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TF 384 Operating Manual - Beta

1. Appendix D ThermoFlour 384 Operating Manual Copyright 2001 3 Dimensional Pharmaceuticals Inc C 3 Confidential May 2001 Artisan Scientific Quality Instrumentation Guaranteed 888 88 SOURCE www artisan scientific com hy Artisan Scientific Quality Instrumentation Guaranteed 888 88 SOURCE www artisan scientific com
2. Construction and Function ThermoFlour 384 Operating Manual Plate Processing System The plate processing system consists of the input and output storage magazines the PlateCrane robotic arm and the bar code reader It is controlled by Total Control for Windows This system loads and unloads microplates for the ThermoFluor 384 instrument Storage Magazines The storage magazines are designed to hold up to 30 microplates in a stack for the PlateCrane The standard run is eight plates which takes 24 hours in up down mode The magazines are held in position by magazine bases that are connected to the ThermoFluor 384 system base plate Four magazine positions are supplied with the system Two are provided to hold plates for the ThermoFluor 384 instrument and two magazines are provided to allow for the integration of a liquid handler This allows for an input and an output stack for the ThermoFluor 384 instrument Figure 2 12 Input output and barcode reader magazines PlateCrane The PlateCrane robotic arm moves 384 well thermal cycler microplates from the input storage magazine to the thermal cycler block in the ThermoFluor 384 instrument s light tight compartment When the experiment is completed the PlateCrane retrieves the plate and places it into the output magazine This process is repeated for up to 30 microplates in each a single session Alternatively a lid can be used as a topper
3. VV INUNVNONUUNVNUNNUUUUuUUUUYUYUVUUY _ w D v w v wow Preface About the Documentation Documentation Overview This preface will familiarize the user with the ThermoFluor 384 system documentation set and explains how to use these documents Specifically this preface includes the following sections e Introduction to ThermoFluor 384 System Documentation e Scope of these Documentls e Standard Conventions e Typographical Conventions e Special Terminology The following table identifies the appropriate section to read in order to learn about specific aspects of the documentation set To learn more about Read the section See page Overview of the ThermoFluor 384 Introduction to ThermoFluor 1 2 System documentation 384 System Documentation 2 Scope of these documents Scope of these Documents Standard Conventions Typographical conventions Typographical Conventions Special terminology Special Terminology Copyright 2001 3 Dimensional Pharmaceuticals Inc 0 1 Confidential May 2001 Artisan Scientific Quality Instrumentation Guaranteed 888 88 SOURCE www artisan scientific com Preface Introduction to ThermoFluor 384 System Documentation ThermoFluor Instrument Operations The ThermoFluor 384 System Documentation is a set of documents covering all aspects of the ThermoFluor 384 system necessary to support executing applications This in
4. Description No filter is used 500 30 nm This is the standard filter that comes with the ThermoFluor 384 instrument 530 20 nm For use in viewing higher wavelength fluorescence 470 30 nm For use in viewing lower wavelength fluorescence Copyright 2001 3 Dimensional Pharmaceuticals Inc Le se 4 13 Confidential May 2001 Artisan Scientific Quality Instrumentation Guaranteed 888 88 SOURCE www artisan scientific com Operation ThermoFlour 384 Operating Manual 5 Enter the appropriate System Parameters These fields are No Replicates The number of images of the well taken by the CCD camera following the shutter closed image during each cycle in the Up Down mode This represents the detection mode of the CCD camera e 1 corresponds to High Signal to Noise Ratio Use for bright plates pes e 2 corresponds to High Dynamic Range Standard use e 3 corresponds to High Sensitivity Use for dim plates The system is set at 2 during installation 6 Enter the appropriate Integration Parameters These values are usually set at installation They determine the area to be integrated when the Acquire 3 0 programs create the INT files These fields are Field The width of the integration field used for each well IBox Y Dimension The height of the integration field used for each well 7 Enter User Comments if any in the text box provided 8
5. e Error log e Current state of the instrument Recovering Plate Data Following System Disruption The Reintegrate command found under the IMAGES menu in the ThermoFluor Acquire 3 0 application can be used to recover data for a plate if the system experienced a disruption after the initial images for a plate were taken but before TCW finished running the method for that plate If the system is disrupted during image collection that data will be lost Action Open the ThermoFluor Acquire 3 0 application Select Read Images from the IMAGE menu The OPEN dialog box appears ta 3DP s ThermoFluor FFAcquite version 3 00 File Subsystems Images En sad Tena poe Experimental Parameters d jis a Data Directory NS Wiite Image Browse Reintegrate p Sample Information Plate ID Look in EN TFAcquire J e al e Eal Comment E gt 1118 10 1 psq Heating Parameters Heating Mode Dom y Initial Final Temperatures C 49 ro Temp Incr Low Temp CH M faa pS High Low Temp Holds sec fiso fo Files of type Photometrics Image Sequences psa Cancel Exposure Time sec Reps 19 a Open as read only Fitters a SC Image Y Gain RH LBox KYE 157 157 Ready Run 1118 10 SC psq Camera Ready Select any image file psq from the series of images files for the desired plate from the corresponding data directory using the OPEN dialog box Press OPEN
6. Sian Add Plate Delete Plate Modify Plate Esc Exit Press Esc Exit button to return to the MAIN menu screen Copyright 2001 3 Dimensional Pharmaceuticals Inc 5 16 Confidential May 2001 Artisan Scientific Quality Instrumentation Guaranteed 888 88 SOURCE www artisan scientific com Maintenance ThermoFlour 384 Operating Manual Repair There are no user repairable items in this system If you suspect instrument repair is required please contact 3 Dimensional Pharmaceuticals Inc at 1 610 458 8959 Copyright 2001 3 Dimensional Pharmaceuticals Inc 5 17 Confidential May 2001 Artisan Scientific Quality Instrumentation Guaranteed 888 88 SOURCE www artisan scientific com Maintenance ThermoFlour 384 Operating Manual Copyright 2001 3 Dimensional Pharmaceuticals Inc 5 18 Confidential May 2001 Artisan Scientific Quality Instrumentation Guaranteed 888 88 SOURCE www artisan scientific com Appendix A Total Control for Windows Overview This appendix contains detailed information about the control software Total Control for Windows used for the ThermoFluor 384 system Specifically this appendix includes the following sections e Total Control for Windows Manual The following table identifies the appropriate section to read in order to learn more about a specific area To learn more about Read the section The Total Co
7. VW Y WWE SY YY WD DY YY Y YY Y YY YOSOY Installation and Calibration ThermoFlour 384 Operating Manual aa wheel will rotate to that position tia IDP s Thermofluor FFAcquiie version 3 0 0 File Subsystems Images Experimental Parameters pj d Data Directory Browse Sample Information Plate ID Comment Heating Parameters Heating Mode Up Down Initial Final Temperatures C 40 70 Temp Incr Low Temp C fi 25 High Low Temp Holds sec 780 65 Image Parameters F Scale Bitmap 0759 0009 1 0 IBox 0 OvteO Exposure Time sec Reps T0 4 Filters None y ei 30 nm Uninitialized 10470 3 30 nm Heator g Uninitializad 24 04 10 12 AM Locate the standard ThermoFluor filter supplied with the instrument and place it in the filter wheel at aioe 1 with the writing cise side aup CAUTION The filter is a sensitive optical item and should be Secure the filter wheel filter cover over the filter Close the optical compartment door and secure with screws Focus the Camera Use the procedure below to focus the camera The system software must be running to Becomplish this task Step Action i a If it is not already open open the optical compartment by removing the screws from the rear panel Place a white piece of paper with small print on the thermal cycler block To do this manually open the light tight door as follows From the Acquire 3 0
8. 888 88 SOURCE www artisan scientific com ODO ARANA TVT TPVT A AAA AA AAA AX Construction and Function ThermoFlour 384 Operating Manual Windows NT Workstation The Windows NT Workstation contains the software that controls the operation of the system and creates data files that are analyzed by the ThermoFluor analysis software The ThermoFluor analysis software is usually installed on a separate analysis workstation The ThermoFluor analysis software will be discussed in the ThermoFluor Analysis Software Manual Total Control for Windows TCW operates the overall system and allows the running of multiple plates It directly operates the plate processing system and it indirectly operates the ThermoFluor 384 instrument through the ThermoFluor Acquire 3 0 control software The ThermoFluor Acquire 3 0 control software interfaces with a CCD camera and an embedded controller in the ThermoFluor 384 instrument The ThermoFluor Acquire 3 0 control software can be used to run a single plate without the use of TCW or the PlateCrane robotic arm The software relationships are illustrated below Analysis Workstation ThermoFluor at Analysis Software Euan Reader Embedded Controller Plate Processina System ThermoFluor 384 Instrument Figure 2 2 Windows NT Workstation software applications Total Control for Windows This software controls the PlateCrane
9. 2 Initial Temperature 35 00 IBox X 16 Temperatura Increment 1 00 IBox Y 16 Final Temperature 26 00 Number of Raplicates 2 Imaging Temperature 25 00 Gain 2 Low Equilibration Time 30 00 Shutter Closed N High Equilibration Time 30 00 Your message here Exposura Time 1 00 laser Delete Eat Figure 4 9 Set up plate parameters from the TCW ThermoFluor Plate setup screen This screen is used to specify which set of pa will be used for each plate to be run Copyright 2001 3 Dimensional Pharmaceuticals Inc 4 11 Confidential May 2001 Artisan Scientific Quality Instrumentation Guaranteed 888 88 SOURCE www artisan scientific com Operation ThermoFlour 384 Operating Manual The system will begin processing Plate 1 using the set of parameters entered for Plate 1 It will then proceed to Plate 2 using the set of parameters entered for Plate 2 It will continue processing each plate with its corresponding set of parameters until it comes to the last designated plate If there are more plates in the stack in the magazine than designated on the ThermoFluor Plate Setup screen then the system will use the last designated set of parameters for the remaining plates Click on the plate number under Stack1 on the screen to view the set of parameters for a given plate The available functions for the screen are listed in the table below Clicking the Add button adds another plate to the bottom of the lis
10. Cleaning the system Focusing the camera 5 3 Changing filters Changing a light source Troubleshooting the system Repairing the ThermoFluor 384 system Copyright 2001 3 Dimensional Pharmaceuticals Inc 5 1 Confidential May 2001 Artisan Scientific Quality Instrumentation Guaranteed 888 88 SOURCE www artisan scientific com Maintenance ThermoFlour 384 Operating Manual System Cleaning The following cleaning procedures should be followed every three 3 months Internal Instrument Cleaning Use the following procedure to clean the interior of the ThermoFluor 384 instrument p_ Action Shut down and disconnect power to the system External Instrument Cleaning Follow the following procedure to clean the exterior of the ThermoFluor 384 instrument Shut down and disconnect power to the instrument Dampen a cloth in glass cleaner solution Wipe all external surfaces on the instrument and around the plate processing system with the damp cloth Avoid any connectors plugs or cables t o p Wipe all external surfaces on the instrument with a dry cloth Wipe all horizontal black anodized surfaces around the plate processing system with a damp cloth 4 5 _ Wipe the stainless steel rods of the plate magazines with a damp cloth 7 Wipe and thoroughly dry all horizontal black anodized surfaces around the plate processing system with a dry cloth Wipe and thoroughl
11. Designed for proteins that irreversibly unfold Continuous Ramp Mode Takes the sample from one temperature value to the next without lowering the temperature Images are taken at each temperature value Designed for proteins that easily refold 4 Initial Final Temperature C This specifies the range of temperatures for the run Initial Temp The starting temperature in C for measurements First high temperature in Up Down mode Final Temp The last temperature in C for measurements Last high temperature in Up Down mode Specify the temperature increments to be taken over the range of temperatures specified above and the Low Temperature for the Temp Incr Low Temp C Up Down heating mode Temp Increments Temperature difference between measurements in C Low Temp The low temperature in Up Down mode that is held while images are taken Not used in ramp mode Specify the High and Low Temperature Hold times for the heating cycle e High Temperature hold The high temperature equilibration or hold time in seconds for the cycle in Up Down mode High Low Temp Holds sec e Low Temperature hold The low temperature equilibration or hold time in seconds for the cycle in Up Down mode Specify the length of exposure for each image and the number of replicate images at each temperature e Expo
12. It should not have to be done again under normal operating conditions To verify that the stack position in the TCW program is correct follow the procedure below Action Select the Calibrate PlateCrane F4 button on the MAIN menu of TCW The Calibration screen will appear CALIBRATION A ape nt a Position Nene R 2 D Current Position rm D MM ME SSR ane R Jog Distance 5 0deg Z Jog Distance 5 0 mm P Jog Distance 5 0deg Change R Jog Change Z Jog Change E Jog o NOTE Teach STACK positions by Jogging E Save Points the Z axis DOWN at 20 SPEED until h ia the LIMIT switch triggers Then jog UP mm then TEACH the position Press lt SPACE BAR to Halt Movement Home the arm by selecting Home Plate Crane button The plate arm will move to its home position Copyright 2001 3 Dimensional Pharmaceuticals Inc 5 12 Confidential May 2001 Artisan Scientific Quality Instrumentation Guaranteed 888 88 SOURCE www artisan scientific com 7 r Maintenance ThermoFlour 384 Operating Manual Select the desired stack location on the Calibration screen For example Stack1 Click the Move to Point button on the Calibration screen Move to Safe Select
13. floor should be sufficiently level to provide for leveling the instrument front to back and sided to side with the supplied leveling feet The ambient temperature should remain between 20 and 24 C The relative humidity should be low enough to ensure a non condensing environment The system operates from a universal power supply that runs on 110 VAC 60 Hz properly grounded outlet and should draw no more than 15 amps The PC is equipped for connection to a network via a 10 100 Ethernet connector if desired Stand Alone Placement and Access The footprint of the system is 24 inches wide by 36 inches deep The system stands approximately 66 inches high A minimum of 18 inches 24 inches recommended should be provided on the side and back of the instrument to ensure adequate access for routine maintenance and operation A minimum of 36 inches should be provided at the front of the instrument See figure 3 1 below Copyright 2001 3 Dimensional Pharmaceuticals Inc 3 3 Confidential May 2001 Artisan Scientific Quality Instrumentation Guaranteed 888 88 SOURCE www artisan scientific com Installation and Calibration ThermoFlour 384 Operating Manual Minimum 60 inches gt i 24 inches I Minimum Minimum Access han e s 90 inches 18 inches E 36 inches fi um t N N fi um Access Access 18 inches 18 inches Minimum Access 36 inches Figure 3 1 ThermoFluor 384 system stand alone place
14. robotic arm movements and interfaces with the bar code reader The method that is used to move the microplates from the input storage magazine to the imaging area then to the output magazine is programmed using this application The program also interacts with the ThermoFluor Acquire 3 0 control software to coordinate actions between the ThermoFluor 384 instrument and the Plate Processing system Refer to Appendix A for details ThermoFluor 384 Acquire 3 0 The ThermoFluor Acquire 3 0 control software provides the user interface for the ThermoFluor 384 system This software allows the user to enter run parameters that are sent to the embedded controller that operates the instrument components It also initiates the experiment and allows the user to designate where the resulting data will be saved The details of this program and its user interface will be discussed in Chapter 4 Operation Copyright 2001 3 Dimensional Pharmaceuticals Inc 2 3 Confidential May 2001 Artisan Scientific Quality Instrumentation Guaranteed 888 88 SOURCE www artisan scientific com Construction and Function ThermoFlour 384 Operating Manual ThermoFluor 384 Instrument The ThermoFluor 384 Instrument actually performs the fluorescence assays It consists of three compartments These are the v e Optical compartment e Light tight compartment e Power and control compartment Optical Compartment The optical compart
15. 0 interface screen It will update the status bar and display the well exposures in the plate imaging display Copyright 2001 3 Dimensional Pharmaceuticals Inc 4 35 Confidential May 2001 Artisan Scientific Quality Instrumentation Guaranteed 888 88 SOURCE www artisan scientific com Operation ThermoFlour 384 Operating Manual a SDP s ThermoFluor TEAcquire version 3 0 0 File Subsystems Images Experimental Parameters Data Directory JesDatay Browse Sample Information fo O OSC KETESEES STG CEO oc EEE REA EA AA AAN Peel fy 22FSSSC SCESECCHCHRE CBE KEHOE Comment LIDIA EEES PADIERNA Heating Parameters OFF SHHSROKSEEHHEEEEECHE DOLLORDO CORRE SOU Heating Mode Up Down POPPSOSSSSaeeeeeceeceeaecgde dial Fi C 77 DIDIER Initial Final Temperatures C ao 50 SLIDE Temp inci Low Temp C 2 30 ISIDORA i i 2osssseeceseseseeesensse 2 A Low TempHolds sech go so LEZ eae FPPPSPRMHAAEARECEREEEGH Image Parameters F Scale Bitmap 0456 0185 1 76 IBox 686 460 HOvf 0 Exposure Time sec Reps D5 fi Filters gt J500 30nm Y Error Run lblEror Heater Camera Error 11 7 00 At the completion of the run remove the plates from the ThermoFluor 384 system output magazine If the ThermoFluor 384 system is not going to be used for an extended period of time shut down the system Analyze t
