the list of pre defined test protocols 47 Lr lt J HN gnis os m m LEE rod m m e sh Erotica is T3 NOVO STD TESTS TS welTST mm ZJ Novo t wl NOVO STD TESTs TST 5X2 Novo wm ru faa NOVO neu TST zd Nwelpip la m Nwellpip tst sied FLU PLATE TST Tpmt ist aa ey DW ea CUTE ria a ire ee a da TE D S om Novo tst E i case ROA eat RES i ume m 2 est Protocols TST z ES HH er re a m Pe Cu wo IDs TL ide wee pr 1 RA zr In zia a ri mu T xt mr E Lx D REALE Toll Ip p RET np S e Tau iE Un NI EAS Tia V T
54. allation from CD ROM Evaluation setup2000 exe for the Excel 2000 XP version 8 2 Error 429 ActiveX component cant create object or return reference to this object If this message appears when you open the Excel evaluation sheet you should use the Fix429 program from the installation CD ROM under FLUOstar OPTIMA V1 xx Installation from CD ROM Evaluation This program checks a registry key responsible for ActiveX data access objects delivered with Microsoft programs Office Excel Visual Basic Internet Explorer regarding presence and correct value Key HKEY_CLASSES_ROOT LICENSES P4FC596D DFPE 11CF 9551 00AA00A3DC45 Value mbomabptebkjcdlgtjmskjwtsdhjbmkmwtrak 2002 11 15 0413F0003A 117 123 FLUOstar OPTIMA Software User Manual BMG Labtechnologies wrong value of this key will cause the error 429 ActiveX component can t create object or return reference to this object at startup of the evaluation software fthe key does not exist it will be created after pressing the Correct Error button f the key exists but has a wrong value the old value will be saved under the name backup in the same key and the key value will be changed to the correct value Should you encounter problems with other programs you can restore the old key value using the Restore button of the Fix429 program Note The patch program Fix429 will be called automatically during the installation of the FLUOstar OPTIMA
55. aluation part see chapter 7 3 of the software Note The values specified here are used as default values for calculation range 1 All intervals behind the entered stop interval are used as presetting for range 2 Comment Here you can enter a short description of the test protocol up to 255 characters The first 100 characters of this description will also be visible later in the comment field of the evaluation sheet of the FLUOstar OPTIMA evaluation software see chapter Fehler Verweisquelle konnte nicht gefunden werden Evaluation Worksheet Check timing Minimum interval time 1 The minimum interval times this time might be different for different kinetic windows can only be calculated by the reader After defining all necessary settings do not forget the filters click the button Check timing The shortest possible interval time will be displayed If you have defined an interval time in any of the kinetic windows which is shorter than the respective minimum time it will automatically be corrected If necessary the start times for all following kinetic windows will also be corrected At the bottom of the test protocol definition window the calculated Total measurement time for the entire plate will be displayed Note The Check timing function is only available if the reader is switched on 2002 11 15 0413F0003A 51 123 FLUOStar OPTIMA Software User Manual BMG Labtechnologies Total measurement time per w
56. an calculate the ratio the product the difference or the sum between the measurement results of two filter pairs The operand and the filter pairs are selectable with dropdown boxes The option Ratio calc off appears as standard Blank Cor calculation between Filter pairs J Averages of replicates 340 10 440 10 3 E Start 1 100 123 0413F0003A 2002 11 15 BMG Labtechnologies FLUOSstar OPTIMA Software User Manual 7 5 Evaluation Worksheet In the evaluation worksheet you can specify the evaluation method It contains tables where you can see calculated data The name of this worksheet is Evaluation followed by the plate format i e Evaluation 96 for a 96 well format You can see the most important settings from the used protocol definition on the top portion of the evaluation sheet You can see all settings in the Protocol Settings Sheet see chapter 7 8 Note The header section of 24 48 and 96 evaluation sheets is fixed for better general view of the results If you use the scrolibar at the left of the window only the tables move Test GROUP A B C STANDARD IMAN 12 08 Hide protocol settings 1 1 2 3 Rhodemine4 Methylumb Coumarin FLUGstar OPTIMA BMG Labtechnologies Fluorescence hate mode equidistant Microplate BG LABTECHNOLOGES Gain 118 048 116 093 Exckation filter 44 355 540 485 Kinetic window Ho of cycles Cycle time s mE Meas stert tine s No of flash
57. ar OPTIMA evaluation part It is also possible to store these IDs together with the measurement results in an ASCII file see chapter 0 In addition it is possible to define dilution factors for all wells which does not contain blanks or standards The dilution factor will be taken into account when calculating the unknown concentrations see chapter 7 7 Standard Curve Worksheet Note f you are using replicates the sample ID and the dilution factor you entered for one well will be used for all replicates Start Test Hur Clear IDs Pressing this button will clear all ID fields Get last IDs By clicking this button you can recall the last IDs used for the selected test even if you did not use the option Automatically enter sample IDs previously used with this test protocol see below Table sort order You can choose between sorting for rows columns or well content The selected sort order will also be used for printing or exporting the sample ID dilution factor list Print ID list You can print out the ID list on any available printer 2002 11 15 0413FQ003A 83 123 FL UOstar OPTIMA Software User Manual BMG Labtechnologies Import IDs Import sample Ds dilution factors from a text file created using the export function or using a text editor like Notepad or from a XLS Excel format file Format for Sample ID text files In the first column of such file there is the well number e g A1 the sample
58. ate names with the left mouse button New Click on New to enter the plate dimensions for a new plate A new window will appear that allows you to enter the necessary coordinates Enter the plate format well number and the mechanical dimensions of the microplate see Edit below Nofe The dimensions should be measured from the center of the wells and need to be very accurate in order for the wells to be positioned exactly during measurements Edit Select a microplate entry that you want to modify second window appears that displays the dimensions and plate format All dimensions are in mm 22 123 0413F0003A 2002 11 15 BMG Labtechnologies FLUOStar OPTIMA Software User Manual Microplate Ed po 7 Me pe STALE DC DN ELE E e sore ae TM A S Tut eI x 5 feel RE xS t Ut ts 3 ERE D E 2 Vs oe Ton EN Az X 9 4 1 1 ur Bc us NN A h 1 i SUD 1 a m n gt Bm na 0 190 rom 128 00 dih 0 90 mmi 8550 Ml So tet wae tes tote Ir UOI t ul XE 14 40 7 Teplett YEE 1120 771 S EE T c i a YRL FE E m ER topi duce MET Sid Lp 16 zo QUT cC ui 1 to Qs S mnm LIU Dads et wo Da U re AM Ao CeBIS2U Py OC bnc t 0t ua Do u Bow tae a l a DIL Soia o at tu 2 i t t MM
59. be analyzed then click the arrow button or on the Multichromatic tab to define the filter combination for each fluorophore to be analyzed Excitation and Emission filter If you only want to analyze one fluorophore you can select the filter combinations directly in this window When you use the pull down menu the list of filters as defined under Setup Filters is displayed 50 123 0413F0003A 2002 11 15 BMG Labtechnologies FLUOstar OPTIMA Software User Manual Gain This value will be used to adjust the sensitivity of the photo multiplier tube PMT A higher gain factor will increase the signal Choose a gain value which will keep the measurement result of higher concentrations within the range of the instrument i e 0 to 65000 relative fluorescence units You can enter a gain manually or perform an automatic gain adjustment before the measurement Integration Start and Time This is for time resolved assays For integration start enter the time in which the measurement should begin after the flash This time should provide the maximum signal from the fluorophore after the flash has dissipated The integration time is the length of the measurement i e how long the PMT reads the emission light way to find the optimal times for the fluorophore is described section 4 8 1 Calculation Range Start Stop You can enter the intervals which you want to use for calculations here This can also be done later in the Ev
60. button then press Download The FLUOstar OPTIMA program will bring the reader into the download mode and then start the download program FlashTools Downloading the new firmware is an automatic process which will last a few minutes Do not interrupt this process by switching the reader or the computer off or by closing the download program After the download is completed the FlashToots program will close automatically Then the FLUOstar OPTIMA needs to be switched off and on again to activate the new firmware If the download process is interrupted the FLUOstar OPTIMA will not work but even in this state it is possible to repeat the download process you need to restart the FLUOstar OPTIMA software after an interrupted download process 2002 11 15 0413F0003A 25 123 FLUOstar OPTIMA Software User Manual BMG Labtechnologies 3 3 Program Configuration Program Configuration allows the user to modify the appearance of the software You can activate a feature by ticking the box next to the statement 3 3 1 Preferences Sheet In the first sheet you can select how the measurement data should be stored Here you can also define some general program behavior settings Fam Save Measurement Data in ASCII Format All measurement data is saved in the specified user directory e g Program Files BMG FLUOoptima lt Username gt Data The data is saved in a dbase file format that can be used with the macros i
61. ction all sheets All or a selection of sheets Pages When using portrait format two sheets will be printed on one page using landscape format only one 44 123 0413F0003A 2002 11 15 BMG Labtechnologies FLUOstar OPTIMA Software User Manual 4 3 2 Time Resolved Test Protocols In the Fitters and Integration box you can define the optimal integration time for a time resolved assay i T EES D hon l M tee ur arr Uo na tal o c 77087 Ia wee a E tUe dor us s RI pc 7 5 SUDO dall vont n Uu eus Soa rl n ett os Vea L Dono ta JO uui 7260 eX 035 40807 EUER ae v ets E VEG XU BuU a7 Nol M IM xot 2S Ealb c iR NO TUS Dae o 7 EE eI LU t Moll MENU ML ts er a ec Ta Da wets i on THEM Sa MD LU TD D ut T m uia ex E Tal f at T IE rt Bee Om LAE k rie rpl aT c UMEN ul cam d My tr e 24 a B Dx TT van 1 um 2 i a 2 eR MERECE ee o Dx 2x 1 da eve nl un QN STD J DOBIT fuu If you have one time resolved fluorophore you can define the same filter setting for up to 8 multichromatic tests and try different integration times see chapter 4 8 1 You can then compare the measurement data to find the optimal time for se
62. ding will continue after reading 1 A12 with B12 and then go back via B11 B10 to B1 if you choose unidirectional reading the plate carrier would move to B1 after A12 and continue reading with B2 B3 It is possible to start the reading from any of the four corners Note The reading direction has no bearing on the measurements it is intended to optimize the amount of time it takes to read the plate 56 123 0413F0003A 2002 11 15 BMG Labtechnologies FLUOstar OPTIMA Software User Manual Possible reading modes bidirectional reading unidirectional reading stat left startright start right pg 2 SS 8 sg 38 OR ru 4 5 1 Using Layout Groups It is possible to use up to 12 independent layout groups sets of samples blanks controls and standards Each group can be individually blank corrected and it is possible to calculate a separate standard curve for each group see 7 7 Standard Curve Worksheet Mate Bade Fluorescence Looe eee Doc at To activate the usage of groups activate the On check box in the groups box Use the pull down box next to this check box to select one of the 12 possible groups A L Now enter samples blanks and standards as described above The group will be shown in the layout grid using different background colors and by inserting the group letter between content type identifier e g B for blank S for standard and X for sample and
63. display Here you can specify how often the display ts updated In well mode the display is updated at least after finishing the measurement of a well in plate mode it is updated at least after each cycle If you select any of the Update every X seconds options there will be updates even for unfinished cycles wells showing the already available measurement values Notes For updating the Current State Overview and the Current State Zooming window a large amount of computing power is necessary especially for tests with a large number of cycles intervals or multichromatics If the computer is too slow to redraw the current state display between two measurement values the update sequence will be lowered automatically The update display modes for well mode tests and plate mode tests are independent 2002 11 15 0413F0003A 89 123 FLUOstar OPTIMA Software User Manual BMG Labtechnologies Polarization mode The measurement results from polarization tests can be displayed as e Raw data from both channels se Polarization values in mP units These values are calculated from the results of channel 1 and channei 2 p Chi k Ch2 Ch1 k Ch2 Anisotropy values in mA units A Chit k Ch2 Ch1 2k Ch2 Use blank corrected values If you use this option all results wil be blank corrected before display Notes f you use this option during well mode tests measurement results cannot be displayed before the first Blank wel
