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AIDA 2D TLC User's Manual

1. Region Determination Toolbox The controls of the Region Determination toolbox allow you to define regions in the image for signal intensity evaluation To display the Lane amp Peak Determination toolbox e On the main menu click Evaluation and choose the Region Determination entry Or e Click the Region Determination button on the toolbar 29 Region Determination Region Determination Region Creation E Region Creation Pane Create Matrix Je os Integration Background Integration Pane r Local bko Integration limits Report Report Pane i Region Label Region Name hove Label Font C None FR Renumber iZ Name Region Label and Name Pane Free Choice Configure i Select Name List Actual Region Configure Intensity 4625 0121 Actual Region Pane Recalc Figure 2 38 Region Determination Toolbox Overview 42 AIDA 2D TLC User s Manual Region Creation Pane The Region Creation pane of the toolbox is used to mark various areas in the image Rectangle Tool Square Tool Region Creation FE Ellipse Tool Gl El El Freehand Drawing Tool Circle Tool Polygon Tool AutoContour Tool Figure 2 39 Region Creation Pane Overview Areas of different shapes rectangles squares ellipses and circles can be marked To create a region 1 Click on the symbol of the desired shape 2 Click at the upper left corner of the area hold down the mouse button a
2. i i Name Free Choice Configure ETT Select Name List Figure 2 45 Region Label and Name Pane Region Label tab You can turn off labeling None radio button select a number as current label Number radio button or display the name of the region instead Name radio button Alternatively you can select a label freely from the column list displayed after clicking the Free Choice option and then clicking the Configure button The contents of this list 1s identical to the one from the Columns Selection dialog box cf the Region Report pane of this manual You can change the position of a region label using the Move Label button to increase visibility To move a label to a desired destination 1 Select the region with a mouse click Figure 2 46 Selecting Region 48 AIDA 2D TLC User s Manual ET 2 Click the Move Label button on the Region Label and Name pane of the Region Determination toolbox Region Label F C None Figure 2 47 Move Label Button The label of the previously selected region turns its color from magenta to red EREN Bl x Figure 2 48 Activated Label 3 Drag the label to its designated location FR raytest Manuals Y To Ima loj xi ES o a8 se te rn a hes aa eo a A den E PE A Teln i A EN i i A ne Figure 2 49 Moving Label Using 2D TLC 49 4 Confirm the action by clicking the right mouse but
3. S Paytest AIDA 2D Thin Layer Chromatography User s Manual Part Number 2004 04 1 1 02004 raytest IsotopenmeBgerate GmbH Benzstr 4 D 75334 Straubenhardt Germany All rights reserved This product or document is protected by copyright and distributed under licenses restricting its use copying distribution and decompilation No part of this product or document may be reproduced in any form by any means without prior written authorization of raytest and its licensors if any RESTRICTED RIGHTS LEGEND Use duplication or disclosure by the government is subject to restrictions as set forth in subparagraph c 1 11 of the Rights in Technical Data and Computer Software clause at DFARS 252 227 7013 and FAR 52 227 19 The product described in this manual may be protected by one or more U S patents foreign patents or pending applications TRADEMARKS AIDA AIDA Image Analyzer AIDA Annotation AIDA AIDA Array Metrix AIDA Array Compare AIDA Multi Labeling AIDA ID Evaluation AIDA ID Densitometry AIDA 2D Densitometry AIDA Easy Array AIDA Whole Body Autoradiography AIDA 1D Thin Layer Chromatography and AIDA 2D Thin Layer Chromatography are trademarks or registered trademarks of raytest inthe United States and may be protected as trademark in other countries All other product service or company names mentioned herein are claimed as trademarks and trade names by their respective companies THIS PUBLICATION IS PRO
4. gt Right Using 2D TLC 39 Sample Definition Panel Use the two buttons of the Sample Defintion panel to create or delete the TLC plate overlay sample Definition Figure 2 35 Sample Definition Panel TLC Plate Definition Panel The various combo boxes of the TLC Plate Definition panel allow you to set the position of the TLC plate overlay by entering the appropriate values for the Position of Corner A the upper right corner of the plate and the horizontal Distance A B and vertical Dimensions Distance A D of the plate together with 1ts Angle TLC Plate Definition Position of corner A y hize 968 Y pixel Dimensions Distance A B pixel 96 Distance A O pixel 91 om co EMS ENS T 3 D Pa a HTF Figure 2 36 TLC Plate Definition Panel 40 AIDA 2D TLC User s Manual Origin Front Panel The Origin Front panel of the toolbox provides you with options to determine the position and dimension of the origin front overlay Enter the appropriate values for the origin Distance A E and Distance A G and the front line Distance G H and Distance E F of the plate Figure 2 37 Origin Front Panel The units of measurement can be changed via the Image Attributes option of the Options menu Origin Front Distance A E piel 97 HAE Distance A G pixel q E Dimensions Distance G H pixel Distance E F pixel Qi EME T Using 2D TLC 41
5. Figure 2 13 Columns Selection Dialog Box In addition each cell of the Region Report has its own contextual menu attached providing commands for grouping of regions and standards as well as displaying hiding of the special row types Sum Total Remainder AS Region Report ZD BM Region Report 2D Calibrati Grp Name Group Regions Ungroup Regions Group standards Hide standards Hide surn Show remainder Show total Show header Figure 2 14 Using 2D TLC Contextual Menu of a Single Region Report Row 25 If multiple rows are selected the contextual menu contains only the grouping commands applicable in this context T Region Report 2D Ca No Grp Name Group Regions Do capas Ungroup Regions Group standards Hide standards Figure 2 15 Contextual Menu of Region Report Rows Multiple Selection The contextual menus of the TYPE NAME STD and cells provide additional menu items for their respective context If a single row is selected the contextual menu of the Type cells contains the additional commands Set as Normal Set as Background as well as Set as Total for changing the region s type Hi Region Report 2D Calibratio Type Area Grp Name Integral Group Regions Ungroup Regions Group standards Hide standards Average Regions Set as normal Sebas background Setas total Hide surn Show remainder Show total Show header Figure 2 16 Contextual Menu o