16. 2 2 amp LE D 2 2 2 A 4 2 4 2 amp amp 4 A YEN NUNUNVNVUUUuUUuUuuUuuE uv VVT VVV VT VVV Appendix C Consumables List Overview This appendix contains a list of consumables that are used in the operation of the ThermoFluor 384 system Specifically this appendix includes the following sections e Consumables List The following table identifies the appropriate section to read in order to learn more about a specific area To learn more about Read the section Recommended Consumables Consumables List Copyright 2001 3 Dimensional Pharmaceuticals Inc om C 1 Confidential May 2001 Artisan Scientific Quality Instrumentation Guaranteed 888 88 SOURCE www artisan scientific com Appendix C ThermoFlour 384 Operating Manual Consumables List Description Manufacturer Plate PCR 384 well PP blk MJ Research Inc Neutral Disinfectant and Detergent Misco Products Company Didecxyl dimethyl ammonium chloride Dimethyl benzyl ammonium chloride Water Glass Cleaner Misco Products Company Isopropyl alcohol 2 Butoxyethanol Anionic surfactant Dye Water Ammonium hydroxide Distilled Water Bar code labels Copyright 2001 3 Dimensional Pharmaceuticals Inc Confidential Mgf Tel No 1 888 735 8437 1 800 548 4568 1 800 548 4568 May 2001 Artisan Scientific Quality Instrumentation Guaranteed 888 88 SOURCE www artisan scientific com
17. Edit Position List F3 Esc Exit TCW Copyright 2001 3 Dimensional Pharmaceuticals Inc _ 4 30 Confidential _ May 2001 Artisan Scientific Quality Instrumentation Guaranteed 888 88 SOURCE www artisan scientific com Be NDA AAA AAA AAA AAA AAA AAA AAA AM UU Operation ThermoFlour 384 Operating Manual 6 Access the THERMOFLUOR PLATE SETUP screen This is done by 6A Clicking the Run Methods button or press the lt F1 gt function key The Menu dialog box appears Select Method to EDIT CREATE A NEW METHOD 031601MT ev Select the appropriate method from the list and click OK The CURRENT METHOD screen appears Current Method THERMO1 Step Selected No Position Current Plate Methods PlateCrane Start Run Esc Click the Start Run button The START RUN dialog appears Start RUN now Copyright O 2001 3 Dimensional Pharmaceuticals Inc 4 31 Confidential May 2001 Artisan Scientific Quality Instrumentation Guaranteed 888 88 SOURCE www artisan scientific com Operation ThermoFlour 384 Operating Manual D Click Yes or press lt Enter gt The CREATE METHOD RUN LIST screen appears Create METHOD RUN LIST Methods to be Run ARTE No Method Name Methods Delete method To Create a RUN LIST CLICK on any of the METHODS listed at RIGHT gt in the ORDER you wish to tun them Select the desired method from t
18. Image This designates a closed shutter image be taken for background subtraction e Gain amplification value 2 This is usually set to two at installation and left 1 can be used for particularly bright plates and 3 for particularly dim plates e Box X Dimension The width of the integration field used for each well e Box Y Dimension The height of the integration field used for each well Click the Run button to begin the run The light tight door closes and the run begins The plate is run and the data files are written to the designated data directory The run can be monitored from the Acquire 3 0 interface screen It will update the status bar and display the well exposures in the plate imaging display area IDPs ThermaFluor TFAcquue version 3 0 0 Elle Subsystems Images Experimental Parameters nant Data Directory da la la Te Free FAData Browse PE fe 06 i 0 4600 Secscssosses APICAL rara ARABIA IICA Plate 1D EE Poh OCS LILI TITRE Comment ORDER eee PPFD SPITIS SVEHROHHBEOH GEES Heating Parameters Sood ete ROKSEEHEREOCEEOEE Heating Made PODIA PON PIDIERA AAA i Ch fo DODPIODORSOS ESS ER mich Final died faq is POPE Temp Incr Low Temp C 2 30 DOPOPSORECRSOTCRESSES 6684 LRHPISPFIAMIOREHEEOEESRSEESEE A ae aad a PODIAN AA si Y Scale Bitmap 0456 0185 1 76 IBox 686 460 Ov l 0 Exposure Time sec Reps 9 5 1 Filters v 500 30nm Err
19. SOURCE www artisan scientific com YPEY FVV VV VVF TVT VTPT VVV VT VVV VTV PVV PUU Installation and Calibration ThermoFlour 384 Operating Manual System installation This section provides information and procedures for installing the ThermoFluor 384 system It includes unpacking and setting the system up The system setup includes Power Up the System System Initialization Install the Filter s Focus the Camera When the installation is complete the system will be ready for operation Unpack the System The ThermoFluor 384 system is packed in two custom crates One crate contains the ThermoFluor 384 instrument and plate processing system attached to the instrument base plate The second crate contains the system stands NT Workstation and universal power supply Please save these crates upon receipt of the instrument This is the factory recommended method of transport for the instrument Inspect for Damage Before installing the system inspect the crates and contents for signs of damage Visually inspect for any external damage Any damage should be immediately reported to the shipping company as well as 3 Dimensional Pharmaceuticals Inc 2 3 If any shock or temperature indicators are on the crate locate and note their status 4 Locate a row of Philips head screws along all four sides of the crates them Remove these screws from all four sides of each crate and store in a safe place approxima
20. Select rather the camera take a shutter closed image with each set of replicate images during data acquisition to use for background correction Select YES or NO for shutter closed background correction images e p Copyright 2001 3 Dimensional Pharmaceuticals Inc Curi ai 4 14 Confidential May 2001 Artisan Scientific Quality Instrumentation Guaranteed 888 88 SOURCE www artisan scientific com AAADAQRRALAADMdADAADAAQRAAAAQADAADADANAADAAANAAAANALADAZA2 EA Operation ThermoFlour 384 Operating Manual Edit Methods This selection is used to set up a Method It should not be used in the normal operation of the ThermoFluor 384 system If more details about this section of the software are desired refer to Appendix A Total Control for Windows for the TCW manual METHOD EDITOR Process Sequence for Method THERMO1 Move From Mova To Lid Holder Plate Type Esc Phone eee ee o E poemas O CI CA LAS El ee E Available Associated Available Process Steps Positions Instruments a S f Add Step Insert Step Delete Step Saye Method As Save Methed Change Methed Figure 4 1 4 The Method Editor s screen is used to setup methods It is i n t used during normal operation Edit Position List This selection should not be used in the normal operation of the ThermoFluor 384 system If more details about this section of the software are desired refer to Appendix A Total Contro
21. allows selection of the desired filter Copyright 2001 3 Dimensional Pharmaceuticals Inc a 4 20 Confidential May 2001 Artisan Scientific Quality Instrumentation Guaranteed 888 88 SOURCE www artisan scientific com RAR AAA LL LA A 4 4 4 G2 G 42 42 4 4 KK LALA DD 4 4 4A A A 4 LA A LB OMS OB VV Nu AAA AA AAA AAA AAA AAA AA AA VVV PUV VVU Operation Setup Dialog Boxes ThermoFlour 384 Operating Manual The buttons in the setup area of the CAMERA Dialog box opens several dialog boxes that allow camera configuration settings to be changed Only appropriately trained personnel should use the features in these dialog boxes for setup and maintenance of the system dm Camera Seltings General CCD Name SenSys 0400 Serial Number 0 FT Capable No MPP Capable Always On Well Capacity 19464 Summing Well Absent Cooling Mode Air or Water Preamp Delay O ms Max Set Temp 10 00 C PreFlash 0 ms Min Set Temp 10 00C Total Ports 1 Current Temp 10 00 C Parallel Dimension Premask 4 Pixel Size nm 9000 Active Rows 512 Pixel Sep nm 9000 Postmask 4 Total Rows 520 Serial Dimension Prescan 14 Pixel Size nm 9000 Active Pixels 768 Pixel Sep nm 9000 Postscan 14 Total Pixels 796 w Camera Configuration Clearing Mode 33 Cycles 2 f2 Set Shutter Mode PRE EXPOSURE z Open Delay ms 5 E Set Close Delay ms 10 10 Set Readout
22. for Windows and 3 Dimensional Pharmaceuticals ThermoFluor Acquire 3 0 control software under Microsoft Windows NT The robotic microplate handler PlateCrane by Hudson Control Group moves sample plates from an input storage magazine to the imaging area of the instrument As the microplate is removed from the input magazine a barcode reader reads the plate s bar code identification number Once the microplate is in the imaging area the ThermoFluor control software initiates a ThermoFluor experiment on the microplate When the experiment is completed the robotic handler then retrieves the plate from the imaging area and places it into an output storage magazine This process is repeated for up to 20 microplates in a single session The PlateCrane robot and plate storage magazines Es are optimized to handle a specified brand and model 384 well PCR plates Once the data have been collected the 3 Dimensional Pharmaceuticals ThermoFluor analysis software i is used to analyze the data to determine melting temperatures and identify TT ALO ADD DD DADDY YY NY YOYYvYYy hits i Copyright 2001 3 Dimensional Pharmaceuticals Inc ee Ed Confidential May2001 Ss Artisan Scientific Quality Instrumentation Guaranteed 888 88 SOURCE www artisan scientific com Introduction i ThermoFlour 384 Operating Manual Instrument Specifications This section
23. gives the specifications of the ThermoFluor 384 system and the conditions necessary for it to function properly Overall System Specifications Height H 65 1575 mm Width W 24 610 mm Depth D 36 915 mm Weight Table Top 160 lbs 73 kg Control Cabinet 300 lbs 136 kg Environmental Conditions Storage Temperature 20 40 C Storage Humidity 20 80 Operation Temperature 20 24 C Operation Humidity 30 50 relative non condensing Power Supply Ratings Line Voltage 110 VAC 10 Frequency 60 Hz 10 Power 15 A Properly grounded outlet Windows NT workstation Hewlett Packard Vectra Pentium II 300 MHz or better 128 MB RAM hard disk with 10 0 GB or better SVGA monitor with 800 x 600 resolution 3 1 2 floppy disk drive CD ROM 250 MB Iomega ZIP Drive 2 available PCI slot one for camera card and one for Rocketport serial expansion card Microsoft Windows NT 4 0 with Service Pack 6 0 Copyright 2001 3 Dimensional Pharmaceuticals Inc gt Li ie a 1 4 Confidential May 2001 Artisan Scientific Quality Instrumentation Guaranteed 888 88 SOURCE www artisan scientific com TV VVTV TVTV VT VT VVTV TVTV VVV VVV VVV TU Introduction ThermoFlour 384 Operating Manual Safety Features The ThermoFluor 384 system should be setup and run by 3 Dimensional Pharmaceuticals Inc factory trained personnel only If you are not factory trained i
24. grounding strap and discharge cable per manufacturer s instructions during all signal cable connections to avoid damage to the equipment by static electricity UV light Avoid looking directly into UV light source as it may cause damage to the eye Labeling Warning labels are located on the instrument to avoid potential dangerous situations to an operator or other lab personnel using the ThermoFluor 384 instrument Normal operational labels are provided to clarify function of the various indicator lights switches and electrical connections used in normal operations Each label its location and a brief description of the instrument operation associated with the label are listed below Copyright 2001 3 Dimensional Pharmaceuticals Inc 1 5 Confidential May 2001 Artisan Scientific Quality Instrumentation Guaranteed 888 88 SOURCE www artisan scientific com or when power is first applied to the instrument Never reach into the access door or place any into access door Provide only 110 VAC 60 Hz single phase electrical connection or unexpected system performance Introduction ThermoFlour 384 Operating Manual Light Tight Front Access Door Label This label is located above the light tight compartment front access door of the ThermoFluor 384 instrument This door provides access to the light tight compartment for plate loading and unloading THE FRONT ACCESS DO
25. hits Qualify hits Preparing the uniform plates Standard Conventions To make the documentation easy to read the manuals use standard guidelines or conventions In other words formatting for specific items within the text will always be presented in the same way For example software dialog box names are printed in Tahoma font and all caps such as the OUTPUT FILE dialog box Tutorial Five Select and Qualify Hits Tutorial Six Screening Libraries Copyright 2001 3 Dimensional Pharmaceuticals Inc 5 06 Confidential May 2001 Artisan Scientific Quality Instrumentation Guaranteed 888 88 SOURCE www artisan scientific com Ma Ma MA D Me de Lee Preface ThermoFluor Instrument Operations A A AA A A A OPEN Typographical Conventions The documents use the following typographical conventions for the analysis and control software applications Depicted as For example Menu name Tahoma font all caps bold The FILE menu Toolbar name Tahoma font all caps bold The PAGER toolbar Menu command Tahoma font initial cap bold The Open file command Toolbar buttons Tahoma font initial cap bold The Selection button Dialog box name Tahoma font all caps Type the desired file name in the PRINT dialog box Dialog box field name Tahoma font initial cap Number Of Copies Text entry Courier PCL6 font Enter the file name Batch int Keyboard keys Tahoma font initial cap en Press and
26. material holding the PlateCrane robotic arm in place ae Open the top back panel and carefully remove the box labeled FRAGILE CCD Camera and place in safe location 16 Unpack computer monitor and universal power supply from their shipping boxes and place in safe location 17 Place all packing materials on the pallets Replace the tops of the shipping crates onto the pallets and secure with the screws The system is now ready for setup Copyright 2001 3 Dimensional Pharmaceuticals Inc 3 9 Confidential May 2001 Artisan Scientific Quality Instrumentation Guaranteed 888 88 SOURCE www artisan scientific com w wewvwvvwvvwvvewvwvvwvvevevwvvvvvvvvuVvVTVvVvuVvueTuUuUuVEUeTueeFVvVTU VY Y Set Installation and Calibration ThermoFlour 384 Operating Manual up the System After the system has been located at an appropriate site it can be set up Start by positioning the system on the floor so that the plate processing magazines are at the front facing toward the operator Adjust the system stand leveling feet so the robotic work area of the instrument base plate is level in both front to back and side to side directions A factory trained field engineer will install system components and connect the necessary cables and power cords Power Up the System Use the procedure below to initially power up the instrument Action Verify that all power switches are in the OFF
27. or blocks the airflow through the gap between the system base plate and bench top created by the leveling feet This opening ensures proper airflow to the instrument It is also necessary to provide a minimum of 6 inches between the side of the instrument with the fan where the cabling exits and any wall or splash plate This ensures adequate clearance for cabling and airflow from the instrument The universal power supply control computer monitor keyboard and mouse are placed to the immediate left of the instrument The power receptacle should be located within six feet of the rear of the universal power supply Optional network connection should also be located at the rear of the system control computer 30 inches 30 inches Minimum 66 inches Minimum Clearance 6 inches Minimum 36 inches Figure 3 3 ThermoFluor 384 system table top placement and access Copyright 2001 3 Dimensional Pharmaceuticals Inc RUE 3 6 Confidential May 2001 Artisan Scientific Quality Instrumentation Guaranteed 888 88 SOURCE www artisan scientific com Installation and Calibration ThermoFlour 384 Operating Manual About 38 inches loa Qe Top Minimum Access 66 inches Figure 3 4 ThermoFluor 384 system table top placement front view Copyright 2001 3 Dimensional Pharmaceuticals Inc 3 7 Confidential May 2001 Artisan Scientific Quality Instrumentation Guaranteed 888 88
28. 0 uM 1 8 ANS overlayed with 3 ul oil Condition 3 Tin 60 C 5 ul of 0 15 mg ml Carbonic Anhydrase in 25 mM NaPIPES 100 mM NaCl 1 mM EDTA pH 7 0 50 uM 1 8 ANS overlayed with 3 ul oil Condition 4 Tn 70 C 5 ul of 0 15 mg ml Carbonic Anhydrase in 25 mM NaPIPES 100 mM NaCl 50 uM ZnCl pH 7 0 1 mM Acetazolamide 50 uM 1 8 ANS overlayed with 3 ul oil The ThermoFluor Instrument parameters for measuring the thermal denaturation in this test should be Ramp Mode heating 25 85 C in 1 C increments High temperature equilibration for 0 25 min 15s Fluorescent intensity averaged over 4 10s exposures Acceptance shall be achieved when deviation across a plate measured as the standard deviation in the Tm of 384 wells is lt 0 2 C Uniform plates for the latter three conditions have a long shelf life and will give identical results after freeze thaw 80 C 1 week These tests can also be run in step mode 0 1mg ml protein is sufficient but it will take longer The lot number for the CAII that 3DP have is 100K9279 100 mgs is 493 50 at 0 15mg ml Sul volume this comes to 350ug plate a 20 excess using the indicated conditions each plate uses 1 72 of protein Copyright 2001 3 Dimensional Pharmaceuticals Inc 3 19 Confidential May 2001 Artisan Scientific Quality Instrumentation Guaranteed 888 88 SOURCE www artisan scientific com Installation and Calibration ThermoFlo
29. 001 3 Dimensional Pharmaceuticals Inc 4 5 Confidential May 2001 Artisan Scientific Quality Instrumentation Guaranteed 888 88 SOURCE www artisan scientific com Operation ThermoFlour 384 Operating Manual Current Method THERMO1 Siep Selected No Position Current Plate Methods PlateCrane Start Run Esc Figure 4 4 The TCW Current Run screen PlateCrane Loads Plate The TCW program then checks the status of the instrument through Acquire 3 0 Once this is done the PlateCrane goes to the input magazine removes the top plate and loads it in the instrument TCW then signals Acquire 3 0 that a plate has been placed in the light tight compartment As the plate passes the barcode reader the barcode is read This information is communicated to the Acquire 3 0 program At the end of the run the Acquire 3 0 program stores it with the experimental data in the INT file in the directory designated when the experimental parameters are entered Heater Cycle The Acquire 3 0 program then signals the embedded controller to begin the run The embedded controller closes the door to the light tight compartment and begins heating depending upon which heating mode Up Down or Continuous Ramp mode is selected Up Down Mode In the Up Down mode the temperature is raised to a high temperature value held there for a set time High Temperature Hold Time then lowered to the Low Temperature setting and held there The p