64. e g the program window position or current state display options are stored in the registry part HKEY_CURRENT_USER There access problems to this part All settings which are important for all users like the selected communication port or reading mode will be stored in the HKEY_LOCAL_MACHINE part of the registry as this part of the registry is intended for non user specific information For unintelligible reasons Microsoft changed the default access permissions for this part of the registry beginning with Windows 2000 On all Windows versions before Windows 2000 every user has read and write access to this general information part of the local registry beginning with version 2000 a standard user non power user by default only gets read access to newly generated keys in this part of the Windows registry This access limitation can cause the Error 0509 The program has not been correctly installed or you are using W2000 XP without appropriate registry access rights at program start if you did not use the option Program should be usable also for non power users during installation see chapter 1 3 2 Control Part installation Solution e Logon as Administrator or a user with administrative rights e Run the program SetPermission from the FLUOstar OPTIMA CD ROM folder FLUOstar OPTIMA Vx xxMInstallation from CD ROM OF e Start the program Regedt32 Windows XP Regedit use the Run from windo
65. e mixed up 2002 11 15 0413F0003A 87 123 FLUOstar OPTIMA Software User Manual BMG Labtechnologies If you want to see the last available measurement value for each well regardless whether this measurement value is from the current cycle or still from the previous cycle add the following setting to the Configuration section of the FLUOstar OPTIMA ini file ClearBeforeUpdateInMValuesMode False You will find this configuration file in the FLUOstar OPTIMA main installation directory usually Program Files BMG FLUOoptima Auto If you use this option you will get numeric values for all tests with only one cycle interval endpoint tests and a curve display for all other tests Two colors If you are only interested in a good bad decision you should choose the option to display different colors for all values under a certain limit and for all values above the limit You can select the two colors and change the limit value Only the results from one chromatic will be displayed but if your test uses multichromatics you can decide which chromatic should be used for the current state display Three colors Same concept as Two colors but here you can also define a range in between to be displayed in a third color Color gradient The measurement values will be displayed in different shades of colors or gray levels mee a a e RS C amen o mrm oe 1 uei
66. e no error messages 8 5 Disabled Plate In Out Buttons If both plate buttons t 2 are disabled and there is no test run active and the communication to the reader is OK then it is most likely that there is a wrong setting in the configuration file Program Files BMG FLUOoptima FLUOstar OPTIMA ini The value behind DisablePlateCmds should be False This parameter will be set to True if the reader is connected to a robotic stacker system and the plate in out movement will be controlled only using the robotic software 118 123 0413F0003A 2002 11 15 BMG Labtechnologies FLUOstar OPTIMA Software User Manual 8 6 Access Rights under Windows NT 2000 XP 8 6 1 File Access Permissions Microsoft changed the default access permissions for all program files newly installed In all Windows versions before Windows 2000 any standard user has access to the files installed using a standard installation program Beginning with Windows 2000 a normal user non power user has only read access by default As we store important information in data base files all users of the BMG software need to have write access to certain files in the program directory usually Program Files BMG FLUOoptima If you have used the option Program should be usable also for non power users during installation see chapter 1 3 2 Control Part Installation the write access rights to the necessary files will be set during the installa
67. eat sims PAM atc t4 CE rl PO DAVE Lyme 00724 7 cog n Loa nl CURA DS Sa IS 7 In L EN BERTH ion lt vC rr he rj Lr rye EJ woe iaa EH val wt De UMS TTA T DUM 54 So UO SUUS ER VR E E nlt weg 017 54 5 Ue tats Loe ORE ony Gat TA amp GgI 0 0M a T Mg t wh 1 1 ose M 1 t 2 1 2r 1 Fo 2 1 TAL P DD Luan pre H mug Cap PE Tebo oto Me Me A ds TATAR I an Q0 3 7 cuc T e M vu VV EE UAR are oc DA wei t QUU 2o fas DAMES re QD om DU e I e Holt ct MM oe sra DA QU LOL woe 1 lus pT rM Un e Mf ERN vuoi toe a elo FE T uc S Mu et As fe n T SU ul 1 1 A i Fr RAW vts eto mu gt l4 7 DU a EE gt el Mort bes z 2 me a QNIN previous plate IDs when same testp omo pci Nac tav Uu 7 ME icc ul fiae EN ae Ve VEZ T Cl lT AMA on DT ivy t ud IU oF i E E SIC E tcl 1 2 PEN e e n Lor is i lh ns 7 T s Options lt protocol gt lt 1 gt lt gt 2002 11 15 o ld De U ar N 1 MPN t zo QUAS no 2t iUi a T a a Tuan executed 85 i SU En E LS E Eu mat VAR
68. ed for all volume groups and for possible additional shaking in the Shaking Options group box on the left side of this sheet Shaking Options Shaklng mode Orbital Shaking mode is circular mixing is more complete especially around the edges of the microplate Double orbital The shaking function is performed as orbital movement The plate carrier makes a figure eight movement Linear Shaking mode is from right to left 2002 11 15 Q413F0003A 59 123 FLUOstar OPTIMA Software User Manual BMG Labtechnologies Shaking width The width linear or diameter orbital of the shaking motion The range is 1 mm to 7 mm The width depends on several conditions Plate format For plates with larger wells such as 6 or 24 well plates you can use a larger diameter slower speed while you should use a smaller diameter faster speed for plates with smaller wells Samples If the wells contain cells the diameter should be larger so the force of the shaking does not stress the cells For viscous solutions you should also use a larger shaking diameter Volume A smaller diameter is suggested for smaller volumes Additional shaking If you want additional shaking other than shaking after injection you can choose in plate mode between shaking before or after each cycle or before after the first cycle In well mode additional shaking is only possible before plate reading Shaking time The duration of time for additio
69. ell This time will be calculated using the settings in the protocol i e the interval time the number of intervals and the start time for all kinetic windows If the defined interval time for the last interval in the last kinetic window is higher than the minimum interval time the calculated total measurement time per well will show a value slightly too high as the reader when the last interval is finished will not wait for the defined interval time before it goes on to the next well If you have defined an injection which will last longer than the last measurement which usually makes no sense this time will be too short The total measurement time calculated by the reader will always be correct After pressing the Check timing button the Timing Overview sheet will become available see chapter 4 9 Click on OK to save the information and to add the test protocol to the list 52 123 0413F0003A 2002 11 15 BMG Labtechnologies FLUOSstar OPTIMA Software User Manual 4 4 2 Kinetic Windows Well Mode It is possible to split the measurement into up to 4 kinetic windows You can define the interval time independently for each kinetic window Therefore it is possible to have denser measurement points on more interesting parts of the kinetic curve Example Calcium influx Ind Kinetic window 4 2 J Start time 6s 185 15 05 No of intervals 3 40 5 interval time 05s 8 25 1s 48 intervals in
70. ell contains select sample standard or blank X The well s content has unknown concentration Standard The wells content has a known concentration and is used to formulate a standard curve in the data reduction Negative N The well s content has known concentrations but will not be Control used for the standard curve calculation It can be used for Positive P comparisons or for special evaluation calculations Control Control C Blank B The well contains water or buffer for measuring background 2002 11 15 0413F0003A 55 123 FLUOstar OPTIMA Software User Manual BMG Labtechnologies Index The Index is the reference number of the sample or standard The index box displays the number that will be used for the next sample or a standard If Increase is selected each sample or standard will be labeled with consecutive numbers With Constant the number will remain the same in the case of continuous replicates In the example picture above the index box shows that the next standard well number is 6 Li shown in the index box is the number of the next well The number remains fixed use this if the samples are identical Replicates Replicates are the number of repeated samples If you have duplicates of a sample on the microplate then you select 2 and whether they are labeled in the horizontal or vertical direction increase For each well the next consecutive number will be u
71. ell i 1 well i Well 1 1 P1 p2 m Example for well mode test with time optimization V1 Injection of volume 1 V2 Injection of volume 2 Pi Volume group 1 injection start time D2 Volume group 2 injection start time Me Measurement start time 64 123 0413F0003A 2002 11 15 BMG Labtechnologies FLUOstar OPTIMA Software User Manual 4 8 Multichromatics When you choose to analyze more than one fluorophore per well well mode or plate mode you must define the filter combinations for each fluorophore First enter the number of fluorophores to be analyzed Basic Parameters sheet and then click the arrow key or the Multichromatic tab A new sheet will appear Now you can use the pull down menu in each row to define the filters The filters shown in the pull down menu are listed as they are defined in the Setup Filter section see chapter 3 1 3 Filters It is possible to define the gain for each filter combination The gain is the amplification of the signal in the PMT This function allows you to optimize the sensitivity for each assay Enter the gain manually from to 4095 or perform an automatic gain adjustment for each filter combination before the measurement see chapter 6 2 Gain Adjustment 2002 11 15 0413F0003A 65 123 FLUOstar OPTIMA Software User Manual BMG Labtechnologies 4 8 1 Multichromatics in Time Resolved Fluorescence Using the Multichromatics function you can deter
72. ent window is specific for each configuration The parameters for different test modes are described on the following pages 6 2 1 Gain Adjustment Fluorescence and Luminescence Mode Stal Test Run pm pa 7 dr bug cen Dou E The filter settings are displayed on the left in luminescence mode there are no excitation filters only emission filters If the protocol is multichromatic then all filter combinations used will be displayed you can perform a gain adjustment for each filter set Automatic Gain Adjustment for one Well Select a well for gain adjustment then click the Gain adjustment well button The instrument samples the well eleven times in 3 seconds to find the optimal gain The optimal gain value appears in the box next to the filter settings 2002 11 15 0413F0003A 79 123 FLUOstar OPTIMA Software User Manual BMG Labtechnologies Stop will terminate the gain adjustment process Automatic Gain Adjustment for the entire Plate There are occasions when a well gain adjustment is not possible for example when the well with the highest intensity is not known You can then do a gain adjustment on the entire plate The instrument finds the well with the highest intensity and determines the gain Click on the blue microplate format number in the top left corner of the layout i e 96 or 384 All wells are selected and the caption of the gain adjustment button changes to Gain adjustment plate Click t
73. ers and the gain settings e Danish style If you use this option there will be no header but in the first column of every data line the plate 101 will be listed This may be useful if you combine data from more than one test run in one ASCII file and search for example for the highest result in any well of all plates When you have found this result you not only see the well name you also see immediately the plate identification Include sample IDs Store the sample IDs see 6 3 Sample IDs also in the ASCII file after the measurement value Calculation Store blank corrected values If this option is selected the measurement values stored in the ASCII file will be blank corrected Use average of all blanks from all groups for blank correction This option is only important when different layout groups see chapter 4 5 1 Using Layout Groups are used If this option is not selected the measurement values of a group will be corrected using only blanks from the same group If you select this option the correction will be done using the average of all blanks from all groups 2002 11 15 0413F0003A 33 123 FLUOstar OPTIMA Software User Manual BMG Labtechnologies Polarization Tests For polarization tests you can decide whether you want the raw data for both channels to be stored or the blank corrected polarization or anisotropy values If you choose the second option the polarization value will be calculated and stored in mP In
74. es Emission titer 590400440520 Volume group 1 Volume Lf 20 injection cyce 1 Shaking after inject s Calculation Startt 1 Stopi 2 Sum Channel Table content 1 Maximum Minimum Average 1 3lope second Slopesminute Time to threshold Time to maximum SD of raw data of raw data Hide protocol settings It is possible to hide the header area and the comment to give better overview for the data by checking the Hide protocol settings checkbox 2002 11 15 0413F0003A 101 123 FLUOstar OPTIMA Software User Manual BMG Labtechnologies Reading Direction icon Legend Comment horizontal vertical reading reading bidirectional reading unidzectional reading start lef start left 59 Bm g v mee a 3 B is LE 5g 58 It is possible to insert comment on worksheet comment is automatically saved in the test run file 102 123 0413F0003A 2002 11 15 BMG Labtechnologies FLUOstar OPTIMA Software User Manual 7 5 4 Calculations on the Three Tables in the Evaluation Sheet The data is presented on three different tables Each table presents different information based on what you choose from the pull down menu on the right side of each table Table 1 56575 20477 9915 1788 4128 265 9 58 epe a TU 20 T BER om ia 28 5 d 88 8 ETAT DSO Raw data blank 1520885 7407 372 3790728 2124215 1196 094 700 246 3511