6. will not be used for integration The choice of coloring the Integrated Area or the Non integrated area to visualize the effect can be selected in the Use Colors for field Report Pane The Report pane of the Region Determination toolbox is used to activate and configure the report that summarizes the integration results Region Report Button Renumbering Tool Ungroup Button Config Table Header Button Group Button Figure 2 44 Report Pane Clicking the Region Report button activates the display of the report same as the Region Report function in the View menu With the Config Table Header button the table header name for the columns Integral Integral Area Quantity can be configured The following three buttons are used to group ungroup and renumber in this order groups and regions Every area can only belong to one group Grouped areas will get a common summary line at the end of all integrated regions in the region report Groups can be formed by selecting multiple regions and clicking the Group button Selected regions can be removed from their group by selecting the Ungroup button Using the Renumbering Tool all groups are renumbered in sequential order This avoids gaps in numbers which are caused by ungroup processes Using 2D TLC 47 Region Label and Name Pane The Region Label and Name pane is used to control the labeling and name of regions Region Label Region Hame hove Label Font None
7. 2D Region Report from the View menu LE xl No Name Type Grp Grp Name Area AF 1 AF 2 Integral Integral Bkg Intec pixel FSL PSL 1 0 GroupO 978 0 O55 0 83 1560 0 1560 0 i 2 of Group 630 0 0 47 0 80 3871 031 ol Group 0 648 0 0 62 0 87 4537 4537 a of Group 740 0 071 ogo 4728 4728 5f of Group 676 0 0 20 oss 5307 5307 cef ol Group 287 0 0 14 ost 2472 2472 7 of Group soo 0 15 oss 5282 5282 ef of Group senoj 013 0 37 15934 15934 f oj Group 458 0 oo 0 34 3621 io of Group 475 0 0 06 0 18 2588 2588 uf of Group 753 0 oo 0 13 4340 4340 sum Sum o Groupojessoo 68280 68280 E AA Figure 2 8 Region Report Window Ed Data Description Available Columns in the Region Report Table 2 1 Columns Available in the Region Report Window Column Short Description No The number of the region Grp The group number to which the region belongs 0 no group selected for this region Name The name of the region as 1t was assigned by the user Grp Name The name of the group assigned by the user to which the region belongs signal unit and length unit units of measurement depending on the type of the imaging device used to obtain the image and the options set in the Image Attributes dialog box concentration unit and quantity unit unit of measurement specified by the user through calibrat
8. Administrator to change the GLP settings If you are logged in as normal user the GLP Supporting Tools toolbox is considerably reduced providing you with the Data Trailing controls only HA aji GLP GLF Supporting Tools Data Trailing View Audit Trail stop Evaluation Figure 2 68 GLP Supporting Tools Toolbox User Account Administration Pane In the Administration pane of the GLP Supporting Tools toolbox you can configure all GLP support settings Administration M apply GLP support proof PCA check sum Reason List Figure 2 69 Administration Pane Initial State Since this is only possible if no evaluation has been started ensure that no image is loaded into AIDA yet To enable GLP support e Click the Apply GLP Support option The Proof PCB Checksum option enables the PCB image authentification feature Note Older PCB images do not incorporate checksums So if you want to apply the GLP Supporting Tools controls to those images make sure that this option is disabled Using 2D TLC 61 The Reason List button opens the Audit Trail Reason List dialog box for predefining evaluation change reasons x codi it Delete a Figure 2 70 Audit Trail Reason List Dialog Box Data Trail Settings Pane The behavior of the GLP recording function is configured using the Data Trail Settings for controls Data Trail Settings for Evaluation Changes Aftenvards Figure 2 71
9. Afterwards dialog box become active after saving the evaluation the first time First Close activates the Changes Afterwards settings when you close the Image window during the initial evaluation Explicit Stop allows you to mark the end of the evaluation anytime you want using the Stop Evaluation button on the Data Trailing pane Using 2D TLC 65 Region Names Pane Region Mames M apply Master Nametable Master Maretable Figure 2 77 Region Names Pane Clicking the Master Nametable button on the Region Names pane opens the Master Name Table dialog box which is used to create or modify master nametables Selecting the Apply Master Nametable option applies a master nametable to subsequent evaluations Existing name region assignments are left unchanged Automatic Export Pane Automatic Export Edit Setting Figure 2 78 Automatic Export Pane The Edit Settings button of the Automatic Export pane allows you to select edit and save settings for automatically exporting the 2D Region Report Region Calibration tables when the evaluation is saved to file Note If you are perfoming evaluations with GLP support disabled you can access the automatic export settings via the Automatic Export command of the Options menu 66 AIDA 2D TLC User s Manual Data Trailing Pane After starting an evaluation the controls of the Data Trailing pane are enabled Data Trailing View Audit Trail Stop Evaluation PC