30. 2 TF Repeat steps 7D and 7E until all the parameters have been designated for all the plates in the Stack1 magazine If a plate needs to be inserted clicking the Insert but will insert a plate above the plate selected Ifa plate needs to be deleted Copyright 2001 3 Dimensional Pharmaceuticals Inc gt 4 34 Confidential May 2001 Artisan Scientific Quality Instrumentation Guaranteed 888 88 SOURCE www artisan scientific com 2 l amp amp 42 A 4 AB BA AAAQAAAY AA AAMAAADADAAMA NANA MAMA NA AA AAA Ms Ms es ER BD a di ED Operation ThermoFlour 384 Operating Manual clicking the Delete button deletes the selected plate from the list Start the run in TCW To do this 8A Exit the THERMOFLUOR PLATE SETUP screen by pressing the lt Esc gt key The CURRENT METHOD screen appears with the message System is PAUSED Current Method THERMO1 Step Selected No Position Current Plate Methods PlateCrane System is PAUSED Terminate Run Press lt SPACE BAR gt to UNPAUSE System Esc 8B Press the lt spacebar gt to unpause the system The IS THE PLATECRANE CLEAR TO HOME NOW Dialog opens ls the PLATE CRANE clear to HOME now Click Yes or press lt Enter gt to begin the run The plates are run and data files are written to the designated data directory If it becomes necessary to terminate the run click the Terminate Run button The run can be monitored from the Acquire 3
31. 3 1 Confidential May 2001 Artisan Scientific Quality Instrumentation Guaranteed 888 88 SOURCE www artisan scientific com Installation and Calibration ThermoFlour 384 Operating Manual Safety instructions The ThermoFluor 384 system should be set up and run by 3 Dimensional Pharmaceuticals Inc factory trained personnel only If you are not factory trained immediately contact 3 Dimensional Pharmaceuticals Inc at 1 610 458 8959 to schedule training This Operating Manual contains information and procedures for the safe installation and operation of the ThermoFluor 384 system Before installing or running the ThermoFluor 384 system completely and thoroughly review the latest version of the Operating Manual If any instructions are unclear or unfamiliar to you immediately contact 3 Dimensional Pharmaceuticals Inc at 1 610 458 8959 for clarification and proper operation Please locate and identify all safety and informational labels on the instrument If any labels are missing smudged or damaged see Chapter 1 Introduction discontinue use and contact 3 Dimensional Pharmaceuticals Inc for replacements Please locate identify and recognize all safety items on the instrument including covers and panels If any items are missing damaged or disabled in any way discontinue use and contact 3 Dimensional Pharmaceuticals Inc for instrument repair Handle with Care To avoid equipment damage handle components carefully
32. Box 0 Ovfle0 Exposure Time sec HReps 0 5 fi Filters 500 30nm Y Eror Run bEror e Experimental Parameters Heater Camera Error 11 7 00 8 45 AM As the program launches it searches for several available components inside the ThermoFluor 384 system There are indicator fields for the heater and camera at the lower left corner of the screen Upon successful initialization the system is ready for the remainder of the setup process Install the Filter Use the procedure below to install the filter The system software must be running to accomplish this task Action If it is not already open open the optical compartment by removing the screws from the rear panel If the filter wheel is already in position 1 go to step 4 If it is not in position 1 click on the arrow for the right hand Filters drop down menu in the image parameters section at the lower right of the ThermoFluor 384 Acquire 3 0 window See Below Select Position 1 500 30 nm from the drop down menu for the standard ThermoFluor filter supplied with the instrument See figure below The filter Copyright 2001 3 Dimensional Pharmaceuticals Inc 3 12 Confidential May 2001 Artisan Scientific Quality Instrumentation Guaranteed 888 88 SOURCE www artisan scientific com HA ARK AAAA BD A BAAAAA AAA A LB AA BB amp AB 4 4 4 AAA amp LB A amp 4 4 DLS amp A
33. CE www artisan scientific com HRA BAD AKAAA AAA AA HH A BB 4 A A YYYY BA A Installation and Calibration ThermoFlour 384 Operating Manual Acceptance Tests This test is ran to validate the proper operation of the ThermoFluor 384 instrument The system and analysis software must be running to accomplish this task For a procedure to prepare and run plates and then analyze the data refer to Tutorial Seven in the Tutorial Manual The most fundamental measurement giving meaningful data using er acl is the AT between a well containing compound and a reference well Tm scomp Tm ref Since the Tm is independent of initial and final fluorescent intensities validation of ThermoFluor instruments should be based on the uniformity of T across the entire heating block Accuracy Test To measure uniformity of Tm across a plate uniform assay plates of bovine Carbonic Anhydrase II Sigma catalog C2522 E C 4 2 1 1 should be made using the conditions listed below These conditions were chosen to give a wide working range of T s 40 C 70 C while using an inexpensive protein and a procedure that can be accomplished relatively quickly 1 day or less Condition 1 Tm 40 C 5 ul of 0 15 mg ml Carbonic Anhydrase in 50 mM NaOAc 100 mM NaCl pH 4 4 1 mM EDTA 50 uM 1 8 ANS overlayed with 3 ul oil Condition 2 Tm 50 C 5 ul of 0 15 mg ml Carbonic Anhydrase in 50 mM NaOAc 100 mM NaCl pH 4 5 50 uM ZnCl 5
34. Handle the CCD Camera with particular care as it is fragile Lift Properly Use proper lifting techniques when lifting shipping containers and system components Four 4 persons positioned at each corner should be used when lifting the system stand and the instrument base plate Electrical Do not plug the universal power supply in until the installation procedure specifically instructs you to plug in the universal power supply Then before plugging in the universal power supply verify that all power switches are in the OFF position Verify that the wall outlet for the universal supply is 110 VAC 60 Hz and properly grounded Use a ESD electrostatic discharge wrist grounding strap and discharge cable per the manufacturer s instructions during all signal cable connections to avoid damage to the equipment by static electricity Copyright 2001 3 Dimensional Pharmaceuticals Inc 3 2 Confidential May 2001 Artisan Scientific Quality Instrumentation Guaranteed 888 88 SOURCE www artisan scientific com Installation and Calibration ThermoFlour 384 Operating Manual Site preparation This section describes the site requirements for the ThermoFluor 384 system to ensure proper function and access It includes the recommended stand alone placement and the alternate bench top placement Overall Site Specifications The ThermoFluor 384 system should be located in a clean dry laboratory environment The
35. OR IS NOT INTERLOCKED Never open the front access door when the system active light is illuminated during a sample run l This door should only be open when plates are being loaded and unloaded from the Plate Processing Area Warning Area Label This label is located above the light tight compartment front access door of the ThermoFluor 384 instrument This door provides access to the light tight compartment for plate loading and unloading Normal operation should never require reaching through the access door into the light tight compartment Such action is hazardous and can result in injury or damage to the instrument Manufacturer s Serial Plate Label This label in located on the right side of the ThermoFluor 384 instrument These connections should be made with no power applied to either the computer or the instrument In addition there is labeling for the power entry module 110 VAC 60 Hz ONLY amp Use 10A 250 VAC Slo Blo Fuses only Other operating voltages and frequencies may cause damage to the instrument and Copyright 2001 3 Dimensional Pharmaceuticals Inc 1 6 Confidential May 2001 Artisan Scientific Quality Instrumentation Guaranteed 888 88 SOURCE www artisan scientific com Introduction ThermoFlour 384 Operating Manual _ MOVAC GOHZ ONY A ground stud label is also provided to indicate the ground stud located on the side instrument panel
36. OROEE ht Flos CC x LTT TE Temp Incr Low Temp C 2 En BIRD ELIITTI TTT TOT t ttet ee High Low Temp Holis sec PSS f CIRIA Pants FF Scale Bitmap 0456 0185 1 76 IBox 686460 Ovil 0 Exposure Time sec HReps 0 5 1 Filters 500 30nm NS E i SClmage Gain R 4 Box KY 157 157 Y Error Run IblError Sample Information Heater Camera rror 11 7 00 8 45 AM Figure 4 15 Experimental Parameters area of Acquire 3 0 user interface Field s Data Directory Description Enter the path and directory for the data psq and int files to be stored or click on browse to open the Set Directory dialog box to select the desired folder See below Si Set Data Directory Data Directory Dive ae ZEA y Program Files AN TFAcquiie Set Directory Copyright 2001 3 Dimensional Pharmaceuticals Inc 4 25 Confidential May 2001 Artisan Scientific Quality Instrumentation Guaranteed 888 88 SOURCE www artisan scientific com Operation ThermoFlour 384 Operating Manual Field s Description Plate ID i The plate ID is an eight digit number that correspond to the barcode number Enter User Comments in the text box Heating Mode Specify the heating mode from the drop down menu The modes are Up Down or Continuous Ramp Up Down Mode Cycles the assay plate between successively higher temperature values and the low temperature value where images are taken
37. Place the assay plates in the input magazine of the ThermoFluor 384 system a Open the ThermoFluor Acquire 3 0 application by double clicking the Copyright 2001 3 Dimensional Pharmaceuticals Inc 4 29 Confidential May 2001 Artisan Scientific Quality Instrumentation Guaranteed 888 88 SOURCE www artisan scientific com Operation ThermoFlour 384 Operating Manual ThermoFluor Acquire 3 0 icon The ThermoFluor 384 Acquire 3 0 program opens The instrument goes through an initialization Observe the screen to verify the heater and camera initialize properly Minimize the ThermoFluor 384 Acquire 3 0 screen LS 3DP s Thembrluor T FAcaue version 3 0 0 gt adh aah ce elena sat wae ero seen ms Experimental Parameters lt on nn P fi Date Directoy E Da Brows i Sample Information o i Plate ID FRET Comment Hevlig Paineis cores Heating Mode Up Down iniia Final Temperatures CJ 40 50 Temp Inct Low Temp CC 2 A High Low Temp Holds sec fo 4 image Pattes FF Scale Bimap 0233 0258 m0 IBom0 momo Exposure Time sec tes AA A lens gt Se Eno Run blEnor Open the Total Control for Windows TCW application by clicking on the TCW icon The Main Menu screen for TCW opens MAIN MENU Run Methods F1 F4 come PlateCrane Edit Methods F2 F5 System Settings Current Speed
38. PlateCrane Label Labels are located on the side of the PowerCrane indicating its DB 9 serial control connection AC power connection and manufacturer s serial number Bar Code Reader Label Labels are located on the side of the Bar Code Reader indicating its DB 9 serial control connection and power connection Ground Labels Ground labels are located throughout the instrument These labels indicate a chassis ground connection point Electrical Warning Labels Electrical warning labels are located on the back instrument panel and inside the instrument There are no user serviceable parts in this panel and opening this panel can expose the user to unsafe voltages Bee Copyright 2001 3 Dimensional Pharmaceuticals Inc ee 1 7 Confidential May 2001 Artisan Scientific Quality Instrumentation Guaranteed 888 88 SOURCE www artisan scientific com Introduction ThermoFlour 384 Operating Manual Plate Processing Labels Several additional labels are best viewed from the top of the instrument Specifically plate orientation labels and input output stack labels PlateCrane robotic arm gu Es AC o og E The plate orientation label locates well A1 on each plate placed in the instrument Connections The ThermoFluor 384 instrument CCD camera SCSI connection and controller serial connection should be made with no power applied to either the computer or the instrument Similar
39. SUBSYSTEM menu select Camera The CAMERA dialog box appears Copyright 2001 3 Dimensional Pharmaceuticals Inc oo 3 13 Confidential 35 May 2001 Artisan Scientific Quality Instrumentation Guaranteed 888 88 SOURCE www artisan scientific com Installation and Calibration ThermoFlour 384 Operating Manual Click the Open Door button The light tight door opens HZ 3DP s ThennoFluer TFAcquire version 3 0 0 File ESE Images Experimental Parameters Data Directory Browse Sample Information Setup Dr grameters Get Info Configure Healing Mode Up Down Exposure SpeedT 2b gt A in s n aal Temperatures C ao zo Se Ui e Light Off 9 mo Low Temp tO ff 5 g ow Temp Holds sec fiso 60 ameters Integrate Image Exposure Time sec F Scale Bitmap Cork e Time sec HReps fio 4 Calibrate Image des ae z None z di l bm o I Create New Calibration File A F Gain 7 H Box RYE m m a Run Light Indicator Heater Camera Ready Idle 41330701 3 32 PM Once the piece of paper is placed on the thermal cycler block close the light tight door by clicking the Close Door button in the CAMERA dialog box From Acquire 3 0 CAMERA dialog box turn ON the UV light source by clicking the Light On button Enter an exposure time one second for the camera in the Integrate Image section of the CAMERA dialog box Click on the Cont button to have the camera start tak
40. Speed Gain Speed Table Entry Readout Rate MHz 1 00 Bit Depth 12 Port 0 Gain gt Other PMode NORMAL Temp Setpoint CJ 10 00 10 00 Set ADC Offset 6100 6100 Set Copyright 2001 3 Dimensional Pharmaceuticals Inc Confidential Get Info The Get Info button on the CAMERA Dialog box opens the CAMERA SETTING pop up window This window displays various camera settings as shown below Configure The Configure button on the CAMERA Dialog box opens the CAMERA CONFIGURATION dialog box This dialog box allows various camera configuration parameters to be set as shown below 4 21 May 2001 Artisan Scientific Quality Instrumentation Guaranteed 888 88 SOURCE www artisan scientific com Operation Setup Exposure Region Definition Serial Dimension Offset 0 E Set Size 768 768 Set Binning Factor 1 TRS Set Parallel Dimension Offset 0 bp Ser Size 512 B2 Set Binning Factor 1 Fo Set Exposure Parameters Defaults Mode TIMED Exposure Time sec 10 j 0 Set Exposures inSeq 1 fi Set ThermoFlour 384 Operating Manual Exposure The Exposure button on the CAMERA Dialog box opens the SETUP EXPOSURE dialog box This dialog box allows various exposure parameters to be set as shown below w Speed Table Readout Rate MHz Copyright 2001 3 Dimensional Pharmaceuticals Inc Confidential SpeedTab The SpeedTa
41. Yes in the MOVE TO SAFE confirmation pop up dialog box The arm should move over the plate at the bottom of the desired stack Verify the grippers are positioned properly over the plate In a position to pick up the plate Click the Close Gripper button on the Calibration screen to verify they grip the plate Select the stack in question on the Calibration screen and click the Move to Point button Select Yes in the MOVE TO SAFE confirmation pop up dialog box The arm should lift the plate and return it to the bottom of the magazine stack vus us suuuuyuuuuuvuuuu VFV uuuvuvuvyuuvuvyuvvywy 0 If this is done successfully there is no problem with the stack position calibration Exit the Calibration screen by clicking the Esc Exit button If the arm doesn t line up over the stack correctly refer to Appendix A Total Control for Windows for the procedure to re teach recalibrate this position to the arm j Verify or Reset Plate Position When the Plate processing system is first set up the position of the assay plates relative to the PlateCrane arm grippers plate position is set When the type of plates are changed or possibly following a crash of the system the plate position may need to be verified or reset To verify or reset information about plate position follow the procedure below Action Step _ desktop Click the System Settings F6 button on the MAIN menu of TCW The PlateCrane Sy
42. an scientific com Operation a ThermoFlour 384 Operating Manual Camera Dialog Box This dialog box provides a means of interacting with the camera and setting camera parameters En st Camera Setup gt Thermofluor Box Get Info Configure Open Door Close Door Exposure SpeedT ab Integrate Image Exposure Time iit sec Cont ie Li Cancel Calibrate Imaae al Emission Filter M Create New Calibration File Ic Embot Move Selection Toot to Each Comer and Click the Pick Button Dialog Area Description Setup Buttons allow access to camera information and camera setup entry dialog boxes Integrate This area allows entry of the image exposure time and commands for Image the camera to take a exposures The Single button instructs the camera to take a single image that is displayed in the Image display area e The Cont button instructs the camera to take a continual series of images e When the Cont button is selected it changes to Cancel after the first image is taken Click Cancel to stop taking images Calibrate This area allows for image calibration See Calibrate Spots in Image Chapter 3 Installation and Calibration ThermoFluor This area allows the user to operate instrument functions These box include e Open and Close Door buttons open and close the light tight door e Light On and Light Off buttons turn the UV lights on and off e The Emission Filter drop down menu
43. and plate image display The menus access the basic commands for manipulating data images and setting subsystem parameters The menus are constructed using standard MS Windows formatting and functions to SD P s ThermoFluor TFAcquire version 3 0 0 File Subsystems Images Experimental Parameters se x Data Directory Evan Browse Sample Information p ienes Plate ID HEEE Comment PA lt Heating Parameters i Heating Mode Up Down 7 Initial Final Temperatures C 40 l 50 Temp Inci Low Temp C 2 Es i High Low Tene Holds ts hoc fp Feo z gt nage Parameters se Y Scale Bitmap 0233 0258 1 0 IBowsd gt Ewpoue Tine se Rep m e Fitters gt 500 30 nm SCimage Y Gain e Box KY fig fe Eror Run IbEnor ASES Heater E ee He rror 11j7 00 8 45 AM Figure 4 14 ThermoFluor Acquire 3 0 user interface Title Bar E 3DP ThermoFluor TFAcquite version 3 0 0 The title bar identifies the ThermoFluor 384 Acquire 3 0 program window and contains standard Windows buttons for minimizing and maximizing the window and closing the application Copyright 2001 3 Dimensional Pharmaceuticals Inc p 4 18 Confidential May 2001 Artisan Scientific Quality Instrumentation Guaranteed 888 88 SOURCE www artisan scientific com ES ia DADA dd DAMMAM A AAA ALA SL LA AA AAA AA AAA AAA AAA EE AAA Operation ThermoFlou