75. essen eme Customized filters Additionally BMG Labtechnologies has special made filters with a bandwidth of 10 nm 5 nm on stock These filters are available for every 10 nm from 330 to 680 nm BMG 10 nm filters are symmetrical and can therefore be used both in the excitation and in the emission position The stokes shift should be at least 40 nm Indicate wavelength with the last three digits in the part number e g part number 010 340 for BMG340 10 nm filter Upon request special filters can be custom made specify wavelength and bandwidth 20 123 0413F0003A 2002 11 15 BMG Labtechnologies FLUOstar OPTIMA Software User Manual 3 1 4 Reader Offsets Every FLUOstar OPTIMA is accurately calibrated at the factory and has individually defined offset values The offset values correspond to the home position of the microplate carrier Correct offset values are important for optimal measurement results The offset values are stored in the reader EEPROM The determination of the offset values should be carried out only by a qualified service technician If the plate carrier is removed or replaced the offset values should be re calibrated The Reader Offsets screen can be reached by selecting Reader Offset in the Setup menu The password to enable changes of the offset values is bmg You can now enter new values Do not perform an Offset determination unless you are qualified to do so The un button w
76. eved by using the prime function The priming procedure needs to be repeated once after each program start Solutions for injections can be placed inside the reagent box d button or choose the menu command Measure To open the prime window use the 2 Prime Note Before priming a pump the injection needle must be removed from the measurement head to prevent contamination of the instrument Pump speed The pump speed selected here will only be used for priming The injection speed for test protocols is defined in the Concentrations Volumes Shaking sheet of the test protocol definition window see chapter 4 6 Priming It is necessary to fil the tubing with the dispensing solution prior to starting a measurement Place the tubing in the solution to be dispensed and place the injection needle in a waste container Do not leave the needle in the measurement head or you will risk contamination of the instrument Select the prime volume from 500 to 4500 ul The volume of the syringe is 500 ul and a prime volume of at least 1000 pl is recommended for priming the tubings and the syringe higher volume can be used for washing the tubing after the measurements are complete After clicking on a Prime pump button a message will appear to remind you to remove the injection needle from the measurement head Repeat the procedure for all pumps you want to use 72 123 0413F0003A 2002 11 15 BMG Labtechnologies
77. flashes are usually adequate Note Increasing the number of flashes also increases the minimum cycle time and therefore the reading time 46 123 0413F0003A 2002 11 15 BMG Labtechnologies FLUOstar OPTIMA Software User Manual Measurement interval time only luminescence mode Defines the measurement time defines how long the light is measured By increasing this time you will get higher and usually more accurate measurement values Cycle time You can define the duration of each cycle from 1 to 10000 seconds 2hr46min40s When the instrument is switched on you can click the Check timing button and the cycle time is automatically validated by the instrument If you want to increase the cycle time if you want a delay between cycles you can enter a time greater than the minimum cycle time calculated by the Check timing function For example if the instrument gives a minimum cycle time of 25 seconds and you change this to 85 seconds there will be a delay of 60 seconds between cycles Equidistant kinetic cycles If you use this option the minimum cycle time for ali cycles is as long as the cycle with the latest longest injection or measurement requires If you do not use this option the t me for cycles with injection can be different from cycles without injection and the minimum cycle time for each kinetic window can be different This allows very short sampling rates in kinetic areas with a fast change in signal
78. gram start there will be no login screen at program start instead you will be logged in as the default user USER It is still possible to use the login function later Setup Login Beep after finishing a test run If you select this option there will be a short acoustic signal after a test run is finished Confirm program exit If you deselect this option there will be no confirmation window when exiting the program 2002 11 15 0413F0003A 27 123 FLUOstar OPTIMA Software User Manual BMG Labtechnologies 3 3 2 View Sheet Using this sheet you can change the program look Progar n Configuration White marble Green marble Brown marble Red marble Use Golor Gradient in Title Bar The border or title bar at the top of the software can be either a solid color or a gradient from dark to light This is only for appearance and does not affect any other feature of the software Note In Windows 98 2000 or XP you define the title bar style using the windows control panel Therefore this option will be grayed out Speed bar background It is possible to define the style of the speed bar Choose any of the pre defined background styles or use one of your own bitmap files BMP GIF JPEG format Main window background similar to the speed bar it is also possible to define the background of the main program window Choose any of the pre defined background styles or use one of your own bitmap files
79. greater the accuracy For fluorescence measurements 10 flashes are usually adequate Note increasing the number of flashes also increases the minimum cycle time and therefore the reading time 2002 11 15 0413F0003A 41 123 FLUOStar OPTIMA Software User Manual BMG Labtechnologies Cycle time You can define the duration of each cycle from 1 to 10000 seconds When the instrument is switched on you can click the Check timing button and the cycle time is automatically validated by the instrument You will see the minimum cycle time below this group box If the cycle time you have defined is smaller than this minimum cycle time it will be automatically corrected to the minimum time If you want to increase the cycle time if you want a delay between cycles you can manually enter a time greater than the minimum cycle time calculated by the Check timing function For example the instrument gives a time of 25 seconds per cycle but you can change this to 85 seconds so that there will be a delay of 60 seconds between cycles Filters and Integration select Fluorescence intensity or Time resolved fluorescence time resolved fluorescence involves a delay time between flash and measurement Number of multichromatics There is the possibility to analyze 8 fluorophores per well Enter the number of fluorophores to be analyzed then click the arrow button or on the Multichromatic tab to define the filter combination for each
80. gs from this window are user specific therefore each user can select his preferences independently 84 123 0413F0003A 2002 11 15 BMG Labtechnologies FLUOstar OPTIMA Software User Manual 6 4 Measurement After entering IDs performing the gain adjustment you have the following options Start test run Begins the measurement using the defined gain Save amp Close saves the results of the gain adjustment the ID settings and the dilution factors and retum to the main menu without performing a measurement Cancel Closes this window without saving the gain adjustment results and ID settings and without starting the test 6 4 1 Time Duration top of the main program window there is a time gauge indicating the elapsed time of the test The remaining time will be displayed above the time gauge It is updated dynamically during the measurement imo p If you move the mouse cursor to the time gauge a small window with additional timing information will appear ee j Total best run 2mnl5s 2 R u Elapsed test run lime 338 D PME 20 AO dus Curent cycle 2013 Total time for cuvert cycle 50 J Ro EP Remaining cycle time 4s 6 4 2 Stopping the Test Run lt is possible to stop a measurement procedure after it has started by pressing the stop button if there is already measurement data available a dialog box will ask if you want to save the inco
81. he data from the first filter set followed by the data from the second filter set etc If you choose the option List sorted by wells you will get the measurement results for all Cycles in one line per well but if your test contains more than one filter setting you will still get a data block for each filter setting Separator Select how the individual raw data number will be separated for example a comma a semi colon or back slash By selecting 9 a tab step will be used Number format Select the number of digits before and after the decimal point position described with a symbol will be a number or a space a position described with a zero will be a number or a Zero For absorbance tests you should specify a format of 0 000 because the OD values can range from 0 000 to 4 500 By adding a to the format string e g 4 0m the numbers will be expressed in milliOD 0 to 4500 32 123 0413F0003A 2002 11 15 BMG Labtechnologies FLUOstar OPTIMA Software User Manual Non measured wells select the appearance of a non measured well for example hyphen backslash or a zero 0 Header It is possible to include a description of the test run e Short header Describes the test run in 5 lines test name date plate IDs number of cycles and number of channels e Long header Contains the information of the short header but also includes the measurement mode layout name filt
82. he plate identifiers that were created before the measurement see chapter 6 1 Plate Identification Date and Time The date and time that the measurement took place Wells Plate format number of wells of the microplate Filename The number of the database file assigned to the test run Select the test run whose results you want to view and double click it You can also select the test run and select one of the options from the FLUOstar OPTIMA pull down menu located in the toolbar 94 123 0413F0003A 2002 11 15 BMG Labtechnologies FLUOstar OPTIMA Software User Manual 7 2 1 FLUOstar OPTIMA Pull Down Menu The following options are located under the FLUOstar OPTIMA menu entry at the top of the Excel worksheets Copy Selection Select the data you want to copy in a worksheet and choose Copy Selection A new workbook is created and the selected data will be copied into a sheet within the new workbook This workbook can be saved under a new name and can be used like any Excel workbook Change Data Path Select a new destination directory for the test runs The change is valid only for the user who is logged in The new directory must be a subdirectory of the user folder Select the drive and directory in the user path window see chapter 3 5 Data Path Login This allows you to change the logged in user The functions are the same as in the control part see chapter 2 1 Login Screen Copy Test Run This feature allows
83. he well with the highest concentration of free fluorophore Because the relationship between channel 1 and 2 determine polarization it is important that the two channels are optimized for the best results Select the desired well and click on Gain adjustment The automatic gain adjustment calculates the gain settings needed to reach the required value in percent of the measurement range for each channel The required value you enter in this window is used as target value for channel 1 An optimal value for channel 2 is calculated automatically based on the entered Target mP see below After performing the gain adjustment the K factor is calculated automatically K Factor The K factor is used for fine adjustment of the two channels During calculation of the polarization and anisotropy values the measurement results of channel two are multiplied by the K factor as a correction factor for the difference in the two channels Target mP It is recommended to use free fluorescein for adjustment of gain and K factor but it is possible to use other fluorophores or labeled biomolecules The theoretical polarization value for free fluorescein is 35 mP Enter this value in the field Target mP In case you use a different fluorophore or a labeled biomolecule you need to use the theoretical mP value of this molecule as Target mP 2002 11 15 0413F0003A 81 123 FLUOstar OPTIMA Software User Manual BMG Labtechnologies Now when you perfor
84. his button and the instrument will retum a gain value in the box next to the filter settings Note An automatic gain adjustment for the entire plate is not possible for 1536 well plates 6 2 2 Gain Adjustment Absorbance Mode In absorbance mode the gain is determined when the instrument measures the full transmission of light 096 absorbance The gain is dependent on the filter wavelength used During the gain adjustment procedure the plate carrier moves out of the way of the optics and the instrument samples the full transmission of fight Start best Huri The gain sets the optimal signal amplification to achieve optimal sensitivity Perform the gain adjustment on the well with the highest intensity It is also possible to perform a gain adjustment for the entire plate as described above Stop will terminate the gain adjustment process 80 123 0413F0003A 2002 11 15 BMG Labtechnologies FLUOstar OPTIMA Software User Manual 6 2 3 Gain Adjustment Fluorescence Polarization Gain adjustment for polarization requires some extra parameters n dor EC cs Bas ae Cau ee UR ni is inse m S YT uu ES 5 t ae UND Vom Unc ene Lt Gain There are two PMTs photo multiplier tubes involved in polarization one for channel 1 vertical light and one for channel 2 horizontal light As with standard fluorescence you want to perform the gain adjustment on t
85. ife i n 1 VS OE Exe PLE at Arn as cra a to Et nee E Reo ra 7 Lm valid Va F 1 ze 1 1 Pe a 3 auno cad ES BEAT e t oou E You can use this number together with the lt 1 gt or lt gt option for defining the ASCIl export filename or as part of a plate ID In this window you aiso see the Total no of executed runs This is the total number of all tests executed using this computer after installing the FLUOstar OPTIMA software 2002 11 15 0413F0003A 35 123 FLUOstar OPTIMA Software User Manual BMG Labtechnologies 3 4 Login The login function is described in chapter 2 1 Login Screen 3 5 Data Path This window allows you to temporarily change the directory for your measurement results to a directory different than the one specified using the Login screen see chapter 2 1 This might be useful for example to store the measurement results from different methods in different subdirectories toT E ee T2 Coda MILLS 36 123 0413F0003A 2002 11 15 BMG Labtechnologies FLUOstar OPTIMA Software User Manual 4 Defining Protocols 4 1 Protocol Selection Window To create or edit a protocol click on menu The Test Protocols window lists all previously defined tests and gives you options for creating new test protocols or modifying existing protocols or select Test Protocol from