10. Change Evaluation Mode E x Available Evaluation Modes 10 Densitometry 10 Censitormetry 10 Evaluation 10 Evaluation ALP 10 Multi Labeling 10 Runlength Determination 10 Runlength Determination 10 Thin Layer Chromatography 0 Densitometry 20 Densitometr GLF 20 Multi Labeling ee 20 Thin Layer Chromatography Annotation Array Metrix Crystallography Material Science Cancel Microarray hul AA Mee ARA ee A a TV x Figure l 1 Changing Evaluation Mode The list entries reflect the installed AIDA application modules 2 Select the 2D Thin Layer Chromatography list item und click OK Note This evaluation mode is only available if you purchased the AIDA 2D TLC module Note further that there are two possible 2D TLC evaluation modes in AIDA 2D TLC one with GLP features indicated by the acronym GLP in parentheses and the other without those functionality The 2D TLC GLP evaluation mode is only available after establishing access control by applying the user registration see the Create User Registration section below 6 AIDA 2D TLC User s Manual Create User Registration Access control to AIDA might be required when working under Good Laboratory Practice GLP conditions AIDA provides three different types of users a System Administrator Administrators and normal Users The System Administrator is a fixed account in AIDA which cannot be changed or deleted When no
11. Determination toolbox 2 Accomplish the region creation by clicking the right mouse button or alternatively pressing the ENTER key 3 Click the Create Matrix button on the Region Creation pane to specify the appropriate matrix layout for the region pattern Matrix options i X Dimensions Cancel Lines Columns Center distance vertical es mm horizontal min Mumbering options bylines l alternatin C by columns Automatic region grouping C bylines none C by columns Figure 3 19 Matrix Options Dialog Box 4 Enter the number of lines 2 in this examples and columns 6 in this examples as well as the distances x and y direction between the center of the regions Performing 2D TLC Evaluations 97 This will be easy for scanners just measure the original and enter the actual distance but difficult for camera images trial and error However the work is worthwhile because all regions will be drawn and assigned at the same time and the same array can be used for another image even if the size of the image is somewhat different from the original one Figure 3 20 Matrix Creation To save the matrix 1 Choose Template from the Export submenu of the File menu 2 Type a name for your matrix template in the text box of the dialog box that appears e g slotblot atf You can apply your saved template to a similar image from the same kind of application by selecting
12. Report window Area 2 Integral pixel FSL me 0 83 EA EEE Figure 3 32 Highlighted Regions 102 AIDA 2D TLC User s Manual En raytest 2 To set the regions to background check the Background option on the Integration pane of the Region Determination toolbox 3 Select all spots for which the local background should be defined together with the designated background regions Figure 3 33 Selecting Local Background Application area You can review the selection in the 2D Region Report window Wi Region Report 2dtlc adf l oj xj Area pixel 2 Fi al ow ato ao on 0 2810 Group ata ost 2472 5100 Grupo 015 048 5282 emo Group o 013 037 15934 o Group a os 0 34 3621 pio of 4750 Grou 0 0 06 018 2588 E TE ED 1770 Gro Ip 0 99 Group O o 6950 0 Group O 6626 0 as Figure 3 34 Selected Region highlighted in the 2D Region Report Window Performing 2D TLC Evaluations 103 h 4 On the Report panel click the Group button to assign the selected regions to a single group Report Figure 3 35 Selecting Group Button Again the changes are reflected in the 2D Region Report window Bi Region Report 2dtlc adf z Oj xj Area Integral pixel PSL soo Grupo 015 048 528 2 eso Group 013 037 15934 eso Group a ER a 476 0 GroupO 006 018 2588 7530 GroupO
13. Selecting the System Defaults button applies the Windows default settings to List Delimiter and Decimal Delimiter Using 2D TLC 13 In addition the parameter settings for the evaluation can be exported by checking the Parameter option If this option is enabled the following parameter information will be written to the export file Table 2 6 Exported Parameter Information Parameter Export Parameters 1 Row 2 Row Export settings Master nametable Desciption Application name e g AIDA Whole Body Autoradiography GLP Type of exported data e g 2D Region Report Identifier for the export parameter settings Identifier for the master nametable comprised of the unique AIDA internal indentifier and an import identifier and name Image File Parameters for Fuji BAS Scanner Parameter file Image data file Original filename Scan date Imaging plate type Scanner Sensitivity Latitude IP Number Project Sample Exposure Comment Digital Resolution Image size Pixel size Scan operator Image parameter file path pcb or inf Image raw data file path 1mg Name defined during the scan process Date and time of scan Type of the imaging plate used Scanner type e g Fuji BAS 5000 Sensitivity parameter of scan Latitude parameter of scan ID of IP plate used entered during the scan process Project name entered during the scan process Sample name entered during the sca
14. User Registration Figure 2 5 Options Menu Opening the Image Overlays Dialog Box 16 AIDA 2D TLC User s Manual After choosing Image Overlays from the Options menu the Image Overlays dialog box opens allowing you to specify the type None Number Name Free Choice font font size and font style bold italic roman typeface on white background for labeling For details see the appropriate sections below Image Overlays 2dtlc adf x 92 Region Label ew Bol None Cancel f Number C Name Free choice Conf Fant Arial Size poo feta print B 7 B Figure 2 6 Image Overlays Dialog Box Using 2D TLC 17 Use the 2D TLC Calibration tab of the Image Overlays dialog box to enable the drawing of the Rf Fronts In addition you can set the font size of the label using the Label Size combo box as well as the Location of the Corner s Labels via the appropriate pull down menu Image Overlays 2dtlc adf Gg x 20 TLC Calibration Bp M Draw RF Fronts Cancel Label Size h0 _ Cancel e Location of Corners Labels Figure 2 7 Image Overlays Dialog Box 2D TLC Calibration Tab 18 AIDA 2D TLC User s Manual Region Report Window All information about the evaluation of the assigned regions can be found and configured to the specific needs in the Region Report The Region Report window is shown by clicking this button ET in the Region Determination toolbox or by choosing