44. and their ligand bound complexes The ThermoFluor instrument measures dye fluorescence in the presence of a protein As the protein unfolds its hydrophobic parts are exposed which creates an environment that causes the dye to fluoresce The more the protein unfolds the greater the intensity of the fluorescence Figure 1 1 The thermal unfolding of the protein allows the dye to enter a more hydrophobic environment and fluoresce In this manner the protein s melting transition can be measured More importantly for the ThermoFluor screening strategy the effects of ligand binding on the protein s melting transitions can be measured See the Application Manual for more details on the theory behind ThermoFluor Figure 1 2 Ligand binding shifts the unfolding temperature of the protein Copyright 2001 3 Dimensional Pharmaceuticals Inc 5 12 Confidential May 2001 Artisan Scientific Quality Instrumentation Guaranteed 888 88 SOURCE www artisan scientific com j MO AR A Mi mn mm Introduction ThermoFlour 384 Operating Manual The ThermoFluor 384 system provides a flexible and modular fluorescence assay system with an automated robotic microplate loading system to perform this function The basic ThermoFluor 384 system is capable of sequentially loading and unloading up to twenty 20 384 well thermal cycler microplates The ThermoFluor 384 system works in conjunction with a personal computer running Total Control
45. aranteed 888 88 SOURCE www artisan scientific com Maintenance ThermoFlour 384 Operating Manual If a lid is used enter or verify the plate lid height in mm default assumes no lid in Enter Plate Lid HEIGHT pop up dialog box Click OK The Enter Gripper Rise HEIGHT pop up dialog box appears Enter Gripper Rise HEIGHT mm Enter or verify the gripper rise height in Enter Gripper Rise HEIGHT pop up dialog box When the arm moves to pick up a plate it lowers until the center of the arm touches the plate stack and the limit switch stops the movement The arm then rises to where the grippers are aligned with the top plate The distance the arm must rise for plate to align with the notches in the grippers is the gripper rise height This height should be 2 0 mm for the current 384 well assay plate 10 Click Yes to save the changes The new plate setup should be visible on the list on the PlateCrane System Setup screen Copyright 2001 3 Dimensional Pharmaceuticals Inc 5 15 Confidential May 2001 Artisan Scientific Quality Instrumentation Guaranteed 888 88 SOURCE www artisan scientific com i Maintenance ThermoFlour 384 Operating Manual PlateCrane System Setup Defined Plate Types Plate Description Format Plate Hgt Lid Hgt Rise Hat EE D Se bial aa ay Plate Current Speed fai Run Speed L PlateCrane XL Ask if Ready to Home
46. ate to that position Remove the filter wheel filter cover from over the filter Grasp the filter by the edge and remove from the filter wheel Place in a safe pu or holder 7 CAUTION Filters are sensitive optics and should be handled by the edge with care Locate the replacement filter grasp it by the edge and place it in the filter wheel Copyright 2001 3 Dimensional Pharmaceuticals Inc 5 5 Confidential May 2001 Artisan Scientific Quality Instrumentation Guaranteed 888 88 SOURCE www artisan scientific com VvvwwvwvvwvwvvwvwvwwvwvwwwvvvvvvvvvevVvTVvEeUuVUEVWTY Maintenance ThermoFlour 384 Operating Manual position with the writing right side up l Filters are sensitive optics and should be handled by the edge with care Secure the filter wheel filter cover over the filter i EA Close the optical compartment door and secure with screws Lg Properly store the removed filter Copyright O 2001 3 Dimensional Pharmaceuticals Inc 5 6 Confidential May 2001 Artisan Scientific Quality Instrumentation Guaranteed 888 88 SOURCE www artisan scientific com Maintenance ThermoFlour 384 Operating Manual Light Source Changes Use the procedure below to change the light source Turn off power to the instrument Open the rear panel of the light tight compartment by removing the screws Disconnect the connectors to the UV light source Grasp the UV light fixture by t
47. ature in C for measurements This is the first high temperature in the Up Down mode or the start of the temperature ramp in the Continuous Ramp mode Final Temp The last temperature in C for measurements This is the last high temperature in the Up Down mode or the end of the temperature ramp in the Continuous Ramp mode The temperature to which the heater returns the plate and maintains in the Up Down mode while images are taken Also referred to as the low temperature before exposures are taken in the Up Down mode High Eq Time The time the high temperature equilibration is held in seconds before returning to the low temperature in the Up Down mode Exposure time Time each replicate shot of the camera is exposed in seconds 3 Select the desired operating mode The options are Options Description Cid Up Down mode Cycles the sample between successively higher temperature values and the low temperature value where images are taken Designed for proteins that irreversibly unfold Imaging Temp Continuous Ramp mode Takes the sample from one temperature value to the next without lowering the temperature Images are taken at each temperature value Designed for proteins that easily refold Refer to Chapter 2 Optimizing ThermoFluor Conditions in the Application Manual for the appropriate use of the different modes 4 Select the desired Emission Filter The options are
48. b button on the CAMERA Dialog box opens the SPEED TABLE dialog box This dialog box allows speed table parameters to be viewed and edited as shown below 4 22 May 2001 Artisan Scientific Quality Instrumentation Guaranteed 888 88 SOURCE www artisan scientific com MBA MA AA AKA AAA AA AA HKAAAAKAABAAHKRAAHBALAAAL ALA LLM 4 4 4 4 4 4 Oo Operation i ThermoFlour 384 Operating Manual Images Menu Contains the following commands for writing reading and reintegrating raw camera data in the form of psq files These are designed for offline use Menu Selection Function Read Image Opens the OPEN dialog box for the selection of a psq file for retrieval Used offline Read Series Opens the SELECT IMAGE SERIES dialog box for the selection of a series of psq files corresponding to the Read Image Read Series Write Image Reintegrate data for a single plate at all temperatures Used offline Write Image Opens the SAVE AS dialog box designate which directory and what file name the current image should be saved to as a psq file Reintegrate Used to reintegrate raw camera image files psq offline Used to recover plate data or process plate data from time critical run modes See the Recovering Plate Data Following System Disruption topic in the Troubleshooting section of Chapter 5 Maintenance Read Image This selection allows raw camera data in the form of a s
49. c Quality Instrumentation Guaranteed 888 88 SOURCE www artisan scientific com Installation and Calibration ThermoFlour 384 Operating Manual Click on the Create New Calibration File box A little green box appears in the window This is the I box Ele REANG Images Experimental Parameters Data Directory i Browse Sample Information Plate ID Setup Thermofluor Bos sremeters Get Info Configure Open Door Close Door Heating Mode Up Down Exposure SpeedTab aA aal Temperatures C 40 70 Light On Light Off Incr Low Temp C fi 25 Integrate Image me Light Indicator ow Temp Holds sec F 80 60 A Exposure Time iff sec a ameters W Scale Bitmap Fe on e Time sec Reps 19 4 Calibrate Image Emission Filter s v None brati mbo T Create New Calibration File W Gain 5 1 Box KY M6 16 Run Heater Camera Ready Move the I box the small green box in the image area to the upper left well Al of the plate view section with the mouse by clicking holding and dragging it Click Pick button in the Calibrate Image section of the Camera dialog box The upper left red dot on the dialog box image should change to green Copyright 2001 3 Dimensional Pharmaceuticals Inc 3 16 Confidential May 2001 Artisan Scientific Quality Instrumentation Guaranteed 888 88 SOURCE www artisan scientific
50. ce should be supplied Refer to the APC Smart UPS Manual for additional details Universal Power 4 Windows NT Supply Workstation Figure 2 16 Cabinet containing the universal power supply and Windows NT Workstation Copyright 2001 3 Dimensional Pharmaceuticals Inc 2 12 Confidential May 2001 Artisan Scientific Quality Instrumentation Guaranteed 888 88 SOURCE www artisan scientific com HRA ALAA A BB 4 4 4 4 BB 4 B A B 4 4 4 LB AA A A A LAL A D A EE 4 2 2 amp 4 4 4 2 Construction and Function ThermoFlour 384 Operating Manual Chapter Three Installation and Calibration Overview This chapter provides information and instructions for installing and calibrating the ThermoFluor 384 system It also describes how to perform an acceptance test for the system Specifically this chapter includes the following sections e Safety instructions e Site preparation e System installation e Calibration of spots e Acceptance test The following table identifies the appropriate section to read in order to learn more about a specific item Safety instructions during installation Preparing a site for the system Installing the system System installation Calibrating the instrument Calibration of spots Performing an acceptance test for the system Acceptance test To learn more about See page Copyright 2001 3 Dimensional Pharmaceuticals Inc
51. ck button in the Calibrate Image section of the Camera dialog box The lower left red dot on the dialog box image should change to green paa Click the Test button in the Calibrate Image section of the Camera dialog box The I box will start to move across the screen covering all 384 well locations m Visually verify that the I box remains centered on the wells as it moves This confirms the program has correctly defined the position of each well a Click Save to save this calibration NOTE As long as the camera or thermal cycler block are not moved this calibration should not have to be done again 119 From Acquire 3 0 CAMERA dialog box manually turn OFF the UV light source by clicking the Light Off button Remove the microplate from the thermal cycler block To do this open the light Copyright O 2001 3 Dimensional Pharmaceuticals Inc 3 17 Confidential May 2001 Artisan Scientific Quality Instrumentation Guaranteed 888 88 SOURCE www artisan scientific com Installation and Calibration ThermoFlour 384 Operating Manual tight door by clicking the Open Door button _ Once microplate is removed from the thermal cycler block manually close the light tight door by clicking the Close Door button Properly store the microplate Copyright 2001 3 Dimensional Pharmaceuticals Inc 3 18 Confidential May 2001 Artisan Scientific Quality Instrumentation Guaranteed 888 88 SOUR
52. com aes Me ww ww werwwewvwwwvewvuwvvwveevveTvvvwvvvuvueTwTeTvvUuVveVTVvVvUuVUVUuVuEUVUUY Installation and Calibration ThermoFlour 384 Operating Manual 5 3D P s ThermoFluor FFAcquire version 3 0 0 File Subsystems Images Experimental Parameters Data Directory Browse Sample Information _ Plate ID iment Setup Thermofluor Box ting Parameters Get Info Configure Open Door i Close Door Exposure SpeedTab eag mereen Mal Final Temperatures Ck 0 70 Light On Li Light Off Integrate Image emp incr Low Temp C fj 25 Light Indicator ligh Low Temp Holds sec fiso joo Exposure Time 7 Parameters F Scale posure Time sec Reps i a Emission Filter Filters x None x Cpmbot v mage Y Gain ZH Box KY M6 16 Camera uy Move the I box to the upper right corner A24 of the plate view section with the mouse Click Pick button in the Calibrate Image section of the Camera dialog box The upper right red dot on the dialog box image should change to green Move the I box to the lower right corner P24 of the plate view section with the Ready idle 1 30 01 3 43 PM mouse Click Pick button in the Calibrate Image section of the Camera dialog box The lower right red dot on the dialog box image should change to green Move the I box to the lower left corner P1 of the plate view section with the mouse ia Click Pi
53. d below ThermoFluor 384 Instrument Plate Processing System Figure 4 1 Operating and control software applications Copyright 2001 3 Dimensional Pharmaceuticals Inc 4 3 Confidential May 2001 Artisan Scientific Quality Instrumentation Guaranteed 888 88 SOURCE www artisan scientific com Operation ThermoFlour 384 Operating Manual A System Initialization After the system is powered up the ThermoFluor 384 Acquire 3 0 software is launched The Acquire 3 0 user interface screen appears see below ta 3DP s ThesmoFluor TFAcquire version 3 00 Eile Subsystems Images Experimental Parameters gt gt Data Directoy i Ji f Data Brows Sample Information Plate ID EE Comment Heating Parameters Heating Mode Up Down Initial Final Temperatures C 40 50 Temp Inex Low Temp CO 2 0 e High Low ten Holds sec feo 60 image Pb Rs E onses Sl w IBor 686 4e0 wowea fe iio Exposure Time sec HReps bs aes A E Filters 500 30mm SCimage I Gain R bBox KY ME ME ERE EE ES EE E a Se ee 11 7 00 8 45 AM Figure 4 2 ThermoFluor Acquire 3 0 man screen ThermoFluor Acquire 3 0 searches for several available components inside the ThermoFluor 384 system These include the Camera and thermal cycler heater The embedded controller will position the filter wheel to its home position an
54. d open the light tight compartment door The camera and heater initialization is indicated at the lower left bottom of the screen see above After this is done the TCW application is started and it s MAIN menu appears See below Copyright 2001 3 Dimensional Pharmaceuticals Inc e 4 4 Confidential May 2001 Artisan Scientific Quality Instrumentation Guaranteed 888 88 SOURCE www artisan scientific com Operation ThermoFlour 384 Operating Manual MAIN MENU Run Methods F1 F4 Calibrate PlateCrane Edit Methods F2 F5 System Settings Current Speed Edit Position List F3 Ese Exit TCW Figure 4 3 Total Control for Windows TCW main menu screen Routine Analysis Next the user enters experimental parameters For a single plate these parameters can be entered using the ThermoFluor Acquire 3 0 user interface screen since TCW is not needed For a routine analysis of multiple plates TCW is used to set up the plate handling for the run and enter experimental parameters which are relayed to the ThermoFluor Acquire 3 0 program The ThermoFluor Acquire 3 0 program communicates the appropriate parameters to the embedded controller If a filter wheel position is changed the embedded controller repositions the filter wheel immediately The user then initiates the run by clicking the Start Run button on the TCW Current Method screen or the RUN button on the Acquire 3 0 interface screen Copyright 2
55. data collection It also includes validation and optimization tutorials You can use this manual when you need information on these common tasks with regard to the ThermoFluor 384 system or to train new users of the system The following table identifies the common tasks described in the ThermoFluor Tutorials of the documentation and which Tutorial to refer to for practice in performing a given task To learn more about Preparing the assay plate Setting up and running the assay plates Tutorial One Initial Protein Screen Viewing the analysis results Determining the best concentration mode and dye Copyright 2001 3 Dimensional Pharmaceuticals Inc 0 5 Confidential May 2001 Artisan Scientific Quality Instrumentation Guaranteed 888 88 SOURCE www artisan scientific com Preface ThermoFluor Instrument Operations Continued from previous page Preparing the assay plate Tutorial Two Buffer Salt Screen Determining the best pH buffer and salt Preparing the assay plate Tutorial Three Uniform Plate Screen Setting up and running the assay plates Analyzing the assay plate data Determining existence of irregularities or systematic errors Preparing the assay plate Setting up and running the assay plates Analyzing the assay plate data Tutorial Four Compound Library Screen Initial identification of potential
56. does not solve the problem contact 3 Dimensional Pharmaceuticals Inc at 1 610 458 8959 for technical support i PlateCrane If the PlateCrane fails to initialize the following error message will appear The PlateCrane is not respondirig Please correct the problem If this happens verify there is power to the barcode reader If there is then restart the system If this does not solve the problem contact 3 Dimensional Pharmaceuticals Inc at 1 610 458 8959 for technical support Contact 3 Dimensional Pharmaceuticals Inc at 1 610 458 8959 for technical support Copyright 2001 3 Dimensional Pharmaceuticals Inc 5 9 Confidential May 2001 Artisan Scientific Quality Instrumentation Guaranteed 888 88 SOURCE www artisan scientific com Maintenance ThermoFlour 384 Operating Manual ThermoFluor Instrument Recovery Upon an Error If the ThermoFluor 384 Instrument should fail because of an error follow the procedure below i A a If the instrument fails record the following information e The exact error message word for word e The state of the instrument e Number of plates run e Remaining number of plates e Ifa plate was left in the instrument or not e Is the door open or closed e Is the UV lights on or off What is the PlateCrane arm position rotary vertical and gripper Additionally record all of the steps that were taken in the inst
57. e embedded controller To ensure proper operation regular bulb service and replacement is necessary See Chapi 5 Maintenance Refer to the light source specifications te for more details Caution Do not look directly into UV light source Figure 2 7 UV lamps viewed as they appear in the instrument Thermal Cycler Block The thermal cycler heats and cools the microplates to the prescribed temperatures The thermal cycler block is designed to work with 384 well microplates to provide efficient and effective temperature control See Thermal Cycler under Power and Control Compartment below Figure 2 8 The top of the Thermal Cycler biok Copyright 2001 3 Dimensional Pharmaceuticals Inc 2 6 Confidential May 2001 Artisan Scientific Quality Instrumentation Guaranteed 888 88 SOURCE www artisan scientific com as LAA A AAA AL BA 4B 4 4 4 4 4 BA LB A A A 4 BG A 4 BL 4 BA 4 4 4A BL BB 4 Dm Construction and Function ThermoFlour 384 Operating Manual Light Tight Door The light tight door provides access to the top of the thermal cycler so the PlateCrane can place the microplates on the heater block The door is opened and closed by the embedded controller Its opening is programmed to work in conjunction with the PlateCrane Caution Do not place hand s into the open door when running in batch mode as it could close and cause injury Thermor