86. ill move the plate carrier to the defined offset position The Offset determination button is for service personnel only Click on OK to save the offset values into the reader EEPROM Note Users with activated Run Only option see 2 1 Login Screen are not allowed to change offset values 2002 11 15 0413F0003A 214123 FLUOstar OPTIMA Software User Manual BMG Labtechnologies 3 1 5 Microplates The FLUOstar OPTIMA software contains a data base with dimensions of microplates from most microplate manufacturers While most plates have the standard spacing and footprint some plates have slightly different dimensions and must be positioned accordingly for optimal results In addition new plates that are not on the current list can easily be defined by selecting Setup Microplates If microplate dimensions are edited the changes will apply to all users RTI TATU PECES 711140 A 11 GN 12760 8550 1440 1120 11320 127 60 8550 1440 1120 113 20 12790 8560 1440 11 20 11350 Taosg 12750 85 60 1430 11 40 11320 DYNATECH 96 12800 858 40 1120 11360 Festa MICROFLIR 128 00 8580 1440 11 20 11380 ERI 128 00 0M 20 113 60 128000 8590 1440 1120 11360 3 D 11340 To select the first microplate beginning with e g B simply press the key B To select more than one microplate press Shift together with 7 or 4 or press Ctrl and click on the desired micropl
87. ilter pairs in numeric style When you move the mouse pointer to the filter setting number column of the table a little hint box containing the filter setting details will appear Current Stale Zooming setts fiy You can click and drag the cursor line to each kinetic point to see the measurement value s of this point It is also possible to move the cursor by using the following keys 1 gt PgUp PgDn Posi and End Using the keys you can zoom into the curve and out again When the option Automatically move cursor to last value when updating s selected the cursor will automatically move to the newest value when the measurement result of a new cycle becomes available The table on the right side will show the new value s numerically If you have defined a Sample ID for the selected well this ID will be displayed below the graph Save Saves the data as bitmap windows BMP or JPEG format on the hard drive Print Creates a print out of the screen on any available printer Close Closes this window 2002 11 15 0413F0003A 91 123 FLUOstar OPTIMA Software User Manual BMG Labtechnologies 6 5 4 Timing Overview After clicking the Timing button in the Current State window you will get a window showing an overview of the timing of the currently running test 8 Lumina Cl ween eres LIP In this window you can see a graphic overview of the measurement and all injections f
88. int could be out of the range of the curve or there could be more than one concentration possible in these cases the table will contain asterisks in the place of the data The signal of the respective sample is out of the signal range of the standards No extrapolation is possible The signal of the respective sample is lower or higher than the asymptote min or max values can only appear if 4 parameter fit is chosen For the signal units there is more than one concentration possible No clear relationship between signal and concentration 2002 11 15 0413F0003A 113 123 Fi UOstar OPTIMA Software User Manual BMG Labtechnologies 7 8 Result List Worksheet This worksheet is only available if the standard curve worksheet is visible This means that standards must be defined in the layout and in the third table of the Evaluation Worksheet one of the following selections must be used Raw data minus blank and Averages minus blank FLUOstar OPTIMA Test 71260021051 Hide parameter settings BMG Lebtechnologies 1D 42 3 T Hide standard curve Fluorescence plate mode Evaluation type Sum Calculation Sati 2 2 Selected Range Table calculation None Curve titting Lineer Regression m 3 31963 1 40382 r 0 980503 Comment DNA Test CONCENTRATIONS Sort contents Sort sample Ia Sort rows Sart columns BoR On eem et DT 2 VT Moe EL er emm use dilution Facko
89. ion software see chapter Fehler Verweisquelle konnte nicht gefunden werden Evaluation Worksheet 2002 11 15 0413F0003A 43 123 FLUOstar OPTIMA Software User Manual BMG Labtechnologies Check timing Minimum cycle time 1 The minimum cycle time can only be calculated by the reader After defining all necessary settings do not forget the filters click the button Check timing The shortest possible cycle time will be displayed If you have defined a cycle time which is shorter than this minimum time it will automatically be corrected At the bottom of the test protocol definition window the calculated Total measurement time for the entire plate will be displayed Note The Check timing function is only available if the reader is switched on After pressing the Check timing button the Timing Overview sheet will become available see chapter 4 9 Limitations for 1536 well plates Your reader has to be specially prepared for measuring 1536 well plates Measuring 1536 well plates is only possible in plate mode The foliowing limitations exist for the test protocol e only one kinetic window only horizontal bidirectional reading starting top left e nomultichromatics e no injection e shaking Printing the Protocol To print the protocol use the key combination Shiit Ctrl P A standard print dialog box will appear Here you can decide whether you only want to print the current sheet Sele
90. is a FLUOstar OPTIMA menu on the far right with some special functions specific to the data reduction package There is also an icon for switching back to the control part of the FLUOstar OPTIMA software Note The worksheets are designed for a screen resolution of 800 x 600 pixels or higher 7 1 The Worksheets The FLUOstar OPTIMA evaluation software will be opened with up to 8 worksheets Test Runs This worksheet is displayed when Excel is opened It lists all test runs sorted by date and time with the newest at the top Raw Data This worksheet gives the raw results from the measurement You can select the type of evaluation for kinetic calculations and select the data you want included in the calculations Signal Curve If working with kinetic you can view the signal curve for a single well or a group of wells It appears only if the measurement has more than one kinetic point Evaluation View the results on three tables you can select how the data is presented on the table i e averages raw data blank subtraction standard concentrat ons etc Sampie IDs Contains a list of all sample IDs This sheet is only available if sample IDs are defined Standard Curve Presents a graph of the standard concentrations and lists the resulting calculated concen trations Protocol Settings In this sheet you can see the used protocol settings 2002 11 15 0413F0003A 93 123 FLUOstar OPTIMA Software User Manual BMG Labtechnologies
91. is opened for the first time please refer to chapter 7 of this manual for bug fixes 10 123 0413F0003A 2002 11 15 BMG Labtechnologies FLUOstar OPTIMA Software User Manual 1 4 Uninstalling the FLUOstar OPTIMA Software An uninstall program has been included in the software package which allows you to delete all components of the FLUOstar OPTIMA software in the registry Select Start Settings Control Panel Add Remove Software Select FLUOstar OPTIMA and click on Remove In the Remove FLUOstar OPTIMA window click on Start iis J tt 47 ar 1 T weis 1 gt Qa T E 2002 11 15 0413F0003A 11 123 FLUOstar OPTIMA Software User Manual BMG Labtechnologies 2 Control Software Overview 2 1 Login Screen When the software is opened a login screen appears The Login Screen function can be switched off see chapter 3 3 Program Configuration You can enter a user name a password and a directory path to your personal user folder This feature allows more than one user to perform test runs on one PC The data and test protocols are stored in an individual user folder The user can also edit test parameters and some evaluation features without the changes applying to all users miim n qe m m nac ee our IH NENNT 1 _ RO T SP ULIS LEAL iioc
92. it ne iy INS ros SM fI wi Tn 2 tan PS rd We c MELS a a cafa n3 ves CATT L aL dI 0 o 0o Von D lA a BM Se tae TU uva t 1 ri tree Pee Ce PE Inlet nM GUST We LINE z REPRE m tere s n ata TUM O EO DAY i 2 Save E Dese t c D D E PH ae t VU VUE farci tlt us ono ag C VU dee T The name of the used test protocol will be entered Consecutive numbers for each test run with the same test name will be automatically entered You can manually change the start number in the space No of executed runs since program start The number of digits to be used can be changed by adding characters i e using 1 222 001 002 003 etc will be generated To use the Total no of executed runs instead of the number of executed runs after program start add T before the e g lt 1 THHH gt To use the No of executed runs for the used test protocol add a P before the e g lt 1 gt You can see a Run Statistics after pressing the button see section 3 3 4 Counting down is also possible use a instead of the Same concept as consecutive numbers using the alphabet Example lt A f gt gt AAA AAC O413F0003A 77 123 FLUOStar OPTIMA Software User Manual BMG Labtechnologies lt date gt The date of
93. kinetic window Therefore it is possible to have denser measurement points on more interesting parts of the kinetic curve If you choose to use more than one kinetic window a new sheet Kinetic Windows see chapter 4 3 3 will become available where you can define the parameters for all kinetic windows To switch to this sheet click the arrow button or on the Kinetic Windows tab If you use only one kinetic window you can define all necessary parameters on the Basic Parameters sheet Kinetic Window 1 Number of cycles This is the amount of times the entire plate will be measured for kinetic window 1 Each well defined in the layout will be measured once per cycle if no multichromatics are used otherwise it will be measured as many times as chromatics are used within each cycle You can define up to 250 cycles 250 is the total number for all kinetic windows Measurement start time Defines the time when the measurement will start for all cycles belonging to kinetic window 1 relative to the time when the measurement position is reached plus positioning delay Defining a measurement start time larger than makes sense when i e injection or shaking is performed before the measurement Number of flashes per well and cycle You can define up to 200 flashes per well and cycle All the measurement values obtained for all flashes for a cycle are averaged for one intensity value per well Therefore the greater the number of fiashes the
94. kup of the existing test protocols the user database the filter table microplate definitions and measurement data when performing an upgrade the end of the installation procedure there will be a window telling you where to find the backup files 2002 11 15 0413F0003A 9 123 FLUOstar OPTIMA Software User Manual BMG Labtechnologies ee eee A l amp wL jop Complete M p E 7 1p a MI EP im v T As long as the database format has not been changed see chapter 1 2 it is possible to copy the backup files back to their original directory in case you want restore previous data But if there has been a format change you can only use the backup files after re installing the old program version 2 Evaluation Part Installation After installation of the control software the main menu will reappear Click on Install Evaluation Part and follow the instructions of the installation wizard to install the data reduction software the installation procedure and options are similar to the control part installation see above When the evaluation software in Excel is opened for the first time the following screen might appear Micra el Then click on Enable Macros The macros are safe for the computer There are several bugs associated with Microsoft programs that effect the FLUOstar OPTIMA software If error messages appear when the FLUOstar OPTIMA software
95. l is measured Therefore it might be a good idea to place at least one blank at the beginning of reading depending on the selected reading direction see chapter 4 5 1 Using Layout Groups if you are interested in the current state display As soon as the measurement values of additional blanks become available the entire current state display will be recalculated and updated If you use this option during plate mode tests the current state display will only be updated when a cycle is finished regardless of the selection under Update display Use average of all blanks from all groups for blank correction This option is only available when different layout groups see chapter 4 5 1 Using Layout Groups are used If this option is not selected the measurement values of a group will be corrected using only blanks from the same group If you select this option the correction wil be done using the average of all blanks from all groups Note All settings from this window are user specific therefore each user can select his preferences independently 90 123 0413F0003A 2002 11 15 BMG Labtechnologies FLUOstar OPTIMA Software User Manual 6 5 3 Zoom Feature After double clicking on a well in the Current State window you get a zoomed view of the measurement values Here the measurement values are always displayed as a curve There is also a table on the right side of the window where you can see measurement results for all used f