15. allows you to reshape the region Figure 3 17 Editing Polygonal Overlays Performing 2D TLC Evaluations 95 Finally the AutoContour tool will connect pixels of similar intensity to encircle an object automatically Figure 3 18 Using the AutoContour Tool To mark a region using the AutoContour tool 1 Select the AutoContour tool 2 Point to a position within the border area of an object that you would like to encircle example above heart of a rat cut autoradiography press the mouse button and shift around carefully within the object until the correct border is selected 3 Release the mouse button On release the border is marked and can be edited as usual You can cancel any overlay region editing action anytime you want by simply pressing the ESC key The numbering of the regions can be configured using the Region Label and Name controls of the Region Determination toolbox for details refer to the Region Label and Name pane in the Region Determination Toolbox chapter of this manual Adjust the settings to your experimental design and personal preferences 96 AIDA 2D TLC User s Manual Using Arrays Matrices It can be helpful to use matrices to evaluate regular patterns of regularly shaped regions e g slot or dot blots multiwell plates etc To create a matrix 1 Mark the first region of the regular pattern in question using the appropriate controls of the Region Creation pane on the Region
16. both run directions Figure 3 2 Run Direction Indicators If the 2D TLC Calibration toolbox is active there are additional letters and helping lines that do refer to the dimension and position combo boxes of the toolbox You may change the location and size of the letters by choosing Image Overlays from the Options menu Here you may also choose if the TLC overlays shall be drawn at all To set the TLC plate and 2D origin front overlay follow the steps below 1 Select the 2D TLC Calibration toolbox 2 In the upper part select the direction of the first run by choosing the appropriate entry from the Ist Run Direction pull down menu 20 TLE Calibration 1st Run Direction Top gt Bottom Right gt Left Left Right Figure 3 3 Selecting Direction for First Run 88 AIDA 2D TLC User s Manual 3 Then select the direction of the second run 2D TLE Calibration 1st Run Direction Top gt Bottom nd Run Direction Lett Right Right gt Left rl 4 Figure 3 4 Selecting Direction for Second Run 4 Click the Define Sample button This will create an overlay for the TLC plate containing a second overlay for the origin and front lines Figure 3 5 Define Sample Button Sample Definition Panel The outer rectangle represents the TLC plate the inner rectangle represents the origin and front lines By default the position of the outer rectangle s edges is congruent with
17. button on the toolbar to open the Export Template dialog box Ela Aj eje Figure 2 88 Main Toolbar Export Template Button 2 Type a name for the export file in the File Name text box and complete the export action by clicking Save Export Template 2D Calibration adf 4 a Save in GG 2D Sample ct EJ File name Save as type alba Template Format Cancel Figure 2 89 Export Template Dialog Box 74 AIDA 2D TLC User s Manual Exporting Region Report To export the Region Report 1 On the File menu click the Export menu item and then choose 2D Region Report from the submenu 2 In the 2D Region Report Export dialog box that appears type a name for the export file 3 Click Save to confirm the export The 2D Region Report Export Options dialog box appears allowing you to set further option for the export of the result table x Fange l Parameter W Table Header Cancel All Rows Selected Rows f All Columns Selected Columns List Delimiter Decimal Delimiter i Tabulator f Point C Comma System Detaults C Comma Semicolon Figure 2 90 2D Region Report Export Dialog Box You can specify the Range of the exported rows and columns Table Header All Rows Columns displayed in the Region Report window or only the Selected highlighted Rows Columns in the Region Report window as well as the List Delimiter and Decimal Delimiter in the respective panes of the dialog box
18. from the list below the text box 4 Click Assign Using 2D TLC 51 5 Optional Click the Apply at Region Creation option to assign the names in the list to any newly created region one after another in the order of their appearance in the list Figure 2 54 Selecting Apply at Region Creation Checkbox To edit the list of names e Click the Select Name List button below the Region Name tab Stomach gt M Apply at Region Creation Figure 2 55 Select Name List Button This opens the Region Nametables dialog box Region Nametables Rat adf x Remove ae Figure 2 56 Region Nametables Dialog Box 32 AIDA 2D TLC User s Manual Using this dialog box you can e create a new list of names by clicking the New button or Region Nametables Rat adf Mametable Editor EN Figure 2 57 Nametable Editor Creating New List of Names Using 2D TLC 53 e edit an existing name table by choosing Modify Region Nametables Rat adf X WEA New Remove Heart Kidney Nametable Editor x Liver List of Names List of Names Cancel Type in your list of names in the edit box on the right each name on a line Type in a tithe for this list in the edit bos above Figure 2 58 Nametable Editor Editing Existing Name Table Note If a master nametable is applied the procedure is slightly different from the one just described Please re
19. in the master name table To assign a name from the master name table to a region you must create a work nametable which contains all or a selection of the names provided by the master nametable see the Creating and Editing Work Nametables section below Master nametables are not part of the evaluation and can therefore be applied ex post The application of a master nametable is always logged to the audit trail 108 AIDA 2D TLC User s Manual Creating and Editing Master Nametables Master nametables are created and applied using the GLP Supporting Tools toolbox The names and identifiers for the master nametable are provided by an import table file The import table file is a text file which contains a semicolon or tab separated list of unique predefined names and identifiers Cells cannot be empty and multiple instances of a value are not allowed Valid end of line control codes are the hexadecimal equivalents for CR Macintosh LF UNIX or CRLF Windows A table name and a table ID can be included in the file using the keywords TABLE NAME and TABLE ID respectively TABLE NAME Master Name Table TABLE ID 2003 01 10 35 FIABLE UNIQUEID 20030110 141325 0396 n001 Regionl n002 Region2 n003 Region3 n004 Region4 n005 Region5 n006 Region6 n007 Region n008 Regiong8 Code Example 3 1 Import Table Example On import the import table replaces an empty or existing master nametable and the value for FTAB