58. ear TFAcquire Techne Thermo SendCmdCommand Timed Out 12 9 99 2 16 28 PM ftmT hermoFluor Form_LoadCould not create heater object 12 9 99 2 16 28 PM If this message appears restart the ThermoFluor Acquire 3 0 program If this does not solve the problem contact 3 Dimensional Pharmaceuticals Inc at 1 610 458 8959 for technical support Camera If the camera fails to initialize the following error message will appear FFAcquire Ea pl_cam_open Error 50 Error code 50 If this message appears follow the procedure below Step Action a ns Copyright O 2001 3 Dimensional Pharmaceuticals Inc 5 8 Confidential May 2001 Artisan Scientific Quality Instrumentation Guaranteed 888 88 SOURCE www artisan scientific com Maintenance ThermoFlour 384 Operating Manual Reboot the NT workstation Launch the ThermoFluor Acquire 3 0 program Observe that the camera initializes in the indicator field at the lower left corner of the Acquire 3 0 screen If this does not solve the problem contact 3 Dimensional Pharmaceuticals Inc at 1 610 458 8959 for technical support Barcode Reader If the barcode reader fails to initialize there will be no beep when TCW is started Also the red light will not come on when the PlateCrane moves a plate in front of the reader If this happens verify there is power to the barcode reader If there is then restart the system If this
59. et in place of the bolts Gently remove the 2 x 4 and set the system stand down on the two front leveling feet Place the 2 x 4 under the back of the system stand and install the back two 2 leveling feet in place of the bolts Gently remove the 2 x 4 and set the system stand down on all four leveling feet Gently lift the system stand off the pallet and place it on the floor where the system is to be located Remove the small shipping box secured to the instrument base plate Inside the box are four stainless steel bolts for attaching the instrument base plate to the system stand Remove the box and put it in safe location Unscrew the four bolts holding the instrument base plate to the pallet They are located on both the right and left side of the instrument base plate The bolts are captured in the pallet so they will only drop half an inch or so from the pallet Lift instrument base plate with the attached instrument and plate processing system onto the system stand Four persons positioned at each corner of the instrument should lift and place the instrument onto the table Proper lifting ease nas should be enol followed during this hat CAUTION j Lifting the instrument base plate onto the system stand a2 Bolt the instrument base plate to the system stand 113 Place the magazines for the microplates onto their bases ree Remove the shipping
60. formation is organized into four separate documents The first document ThermoFluor Instrument Operation described above provides basic information on how to install and use the ThermoFluor 384 System The second document ThermoFluor Analysis Software provides basic information on how to use the ThermoFluor analysis software This Software provides a powerful way of analyzing the data generated by the ThermoFluor 384 System The third document ThermoFluor Applications covers the use of the ThermoFluor 384 instrument and Acquire version 3 0 analysis software for applications in general The fourth document ThermoFluor Tutorials is a series of tutorials to help anew user learn how to perform common tasks These tutorials are arranged in the order of increasing complexity They are covered in detail during ThermoFluor training The Scope of these Documents These documents are designed with 3 Dimensional Pharmaceutical s training class to provide instructions and information needed to facilitate someone with background knowledge in screening to use the ThermoFluor 384 system They should enable the user to perform various tasks and develop an understanding of how the system works ThermoFluor 384 System Instrument Operation This document describes the basic construction and function of the ThermoFluor 384 system It also describes the installation operation and maintenance of the ThermoFluor 384 syste
61. ging Close Gripper Save Points the Z axis DOWN at 20 SPEED until the LIMIT switch triggers Then jog UP mm then TEACH the position Home Plate Crane Press lt SPACE BAR gt to Halt Movement Esc Exit Figure 4 12 The Calibration screen is used to establish the PlateCrane positions This screen is not used during normal operation System Settings This selection opens the PlateCrane System Setup screen See below The position of the assay plates relative to the PlateCrane arm grippers plate position is set up from this screen These positions should be established when the system is initially installed and should not have to be adjusted or changed during normal operation of the ThermoFluor 384 system However if new plates are used this position should be verified and may need to be reset To verify or reset the plate position refer to the Troubleshooting section of Chapter 5 Maintenance If more details about this section of the software are desired refer to Appendix A Total Control for Windows for the TCW manual Copyright 2001 3 Dimensional Pharmaceuticals Inc 4 16 Confidential May 2001 Artisan Scientific Quality Instrumentation Guaranteed 888 88 SOURCE www artisan scientific com Move to Point FEYT YY YTY YYTYYYYTYYTYYYYYYYYYTYYTYYYTYYYEYEYEYTTETTYTETETEET Operation ThermoFlour 384 Operating Manual PlateCrane System Setup med Defined P late Type
62. he available methods list It will appear under the Methods to be Run list If the wrong method is entered selecting it in the Methods to be Run list and clicking the Delete Method button will delete it Click Yes or press lt Enter gt The THERMOFLUOR PLATE SETUP screen appears Copyright 2001 3 Dimensional Pharmaceuticals Inc a 4 32 Confidential May 2001 Artisan Scientific Quality Instrumentation Guaranteed 888 88 SOURCE www artisan scientific com Operation l ThermoFlour 384 Operating Manual ES a AAA M Er En 4 Stack 1 ThermoFlour Plate Setup A Plate 1 2 Plate 2 Data Directory cMcwi3dpidatal Emission Filter 3 Plate 3 4 Plate 4 ajo Operating Mode 1 Excitation Filter 2 initial Temperature 25 00 Box X 16 A Temperature Increment 1 00 IBox Y 18 Final Temperature 26 00 Number of Replicates 2 Imaging Temperature 25 00 Gain 2 Low Equilibration Time 30 00 Shutter Closed N High Equilibration Time 30 00 Your message hers Exposure Time Add Insert Delete Edit Esc Designate the plates and plate parameters to be run in TCW This is done by pt Click the Add button to add Platel to the Stack list If all the plates in the 7 7A B Stack magazine will use the default parameters go step 8 7 To change the parameters for Plate1 click the Edit Button The THERMOFLUOR SETUP Screen appea
63. he data files using the ThermoFluor Analysis software See ThermoFluor 384 Analysis Software Manual Performing a Single Plate Run with Acquire 3 0 There may be times when it is necessary to run only a single plate and manually load it into the ThermoFluor 384 instrument This can be done from the Acquire 3 0 program directly To do this follow the procedure below Complete the necessary run sheet E Turn on the ThermoFluor 384 System in the following order N e Universal power supply e Boot up the NT workstation The startup screen appears see below e Turn on the instrument Copyright 2001 3 Dimensional Pharmaceuticals Inc 4 36 Confidential May 2001 Artisan Scientific Quality Instrumentation Guaranteed 888 88 SOURCE www artisan scientific com AHA BAA AKA AAA AKA AAAMAAAAAA AAAAA LA GHLAAAAA KAA AA LAAA HM ADM e Operation ThermoFlour 384 Operating Manual A A A PP PI O I Start 33 Exploring 3DPnew Open the ThermoFluor Acquire 3 0 application by double clicking the ThermoFluor Acquire 3 0 icon The ThermoFluor 384 Acquire 3 0 program opens ty 3 28 PM e The instrument goes through an initialization Observe the screen to verify that the heater and camera initialize properly Place the assay plate on of the ThermoFluor instrument heater block VY VV NN vuuu vu vu VVE VVV PPO VV VV Copyright 2001 3 Di
64. he supports and pull backwards The unit should s __ Reconnect the connectors to the UV light source O s E Close the optical compartment door and secure with screws Turn on power to the instrument Copyright 2001 3 Dimensional Pharmaceuticals Inc 5 7 Confidential May 2001 Artisan Scientific Quality Instrumentation Guaranteed 888 88 SOURCE www artisan scientific com Maintenance ThermoFlour 384 Operating Manual Troubleshooting This section discusses some of the ThermoFluor 384 system specific problems that might occur and their possible solutions These problems include e Failure of a component to initialize e Recovering plate data following system disruption e Plate crane doesn t pick up plates For additional information about problems with specific components of the system refer to their documentation that is supplied with the system Component Fails to Initialize As the ThermoFluor Acquire 3 0 program launches it searches for the several available sub systems inside the ThermoFluor system These are the CCD camera the PlateCrane thermal cycler heater and the bar code reader There are indicator fields for the heater and camera at the lower left corner of the Acquire 3 0 screen If any of these fail to initialize the system will not function properly Thermal Cycler Heater If the heater module fails to initialize the following error message will app
65. hermoFlour 384 Operating Manual Continuous Ramp Mode In the Continuous Ramp mode the temperature is raised to a temperature value and held there for a set time High Temperature Hold Time to allow equilibration and then while the camera records images Then the temperature is raised to the next temperature value and held there to allow equilibration and then while the camera takes shots The embedded controller repeats this process for each temperature point in the selected range The embedded controller begins the first heating cycle Continuous Ramp mode by signaling the heater to raise the temperature of the plate to the initially selected High temperature value When the controller senses this temperature has been reached it signals the heater to maintain the High temperature and begins measuring the High temperature hold time See below TEMPERATURE Shutter Closed Image for background correction UV light ON prior to End of High T Hold Command issued e UV light off e Next cycle begins Signal at End of High T Hold TIME First High T Hold Number of replicate images specified recorded Figure 4 6 Continuous Ramp mode sequence of events Prior to the end of the high temperature hold the controller turns the UV light on so it can stabilize before images are taken When the high Temperature Hold time has elapsed the controller signals the Acquire 3 0
66. hold the lt Ctrl gt and closed in lt gt lt Shift gt keys Special Terminology The following table provides definitions for some of the terms used in conjunction with ThermoFluor Hit A well on a screening plate that exhibits a signal strength that exceeds the Tm shift threshold set as a significant response for the run int INT filee File created by the ThermoFluor Acquire 3 0 application which contains integrated intensity values for wells in a plate psq PSQ file File that contains raw camera exposure data The ThermoFluor Acquire 3 0 application creates int integration files from the psq files that correspond to the replicate exposures for a well tf TF file The ThermoFluor Analysis Software creates this file It contains the fitted data along with other values that are calculated and or downloaded from a data base for each well on a plate The table below lists common acronyms that are used in conjunction with ThermoFluor Acronym Definition TCW Total Control for Windows program developed by Hudson Control Group Copyright 2001 3 Dimensional Pharmaceuticals Inc 0 7 Confidential May 2001 Artisan Scientific Quality Instrumentation Guaranteed 888 88 SOURCE www artisan scientific com w W v wv w v v v v vyv w Poa Preface ThermoFluor Instrument Operations Copyright 2001 3 Dimensional Pharmaceuticals Inc Confidential Artisan Scien
67. idential May 2001 Artisan Scientific Quality Instrumentation Guaranteed 888 88 SOURCE www artisan scientific com Construction and Function ThermoFlour 384 Operating Manual Overall Construction The ThermoFluor 384 system is a flexible and modular fluorescence assay system with an automated plate processing system The basic ThermoFluor 384 system is capable of sequentially loading and unloading up to twenty 20 384 well thermal cycler microplates The ThermoFluor 384 instrument works in conjunction with a Windows NT workstation running 3 Dimensional Pharmaceuticals ThermoFluor Acquire 3 0 control software to perform the fluorescence assay The plate processing system has a PlateCrane robotic arm that moves sample plates from an input storage magazine to the imaging area of the instrument after which the ThermoFluor Acquire 3 0 control software program initiates a ThermoFluor experiment on the microplate When the experiment is completed the Plate Crane retrieves the plate from the imaging area and places it into an output magazine The various components or the system are powered by a universal power supply Plate ThermoFluor 384 Processing Instrument System Windows NT Workstation Universal Power Supply Figure 2 1 The ThermoFluor 384 system Copyright 2001 3 Dimensional Pharmaceuticals Inc e 22 Confidential May 2001 Artisan Scientific Quality Instrumentation Guaranteed
68. ing a continuous series of images with the given exposure time From the rear of the instrument adjust the bottom adjustment ring on the lens until the image is sharp Close the optical compartment door and secure with screws Remove the white piece of paper from the thermal cycler block To do this open the light tight door by clicking the Open Door button in the CAMERA dialog box as done above Once the piece of paper is removed from the thermal cycler block close the light tight door by clicking the Close Door button Copyright 2001 3 Dimensional Pharmaceuticals Inc 3 14 Confidential May 2001 Artisan Scientific Quality Instrumentation Guaranteed 888 88 SOURCE www artisan scientific com D HAA AK AAAKA KZA DR AAHAAAA BABA AAAAAALAALALALA MM Installation and Calibration ThermoFlour 384 Operating Manual Calibration of Spots Before the system can properly measure microplates it must determine where the wells in the plates are located The system software must be running to accomplish this task First an image of a microplate with standard florescent beads in the wells is taken Then the corner wells are identified for the Acquire 3 0 program After this the program calculates the position of each of the 384 wells and scans the plate so the user can verify that the program has properly defined the well positions Follow the procedure below to perform this calibration Action 1 From Acq
69. ingle psq to be viewed in the Image display area of the Acquire 3 0 interface An Open dialog box with psq files already selected allows a directory and file to be selected Look in 239 TFAcquire ex 1118 10 1 psq 1118 10 2 psq 1118 10 3 psq 111810 4 psq 1118 10 SC psq Files of ype Photometrics Image Sequences psq y Cancel M Open as read only Read Series This selection allows viewing of raw camera data in the form of a series psq files corresponding to the data for a single plate at all temperatures The SELECT IMAGE SERIES dialog box opens to allow selection of a psq file from the series By then clicking the Select Series button the series of psq files corresponding to the selected file are retrieved This feature is also used is used in conjunction with the reintegrate feature See the Recovering Plate Data Following System Disruption topic in the Troubleshooting section of Chapter 5 Maintenance _ Copyright 2001 3 Dimensional Pharmaceuticals Inc 2224 23 Confidential May 2001 Artisan Scientific Quality Instrumentation Guaranteed 888 88 SOURCE www artisan scientific com Operation na ThermoFlour 384 Operating Manual Fm Select Image Series Image Series 2 00 22 moo o Drive Je SA CA EN Program Files AN TFAcquire 1118 10 1 psq 1118 10 2 psq 1118 10 3 psq 1118 10 4 psq 1118 10 SC psq Cancel Write Image This selection allow
70. ion Guaranteed 888 88 SOURCE www artisan scientific com Operation ThermoFlour 384 Operating Manual Image Display Area This area of the screen displays the camera images This allows the run to be monitored from the Acquire 3 0 interface screen It will also display psq files read offline using the Read Image and Read Series selections of the IMAGES Menu a 3DP s ThermoFluor TFAcquire version 3 0 0 Experimental Parameters Data Directory o an Brome soso as cercare PEO CSE SOO SD Shae mn rod f ooo TTT comment aka a A ALIATS i A RN Heating Parameters PALIAR ipo 3 PIDA ACACIA Heating Mode Up Down POPP SPPSERCVPMSKCEHECSEE CHOY ja Fj ct SPFPFPISSPAHESCESE CHES BEES E Initial Final Temperatures C J40 50 SFHSPFPFSISMQFSCSOSSRese tee 66806 Temp Incr Low Temp C 2 80 pe a i f PELEADO Z nro High Low Temp Holds sec 60 60 Y image Parameters E sa Vrrces eee 8 ere Exposure Time sec Reps 05 Fr Filters 500 30nm SCimage Y Gain Ry Box X Y he fe Eror Run blEror e Ae Heater Camera Error 11 7 00 8 45 AM Figure 4 16 Image Display area of the Acquire 3 0 user interface The intensity of an individual pixel can be viewed by placing the cursor over the particular well The intensity reading is displayed as I under the image At the beginning of a run the intensity of some pixels can be checked to verify they do not saturate
71. l for Windows for the TCW manual Calibrate PlateCrane This selection opens the Calibration screen See below The various positions the PlateCrane moves to are set up from this screen These positions should be established when the system is initially installed and should not have to be adjusted or changed during normal operation of the ThermoFluor 384 system It is possible that a crash of the system could cause the Stack Position to be altered or lost This could affect the PlateCrane s ability to pick up plates In this case the Stack Position would have to be re taught re calibrated to the PlateCrane arm Refer to Appendix A Total Control for Windows for the TCW manual to do this To verify the Stack Position is correct refer to the Troubleshooting section of Chapter 5 Maintenance Copyright 2001 3 Dimensional Pharmaceuticals Inc 4 15 Confidential May 2001 Artisan Scientific Quality Instrumentation Guaranteed 888 88 SOURCE www artisan scientific com Operation ThermoFlour 384 Operating Manual CALIBRATION Position Nune R 2 ka Current Po sition Teach Point E ES Rotate CW Jog Up Gripper CW Teaching Speed Rotate CCW Jog Down Gripper CCW Speed A di R Jog Distance 5 0deg Z Jog Distance 5 0 mm P Jog Distance 5 0deg Change R Jog Change Z Jog Change P Jog NOTE Teach STACK positlons by jog