96. m a K factor adjustment on the well with the highest concentration of free fluorophore the result of that well will become equal to the target value entered so for fluorescein the polarization value of the well is equal to 35 mP After a gain adjustment the K factor is automatically calculated but it is also possible to perform a K factor estimation without a preceding gain adjustment using the predefined gain values by pressing the Get K Factor button The two intensity values of channel 1 and channel 2 are measured using the two gain values and a K factor is given To reduce the influence of intensity deviations it is recommended to use a factor near 1 0 To increase the K factor you should decrease the gain value of channel 2 to decrease the K factor you should decrease the gain value of channel 1 Custom Target mP value If you are using a different fluorophore or a fluorescein labeled peptide bound fluorescein you must change the Target mF value If you do not know what this value should be you can start with for example 35 Perform a gain adjustment K factor estimation as described above Start the measurement After the measurement is complete look at the Excel worksheets for the test run If the results are negative then you must increase the target value Increasing the target value will shift the results to the positive range 6 2 4 Required Value The measurement range for fluorescence is 0 to 65 000
97. microplate beginning with e g B simply press the key B after opening the pull down box using e g 4 General Settings Positioning delay You can define a waiting period after the microplate moves a well to the measurement position and before the measurement begins The delay time allows the liquid to settle and the surface to become stable so that the measurement is more accurate For homogeneous fluorescence assays BMG Labtechnologies recommends a delay time of 0 25 For fluorescence measurements of cell assays a delay time of 0 55 is adequate Liquid movements and hence delay time are influenced by viscosity Number of kinetic windows You can split the measurement into up to 4 kinetic windows You can define the number of intervals the interval time and the start time independently for each kinetic window Therefore it is possible to have denser measurement points on more interesting parts of the kinetic curve If you choose to use more than one kinetic window a new sheet Kinetic Windows see chapter 4 4 2 will become available where you can define the parameters for ali kinetic windows To switch to this sheet click the arrow button or on the Kinetic Windows tab If you use only one kinetic window you can define all necessary parameters on the Basic Parameters sheet Kinetic Window 1 Measurement start time Start time for kinetic window 1 Defines the time when the measurement will start relative to the
98. mine the optimal integration time for a time resolved assay Using one time resolved fluorophore you can define the same filter combinations for all 8 filter settings and define 8 different integration times You can then compare the data in the evaluation part to find the optimal time for the fluorophore For example if you were using Eu you would define all 8 filter settings as TR EX and TR EM You can keep the integration time constant and vary the integration start time in order to find the optimal start time You can then keep the integration start time constant and vary the integration time to find the optimal measurement time Plate Mode EF ampri eere iE m mm b i 4 F i F 66 123 0413F0003A 2002 11 15 qr m BMG Labtechnologies FLUOstar OPTIMA Software User Manual 49 Timing Overview After clicking the Check timing button lower left corner of the test protocol definition window the following sheet will become available mL T latae Ne In this sheet you can see a graphic overview of the measurement and all injections for one well For plate mode tests all actions are displayed in the timing overview of one cycle The displayed injections may occur in different cycles as defined under Injection Timing The cycle used will be listed behind each injection bar If there is a timing problem e g overlapping injections using the same
99. moving data if you want to eliminate the results of a well from the data reduction highlight it in one of the three tables and press Delete The content name of this well will now appear in lower case letters Its value will not be used in calculations Pressing Delete again will restore the data value Save changes If you remove data in the described way a save button at the far right below the evaluation sheet will appear Click on this to save the changes you have made to the data permanently 2002 11 15 0413F0003A 109 123 FLUOstar OPTIMA Software User Manual 7 6 Sample IDs Worksheet BMG Labtechnologies This worksheet is only available if you have defined Sample IDsbefore starting the test run see section 6 3 It contains a list with all sample IDs You can choose between sorting for rows columns well content or sample IDs Sort column up Sot column down Sort row up Sort row down Sort up Sort down C Sort up Well Contents 1 SamptelDs j Act 51 Re 100 pot 52 RG 100 rot 153 ARGS 100 por 54 RG 100 55 RG 100 01 56 RG 100 B Blank Ht 0 AU 1 Probe X3217581 B02 xg Probe 321752 coz x17 Probe X3Z2 006 002 X25 Probe X322 018 E02 X33 Probe X322 030 It is possible to change the sample IDs here by editing the respective fields in the Sample ID column You can store these changes permanently by using
100. mperature Results Excel Help Contents Help Contact Addresses Heip BMG Web Page Help About FLUOstar OPTIMA Help System Information 16 123 0413F0003A BMG Labtechnologies Function Moves microplate carrier out of the instrument Moves microplate carrier into the instrument Performs a measurement using a pre defined test protocol Before the measurement starts you can enter plate IDs and perform a automatic gain adjustment Stops the active test run after finishing the current cycle only available for plate mode tests Stops the active test run Prepares the pumps for injection if pumps are installed Define pump speed Defines the temperature of the incubator Opens the evaluation software for data reduction Test runs raw data are automatically stored in an Excel template Opens the online help Shows BMG s contact addresses Starts an internet browser and makes a connection to the BMG web pago Shows the software and firmware reader EPROM version Shows information about the operating system the CPU speed the current Excel version and the amount of memory 2002 11 15 BMG Labtechnologies FLUOstar OPTIMA Software User Manual 3 Setup 3 1 Instrument Setup The configuration settings for the instrument include the reading mode communication port filter definitions instrument offsets definition of microplates and the data path You can use the Setup
101. mplete data 2002 11 15 0413F0003A 85 123 FLUOstar OPTIMA Software User Manual BMG Labtechnologies 6 5 Current State Graphics 6 5 1 Current State Overview When the measurement has started the Start Measurement button will change to the Current State Graphics button This function allows you to view raw data graphically as the measurement occurs wa Stal ar RE 1 4 irt 4 vast Tr Li tiny TA wen ui T al m d 1 3 mat Pan PAY Srt mag E UL Hae ta za 1 Ug 2 oh rate at n Ou a DES i I i e TEMP x E ARE TS The Current State window displays a grid of the microplate format The measurement results can be displayed as curves Each measurement value is represented by a dot and you can see the relative position of the values You can choose between a curve of points or lines It is also possible to display the measurement values of the last measured cycle interval numeric or use colors for a fast overview see 6 5 2 Current State Options Current cycle Shows the number of the cycle currently being measured only for plate mode tests Well Shows the name of the current well only for well mode tests Display cycles intervals Kinetic points that will be displayed This can be changed manually otherwise the default number of cycles intervals as defined in the te
102. n reference to this object 3 Error 1004 VBA initialization failed 4 Error 40009 No current row 5 Disabled Plate In Out Buttons 6 Access Rights Problems under Windows NT 2000 XP For solutions see the following chapters 8 1 Error Cannot open Data Path This error can occur if there is a problem with the Microsoft JET engine or if there is a language conflict between the Windows operating system and Excel Installing a new service pack for the operating system or installing a new service release of Excel may sometimes help but we strongly recommend not to use different language versions of Excel and Windows The BMG software is tested with English Windows and Excel versions Windows 98 ME NT 2000 and Excel 97 2000 XP and with German Windows Excel versions We can not guarantee that our software will run without problems on computers with Windows Excel in other languages especially on mixed language systems Note This error can also occur when you have installed the evaluation part for Excel 97 and upgrade to Excel 2000 XP or vice versa In this case please reinstall the evaluation part The installation program will automatically install an evaluation part optimized for the Excel version used Altematively you can manually start FLUOstar OPTIMA Vx xx x Installation from CD ROM Evaluation setup97 exe for the Excel 97 optimized evaluation program version and FLUOstar OPTIMA Vx xx x Inst
103. n the evaluation software If you would rather use a different evaluation software package which will not work with dbase formats then it is also possible to store the data in ASCII format If you select this option then the Define Format for ASCII Export window becomes available see chapter 3 3 3 Save Measurement Data in dBase Format If this option is selected all measurement data is stored in a set of dbase files in the data directory of the user logged in The data is saved in a dbase file format that can be used with the macros in the evaluation software If you would rather use a different evaluation software package which will not work with dbase formats then it is possible to get an ASCII file see above Note lt is possible to use both formats simultaneously Retum to selection window after editing microplates or test protocols The selection window for tests and microplates contains previously defined setups and options to edit export import etc By default you can return to the main control software after creating or editing a test or microplate definition without returning to the selection window If you wish to see the selection window for the microplates or test protocol definitions again check the box in the Program Configuration window 26 123 0413F0003A 2002 11 15 BMG Labtechnologies FLUOstar OPTIMA Software User Manual Auto Login If you choose the option Automatically login as user USER at pro
104. nal shaking is defined in seconds The maximum shaking time is 300 seconds 5 minutes Limitations for injection The maximum amount per well Volume 1 Volume 2 Volume 3 Volume 4 is 500 ul 6 12 24 and 48 well plates 350 ul for 96 well plates and 100 nl for 384 well plates respectively Injection is not possible in 1536 well plates Note It is necessary to prime all used pumps before performing a test with injections see chapter 5 1 Priming Shaking after Injection without Injection If you need shaking at a certain time but no injection at this time you can use any of the four volume groups Please set all injection volumes in the table column belonging to this group to 0 but select a pump which will not be used You can then specify the shaking duration at the bottom of the table The start time of this shaking procedure can be defined under Volume group X injection start time in the Injection Timing sheet see chapter 4 7 60 123 0413F0003A 2002 11 15 BMG Labtechnologies FLUOstar OPTIMA Software User Manual 4 7 lnjection Timing 4 7 1 Injection Timing Plate Mode Made aL luores scence mc be ee Aa t eer gd a cmm un e 1 M UU Veto Te itta Vogt tt BR Dui r co o c n co adve AUI 2 MC PR fu t 1 ek E rope AU Soc etc MNT Te re np IMS ua ES Yo trt NI PEE
105. nal up to 60 C The chosen temperature must be higher than the ambient temperature The temperature can be set in 0 1 C increments The temperature can also be monitored without activating the incubator see Temperature Monitoring Feature below You can enter the temperature manually or toggle to the desired temperature using the arrow buttons Temperabure Coomtial ee Te Click on Incubator on The temperature indicator in the toolbar of the control software will be activated The indicator will be red unti the selected temperature is reached then the indicator turns green it is possible to perform a measurement before the target temperature is reached In the Excel data reduction the current temperature for each kinetic point during the measure ment is displayed in the raw data worksheet The Incubator on button changes to Set new temperature if you select a new target temperature during incubation 5 2 2 Temperature Monitoring Feature It is possible to monitor the instruments temperature without using the incubator The temperature sensor of the incubator will be activated and update the temperature display Click the Temperature monitoring without incubation button The temperature will appear on the temperature display in the control software When using this function the indicator color will be cyan The temperature will also be stored with the measurement data and displayed in the Raw Da
106. nge will be listed in the tables of the evaluation sheet Minimum Finds the minimum value for each well for a set of intervals Select Minimum in the pull down menu The Minimum of the selected range will be listed in the tables of the evaluation sheet Average Finds the average value for each well for a set of intervals Select Average in the puli down menu The average of the selected range will be listed in the tables of the evaluation sheet Slope second Calculates the linear regression curve and gives the corresponding slope per second for each well The slope of the selected range will be displayed in the tables of the evaluation sheet 104 123 0413F0003A 2002 11 15 BMG Labtechnologies FLUOstar OPTIMA Software User Manual Slope minute Calculates the linear regression curve and gives the corresponding slope per minute for each well The slope of the selected range will be displayed in the tables of the evaluation sheet Time to threshold When you select threshold over time an additional box appears next to the pull down menu Time to threshold v You must enter the threshold that you are interested in in this field The time that the threshold is reached is indicated in the tables of the evaluation sheet Time to maximum Calculates the time until the maximum is reached for each well SD of raw data Calculates the standard deviation of the raw data for each well 5 n ar 96 CV of raw data