20. of the overlay to adjust the shape of the region overlay Figure 3 12 Adjusting Region Shape 5 Click in the middle of the region to adjust the position of the entire region Figure 3 13 Adjusting Region Overlay Position Performing 2D TLC Evaluations 93 To rotate a region overlay 1 Click the left mouse button to switch to rotation mode Figure 3 14 Activating Rotation Mode 2 Drag on a handle to rotate the overlay Figure 3 15 Rotating an Overlay 3 Click the left mouse button again to go back to the relocation mode You might need multiple rounds of adjusting in the beginning 4 After the region is correctly assigned click the right mouse button or press the ENTER key to save it The same strategy is followed for the squares circles and ellipses The ellipses tend to be the most difficult to align The Polygon tool and the Freehand Drawing tool work similar as for known graphic programs 94 AIDA 2D TLC User s Manual E Dk sy Z y Using the Ploygon tool every edge of the new region is clicked whereas using the Freehand Drawing tool the mouse button is hold during the manual encircling Figure 3 16 Using the Polygon Tool To edit these polygons a third mode exists besides the relocation and turning mode In this third mode all handles edges of the region will be displayed and their position can be changed This
21. oo 013 4340 Sum ETRE E FR Z sota al cer ICI EEE 0 90 pef aee eis 08 a Jog REO 0 50 1 Sum 2996 0 1 28735 Hee Figure 3 36 Reviewing Grouping in 2D Region Report Window Note All members of the newly created group have the same Grp Name value assigned The previously performed local background setting now applies only to the domain of its group members 104 AIDA 2D TLC User s Manual Performing Calibrations The signal intensity of a region can be compared with the signal intensity of other regions relative comparison or normalized by standards in the same image or even in other samples of the same series For the normalization calibration it is necessary to define reference regions for the creation of standard curves To assign standards 1 Select the Manual Assignment tool from the Region Calibration toolbox 2 Click on a region The Region Calibration dialog box pops up and prompts you to enter the standard value und unit of measurement Region no Standard value 3 125 _ _ Unit ng OK Cancel Figure 3 37 Entering Standard Values and Unit of Measurement In the example shown below the regions 1 to 6 are a series of a standard Region 7 was assigned as a background region 10 x eae 3 4 5 6 7 Region s labels Figure 3 38 Region Calibration Example 3 Enter the appropriate standard value und click OK to assign it to the region Performing 2D TL
22. operator e g on print outs It cannot be longer than 63 characters The user s Password must contain at least 4 characters maximum 8 characters Note In previous AIDA releases the Password text field allowed to enter passwords with more than 8 characters without warning you that internally only the first 8 characters of the password are processed In this release the behavior of the text field has been adjusted to reflect this fact properly by restricting the input of password to at least 4 and at most 8 characters Users whose old password exceeds 8 characters can use the first 8 characters of the old password as a valid new password without changing anything else just type in the characters until the text field warns you with a beep 3 Specify the user s permission level Currently two permission levels Administrator and User see above are available 4 Enter an optional comment for the account The Comment for a user account appears only within the user registration database 5 Click OK to apply the changes Introduction 9 To modify an existing user account 1 On the AIDA Registration User Management dialog box click Modify to open the Modify User dialog box User name Full mame Permissions Password Confirm pazzword Comment Cancel Help Figure 1 6 Modifying Users 2 Edit the database entries you want to change 3 Click OK to confirm the changes If the opera
23. password is set for the System Administrator the user registration in AIDA is deactivated Administrators have the permission to create modify and delete users or administrators For GLP relevant modifications of the program settings general administrator rights are required in AIDA Users can modify their own password They have access to the normal functionality of AIDA When the user registration is activated you must type your user name and password before you get access to the program AIDA Registration Login xX Pleaze enter user name and password User name Administrata Password Cancel Figure 1 2 AIDA Registration Login Dialog Box To activate the user registration in AIDA the first time you must enter a password for the system administrator account To enter the password for the system administrator account 1 Choose Create User Registration from the Options menu The AIDA Registration User Management Setup dialog box appears Introduction 7 pl Ge 2 Type a password for the system administrator account in the Password text field of the Aida Registration User Management Setup dialog box and confirm the new password by retyping it in the Confirm Password field AIDA Registration User Management Setup X This dialog will actiwate the user management of this program From now on the program will require a user login on each start Pleaze enter an administrator password for the u