72. l laboratory equipment follow all standard operating procedures and policies An operations and maintenance logbook should be maintained at the instrument s location WARNING Do not place hand into the open Light Tight Compartment door If it were to shut on the hand it could cause serious injury Remember above all safety first Copyright 2001 3 Dimensional Pharmaceuticals Inc aii 4 2 Confidential May 2001 Artisan Scientific Quality Instrumentation Guaranteed 888 88 SOURCE www artisan scientific com Operation ThermoFlour 384 Operating Manual Operating Software and Controls As mentioned in Chapter 2 Construction and Function the Windows NT Workstation contains the software that controls the operation of the system The Total Control for Windows TCW program coordinates the overall operation of the system while the ThermoFluor Acquire 3 0 program provides the user interface to the ThermoFluor 384 instrument Total Control for Windows directly operates the plate processing system and it indirectly operates the ThermoFluor 384 instrument through a DDE connection with the ThermoFluor Acquire 3 0 control software The ThermoFluor Acquire 3 0 control software interfaces with the CCD camera and an embedded controller Zworld in the ThermoFluor 384 instrument that controls the filter wheel UV light source light tight compartment door and the thermal cycler heater This is illustrate
73. late is held at the low temperature for a set time Low Temperature Hold Time to allow equilibration and then while the camera records images The embedded controller repeats this process for each temperature point in the selected range The embedded controller begins the first heating cycle Up Down mode by signaling the heater to raise the temperature of the plate to the initially selected High temperature value When the controller senses this temperature has been reached it signals the heater to maintain the High temperature and begins measuring the High ae hold time See below Copyright O 2001 3 Dimensional Pharmaceuticals Inc 4 6 Confidential May 2001 Artisan Scientific Quality Instrumentation Guaranteed 888 88 SOURCE www artisan scientific com A 2 2 2 2 BA GB BB AA BA AAAAAA AMA AA ARANRAAAMA AA AHKAMAAARAAALA LL LL T Y Nu uv uv uv VVV VV VV YPU vuuy Operation ThermoFlour 384 Operating Manual _ eee AMOS 28 Uperating Manual TEMPERATURE Shutter Closed Image for background correction EC CG CU UV light ON prior to End of Low T Second High T Hold High T Hold Command issued e UV light off e Next cycle begins Cycle begins Low T Hold Signal at End of Low T Hold AAA E AE EA Number of replicate images TIME Figure 4 5 Up Down mode sequence of events When this time has elapsed the contro
74. le F 9 2 F Gain 2 180 KT fie fie Run Camera Ready Idle 1 30 01 3 32 PM Heater Once the piece of paper is placed on the thermal cycler block close the light tight door by clicking the Close Door button in the CAMERA dialog box From Acquire 3 0 CAMERA dialog box turn ON the UV light source by clicking the Light On button 5 Enter an exposure time one second for the camera in the Integrate Image section of the CAMERA dialog box Click on the Cont button to have the camera start taking a continuous series of images with the given exposure time Copyright 2001 3 Dimensional Pharmaceuticals Inc 5 5 3 Confidential May 2001 Artisan Scientific Quality Instrumentation Guaranteed 888 88 SOURCE www artisan scientific com Maintenance ThermoFlour 384 Operating Manual From the rear of the instrument adjust the bottom e ring on the lens yanl the image is siap T i Turn OFF the UV light source by clicking the Light Off bedak Remove the white piece of paper from the thermal cycler block To do this open the light tight door by clicking the Open Door button in the CAMERA dialog box as done above Once the piece of paper is removed from the thermal cycler block close the light tight door by clicking the Close Door button Copyright 2001 3 Dimensional Pharmaceuticals Inc ee Es err 5 4 Confidential May 2001 Artisan Scientific Quality Instru
75. ller signals the heater to lower the temperature of the plate When the controller senses this temperature has been reached it signals the heater to maintain the Low temperature and begins the Low temperature hold time Prior to the end of the Low temperature hold the controller turns the UV light on so it can stabilize before images are taken When the Low Temperature Hold time has elapsed the controller signals the Acquire 3 0 program During this time the Acquire 3 0 program polls the embedded controller for the signal that the low temperature hold time is complete When it finds this signal the Acquire 3 0 program signals the camera to first acquire a shutter closed image if selected for background correction It then directs the camera to take the number of replicate images typically 4 with specified exposure times as indicated by the previously communicated experimental parameters When these are completed the Acquire 3 0 program sends a command to the embedded controller When the embedded controller receives the command it turns off the UV light and begins the next heater cycle at the next higher temperature increment These cycles are continued until the cycle for the final temperature is completed Copyright 2001 3 Dimensional Pharmaceuticals Inc 4 7 Confidential May 2001 Artisan Scientific Quality Instrumentation Guaranteed 888 88 SOURCE www artisan scientific com Operation T
76. luor Figure 2 9 The light tight door and it s drive motor Copyright O 2001 3 Dimensional Pharmaceuticals Inc 2 7 Confidential May 2001 Artisan Scientific Quality Instrumentation Guaranteed 888 88 SOURCE www artisan scientific com Construction and Function ThermoFlour 384 Operating Manual Power and Control Compartment The power and control compartment contains the rest of the thermal cycler assembly and the embedded controller Because of the presence of high voltage components and the embedded controller access to this compartment is limited to factory technicians Thermal Cycler The thermal cycler assembly raises lowers and maintains the temperature of the microplates as directed by the embedded controller It is designed to work with a 384 microplate The temperature is programmable from 4 to 99 C CEE Embedded Controller The embedded controller performs real time control functions for the ThermoFluor 384 Instrument It opens and closes the light tight door turns the UV light source on and off operates the filter wheel and controls the temperature of the thermal cycler The user interfaces with the controller through the ThermoFluor 384 Acquire 3 0 software Copyright 2001 3 Dimensional Pharmaceuticals Inc ae 2 8 Confidential May 2001 Artisan Scientific Quality Instrumentation Guaranteed 888 88 SOURCE www artisan scientific com ee
77. ly the plate processing PlateCrane and bar code reader cables should be connected with no power applied to either the computer or the instrument The UPS inside the workcell must be turned off even if no external power is applied Copyright O 2001 3 Dimensional Pharmaceuticals Inc 1 8 Confidential May 2001 Artisan Scientific Quality Instrumentation Guaranteed 888 88 SOURCE www artisan scientific com AAA ADA AAA AAA AAA AA AAA AAA VUV VU Introduction ThermoFlour 384 Operating Manual Chapter Two Construction and Function Overview This chapter will familiarize the user with the construction of the ThermoFluor 384 system and the function of its major components Specifically this chapter will include the following sections e Overall system construction e Windows NT workstation PC e ThermoFluor 384 instrument construction e Plate processing system construction The following table identifies the appropriate section to read in order to learn more about the construction of the ThermoFluor 384 system To learn more about Read the section See page Overall system construction Overall Construction 2 2 Windows NT workstation PC Windows NT Workstation 2 3 ThermoFluor 384 instrument construction ThermoFluor 384 2 4 Instrument Plate Processing System Plate processing system construction Copyright 2001 3 Dimensional Pharmaceuticals Inc 2 1 Conf
78. m In addition it contains background information in the Appendix sections You can use this manual when you need information on how to operate the system or to train new users of the system The following table outlines the information contained within the Instrument Operation section of the documentation and which chapter to refer to for more information Safety features Plate processing system Chapter One Introduction Chapter Two Construction and Function Copyright O 2001 3 Dimensional Pharmaceuticals Inc 0 2 Confidential May 2001 Artisan Scientific Quality Instrumentation Guaranteed 888 88 SOURCE www artisan scientific com Preface ThermoFluor Instrument Operations Continued from previous page gt 7 Chapter Three Installation and Calibration Chapter Four Operation Instrument Operation Chapter Five Maintenance Hudson Group Plate Crane Manual Appendix B Consumables List Appendix C ThermoFluor Analysis Software This document describes how to analyze ThermoFluor 384 system data using the ThermoFluor Analysis Software It describes basic ThermoFluor theory software navigation and data analysis It also describes the data exporting batch processing of data and DB sheet data browsing You can use this documentation when you need information on how to use the ThermoFluor Analy
79. mage is exposed in seconds in Up Down mode 3 Select the desired operating mode by clicking on the corresponding box Green indicates the selection 4 Select the desired Emission Filter The 500 nm is standard equipment with ThermoFluor 384 instrument 5 Enter the appropriate System Parameters in the fields e No Replicates The number of images of the well recorded during each cycle in the Up Down mode e Gain amplification value This is usually set at installation 1 can be used for particularly bright plates and 3 for particularly dim plates 6 Enter the appropriate Integration Parameters in the fields These values are usually set at installation e Box X Dimension The width of the integration field used for each well e Box Y Dimension The height of the integration field used for each well Enter User Comments if any in the text box Select whether the shutter is opened or closed Select YES for closed and NO for Open Green indicates the selection Usually Closed at beginning of run on When complete press the lt Esc gt key The THERMOFLUOR PLATE SETUP screen appears Click the Add button to add Plate2 to the Stack list If all the remaining plates ___ in the Stackl magazine will use the default parameters go step 8 To change the parameters for Plate2 click the Edit Button The THERMOFLUOR ___ SETUP Screen appears Repeat step 7C to designate the parameters for Plate
80. mensional Pharmaceuticals Inc 4 37 Confidential May 2001 Artisan Scientific Quality Instrumentation Guaranteed 888 88 SOURCE www artisan scientific com Operation ThermoFlour 384 Operating Manual 5 3DP s ThemoFluor TFAcquire version 3 0 0 File Subsystems Images gt Experimental Parameters nr ES y Data Directory lt Ik FADetes nl il y Sample nd Ju Plate ID 11254 Comment J f Heating Parameters gt FF E Healing Mode Up Down gt E hi Initial Final Temperatures C ao En i gt Temp incr Low Temp Ck 2 o ii High Low Tene Holds sec jo 1 i et r Image Parameters r E Scale Bitmap 0233 0258 1 0 IBox 0 414 Exposure Time sec Reps UE fi PETO et aa Sane E aaah A gt a i Filters ARS paa image a Gain E Box ad Fe FE Error bE or Heater Camera Error Enter the experimental parameters using the Acquire 3 0 interface screen This is done by Enter the path and directory for the data PSQ and INT files to be stored By clicking on the Browse button the SET DATA DIRECTORY dialog box appears to select the desired folder Enter the plate ID number This is an eight digit number that would correspond to the barcode number Enter User Comments if any in the text box Specify the heating mode from the drop down menu The modes are Up Do
81. ment and access e Copyright O 2001 3 Dimensional Pharmaceuticals Inc a E Confidential May 2001 Artisan Scientific Quality Instrumentation Guaranteed 888 88 SOURCE www artisan scientific com VINS vuuvuvyvyuyuvu Installation and Calibration ThermoFlour 384 Operating Manual About 66 inches Minimum Access 18 inches 1 30 inches Network Connection 110 VAC 60 Hz 15 Amp Minimum Access 36 inches Figure 3 2 ThermoFluor 384 system stand alone placement side view The power receptacle should be located within four feet of the rear of the system Optional network connection should also be located at the rear of the system Copyright 2001 3 Dimensional Pharmaceuticals Inc 3 5 Confidential May 2001 Artisan Scientific Quality Instrumentation Guaranteed 888 88 SOURCE www artisan scientific com Installation and Calibration ThermoFlour 384 Operating Manual Alternate Table Top Placement and Access An alternative is to install the system base plate on a tabletop rather than the supplied stand In this configuration the system pase plate with the ThermoFluor 384 instrument and the plate processing system attached is located on a table with the front of the instrument facing to the right The system base plate must have the leveling feet installed on the corners and be leveled in both front to back and side to side directions It is very important that nothing restricts
82. ment contains the optical components that measure the fluorescence These include the CCD camera lens and filter wheel This compartment is accessible by the user in order to change filters Figure 2 3 Back of ThermoFluor 384 instrument CCD Camera The CCD camera records the plate fluorescence images It is a Roper Scientific camera model Sensys 0400 It has a half inch mega pixel format The CCD camera comes with its own power supply that is located in the optical compartment and plugged into the system Universal power supply Refer to the Photometric s SenSys User Manual Advanced Camera Operation Manual and Software Guide Manual for more details Figure 2 4 CCD camera mounted in instrument power cable This could damage the camera s electronic components Verify Lower Supply before disconnecting its power cable Copyright 2001 3 Dimensional Pharmaceuticals Inc 2 4 Confidential May 2001 Artisan Scientific Quality Instrumentation Guaranteed 888 88 SOURCE www artisan scientific com CAUTION The camera should never be connected or removed from a live l the camera s power supply is switched OFF and unplugged from the Universal Construction and Function ThermoFlour 384 Operating Manual Lens The Lens focuses the image in the camera It is a 25 mm F 0 95 C mount lens with aperture and focus adjustment The aperture adjustment should always be set to wide open The foc
83. mentation Guaranteed 888 88 SOURCE www artisan scientific com LABO ALA LA A BG BG M A 4 amp AA 4 4 2 4 4 amp 4 GA 4 4 4 A 4 amp A A Maintenance ThermoFlour 384 Operating Manual ASES iS lin eee Filter Changes Use the procedure below to change a filter The system software must be running to accomplish this task Step Action CC 1 Open the optical compartment by removing the screws from the rear panel If the filter wheel is already in the desired position go to step 4 If it is not in the desired position click on the arrow for the right hand Filters drop down menu in the images parameter section at the lower right of the ThermoFluor 384 Acquire 3 0 window See Below LS 3DP s ThermoFluor TFAcquite version 3 0 0 Eile Subsystems Images Experimental Parameters Data Directory Browse Sample Information Plate 1D Fe Comment Heating Parameters Heating Mode Up Down Initial Final Temperatures C jo 70 a Temp Incr Low Temp C fi 25 High Low Temp Holds sec 180 60 f Image Parameters F Scale Bitmap 0759 0009 0 IBox 0 Exposure Time sec Reps ig a Filters ES SCimage Y Gain R 1B ur pee 530 20 nm Uninitialized Run IN470 3 nm Hester it 241 10 12 AM Select the desired position from the drop down menu by clicking that position on the listing See the above figure The filter wheel will rot
84. mentation Guaranteed 888 88 SOURCE www artisan scientific com Operation ThermoFlour 384 Operating Manual Safe Operation The ThermoFluor 384 system should be set up and run by 3 Dimensional Pharmaceuticals Inc factory trained personnel only If you are not factory trained contact 3 Dimensional Pharmaceuticals Inc at 1 610 458 8959 to schedule training he coe HAAAAA RA AMAA AAAAAAAAA DALAL a The Operating Manual contains up to date information on the safe installation and operation of the ThermoFluor 384 system Before installing or running the ThermoFluor 384 system completely and thoroughly review the latest version of the Operating Manual If any instructions are unclear or unfamiliar to you contact 3 Dimensional Pharmaceuticals Inc at 1 610 458 8959 for clarification and proper operation Please locate and identify all safety and informational labels on the instrument If any labels are missing smudged or damaged discontinue use and contact 3 Dimensional Pharmaceuticals Inc for replacements Please locate and identify all safety items on the instrument including covers and panels If any items are missing damaged or disabled in any way discontinue use and contact 3 Dimensional Pharmaceuticals Inc for instrument repair Abnormal instrument operation should be immediately noted and instrument use discontinued until examined by a representative from 3 Dimensional Pharmaceuticals Inc As with al
85. mmediately contact 3 Dimensional Pharmaceuticals Inc by e mail at engsupport 3dp com to schedule training This Operating Manual contains information and procedures for the safe installation and operation of the ThermoFluor 384 system Before installing or running the ThermoFluor 384 system completely and thoroughly review the latest version of this document If any instructions are unclear or unfamiliar to you immediately contact 3 Dimensional Pharmaceuticals Inc by e mail at engsupport 3dp com for clarification and proper operation Abnormal instrument operation should be immediately noted and instrument use discontinued until examined by a representative from 3 Dimensional Pharmaceuticals Inc As with all laboratory equipment follow all standard operating procedures and policies An operations and maintenance logbook should be maintained at the instrument s location Handle with Care To avoid equipment damage handle components carefully Handle the CCD Camera with particular care as it is fragile Lift Properly Use proper lifting techniques when lifting shipping containers and system components Four 4 persons positioned at each corner should be used when lifting the system stand and the instrument base plate Electrical Ensure the system s universal power supply is unplugged and that all power switches are in the OFF position before performing maintenance Use an ESD electrostatic discharge wrist
86. ng HTS It also describes some general data analysis methods You can use this document when you need information on these applications with regard to the ThermoFluor 384 system or to train new users of the system Appendix A Dropout and Edge Detection The following table outlines the information contained within the ThermoFluor Applications Guide of the documentation and which chapter to refer to for more information Copyright 2001 3 Dimensional Pharmaceuticals Inc mS 0 4 Confidential May 2001 Artisan Scientific Quality Instrumentation Guaranteed 888 88 SOURCE www artisan scientific com SAA RK AARKARAAARAAAAAAAAAHRABRARAAKAAAAAAAAAAADA Preface Analyzing the assay plate data ThermoFluor Instrument Operations Overview of ThermoFluor applications guide Chapter 1 Oo ask E Introduction to ThermoFluor optimization Chapter 2 en Yl Optimizing ThermoFluor Conditions Secondary Confirmations Introduction to ThermoFluor High Throughput Chapter 3 Screening High Throughput Screening ThermoFluor High Throughput Screening ThermoFluor 384 assay setup Chapter 4 General Data Analysis Hit re testing Secondary assays ThermoFluor Tutorials This document provides procedures and exercises for common tasks associated with the ThermoFluor 384 system It includes tutorials on protein characterization sample preparation and