107. nsitivity 2002 11 15 0413F0003A 45 123 FLUOstar OPTIMA Software User Manual BMG Labtechnologies 4 3 3 Kinetic Windows Plate Mode It is possible to split the measurement into up to 4 kinetic windows You can define the cycle time individually for each kinetic window Therefore it is possible to have denser measurement points on more interesting parts of the kinetic curve r n nomena MET Phat Hole seere Number of cycles This is the amount of times the entire plate will be measured Each well defined in the layout will be measured once per cycle if no multichromatics are used otherwise it will be measured as many times as chromatics are used within each cycle You can define up to a total of 250 cycles divided into up to 4 kinetic windows Measurement start time Defines the time when the measurement will start for all cycles belonging to the respective kinetic window relative to the time when the measurement position is reached plus positioning delay Defining measurement start time larger than 0 makes sense when i e injection or shaking is performed before the measurement Number of flashes all modes except luminescence You can define up to 200 flashes per well and cycle All the measurement values obtained for all flashes of cycle sent to one well are averaged for one intensity Therefore the greater the number of flashes the greater the accuracy For fluorescence measurements 10
108. ols or if he is only allowed to run pre defined protocols 7 Run Only mode 14 123 0413F0003A 2002 11 15 BMG Labtechnologies FLUOstar OPTIMA Software User Manual 2 3 Icons and Menu Commands The main menu is displayed across the top of the screen Click the item to display the pull down menu for additional options You can also use the corresponding buttons of the tool bar that perform the same functions as the main menu items The instrument must be turned on and connected for certain functions to be available FP tMastar X Wr ETE ons Em uml c DESI i e tar pH Hor TET aL IP Menu command Menu for configuring the instrument and program settings Select the measurement mode reader configuration filters change offsets values and select a directory for measurement data Setup Changes program behavior define options for Program Configuration ASCII export Setup Exit P Exits FLUOstar OPTIMA control software Test Setup Test Protocol ELE Defines the test parameters e g number of flashes intervals cycles content of the wells in the microplate standard concentrations injection volumes and times etc 2002 11 15 0413F0003A 15 123 FLUOstar OPTIMA Software User Manual Menu command icon Measure Piate Out Measure Plate In Measure Measure Measure Pause after current Cycle Measure Stop Test Run Measure Prime Measure Te
109. or one well For plate mode tests all actions are displayed in the timing overview of one cycle The displayed injections may occur in different cycles The used cycle will be listed behind each injection bar In addition you will see tables containing the measurement and the injection times Note The listed pump action times include the time for shaking after injection An injection action can contain different steps E ERR Injection Shaking 92 123 0413F0003A 2002 11 15 BMG Labtechnologies FLUOstar OPTIMA Software User Manual 7 Evaluation Software Data Reduction The data reduction package provides powerful Excel macros for easy data calculations as weil as all the functions possible with the Excel software Once a run is performed the data is automatically saved as a database file in Excel The test run will be present in a list of all saved test runs on the first worksheet in Excel You can access the data by clicking on the Excel aj icon in the tool bar or selecting Results Excel from the main menu bar you can go directly to Excel from the Windows Start menu Programs BMG FLUOoptima FLUOstar OPTIMA Evaluation If the evaluation software is opened from the start menu then a login screen will appear as with the control software The same user path and password applies At the top of every worksheet is the normal Excel menu with ail the functions from the Excel program In addition there
110. r Up C Dom C Up Down Up Down C Up C Down V Content Sample Well D ution Rawdata Avgeof 5007 C Calculated factor replicates replicates concentr 81 1 70 25 65 25 5 7 662635 006193665 31 A02 80 25 The worksheet contains a list of all raw data based on the selection you made for the third table on the evaluation sheet The data values are grouped by replicates Also the calculated concentrations are shown using the curve fitting method of the current standard curve worksheet In the header appear the most important measurement parameters the parameter table for the standard curve and the graph of the standard curve Both can be hidden by checking the checkboxes Hide parameter settings and Hide standard curve to get a better overview of the data 114 123 0413F0003A 2002 11 15 BMG Labtechnologies FLUOstar OPTIMA Software User Manual The data can be sorted by plate rows plate columns well contents or sample lds in upward or downward direction using the corresponding checkboxes If you check the box Use dilution factor the raw data results are multiplied with the corresponding dilution factor The calculated concentration will be recalculated from the new raw data results FLUOstar OPTIMA Test Name FSS 7 26 02 10 51 Hide parameter settings BMG Labtechnalogies 101 23 Hide standard curve Sort contents Sort sample IDs Sort rows Sort columnis Lp Down Gun C Do
111. ration section of the PLUOstar OPTIMA ini file MinVolumeStep 6 for minimum steps of 1 ul use MinVolumeStep 1 You will find this configuration file in the FLUOstar OPTIMA main installation directory usually Program Files BMG FLUOocptima Note Exporting this table into a XLS Excel text or HTML file is possible after right clicking on the content column of the table Pump to use Select which pump should be used to inject the defined volume s It is possible to use the same pump in different volume groups Pump speed The pump can dispense at different speeds The injection speed is defined in ul second The default speed is 310 second which should be appropriate for most assays There are pre defined speeds available by using the pull down menu Factors determining pump speed Viscosity Liquids such as water and buffer can be dispensed at higher speeds For highly viscous solutions you should use a slower speed to ensure higher precision and lower the risk of air bubbles Celis Solutions containing celis should have a lower speed because it causes less stress on the cells Volume Higher speeds are necessary for small volumes below 3 5 pl to ensure the best performance Shaking time If you want shaking after an injection add the desired shaking time here It is possible to define different shaking times for different volume groups The shaking width and shaking style orbital or linear will be defin
112. red individually for the defined number of intervals For example a test can be defined with 3 intervals and an injection Therefore well A1 moves to the measurement position a measurement is performed then an injection followed by two more measurements When all defined measurement intervals have been performed on well A1 the microplate carrier moves to the next defined weli for the same protocol Once all the defined wells have been measured the assay is finished Key words Intervals Number of times the well is measured Up to 250 intervals per well can be defined Interval time The length of time in seconds for each measurement interval This includes the flashes and the time for measuring the emission tight Total measure The amount time it takes to perform all the measurement intervals ment time per including any injections on one well It is calculated by multiplying well the number of intervals by the interval time and taking into account all used kinetic windows and the measurement start times 4 4 1 Basic Parameters Well Mode N Hod Daret seier 48 123 0413F0003A 2002 11 15 BMG Labtechnologies FLUOstar OPTIMA Software User Manual Test name Assign a test name as you would like it to appear on the list of test protocols Microplate Use the pull down menu to select the microplate used in the assay All microplates defined under Setup Microplates will be listed To select the first
113. roup 1 2 3 or 4 is initiated relative to the time when the measurement position is reached plus positioning delay Example The following scenario would be possible with this method e Inject the volumes defined in volume group 1 in the first cycle at 0 7 s An incubation time of 10 minutes could be realized by setting the cycle time of the first cycle kinetic window 1 for 600 s e Inject the volumes from group 2 in the second cycle 10 minutes later at 0 s and the volumes from group 3 in the same cycle at 2 s which then will take place just after the volume group 2 injection e The measurement could be started at 4 s This will then be directly after injection 3 the measurement will start in all kinetic cycles at the same time Legend B Measurement aes Yoluma 1 BE Volume goup 2 Bea Volume aoup 3 62 123 0413F0003A 2002 11 15 BMG Labtechnologies FL UOstar OPTIMA Software User Manual 4 7 2 Injection Timing Well Mode Click on the Injection Timing tab in order to define the injection times well Mode Fluarescence E i ioa ue 0 I xl un m Meas start 1200s PE S Vol group 1 in time 7 03 Vol group Zini time 20s 0 Wn t E AU zn Mee an elle aver Sw MENT e mE GG I M CP PLANA 2 MEE Waar es d eB ee II A a wx E a PL hg oa M 1 i aa i ra Een LT
114. rt to a disk or to a different directory A new window will ask for the destination drive and directory The file name will get an extension MPL Import Imports microplate definition from a disk or another directory and adds it to the existing data base The extension for the microplate file must be MPL 2002 11 15 0413F0003A 23 123 FLUOSstar OPTIMA Software User Manual BMG Labtechnologies Delete Deletes an existing microplate and its dimensions from the data base Glose Returns to main menu Notes Exporting the microplate table into a XLS Excel text or HTML file is possible after right clicking the table Users with activated Run Only option see 2 1 Login Screen are not allowed to edit copy or import microplate definitions 24 123 0413F0003A 2002 11 15 BMG Labtechnologies FLUOstar OPTIMA Software User Manual 3 2 Firmware Update This function allows you to update the reader firmware content of the built in Flash EPROM E THANE Update LIC VAT we t c2 mir ma Tute n lho i S SU Cote th os S RIED md ene 7 ule D BST tz t 1 ZEE SPA s t1 M E If you received a new firmware file from BMG you should copy this file into the folder Program Files BMG FLUOoptima Firmware Make sure that the reader is connected to the computer and switched on After typing in the correct password you will get the password with the new firmware select the firmware file using the S3 Al
115. rvice bmglabtech com You can also use our on line bug report form http www bmglabtech com html support bugreport cfm 2002 11 15 0413F0003A 123 123
116. s will be displayed SD of replicates The standard deviation of the replicates and the blanks will be displayed SD n2 x 2 x 106 123 0413F0003A 2002 11 15 BMG Labtechnologies FLUOstar OPTIMA Software User Manual 26 CV of replicates The CV standard deviation average in percent of the replicates and the blanks will be displayed Regression coeff r The regression coefficient as calculated from the linear regression equation is displayed Appears only if Slope second minute is chosen in the calculation pull down menu Layout The contents standards samples blank as defined in the layout section will be displayed Standard concentration The concentrations of the standards that were defined in the layout Concentrations and Volumes window will be displayed Volumes 1 2 3 4 The injection volumes for each pump as defined in the layout Concentrations and Volumes window will be displayed Sample IDs Shows the Sample ID for each well Note If you use very long sample IDs you will only see part here approximately up to 10 characters To see the full sample ID use the Sample Ds worksheet Dilution factors Shows the dilution factor for each well Multichromatic Data if more than one filter setting was used in the test protocol multichromatic it is possible to view the data for each setting by using the small pull down box The box contains numbers corresponding to the order of
117. se multichromatics the minimum interval time is equal to the defined measurement interval time multiplied by the number of m ultichromatics plus the time necessary to switch the filters If you want to increase the interval time if you want a delay between the intervals you can enter a time manually If you have defined an interval time in any of the kinetic windows which is shorter than the respective minimum interval time it will automaticaily be corrected to the shortest possible value If necessary the start times for following kinetic windows will also be corrected 2002 11 15 0413F0003A 69 123 FLUOstar OPTIMA Software User Manual BMG Labtechnologies For example the instrument gives a minimum interval time of 0 1 seconds but you can change this to 0 2 seconds so that there will be a delay of 0 1 seconds between intervals Time to normalize the results As the measurement values depend on the measurement interval time it is usually a good idea to normalize the measurement values This will allow you to compare the measurement values of test runs using different measurement interval times This will also allow you to compare the results of different kinetic windows using different measurement times To switch off the normalization enter 0 here Example When you set the Time to normalize the results to 1 0s to get the results in counts per second and use a Measurement interval time of 0 1s the raw measurement values will be m