24. replace the current one the changes are logged in the audit trail report upon creation of a new region Audit Trail rat glp adf JO xj 4 Thursday March 18 2004 22 18 11 01 00 System Administrator Administrator al D Thin Layer Chromatography GLP evaluation started 5 Thursday March 18 2004 22241 7 01 00 System Administrator Administrator Master Nametable 20030113 162545 8427 Master Name Table 2002 06 16 35 ts applied Figure 3 62 Audit Trail Logging Master Nametable Replaced Note Changes of the master nametable are always logged regardless of the Data Trail Settings Changing replacing the master nametable does not change existing name assignments The new master nametable is applied to newly created regions assignments only Performing 2D TLC Evaluations 123 124 AIDA 2D TLC User s Manual
25. rete a A 5 a PS Lm 604 34 al Region Report 2D Calibrat Integral Bkg DES fleet torte PSLemnr T Region Report ZD Calibrat Integral Srea Bkg Integral Bk g PSLe mpn 5 Figure 2 9 Changing Column Order To change the column width 1 Position the mouse pointer over the border of the column whose width you want to change 2 Click and drag the border in the appropriate direction 3 Release the mouse button PT Region Report 2D E Mo Type 1 H Region Report 2D C No Type Std 1 iT Region Report 2D C No Name Type rea Std Figure 2 10 Changing Column Width Using 2D TLC 23 Contextual Menu of the Region Report Window Each column in the Result Table has a contextual menu attached To open the contextual menu right click on the column heading gray area Hi Region Report ZD Calibratic Mo Grp Mame Type E w Sort ascending by Mo Sort descending by No Select No Hide No i One lined Header Select Columns Figure 2 11 Contextual Menu of the Region Report Window With the first two commands of the contextual menu you can sort the table of the Region Report by any value column in ascending order A to Z or zero to 9 or descending order Z to A or 9 to zero Just choose the appropriate menu item Sort Ascending by Sort Descending by The Select Unselect commands of the contextual menu are used to select deselect a column
26. slowly to the side of the object 2 Try to drag the cursor around with the mouse button pressed until the area is completely encircled 3 Release the mouse button 4 Remember to press ENTER or the right mouse button after the area has been correctly assigned To place multiple identical objects on top of the image use the Create Matrix button to open the Matrix Options dialog box This function is useful for the analysis of multi well plates or dot blots were signals of identical shape and distance have to be analyzed OR Op Or Or Or Or Or Crd Ord 0 Ke KOTA Os Osta Oats Oria Ori Orta Opa Opa Ops Matrix options Ls xi Opes Onde Opbr Oba Onda Ordo Ort Ona Ong C r Dimensions Oks Oksa Ogs Opto Orar Ordo Oksa Okaa Okas C Lines Opus 0 00 Oksa Oros Okse Orar C Columns Cancel Oksi O Orea Ok h Opos Orea Ope Orea Ores C OR OR Oe Os Oy OM OY OM Oc p Center distance OR Oy Oy Oy OM On Of Of Oxf c vertical B pixel Horizontal pixel Mumbering options i by lines FT alternatin by columns 2 Automatic region grouping O by lines i none by columns Figure 2 40 Matrix Options Dialog Box With Application Example Dimension In the Dimension pane of the Matrix Options dialog box you can enter the number of objects to be created in x Lines and y Column direction of the matrix Center Distance In the Center Distance part of the dialog box enter the Vertical and Horiz
27. the Template command from the Import submenu and then choosing the appropriate template from the displayed file list in the dialog box that appears The matrix region will be displayed in one single frame which can be moved and reshaped as if it would be only one region Figure 3 21 Matrix Creation Completed You can relocate the matrix template by dragging the frame in the appropriate direction Figure 3 22 Relocating Matrix Template 98 AIDA 2D TLC User s Manual Figure 3 23 Rotating Matrix Template Finally accomplish your adjustments by right clicking the matrix or alternatively pressing ENTER Figure 3 24 Matrix Template Adjustments Completed Performing 2D TLC Evaluations 99 Defining a Background Signal It is advisable to determine the background signal before quantifying the signals Since in many applications the background can vary considerably from location to location the possibility to assign an individual local background around a region can help to evaluate the signals Note that local backgrounds can be defined for rectangular and ellipsoidal regions only It should also be noted that the background around the region must be clearly separated from the signal If e g a 2D gel spot is assigned it is likely that the outer borders of the signal come to lie under the background area because they are very difficult to see for the human eye To define a global background region 1 Mark an image are
28. the dialog box shows the current settings parameters whereas the second displays the current settings for the automatic export of the 2D Region Report Region Calibration Click on the toolbar to switch between the settings pages 82 AIDA 2D TLC User s Manual 2 Click Edit Settings The Edit Automatic Export Setting dialog box opens Edit Automatic Export Setting X 2D Region Report Export on Save C No Automatic i PromptFor Decimal Delimiter W Parameter i Point W Table Header C Comma Setting for Report Columns Intensity Area Bkg M Included in Export Decimal Digits 2 E List Delimiter i Tabulator C Comma OF a Cancel Semicolon Figure 2 96 Edit Automatic Export Setting Dialog Box The dialog box provides you with the following option e Export on Save Automatic enables the automatic export and generates the export filename automatically from the evaluation filename No disables the automatic export If you select Prompt for you will be prompted for an export data filename when you save an evaluation e Parameter enables disables the export of the result table s parameter pane e Table Header enables disables the export of the table header e List Delimiter allows you to select a delimiter for the columns e Decimal Delimiter allows you to select a decimal delimiter format e Settings for Report Columns select columns subsequently and click to sel