87. ntrol for Windows control Total Control for A 2 software Windows Manual Copyright 2001 3 Dimensional Pharmaceuticals Inc eds A 1 Confidential May 2001 Artisan Scientific Quality Instrumentation Guaranteed 888 88 SOURCE www artisan scientific com Copyright 2001 3 Dimensional Pharmaceuticals Inc _ A 2 Confidential May 2001 Artisan Scientific Quality Instrumentation Guaranteed 888 88 SOURCE www artisan scientific com Appendix B Hudson Group Plate Crane Manual Overview This appendix contains the Hudson Group PlateCrane Manual with detailed information about the Hudson Group PlateCrane used in the ThermoFluor 384 system Specifically this appendix includes the following sections e PlateCrane Manual The following table identifies the appropriate section to read in order to learn more about a specific area Read the section See page PlateCrane Manual To learn more about The Hudson Group PlateCrane Manual Copyright 2001 3 Dimensional Pharmaceuticals Inc B 1 Confidential May 2001 Artisan Scientific Quality Instrumentation Guaranteed 888 88 SOURCE www artisan scientific com Copyright 2001 3 Dimensional Pharmaceuticals Inc B 2 Confidential May 2001 Artisan Scientific Quality Instrumentation Guaranteed 888 88 SOURCE www artisan scientific com aa G amp A GO 2e 2 B 2 42 2 42 A D
88. om Pi A w vyv vY YV V V V V VVV y y Installation and Calibration ThermoFlour 384 Operating Manual BS 3 Start Launch the ThermoFluor 384 Acquire 3 0 software by clicking on its icon See above The ThermoFluor 384 Acquire 3 0 screen will appear z Accessories az HP eDiagT ools z Startup gE Internet Explorer 5 Outlook Express ES TestController 2 Windows NT Explorer Ej Administrative Tools Common S Comttol RocketPort RocketModem S HP Lock 3 Photometics Startup J Total Control for Windows 5 Windows NT Update 10574M Launch the ThermoFluor 384 Acquire 3 0 software by clicking the Start menu button then Programs then TFAquire See above The ThermoFluor 384 Copyright 2001 3 Dimensional Pharmaceuticals Inc 3 11 Confidential May 2001 Artisan Scientific Quality Instrumentation Guaranteed 888 88 SOURCE www artisan scientific com Installation and Calibration ThermoFlour 384 Operating Manual Acquire 3 0 screen will appear 5 3DP s FhermoFluor TFAcquire version 3 0 0 File Subsystems images Data Directory fADatai He Li Sample Information Plate ID RE Comment Heating Parametrs Heating Mode Up Down initial Final Temperatures C fao 50 Temp incr Low Temp C A 30 High Low Temp Holds sec Jeo fo a Image Parameters F Scale Bitmap 0233 0258 1 0 I
89. on the stack if desired to prevent sample contamination Copyright 2001 3 Dimensional Pharmaceuticals Inc 2 9 Confidential May 2001 Artisan Scientific Quality Instrumentation Guaranteed 888 88 SOURCE www artisan scientific com Construction and Function ThermoFlour 384 Operating Manual The PlateCrane has three degrees of motion It rotates horizontally 345 about the R axis moves vertically19 inches along the Z axis and the gripper rotates horizontally 350 about the P axis A stepper motor with encoder drives the movement in each axis Each of these have a mechanical switch to determine the Home position The R and Z axis have hard stops A serial cable from the rear of the PlateCrane connects DB 9 connector to a serial port on the Rocketport serial expansion card of the NT Workstation for communications A rocker type power switch is located on the PlateCrane base Power is ON when the switch is illuminated See Appendix B for details about the PlateCrane The PlateCrane gripper has two custom designed hands to securely grip the microplates Two guides assist in centering the plate when it is picked up A mechanical switch in the gripper indicates when the plate is located The lip on the microplate activates this switch Figure 2 13 The PlateCrane is the main component in plate processing Copyright 2001 3 Dimensional Pharmace
90. or Run IbiEror Sample Information 11 7 00 8 45 AM Copyright O 2001 3 Dimensional Pharmaceuticals Inc di 4 39 Confidential May 2001 Artisan Scientific Quality Instrumentation Guaranteed 888 88 SOURCE www artisan scientific com Operation ThermoFlour 384 Operating Manual At the completion of the run the light tight door will open Remove the plate from the ThermoFluor instrument heater block If the ThermoFluor 384 system is not going to be used for an extended period of time shut down the system Analyze the data files using the ThermoFluor Analysis software See ThermoFluor 384 Analysis Software Manual Copyright 2001 3 Dimensional Pharmaceuticals Inc _ 4 40 Confidential May 2001 Artisan Scientific Quality Instrumentation Guaranteed 888 88 SOURCE www artisan scientific com Operation ThermoFlour 384 Operating Manual Chapter Five Maintenance Overview This chapter provides information and instructions for the routine maintenance of the ThermoFluor 384 system i Specifically this chapter includes the following sections e System Cleaning e Camera Focusing e Filter Changes e Light Source changes e Troubleshooting e Repairs The following table identifies the appropriate section to read in order to learn how to perform specific maintenance tasks or troubleshooting To learn more about See page
91. or double click the file listing e The appropriate information is automatically entered in the Experimental Parameter panel of the ThermoFluor Acquire 3 0 screen e The first replicate of the first temperature increment for the plate is displayed in the Plateview panel The first replicate image of each temperature increment can be viewed using the scroll bar Select Reintegrate from the IMAGE menu A check will appear next to the Copyright 2001 3 Dimensional Pharmaceuticals Inc 5 11 Confidential May 2001 Artisan Scientific Quality Instrumentation Guaranteed 888 88 SOURCE www artisan scientific com fe DANNA RANA RARA TVT AAA AA A AAA AAA AA AAA AM Maintenance ThermoFlour 384 Operating Manual command on the menu Click Run at the bottom of the Experimental Parameter panel of the ThermoFluor Acquire 3 0 screen The application runs the integration on the raw image data for the plate and writes the corresponding int files These files can then be analyzed by the ThermoFluor Analysis software application Plate Crane Doesn t Pick up Plates This is an indication of a mechanical problem Examine the PlateCrane arm and grippers for abnormalities Also if the system crashed the stack storage magazine or plate position in the TCW program could have been altered Verify Stack Position The stack position is set up by the technician when the system is initially installed
92. position Plug the universal power supply into the 110 VAC wall outlet Step Turn on the universal power supply Turn the PlateCrane power switch to the ON position The PlateCrane grippers will open and close When the system initialization is complete turn the computer power switch to the ON position 6 Turn the instrument power switch to the ON position When the computer completes the Windows NT boot up the control computer and system are ready for operation System Initialization Once the ThermoFluor 384 system is set up powered up and the control software installed the system is ready for initialization and synchronization with the control computer As the ThermoFluor 384 Acquire 3 0 software launches it searches for several available components in the ThermoFluor 384 system These include the Camera thermo cycler heater and barcode reader If any of the components fail to initialize an error message will be generated see Troubleshooting in Chapter Five Maintenance Use the following procedure to initialize the system To run the system first boot up the NT workstation A Startup screen similar to _ the following will appear Log on as specified by the System Administrator Copyright 2001 3 Dimensional Pharmaceuticals Inc 4 3 10 Confidential May 2001 Artisan Scientific Quality Instrumentation Guaranteed 888 88 SOURCE l www artisan scientific c
93. program During this time the Acquire 3 0 program polls the embedded controller for the signal When it finds the signal the Acquire 3 0 program signals the camera to first acquire a shutter closed image if selected for background correction It then directs the camera to take the number of replicate images typically 4 with specified exposure times as indicated by the previously communicated experimental parameters When these are completed the Acquire 3 0 program sends a command to the embedded controller When the embedded controller receives the command it turns off the UV light and begins the next heater cycle at the next Su Copyright 2001 3 Dimensional Pharmaceuticals Inc 4 8 Confidential May 2001 Artisan Scientific Quality Instrumentation Guaranteed 888 88 SOURCE www artisan scientific com Operation ThermoFlour 384 Operating Manual higher temperature increment These cycles are continued until the cycle for the final temperature is completed abc a Plate Done When the Final temperature cycle for the plate has been completed the embedded controller brings the heater block temperature back to 25 C if it is not already there opens the light tight compartment door and indicates the plate is done The Acquire 3 0 program recognizes the indication the plate done stops polling the controller and communicates that the plate is done to the TCW program Complete Run The TCW program then direct
94. r 384 Operating Manual Status Bar Heater Camera Error OO 28m O S O The Status Bar is located at the bottom of the window It gives the status of the instrument heater and camera Bold letters for the heater or camera indicate they are operational The Status Bar also displays system information and error messages In addition it displays the current date and time File Menu Contains the command for exiting the program Menu Selection Function Quit Quit the application ES Subsystems Quit Ctri F4 Subsystem Menu Contains the following commands Menu Selection Function TF Controller Opens the TF CONTROLLER dialog box which displays the current parameters of the embedded controller and the Test Controller button for accessing controller test functions Camera Opens the CAMERA dialog box for making adjustments to the camera parameters The CAMERA dialog box also contains some instrument functions Subsystems w TF Controller Camera TF Controller pop up window This window displays general information stored in the embedded controller for the current run si TF Controller gt General Heat Mode Up Down Initial Temp 40 Final Temp 70 Temp Incr 1 Image Temp 25 High Eq Time 180 LowEq Time 60 Copyright O 2001 3 Dimensional Pharmaceuticals Inc f 0 2 4 19 Confidential May 2001 Artisan Scientific Quality Instrumentation Guaranteed 888 88 SOURCE www artis
95. rs ThermoFluor Setup Screen ctcwi3idpidatai Initial Temp Low Eq Time 35 BE Up Down Temp increment Tr High Eg Time Final Temp Exposure Time Imaging Temp es ES Continuous Ramp ES 530 20 nm No Replicates Your message here BH 500 30 nm 470 30 nm Salh Box X Dimension Box Y Dimension Copyright O 2001 3 Dimensional Pharmaceuticals Inc ML SA 5 433 Confidential May 2001 Artisan Scientific Quality Instrumentation Guaranteed 888 88 SOURCE www artisan scientific com Operation ThermoFlour 384 Operating Manual DR A Complete the ThermoFluor Setup Screen 1 Enter the path and directory for the data PSQ and INT files to obs stored 2 Enter the appropriate Imaging Profile Parameters in the fields e Initial Temp The starting temperature in C for measurements First high temperature in Up Down mode e Temp Increments Temperature difference between measurements in C e Final Temp The last temperature in C for measurements Last high temperature in Up Down mode e Imaging Temp The low temperature in Up Down mode that is held while images are taken e Low Eq Time The low temperature equilibration or hold time in seconds for the cycle in Up Down mode e High Eq Time The high temperature equilibration or hold time in seconds for the cycle in Up Down mode e Exposure time Time each replicate i
96. rument recovery process Prior to closing the program A Open the FOLDER named TFAquire on the C drive B Copy the file named TFAquire log C Paste it into a dated folder MMDDY YTf in the designated Test folder in the ThermoFluor folder on the D drive on the same unit with the date on it If the Test folder does not exist create one in the manner outlined Rename the freshly pasted TFAquire log to TFAquireMMDDYY Log Close out of the program and restart it This will reinitialize the instrument Often a camera issue will ensue if this is the case see step 6 If this still does not work A Close the program B Shut down the instrument and wait 30 sec C Start the instrument wait for it to finish its start up routine D Then restart TFAquire If the instrument still does not function properly repeat step 7 However prior to restarting the instrument reboot the computer After a successful reboot continue as normal A Restart the instrument B Then restart TFAquire Upon success or failure notify others of the activity Include the e Specific TF unit e Error message that was recorded Copyright 2001 3 Dimensional Pharmaceuticals Inc 5 10 Confidential May 2001 Artisan Scientific Quality Instrumentation Guaranteed 888 88 SOURCE www artisan scientific com Maintenance ThermoFlour 384 Operating Manual Steps taken to address the error
97. s Far Hgt Lid Hgt Current Speed 0 Run Speed C PlateCrane XL Ask if Ready to Home Er Modify Plate Add Plate Delete Plate Figure 4 13 The PlateCrane system setup screen is used to establish the position of the microplates This screen is not used during normai operation Esc Exit EN AAE E Current Speed This indicates the current speed of the PlateCrane robotic arm Copyright O 2001 3 Dimensional Pharmaceuticals Inc i 4 17 Confidential May 2001 Artisan Scientific Quality Instrumentation Guaranteed 888 88 SOURCE www artisan scientific com Operation ThermoFlour 384 Operating Manual ThermoFluor 384 Acquire 3 0 The ThermoFluor Acquire 3 0 control software provides the user interface for the ThermoFluor 384 instrument It allows the user to enter run parameters that are sent to the embedded controller that operates the ThermoFluor 384 instrument components It also accepts parameters from the TCW program It initiates the instruments operation and allows the user to designate where the resulting data files are saved In addition the Acquire 3 0 program provides an interface for the user to view the plates as they are exposed ThermoFluor Acquire 3 0 User Interface This is the Acquire 3 0 user interface main window It consists of a Title bar drop down menus status bar and a screen for experimental parameter entry
98. s the PlateCrane to remove the plate from the heater block and place it in the output storage magazine It then directs the PlateCrane to take the next plate in the input magazine and load it onto the heater block As the plate passes the barcode reader it is read and this information is communicated to the Acquire 3 0 program that stores it with the experimental data TCW then signals Acquire 3 0 that a plate has been placed in the light tight compartment and the Acquire 3 0 program signals the embedded controller to begin the measurements for the next plate The embedded controller then repeats the above steps for this plate This process is repeated until all the plates are measured Copyright 2001 3 Dimensional Pharmaceuticals Inc _ Es 4 9 Confidential May 2001 Artisan Scientific Quality Instrumentation Guaranteed 888 88 SOURCE www artisan scientific com Operation tt ThermoFlour 384 Operating Manual Total Control for Windows The Hudson Control Group has developed the Total Control for Windows software When Total Control for Windows TCW is started by double clicking the PlateCrane icon on l the MS Windows desktop the TCW Main menu screen appears See below This screen provides access to the various functions of the program In this section we discuss those functions that might be used during the operation of the ThermoFluor 384 System MAIN MENU Run Methods Fi F4 ES PlateCrane Edi
99. s the current image displayed in the Image Display area to be written to a file The SAVE AS dialog box opens to allow the designation of the directory and naming of the psq file By then clicking the Save button the image is saved as a psq file in the designated directory Save in y TFAcquire h er 1118 10 1 psq j 1118 10 2 psq 1118 10 3 psq 1118 10 4 psq 1118 10 SC psq Save as type Photometrics Image Sequences psq he Cancel T Open as read only Copyright 2001 3 Dimensional Pharmaceuticals Inc Hole TP ne tide S 4 24 Confidential May 2001 Artisan Scientific Quality Instrumentation Guaranteed 888 88 SOURCE www artisan scientific com Operation ThermoFlour 384 Operating Manual Experimental Parameters This area of the Acquire 3 0 interface screen allows for the user to enter experimental parameters These fields can be filled in directly by the user or the information can be relayed from the ThermoFluor Setup Screen in TCW 3 3DP s ThermoFluos T FAcquire version 3 0 0 File Subsystems Images Experimental Parameters Data Directory CEEP ETETETT cerros fatat eS P aoOaa Sack ocaucsceuceass cll 222 babSDRTZHESHECR REECE KO HEEE Prete ID EE APARECE ICAA Comment V0 DV S SSE d d ess eee eee Heating Parameters PRSIPDISHFSHOTHSTE AAA ti UpDom y Dosddsosese eresssesecen Heating Mode Up Down PPPFFSSSPSRASHSESHCHEESEORES fo PPPSSHOPESHSSHERESEHR
100. sis Software or to train new users of the system The following table outlines the information contained within the ThermoFluor Analysis Software section of the documentation and which chapter to refer to for more information To learn more about Read Introduction to the ThermoFluor analysis software Getting Started Copyright 2001 3 Dimensional Pharmaceuticals Inc bE 0 3 Confidential May 2001 Artisan Scientific Quality Instrumentation Guaranteed 888 88 SOURCE www artisan scientific com Layout table _ Requery plate info Preface _ThermoFluor Instrument Operations Continued from previous pag To learn more about ThermoFluor graphical user interface e Chapter 3 Navigating Analysis Results Results table Summary table Averaging replicates i Modifying analysis settings Specifying reference wells Excluding data points Manual data analysis Saving into a file Batch analysis Batch registration Chapter 4 Customizing Analysis Chapter 5 Output Options Chapter 6 Miscellaneous Options Edge detection Dropout detection ThermoFluor Applications Guide This document provides fundamental information for basic applications associated with the ThermoFluor 384 system It describes the basics of protein preparation and high thru put screeni