118. sed The number Methods of Labeling 1 Select the appropriate content from the contents box and then double click on each well of that type Select in the index whether the samples should be labeled with increasing numbers or with the same number 2 f the samples are in successive rows or columns select increase if the samples should be labeled with consecutive numbers or choose constant if they are continuous replicates Click on the first well with the left mouse key and drag across the wells containing the samples standards controls or blanks 3 Ifa row or column contains the same contents samples standards click the row letter or the column number and all wells of that row column will be labeled 4 To fill the entire microplate select the appropriate content samples standards and click on the format number e g 96 in the top left corner Note Exporting the layout grid into a XLS Excel text or HTML file is possible after right clicking the grid Reading direction After pressing the reading direction button 3 you can choose between horizontal or vertical reading horizontal the plate carrier will move from ieft to right reading across successive columns vertical the plate carrier will move up and down reading in successive rows It is also possible to select a bidirectional or unidirectional reading mode Example Using a bidirectional horizontal reading of a full 96 well plate the rea
119. st protocol will be used Options Opens the Current State Options dialog box Timing Opens the Timing Overview window Save saves the data as bitmap windows BMP format or JPEG format Print Prints the screen on any available printer Close Closes the current state window 86 123 0413F0003A 2002 11 15 BMG Labtechnologies FLUOstar OPTIMA Software User Manual If you are using layout groups see chapter 4 5 1 the layout grid will be displayed using the background colors belonging to the layout groups used When you move the mouse pointer over a well the well content e g SA1 will be shown for a few seconds You can double click on a square to get a zoomed view of the measurement values see chapter 6 5 3 6 5 2 Current State Options After clicking the Options button in the Current State window the following dialog box will appear Cunent State Display Ophons IO ae 1 ee SEM kl 1 worn te 7 reet Spats 2 Measurement values of last measured cycle interval cua i auto values if ony one cycle interval exists otherwise curve Um d Two colors good bad num SOIL e g Sz t tale toot ua te SE E TEN ae gt a EE pra e t Aoc neo xr s p h qe E 4 F SS Pi Roc a E Nu DOE Loc aun s P F MELLE Dendi ni Reading order af the wells ii TE yae oo HERD Ta CEbUSU
120. ta Worksheet of the evaluation software see chapter 7 3 74 123 0413F0003A 2002 11 15 BMG Labtechnologies FLUOstar OPTIMA Software User Manual 5 2 3 Auto Power On Incubation When you click the Use above setting after auto power on the currently selected temperature value will be used as the default target temperature for the incubator If you have defined a value within the allowed temperature range the built in incubator will be automatically switched on the next time the reader is switched on Using this function the incubator will be turned on even without starting the FLUOstar OPTIMA software By using a target temperature of 00 1 C selected e g by clicking the Temperature monitoring without incubation button only the temperature monitoring function is switched on by default the heating plates will not be used To switch off the auto power on incubation function select a target temperature of 0 C either by clicking the Off button or by manually entering this value and click the Use above setting after auto power on button 2002 11 15 0413 00034 75 123 FLUOstar OPTIMA Software User Manual BMG Labtechnologies 6 Performing a Measurement Test runs are performed by selecting the menu command Measure Measure or by using the 24 button A window displays all the defined protocols for the selected configuration i e fluorescence mode ecl AER Protucols me ree nin
121. talls the software necessary for configuring the instrument for setting up test parameters and for executing measurement procedures Evaluation setup installs the data reduction program Excel macros In a few words the control part is used to get the results and the evaluation part to look at them Place the CD into the CD ROM drive This should start the program automatically but if CD auto start is disabled then execute the program Start from the CD drive An installation directory will appear 1 EE LESE ES Alc i oiai Powealustivin Ps Note If you are using Windows NT Windows 2000 or Windows it is necessary that the installation is performed by a user with administrative rights 2002 11 15 Q413F0003A 7 123 FLUOstar OPTIMA Software User Manual BMG Labtechnologies 1 Control Part Installation Click on Install Control Part and follow the instructions from the installation wizard Setup wpe TT DUE NETS T am y E TRIAL RIPE xU HE i EPA IN IL Sen man me 1 ait doces M p You can choose either Full Installation or Custom Installation e Full installation will install the programs with all options This is the installation option recommended for most users Custom installation allows you to choose the options you want to install and is recommended for advanced users If you perform the installation as an user with administrative rights using Windows NT 2000 X
122. the Test Setup f Test Protucals uci Vu a SER TI MG LASTECHNOL GIES 56 7 Wel Fugrescence BMG LABTECHNOLOGIES S5 Fluqwescence du POMS 384 ER TEST GHEINER FOIE 394 Plate Fly Fluorescence WE AEQUORIN TEST NUNC MAXISORP 96 Wel Luminescence i tu LUCIFERASE BMG LABTECHNOLOGIES 96 Wel Luminescence TOM S TEST BMG LABTECHNOLOGIES 96 Plate Luminescence The FLUOstar OPTIMA can measure fluorescence intensity time resolved fluorescence and optional absorbance and luminescence In addition the POLARstar OPTIMA can read in fluorescence polarization mode Before defining a new test it is necessary to select the correct measurement mode in the Setup Reader Configuration see section 3 1 1 New Define a new test protocol See section 4 2 1 Edit Modify a previous test protocol Select the test and click on Edit or double click on the test name Copy Select the protocol you want to duplicate A new screen appears and asks for a name for the copy The duplicate will appear on the test protocol list under the new name Making a duplicate protocol means that you can modify a protocol without changing the original Export You can copy a test protocol to a disk or another directory Select the protocol s you want to export then click on Export new screen will ask for the destination drive and directory and a name for the file 2002 11 15 0413F0003A 37 123 FL
123. the Save changes button 110 123 0413F0003A 2002 11 15 BMG Labtechnologies FLUOstar OPTIMA Software User Manual 7 7 Standard Curve Worksheet After the data is selected in table three on the Evaluation worksheet click on the tab for Standard Curve at the bottom of the screen The worksheet contains the graph of the data optional curve fits and tables of known and unknown concentrations You see the parameter table where the parameters for each group are listed at the top of the sheet You can select the groups you wish to see by clicking the corresponding checkbox above the parameter table Note In this example there are group A and B selected You see the regression lines for these groups 4 Parameter FE Y a di my b Evaluation Type Sum Ma Ge Oc Oo a 29ES 54981 10D0E2 b BE 18220 12300 283 5 2BB8E 8 230 8 a 93005 192883 150204 R 0 989453 0 397037 0 988636 Edit statt parameters for curve fit Lin Log 0 0 0000002 0 000064 0 0000006 0 0000008 0 000001 6 000001 2 CONCENTRATIONS Standard concentrations Linear Regression Segmental Regression Point ta Point gt The calculated unit values for the standard concentrations defined in the test setup plotted in a Standard curve The graph can be plotted on linear or logarithmic scale in x or y direction by clicking the Lin or Log button of the correspondent axis 2002 11 15 041
124. the filter combinations The number is in the order in which the filters were defined in the test protocol see chapter 4 8 Multichromatics Choose the number in the box that corresponds to the raw data you want to view You can choose different numbers for each of the three tables to make comparisons of the data Which filter setting correspondent to which position can be seen in the header of the evaluation sheet The pull down box will be empty if only one filter pair has been used 2 3 4 6 B 2002 11 15 0413F0003A 107 123 FLUOstar OPTIMA Software User Manual BMG Labtechnologies 7 5 3 Data for Standard Curve Table 3 is used for defining the data used for the Standard Curve If you choose Raw data blank then the results if valid will be plotted on Standard Curve worksheet This standard curve will be the basis for calculating the unknowns The data selection that is valid for plotting the standard curve includes raw data minus blank and averages minus blank A standard curve cannot be formed from volumes or layout information If the data from table three cannot form a graph the standard curve worksheet will be blank If a value in table 3 is negative the logarithmic scale for the standard curve cannot be selected Calculations in the three tables Responsible for standard curve Table2 Tablei 885 912 9864 Tablet Table2 T3ble2 Table There is an additional pull down menu on
125. the injection for volume group 1 2 3 or 4 is initiated relative to the time when the measurement position is reached plus positioning delay The time can be before the beginning of the measurement Measurement start time gt Volume group X injection time it can be during or after the measurement You can only define the injection time for those volume groups where volumes are defined and a pump is selected in the Concentration Volumes Shaking sheet Time optimized measurement If a kinetic test run with time optimization is defined then two consecutive wells are processed at the same time This mode is especially useful when there is a long incubation time between e g an injection and the measurement An injection into a well is in this case immediately followed by an injection into the next well Then the first well is measured followed by the second well By injecting into the next well in the sequence the incubation time of that well is underway during the measurement of the first well The ability to use Time optimized measuring is contingent on the injection and measurement start times as well as on the duration of each action This mode is only possible when there is a lag time between different actions this delay time must be longer than the time necessary to perform all actions 2002 11 15 0413F0003A 63 123 FLUOstar OPTIMA Software User Manual BMG Labtechnologies Well i move to mova to m p2 w
126. this case and for anisotropy values stored in you should use a number format with enough space for decimal numbers e g THEHFO 000 Polarization values p Chi k Ch2 k Ch2 Anisotropy values a Chi k Ch2 Ch1 2k Ch2 Number of Executed Runs The counter tracks the number of measurements that were performed after the program was started You can manually type in a starting number This number will be used in the filename options lt 1 gt and lt A gt To see a Run Statistics press the 18 button Note All settings from the Define Format for ASCII Export window are user specific therefore each user can select his preferences independently 34 123 0413F0003A 2002 11 15 BMG Labtechnologies FLUOstar OPTIMA Software User Manual 3 3 4 Run Statistics In this window you see a list of all used test protocols together with a number showing you how often a protocol was executed It is possible to edit these numbers or to delete a protocol from the list Hun 5Latisliex 1536 ER TEST JABER TEST FAST PLATE TOM S PLATE MODE TST 5 TEST Alva s WELL MODE TST Dc 5 B Sie So Mel no ET EP v LP tora ot tai 1 ue gE en De ON is Pe o laec tat Ma Mfr E i foe rS whe done oer wee LB BIG ease at Far gesedeer 0 erg DC VR ies 22 DMo eneDEG T LUNA V 1 SO a 5 IBUM n S LS
127. time when the measurement position is reached plus position delay Example measurement start time 5 seconds After the reader plate carrier reaches the measurement position the defined positioning delay time starts After th s there will be an additional delay of 5 seconds before the measurement itself starts Using this measurement start time it is possible to start the measurement after an injection Number of intervals This is the amount of times a measurement will be successively taken on each well equals the number of readings on each well You can define up to 250 intervals 250 is the total number for all kinetic windows This is typically used for kinetic assays in which the dynamics of a reaction changes very quickly over time Each interval is plotted as a kinetic point displaying the change over time 2002 11 15 0413F0003A 49 123 FLUOstar OPTIMA Software User Manual BMG Labtechnologies Number of flashes well and Interval You can define up to 200 flashes per measurement interval All the measurement values from the flashes defined for one interval are averaged to produce one intensity value per well The greater the number of flashes the greater the accuracy For fiuorescence measurements 10 flashes are usually adequate Note Increasing the number of flashes also increases the reading time Interval time You can define the duration of each interval from 0 02 to 25 0 seconds When the instrument is acti
128. ting of files where write access is necessary The following files directories need to have full access to write temporary data AProgram Fites BMG FLUOoptima FLUOstar OPTIMA ini Configuration file FLUOstar OPTIMA ms Run Statistik File FLUOstar OPTIMA log Log file AProgram Files BMG FLUOoptima Temp Directory for temporary files Program Files BMG FLUOoptima User Subdirectories for test protocol definitions and measurement data of standard user AProgram Files BMG FLUOoptima Admin Subdirectories for test protocol definitions and measurement data of administrator AProgram Files BMG FLUOoptima Subdirectories for test protocol lt UserName gt definitions and measurement data of defined users AProgram Files BMG _ Flashtools Used by Fiash EPROM Update Program 120 123 0413F0003A 2002 11 15 BMG Labtechnologies FLUOstar OPTIMA Software User Manual ata TEE D cM r i T AL ney 7 E xut t i Weed Sir i mi T u H TL pA SUN Program Files Borland Common Files BDE idapi32 cfg BDE properties idapi32 bak Backup of idapi32 cfg pdoxusr net Paradox network control file You can use the BDE Administrator to change the location of the pdoxusr net file Configuration Drivers Native Paradox Net Dir 8 6 2 Registry Access There are two places in the registry where the BMG Labtechnologies software stores information All user specific things like program settings