29. those selectable from the various panes of the Publishing Layout toolbox However they are not stored with the data file The Preview option of the Publishing Layout toolbox allows you to preview all pages as configured in the Publishing Layout toolbox before they are actually printed Clicking the Preview button opens the Preview window directly because all layout setting are already made through the Print Protocol pane He Aida 2D Thin Layer Chromatography Preview 2dtlc adf i i S laj x File Edit Evaluation Options wiew Window Help Page 1 of 3 20 Thin Layer Chromatography Trays Mara VST LO mages Di tc aitimodited Parame kr Page 2 of 3 20 Thin Layer Chromatography Tuesday April 17 2001 602 23 PM Trays Manuals V ITLC mage Di ic att imodited Page 2073 Figure 2 87 Preview Example Using 2D TLC 73 Export and Import In AIDA 2D TLC various tables and graphs can be exported to be imported in other documents or programs In addition the calibrations and templates can be exported to be applied to other images The various functions can be found in the Export and Import submenus of the File menu Exporting Templates To export your own evaluation templates 1 On the File menu click the Export menu item and then choose Template from the submenu 2 In the Export Template dialog box that appears type a name for the export file and confirm the action by clicking Save Or 1 Click the Export Template
30. whatever is available The percentage of the background corrected intensity of the region compared to the reference row in the group of the region The intensity per area The percentage of the intensity per pixel of the region compared to the reference row in the group of the region The background intensity is subtracted from the calculated intensity of the region and this signal intensity 1s divided by the area signal unit and length unit units of measurement depending on the type of the imaging device used to obtain the image and the options set in the Image Attributes dialog box concentration unit and quantity unit unit of measurement specified by the user through calibration 20 AIDA 2D TLC User s Manual Table 2 1 Columns Available in the Region Report Window Column Integral Area Bkg Std Quantity quantity unit Std Quantity Recalc Quantity quantity unit Recalc Quantity Std Conc concentration unit Std Conc Recalc Conc concentration unit Recalc Conc RF 1 RF 2 Short Description The percentage of the background corrected intensity per pixel of the region compared to the reference row in the group of the region The quantity of the region as entered for the calculation of the standard curve With the Quantity Standards the background corrected Intensity values of the regions are calibrated The percentage of the quantity standard
31. 1 Method Drop Down Menu Selecting Quantity will result in the analysis of integrated intensities of all pixels in the defined region whereas Concentration will compare the average intensity in the regions Standard Pane Using the controls of the Standard pane you can assign standards to selected regions standard Standard Selection Drop Down Menu fre st Source Array high i ll List of Standard Values AutoAssign Tool 4240000 112000 0 Manual Assignment Tool 414500 0 13350 0 Delete Standard Assignment Button oa 3950 0 Edit Standard Button Figure 2 62 Standard Pane From the Standard drop down menu a predefined list of standard values can be selected The specified standard values are displayed in the pane below the Standard drop down menu Beside this pane there are four buttons 56 AIDA 2D TLC User s Manual AutoAssign Tool This button allows you to subsequently assign all values in the list by clicking with the mouse on the appropriate region Thus the first value 1107000 0 is assigned to the first clicked region the second value 424000 0 to the secondly clicked region and so on Manual Assignment Tool The second button allows you to assign any value to the clicked region by entering the appropriate value and unit for the selected region in the Region Calibration dialog box that appears Region Calibration k X Region no 4 Standard value 41500 Unit Figure 2 63 Region
32. 4 Exporting Templates iio cued 6 ban Sabha ene ta eesoten toed oe A 74 Exporting Region EDO om ho des Park ded eee BS 75 Exporting Region Calibration 0 0 00 cc teen eee 78 Export AGG Vial si Vath Son ieee ae see 80 Automatic Export of the Region Reports Region Calibrations 81 Importing Region Calibration 0 0 0 ooo 85 Performing 2D TLC Evaluations 87 Setting the TLC Plate and 2D Origin Fron Overlay 0 0 0 0 0 eae 87 ASSIONINO Te COLON aii bok ik Getcha ey ar diia eet Gg betes 92 Usime Atrays Matices crias daras ido 97 Metinine a Backeround SIA A A bee dees 100 Performing Calibrati0MS o o o ooooo ooor nens 105 Usmo Name tables ni E As 108 Creating and Editing Master Nametables o ooooooooooooooooo 109 Activating Master Nametables o o ooooooooooooomomornnn 111 Exporting Master NametablesS o oooooooooooronannno o 112 Creating and Editing Work Nametables o oooooooooooooooooo 114 Assigning Names to Regions o oooooooooo teens 118 Andit Irak IOS ING tao Cia os 123 AIDA 2D TLC User s Manual Introduction 1 Overview The AIDA 2D Thin Layer Chromatography module henceforth 2D TLC was designed for fast and efficient evaluation of signal images obtained from TLC plates which have been developed in one direction in a first step and in a direction perpendicular to that in a subsequent step At the beginning the direct
33. 5 0 13350 1340011 3950 3908 4 Figure 2 22 Region Calibration Window Example 4 2 T 7 The Region Calibration window is bipartite the upper pane displays the curve of the calibration function the underlying function the correlation coefficient and the x axis intercept The lower one contains a table listing the standards their given and recalculated standard values etc Additional columns can be displayed Just right click on the table heading and choose the Select Columns command from the contextual menu that appears The Columns Selection dialog box opens allowing you to select the desired columns from the list 30 AIDA 2D TLC User s Manual Graphics Pane of the Regions Calibration Window The curve shown in the upper part of the Region Calibration window in this example displays the background corrected intensity per area as a function of the quantity concentration of the standard For a more detailed view of the plot you can activate the zoom function To zoom one of the plot s axis 1 On the toolbar click the Select button o Window Help AO Figure 2 23 Select Button Main Toolbar 2 Click and drag one of the axis with the left mouse button pressed Region Calibration 2D Calibration adf loj xj JE Region Calibration 2D Calibration adf loj xj Log weighted Regression log y 0 9691 9 log 3 06108 Log weighted Regression log y 0 9691 9 log 3 06108 Correlation Coefficien