101. stem Setup screen appears Click Add to add new plate dimensions 3 4 Enter the plate name in the ENTER THE NEW PLATE NAME DESCRIPTION pop up dialog box Click OK Copyright 2001 3 Dimensional Pharmaceuticals Inc oe 5 13 Confidential May 2001 Artisan Scientific Quality Instrumentation Guaranteed 888 88 SOURCE www artisan scientific com Maintenance E ThermoFlour 384 Operating Manual Enter the new plate Name Description The MENU dialog box appears to enter plate specifications format Default designations for 96 384 and 1536 well plates are shown Select the desired plate specifications format and click OK The ENTER THE PLATE TO PLATE HEIGHT pop up dialog box appears Select FORMAT Enter the Plate to Plate HEIGHT mm 13 200000 OK __w Enter or verify the plate height in the ENTER THE PLATE TO PLATE HEIGHT pop up dialog box Stack a minimum of 10 plates and measure their height in millimeters then divide the height by the number of plates to get the average height of the plates Enter the average plate height value in mm and click OK Caution This is s very important parameter The measurement should be done accurately lt The Enter Plate Lid HEIGHT pop Enter Plate Lid HEIGHT mm 2 000000 OK Copyright 2001 3 Dimensional Pharmaceuticals Inc sr 5 14 Confidential May 2001 Artisan Scientific Quality Instrumentation Gu
102. sure time Time each replicate image is exposed in seconds in Up Down mode Exposure time sec Reps e No Reps The number of images of the well taken during each cycle in the Up Down mode Using an average of replicate images may improve the overall signal to noise ratio Copyright 2001 3 Dimensional Pharmaceuticals Inc 4 26 Confidential May 2001 Artisan Scientific Quality Instrumentation Guaranteed 888 88 SOURCE www artisan scientific com Operation ThermoFlour 384 Operating Manual Select the desired emission Filter from the drop down menu The 500 nm is standard equipment with ThermoFluor 384 instrument This is selected to designate a shutter closed image tobe taken for background subtraction Gain This represents the detection mode of the CCD camera e 1 corresponds to High Signal to Noise Ratio Use for bright plates e 2 corresponds to High Dynamic Range Standard use e 3 corresponds to High Sensitivity Use for dim plates The system is set at 2 during installation Specify the size of the integration area by designating IBox X and IBox Y IBox X Dimension The width of the integration field used for each well IBox Y Dimension The height of the integration field used for each well Copyright O 2001 3 Dimensional Pharmaceuticals Inc 4 27 Confidential May 2001 Artisan Scientific Quality Instrumentat
103. t selected list that is used for changing the parameters for the selected plate Clicking the Esc button Returns the user to the Current Method screen To change the parameters for a plate select the Edit button The ThermoFluor Setup Screen appears See below ThermoFluor Setup Screen c cwiidpidatal Initial Temp Low Eq Time 35 Up Down i High Eq Time Temp Increment E qn is Continuous Ramp Final Temp Exposure Time Imaging Temp 25 ATINA TR RE ie rent ES None ES 530 20 nm No Replicates 4 Gain 2 Your message here ES 500 30nm BB 470 30 nm Box X Dimension 1Box Y Dimension Bl ves Eno Esc q e E AAA y Figure 4 10 Enter parameters fora plate on the ThermoF luor Setup screen This screen allows the user to 1 Enter the path and directory for the data psq and int files to be stored The psq files s Copyright 2001 3 Dimensional Pharmaceuticals Inc TD 4 12 Confidential May 2001 Artisan Scientific Quality Instrumentation Guaranteed 888 88 SOURCE www artisan scientific com Operation ThermoFlour 384 Operating Manual are the raw camera data and the int files are integration files created by the Acquire 3 0 program The folder in which these files are to be stored is designated here 2 Enter the appropriate Imaging Profile Parameters These fields are Initial Temp The starting temper
104. t Methods F2 F5 System Settings gt Current Speed Edit Position List F3 Esc Exit TCW Figure 4 7 Total Control for Windows TCW main menu screen If more details about this software are desired refer to Appendix A Total Control for Windows for the TCW manual Run Methods This selection is used to set up and start a run The specific procedure for performing a run including setup is discussed in the ThermoFluor 384 System Operation section of this chapter When Run Methods is selected the Current Method screen appears Copyright 2001 3 Dimensional Pharmaceuticals Inc 2 4 10 Confidential May 2001 Artisan Scientific Quality Instrumentation Guaranteed 888 88 SOURCE www artisan scientific com Operation ThermoFlour 384 Operating Manual Current Method THERMO Step Selected No Position Current Plate Methods PlateCrane Start Run Esc Figure 4 8 The TCW Current Run screen The title of the run is displayed after the Current Run screen title In the case above the current run is THERMO1 Clicking the Start Run button allows the user to select which method to run create a method run list and access the ThermoFluor Plate Setup screen shown below __ Stack ThermoFlour Plate Setup _ 1 Plate HSE Cree ea Data Directory ax tewQdpldatal Emission Filter 2 C3 Pilate 3 7 4 TRA 4 Operating Mode y Excitation Filter
105. tely 5 inches from the floor Black circles around each screw identify JN Gently lift the top of the crates straight up over and off the instrument Caution the contents are still attached to the bottom of the pallet __ Inspect the contents of the crates for any signs of damage _ Remove Contents from Pallets Remove the contents of the crates from the pallets Install leveling feet on the system stand and attach the instrument base plate to the system stand Then place all packing materials back in the crates and store for future use Step Action the pallet and put them in safe location Remove the small shipping box secured to the system stand and pallet Inside the __ box are cabling manuals tools and four 4 stainless steel leveling feet for the Copyright 2001 3 Dimensional Pharmaceuticals Inc 3 8 Confidential May 2001 Artisan Scientific Quality Instrumentation Guaranteed 888 88 SOURCE www artisan scientific com he Installation and Calibration ThermoFlour 384 Operating Manual Ld system stand Remove the box and put it in safe location Unscrew the four bolts holding the system stand to the pallet They ar are located on both the right and left side of the system stand The bolts are captured in the pallet so they will only drop half an inch or so from the pallet Prop up the front of the system stand with a 2 x 4 and install the front two 2 leveling fe
106. the camera Moving the cursor over the wells does this by verifying that the intensities are less than 4096 Copyright 2001 3 Dimensional Pharmaceuticals Inc 4 28 Confidential May 2001 Artisan Scientific Quality Instrumentation Guaranteed 888 88 SOURCE www artisan scientific com O LAMA KAA AAKAKALA ALL 4 LL 4 4 4 4A BANDA AA B A G 2 G2 GB 42 2 A amp 4B YSE Operation ThermoFlour 384 Operating Manual AAA APRO OA ene anal ThermoFluor 384 System Operation The ThermoFluor 384 system should be turned on at least 20 minutes before a run to allow the system to warm up After the run sheet is completed and the assay plates are placed in the input magazine the screening run is set up in Total Control For Windows TCW The plates are then run and data files written to a designated directory The ThermoFluor Analysis Software then analyzes these data files Typical Procedure for Performing a Screening Run The following procedure represents the way a typical screening assay is conducted This procedure uses TCW to set up a run of multiple plates Complete a screening run sheet or other documentation of the run Turn on the ThermoFluor 384 System in the following order Universal power supply Boot up the NT workstation The startup screen appears see below Turn on the instrument Turn on the PlateCrane Start 2 Exploring 3DPnew g EN
107. tific Quality Instrumentation Guaranteed 888 88 SOURCE www artisan scientific com 0 8 May 2001 HBA RAAAAAAAAAAAAAAARAAAAAAARANAAAARAAAAAAABAALA LD DODO Table of Contents CHAPTER ONE Introduction Overview Introduction to the ThermoFluor 384 System Instrument Specifications Safety Features CHAPTER TWO Construction and Function Overview Overall Construction Windows NT Workstation ThermoFluor 384 Instrument Plate Processing System Universal Power Supply CHAPTER THREE Installation and Calibration Overview Safety instructions Site preparation System installation Calibration of Spots Acceptance Tests CHAPTER FOUR Operation Overview Safe Operation Operating Software and Controls Total Control for Windows ThermoFluor 384 Acquire 3 0 ThermoFluor 384 System Operation CHAPTER FIVE Maintenance Overview System Cleaning Camera Focusing Filter Changes Light Source Changes Troubleshooting Repair Copyright 2001 3 Dimensional Pharmaceuticals Inc nn z 0 9 Confidential May 2001 Artisan Scientific Quality Instrumentation Guaranteed 888 88 SOURCE www artisan scientific com 2 1 2 1 2 2 2 3 2 4 2 9 2 12 4 10 4 18 4 29 5 1 5 1 5 1 5 2 5 3 5 7 5 8 5 17 Copyright 2001 3 Dimensional Pharmaceuticals Inc z 0 10 Confidential May 2001 Artisan Scientific Quality Instrumentation Guaranteed 888 88 SOURCE www artisan scien
108. tific com f amp amp 4 2 4 A A A 4 amp amp BA 4 4 4 4 BA 4 amp 4B amp amp 42 amp ARR MR BA 4 MD D amp A 4 4 4 O Oe nn Chapter One Introduction Overview This chapter will familiarize the user with the ThermoFluor 384 system and its safety features Specifically this chapter includes the following sections e Introduction to the ThermoFluor 384 System e Instrument Specifications e Safety Features The following table identifies the appropriate section to read in order to learn how to perform a specific task Read the section See page ThermoFluor 384 system Introduction to 1 2 ThermoFluor 384 System Instrument Specifications Safety Features Safety Features Copyright 2001 3 Dimensional Pharmaceuticals Inc i o sde Confidential May 2001 Artisan Scientific Quality Instrumentation Guaranteed 888 88 SOURCE www artisan scientific com f Introduction ThermoFlour 384 Operating Manual Introduction to the ThermoFluor 384 System The ThermoFluor 384 system performs miniaturized fluorescence based thermal shift assays for the high throughput screening of compound libraries The ThermoFluor screening strategy has several advantages over the more conventional HTS methodologies the most important of which is that it relies on a general physical process common to most if not all drug targets thermal unfolding of biomolecules
109. uire 3 0 open the light tight door as follows From the Acquire 3 0 SUBSYSTEM menu select Camera The CAMERA dialog box appears Click the Open Door button The light tight door opens Hi SDP s EhemoFluor TFAcquire veision 3 0 0 Fi mages Experimental Parameters Data Directory Browse Sample Information Plate 1D Py Setup 8 Thermofluor Box l gt aameters Get Info Configure Open Door se Door ry Heating Mode Up Dawn y Exposure Speed ab ss 2 el Temperatures C 40 70 Light Of D Incr Low Temp C f Ps ntegrate Image AS el i ow Temp Holds sec fiso 60 Exposure Time ffi sec Incas ameters F Scale Bitmap Cort 3 e Time sec HReps fio a Calibrate Image E None y mdo v Er FT Create New Calibration File y F Gein ET Box KY fe T Run Heater Camera Ready 19001 3 32PM Place the microplate with standard florescent beads on the thermal cycler block 3 Close the light tight door by clicking the Close Door button in the CAMERA dialog box type CLOSE and press lt Enter gt Turn ON the UV light source by clicking the Light On button Enter an exposure time ten seconds for the camera in the Integrate Image section of the CAMERA dialog box Click on the Single button to have the camera take an image with the given exposure time Copyright 2001 3 Dimensional Pharmaceuticals Inc 3 15 Confidential May 2001 Artisan Scientifi
110. ur 384 Operating Manual Precision Test To measure the precision of the instrument as well as the accuracy a test is run as follows A total of three plates are run using the third set of conditions described for the accuracy tests above on two consecutive days with deviations in absolute Tm on the same instrument to be lt 1 0 C A minimum of one plate will be run under the third set of conditions in step mode Copyright 2001 3 Dimensional Pharmaceuticals Inc A l 3 20 Confidential May 2001 Artisan Scientific Quality Instrumentation Guaranteed 888 88 SOURCE www artisan scientific com HRA AAA AAAAAA AAA BAAAAAARAAAK BARA LAB LB A HB 4 LES Installation and Calibration ThermoFlour 384 Operating Manual Chapter Four Operation Overview This chapter will familiarize the user with the basic operation of the ThermoFluor 384 system Specifically this chapter includes the following sections e Safe Operation e Operating Software and Controls e ThermoFluor 384 Acquire 3 0 e ThermoFluor 384 System Operation The following table identifies the appropriate section to read in order to learn how to properly operate the ThermoFluor 384 system To learn more about Read the section See page the system Controls Windows 3 0 Operation Copyright 2001 3 Dimensional Pharmaceuticals Inc aot 4 1 Confidential May 2001 Artisan Scientific Quality Instru
111. us adjustment will be discussed in Chapter 3 Installation and Calibration and Chapter 5 Maintenance Figure 2 5 25 mm lens for CCD camera Filter Wheel The filter wheel allows for various optical filters to be used in the instrument to provide the capability of measuring images from more specific regions of the UV spectrum The filter wheel can hold 4 filters The ThermoFluor 384 instrument is supplied with a standard ThermoFluor filter in position 1 Oriel 50 mm by 4 mm filters are recommended The embedded controller discussed below controls the positioning of the filter wheel Figure 2 6 Filter wheel in position 1 Copyright 2001 3 Dimensional Pharmaceuticals Inc 2 5 Confidential May 2001 Artisan Scientific Quality Instrumentation Guaranteed 888 88 SOURCE www artisan scientific com Construction and Function ThermoFlour 384 Operating Manual Light Tight Compartment The light tight compartment contains an UV light source and the thermal cycler s block that heats the microplates A light tight access door opens and closes to allow the PlateCrane to place the microplates on the Thermal cycler s block and then remove them Rear access to this compartment is limited to those trained in instrument maintenance UV Light Source The UV light source provides a uniform consistent source of UV light between 350 and 410 nm The light source is turned on and off by th
112. uticals Inc 2 10 Confidential May 2001 Artisan Scientific Quality Instrumentation Guaranteed 888 88 SOURCE www artisan scientific com Nee Construction and Function ThermoFlour 384 Operating Manual Bar Code Reader This is a standard Keyence BL 185 programmable bar code reader that reads 2 of 5 interleave Each microplate has a bar code placed on it that identifies it to the system Figure 2 14 384 well microplate with barcode label This helps to ensure proper correspondence of plates to data The barcode reader communicates with the NT Workstation though a serial cable connected to the control computer s Rockport expansion card RE Figure 2 15 Barcode reader records plate identification numbers Copyright 2001 3 Dimensional Pharmaceuticals Inc 2 11 Confidential May 2001 Artisan Scientific Quality Instrumentation Guaranteed 888 88 SOURCE www artisan scientific com i 4 i i Construction and Function ThermoFlour 384 Operating Manual Universal Power Supply An APC Smart UPS universal power supply is used to provide stable uninterrupted power to the system All system components should be plugged into this power supply to ensure proper operation The power supply is then plugged into a standard 110 120 VAC outlet The UPS is sized to provide 10 to 15 minutes of operating time For continuous uninterrupted operation an emergency backup power sour
113. wn or Continuous Ramp Enter the appropriate Heating Parameters in the fields e Initial Temp The starting temperature in C for measurements First high temperature in Up Down mode e Final Temp The last temperature in C for measurements Last high temperature in Up Down mode e Temp Increments Temperature difference between measurements in C Low Temp The low temperature in ene mode that is held while images are taken High Temperature hold The high temperature equilibration or hold time in seconds for the cycle in Up Down mode Low Temperature hold The low temperature equilibration or hold time in seconds for the cycle in Up Down mode Copyright 2001 3 Dimensional Pharmaceuticals Inc E l o 4 38 Confidential May 2001 Artisan Scientific Quality Instrumentation Guaranteed 888 88 SOURCE www artisan scientific com VW WWW DDD DD DDD VTV Y DY YY YD DY Y UVVP VVO VU VVV Operation ThermoFlour 384 Operating Manual Enter the appropriate Imaging Parameters in the fields e Exposure time Time each replicate image is exposed i in seconds in Up Down mode e No Reps The number of images of the well recorded during each cycle in the Up Down mode Select the desired emission Filter from the drop down menu The 500 nm is standard equipment with ThermoFluor 384 instrument The remaining fields are usually set at installation They are e SC
114. y dry the stainless steel rods of the plate magazines with a dry cloth Properly dispose of all clothes and cleaning materials when done 5 ea Copyright 2001 3 Dimensional Pharmaceuticals Inc 5 2 Confidential May 2001 Artisan Scientific Quality Instrumentation Guaranteed 888 88 SOURCE www artisan scientific com Maintenance ThermoFlour 384 Operating Manual Camera Focusing Use the procedure below to focus the camera The system solar must be running to accomplish this task mpe If it is not already open open the optical compartment by removing the screws from the rear panel Place a white piece of paper with small print on the thermal cycler block To do this manually open the light tight door as follows From the Acquire 3 0 SUBSYSTEM menu select Camera The CAMERA dialog box appears Click the Open Door button The light tight door opens 3DP s TheimoFluor TFAcquire version 3 0 05 0000 Ele BAC Images Experimental Parameters e Data Directory Browse Sample Information _ Plate iD arameters Get Info Configure E 1 Clos rT Heating Mode Up Down y Exposure SpeedT ab 5 m te el Temperatures C 40 Fo q e Li J Incr Low Temp C 1 25 Light indeaor ae ow Temp Holds sec 790 60 Exposure Time sec ameters M Seale Bitmap con i e Time sec Reps 0 4 ae were a E A E Integrate Image Se Combo T Create New Calibration Fi

TF 384 Operating Manual - Beta

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