129. tion If you did not use this option or if you have added new users user groups after installation the following error might appear CIR EGENT RIDE TT n OIGZRUICg ee aS epee R NEN Ree S See rea cee NDA ETAT RR ee RE E n T GERE TIC Ra CE E Solution Logon as administrator or user with administrative rights Run the program SetPermission from the FLUOstar OPTIMA CD ROM folder FLUOstar OPTIMA Vx xx Installation from CD ROM Alternatively you can also manually change the permissions Open the Windows Explorer and right click the folder where the BMG Labtechnologies software is installed usually Program Files BMG FLUOoptima Choose Properties then Security Check the Modify permission Write permission will be checked automatically Uncheck the box Allow inheritable permissions from parent to propagate to this object Now all users should be able to write to the BMG Labtechnologies directory and work with the software 2002 11 15 0413F0003A 119 123 FLUOStar OPTIMA Software User Manual BMG Labtechnologies Default settings Customized settings AMT ABF e Hi nper ies dl pu pe Properties E f MEE TINGZ Adasan MEETING Administrators IEEE Bcrearon owner e creator owner Ji SYSTEM sree Lis
130. tion without Microplate Definitions and Filter Table to keep your definitions when upgrading from version 1 10 0 The format for test protocols and for the measurement results database has changed with version 1 20 0 Therefore you should not deselect the groups Test Protocols and Measurement Data during the first installation of FLUOstar OPTIMA V1 2x x 2002 11 15 0413F0003A 5 23 FLUOstar OPTIMA Software User Manual BMG Labtechnologies 1 3 Software Installation The software needs to be installed in the following order 1 Excel 2 FLUOstar OPTIMA Control 3 FLUOstar OPTIMA Evaluation Please follow the next set of instructions carefully to ensure proper installation of the software 1 3 1 Excel Installation The Excel software should be installed prior to the FLUOstar OPTIMA software There are certain components that must be included in the installation If Excel 97 Excel 2000 or Excel XP has not been previously installed follow the instructions below for complete installation If Excel 97 is already installed follow the instructions below to ensure the necessary components have been installed e Complete installation of Excel 97 2000 XP Start the Office 97 2000 XP or Excel 97 2000 XP installation program Choose Custom or Complete Installation this option installs ail Excel components Be sure that all components are ticked you can be sure of this if click on select all The Excel wizard will ask
131. to select the microplate used in the assay All microplates defined under Setup Microplates will be listed To select the first microplate beginning with e g B simply press the key B after opening the pull down box using e g 4 40 123 0413F0003A 2002 11 15 BMG Labtechnologies FLUOstar OPTIMA Software User Manual General Settings Positioning delay You can define a waiting period after a well of the microplate moves to the measurement position and before the measurement begins The delay time allows the liquid to settle and the surface to become stable so that the measurement is more accurate For homogeneous fluorescence assays BMG Labtechnologies recommends a delay time of 0 25 For fluorescence measurements of cell assays a delay time of 0 55 is adequate Liquid movements and hence delay time are influenced by viscosity Flying mode This is a time optimized parameter for plate mode When this function is selected the measurement using 1 or 3 flashes only will occur at the exact moment that the center of the well is under the measurement head The plate carrier does not stop as the well passes the measurement position If you have defined injections the injection cycles will be performed in non flying mode Number of kinetic windows You can split the measurement into up to 4 kinetic windows You can define the number of cycles the measurement start time the number of flashes and the cycle time independently for each
132. tton on the table headline Concentration or Volume 1 Volume 4 The calculations are done automatically You can also select a specific set of wells by finding the first well and clicking and scrolling down with the right mouse button or using the cursor keys 4 and 7 together with Shift Goncentration The auto function will automatically calculate the concentrations using the given start concentration and a number to be multiplied factor added increment or subtracted decrement Click on Concentration at the top of the table or select a range of standards with the right mouse button and the calculations will automatically be entered Volume Enter the starting injection volume Indicate whether the injection volumes for the wells increase or decrease by a certain amount Leave the factor as 1 if the volume is the same for every well With the right mouse button click the desired field in the table title row Volume 1 for volume group 1 and the volumes are entered automatically in all columns of this row or select the desired columns using the right mouse button It is not necessary to use ail volume groups consecutive you can for example use only volume 2 and 4 58 123 0413F0003A 2002 11 15 BMG Labtechnologies FLUOstar OPTIMA Software User Manual You can enter the volumes in steps of 0 5 ul If you prefer steps of 0 166 ul 1 6 ul minimum step of the injectors add the following line to the Configu
133. tware User Manual New Save Click on New and enter the user name and desired password Select the directory for the user s data and test protocols For directory options click on the far right side under Directory and a button appears After clicking this button a user path dialog box appears where you can select the drive directory etc see section 3 6 After the information is entered click on Save and the information is stored in the user data base When the information is saved two new folders are automatically created in the new user s directory one for storing test protocols and one for storing data see User Directories below Delete When an existing user entry needs to be deleted highlight this entry and click Delete Cancel If you click Cancel without clicking Save ail changes made after the fast save will be discarded Run After clicking Run the FLUOstar OPTIMA software will start using the test protocol and measurement resuits paths of the selected user For ali users other than the default user USER you need to enter the password first Note Exporting the user table into a XLS Excel text or HTML file is possible after right clicking on the table The passwords will only be visible when the export function is used by the administrator 2 1 1 User Directories After the initial login for a new user two directories will be automatically created lt Username gt
134. ultiplied by 10 Emission filter It is possible to choose an emission filter or use an empty position to measure without filter 70 123 0413F0003A 2002 11 15 le LEGNOANPTTTM m 6 BMG Labtechnologies FLUOstar OPTIMA Software User Manual 4 12 Fluorescence Polarization Tests Before defining a fluorescence polarization test protocol you must select the fluorescence polarization mode in the Reader Configuration window see section 3 1 1 Fluorescence polarization can be measured in plate or well mode Most parameters are similar to fluorescence protocols see chapter 4 3 or 4 4 Multichromatic measurements and injections are not possible gt via ci si ef a LIIS IP AE anm n CETERI RE mnm va rans TL ri REIS qa EE EL Hn 1 Des En Cy Hm P us aM DASS S215 DB TIPE hl EM E LEN fc e eflfe L E AENG Teao lec DU Imam eM t Nam F tI E UB SC gt nr Lou e UNA sc UM ox TT E cto Qa OE La Mat i a d to eet jus mM o due UH T2 cl test E I GM to Moe O rne LM EE E 1 TT E a D QUA MI E ELA b 0 Deu DA Mor ot x Va v6 misc ern T DD E 1 17 PILLE Mtm tQ ce E EX lui ua Unc uti gE DT un
135. ve you can click the Check timing button and the interval time is automatically validated by the instrument If you want to increase the interval time if you want a delay between the intervals you can manually enter a time If you have defined an interval time in any of the kinetic windows which is shorter than the respective minimum interval time it will automatically be corrected to the shortest possible value If necessary the start times for following kinetic windows will also be corrected The minimum interval time depends on the number of flashes 1 flash equals 0 02 seconds 10 flashes equals 0 2 seconds etc and on the usage of multichromatics For example the instrument gives a minimum interval time of 0 4 seconds but you can change this to 5 seconds so that there will be a delay of 4 6 seconds between intervals End time of kinetic window 1 This value is automatically calculated Measurement start time plus Number of intervals multiplied by Interval time The start time for kinetic window 2 needs to be higher than or equal this end time Filters and Integration Select Fluorescence intensity or Time resolved fluorescence time resolved fluorescence invoives a delay time between flash and measurement No of multichromatics There is the possibility to analyze up to 8 fluorophores per well In well mode all fluorophore settings will be measured in one cycle one well at a time Enter the number of fluorophores to
136. wheel Click on Setup and Filters and enter the filter wavelength values in the corresponding spaces An empty position should be named on the emission side for the luminescence and absorbance mode Nofe The table is common to all users gt 3 4 a 7 8 Clear After clicking this button the table contents will be removed Standard After clicking this button standard filter values see figure will automatically be entered into the table Load Loads filter information from an ASCII or Excel xls file into the table This might be useful if you are using more than one set of filter wheels Save Saves filter information from the table into an ASCII or Excel xls file OK Saves the filter positions and retums to the main menu Cancel Closes this window without saving changes Note Users with activated Run Only option see 2 1 Login Screen are not allowed to change the filter settings 2002 11 15 0413F0003A 19 123 FLUOstar OPTIMA Software User Manual BMG Labtechnologies Available Filters BMG Standard Filters Excitation and emission filters are optimized in pairs Transmission profiles of the standard filters are not symmetrical and excitation and emission filters are not interchangeable Excitation Absorbance Wavelength _ BE D Wavelength ge tee fee em emm Im _ cem m Lem Mm _ uem sem som _ sem 9m _ Sum Sem _ TREK meni Semen etm
137. wn C Up C Down Up Down use dilution factor Content Sample Well Dihtion Raw Awgeof Sof Calculated ID factor blank replicates replicates concentr Si ACT 1 70 25 8525 5 7 662825 006193665 St 02 80 25 i 52 0015 131528 133025 45 141278508 044309318 BO 1345 25 01 1 2863025 312525 495 15 83873 085394378 53 coz 3820 25 84 000 1 327725 324725 30 0 923859 1 02070352 S4 002 3217 25 55 Em 1 504625 872025 1674 24390979 206715147 S5 E02 839425 SE 01 2 1 787325 814875 1755 2153705 249757211 SB F02 8324 25 GDi 4 1106825 1545975 43915 2840602 470044596 57 G02 19851 25 58 4 1231825 1554175 2235 1438062 472515334 58 H02 15765 25 x 1 503325 500225 31 0619721 154850172 K1 4971 25 M2 Bd 1 896525 8900925 104 1146732 277482751 X2 B 04 9173 25 OX COS 1 194125 196375 225 1445767 083387276 x3 C 4 4996 25 x4 1 214025 214225 2 0 09336 058775851 04 2144 25 X5 E03 3783 25 280475 415 1479633 088737406 X5 E04 2846 25 XB 1 28519 25 2872575 206 5 0 718857 669761798 XB F 4 2893225 2002 11 15 0413F0003A 115 123 FLUOstar OPTIMA Software User Manual BMG Labtechnologies 7 9 Protocol Settings Worksheet Here you can see all settings defined in the used protocol FLUOstar OPTIMA Test name GROUP 4 6 C STANDARD 2001409712 12 09 07 BMG Labtechnalogies D
138. ws start menu e Goto HKEY_LOCAL MACHINE Software BMG Labtechnologies e Select Permissions e Add read and write rights for everyone to this key and all sub keys 2002 11 15 0413F0003A 121 123 FLUOstar OPTIMA Software User Manual 8 7 Run Log Window The Run Log window control part shows all commands sent to the FLUOstar OPTIMA and all responses You can open the Run Log window using the key combination Shift Ctri L from the program main window 16 39 07 Run Log started 17 10 01 at 15 29 07 45 33 07 nit command 16 33 08 busy 115 39 08 Processing init cmd 116 3910 standby 16 39 10 Sending to reader 02 00 0C 05 05 07 1500 30 00 00 00 Hun I Ug 16 38 07 Sending to reader 02 00 04 05 01 00 00 00 12 00 vom as e 1 ut P ILL ae ij ui ty HE i ma 16 33 10 EEPROM data values command 16 39 11 Reader response 02 01 00 05 07 88 2 06 00 00 01 00 00 00 00 00 00 00 00 00 1 00 00 00 00 00 00 00 00 00 00 00 00 00 1 00 00 00 00 00 00 00 00 00 00 00 00 00 1500 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 78 00 78 00 01 00 EE 02 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00
139. y Note It is also possible to import test protocols created using FLUOstar 403 old series FLUOstar Galaxy and LUMIstar Galaxy If you import a test protocol from one of these programs the FLUOstar OPTIMA import function will ask you to import the layout definitions used by these tests immediately after importing the test definitions In FLUOstar OPTIMA the layout information is stored with the other test parameters the above mentioned programs use different data bases for layout and test definitions It is also possible to import test protocols from NOVOstar Delete Remove a test protoco from the list by selecting the protocol and clicking on Delete OK Retum to the main menu Notes To select the first test protocol beginning with e g T simply press the key T To select more than one protocol e g for export use Shift together with 1 or 4 or press Ctrl and click with the left mouse button Exporting the test protocols table into a XLS Excel text or HTML file is possible after right clicking the table Users with activated Run Only option see 2 1 Login Screen are not allowed to edit copy or import test protocols 38 123 0413F0003A 2002 11 15 BMG Labtechnologies FL UOstar OPTIMA Software User Manual 4 2 Creating a New Test Protocol Click on NEW the next dialog box will ask for plate mode or well mode Select the method that is appropriate for the assay The following sections 4 3 Plate

FLUOstar* OPTIMA Software User Manual

Contents

Download Pdf Manuals

Related Search

Related Contents