34. AA A 39 Sample Definition Panel cid A A E isa 40 TEC Plate Detinition Panel A A RAGES 40 Ono Eront Pamela arge cho ada deeded AA Sue RR A 41 Region Determination Toolbox oooooooooooooornan tee eens 42 Resion Creation Pane sedisse nadt ees Bee A A AAA 43 Inte ration P ame lt 2 35 ca cee wea ams one So Bare ork ie ee we eS 45 R pO Palena dS oi he a hh aden pA de Sel a Soe 47 Resion Label and Name Pane 4 09093 oaeth in Fa tai Fe 48 Act al Rezin PANS A ii Wh dete wae stihl eid tore tact ec 55 Region Calibration Toolbox 0 EA A A 55 Nethod Bane cuna matado dd hk SEES Ese KR 56 Standard Pa o taria AS 56 111 iv Calibration Curve Pame 58 GLP Supporting Tools Toolbox 2 A eee eee 60 NGM SRAM OM PANG cast bud bans deh rta dada dos ai 61 Data TralloScttinss Pane iia Sarg a plane ae Sa cee Phas Pee eee 62 Finish Evaluation Pane eetere steres ein a decade ia de hake Pek des 65 Resion Names Pane caries SAS AS Swi ee eee aes Hee 66 Automatic Export Pan 532 cae eh Gob ib ed A a chew 66 Data Trata PANG ar A a Ga wae 67 Publishing Layout Toolbox mM 2D TiC 275 60 eek ie nes beaten oman a 68 Print Options for Region Report 0 0 69 Print Options for Region Calibration 0 0 0 0 0 ccc eee eens 69 Print Options tor Audit Trail taste tare Gacae ene 70 aia sees Slot cael Stal apace SE eae al iG ee BBS RA ea he a a Gee ana O ass 71 A A al SNE wisn mica mee ee REGA ERR ease 73 FOR and IMPO teak ae bears Bae a CR 7
35. B check sum not proofed Evaluation is in process Finish evaluation by first save close the image explicit stop Figure 2 79 Data Trailing Pane Evaluation in Progress The View Audit Trail button is used to open the Audit Trail window Use the Stop Evaluation button to mark the end of an evaluation anytime you want In the lower part of the Data Trailing pane the status of the evaluation as well as the settings for ending the evaluation depending on the checked Finish Evaluation by options are given In addition if the PCB check sum option is disabled a warning is displayed below the Stop Evaluation button After stopping the evaluation the action which marked the evaluation s end is displayed in this example by explicit stop via clicking the Stop Evaluation button Data Trailing View Audit Trail stop Evaluation PCB check sum not proofed Evaluation is finished by explicit stop Figure 2 80 Data Trailing Pane Evaluation Aborted by User Using 2D TLC 67 Publishing Layout Toolbox in 2D TLC In AIDA 2D TLC the standard AIDA Publishing Layout toolbox is expanded by three additional buttons for specifying the print options for the Region Report Region Calibration and Audit Trail aja ajxjze sr Publishing Layout Annotate Z ai aleo o Protocol Print E Page Layout ir Edges i Top 20m 4 Bottom 20m 4 Left 20m 4 Right 20cm 4 Region Report Options Button Re
36. C Evaluations 105 In the Region Calibration toolbox the entered standard values are listed below the Standard drop down menu L4 Region Calibration Method Concentration Standard Test Source Array thigh 11 1107000 0 Calibration Curve Linear Regression HF 0 0 origin PsLimirrr domme Corr Coeff f 0 998526 Figure 3 39 Region Calibration Toolbox With Entered Standard Values Displayed 106 The list of standard values can be saved for later use as a standard list Standard values from saved lists can be used to assign all standards by merely clicking at each region subsequently 4 Select a Calibration Curve type from the Calibration Curve drop down menu The type of the calibration curve can be selected on the basis of theoretical considerations or simply to fit the observed situation The resulting calibration curve can be immediately evaluated and the r value will give a parameter to judge the fit should be next to 1 5 Turn on the 0 0 Origin option if useful AIDA 2D TLC User s Manual lrdensny Bkg a u 10 Linear Regression y 1 16x 4 4 1 Correlation Coedicen f 0 9991 Infercept an xig 3 74 20 2 dm Eb 70 Quantity rel Conc Figure 3 40 Calibration Graph Example As mentioned above a correlation coefficient value of 1 identifies a perfect correlation a value of 0 indicates full independence of values Good standard curves should have value ver
37. Calibration Dialog Box Delete Standard Assignment Button By clicking this button all calibration values of selected regions can be canceled Edit Standard Button The last of the four buttons is used to enter new lists of standard values Clicking this button opens the Quantity Standards or Concentration Standards dialog box which is used to enter a new list or to modify an existing one To create a new list name the list in the Standard text box and specify its Values Unit and Precision displayed fraction digits Quantity Standards N x Standard Unit hg Precision 0 E Default alue 50 Add Modify Delete SEM Figure 2 64 Quantity Standards Dialog Box Using 2D TLC 57 c PS SS abt raytest The Add Modify and Delete buttons are used to alter lists in the same box All used standards should be saved for future use This is also true for one time standards because evaluation get frequently re done which means that the standard values are lost unless entered in the list Calibration Curve Pane In the Calibration Curve pane of the Region Calibration toolbox the mathematical calculation of the standard curve can be selected and the result is displayed in the graph at the lower end of the toolbox Calibration Curve Log weighted Regression F linear fit PSLirnrrt domme Corr Coeff f 0 999865 Figure 2 65 Calibration Curve Pane The Calibration Curve drop down menu p
38. Data Trail Settings Pane 62 AIDA 2D TLC User s Manual 1 Click the Evaluation button to set the recording options for changes during evaluation The Audit Trail Configuration Evaluation dialog box appears ll Audit Trail Configuration Evaluation g X Annotation Metal Create IY Modify Y Delete Measurement M Template Import FT Auto Export Parameter TLC Calibration sample Create M Modify M Delete Region Create IY Modify M Delete Background FT Integration Limits FT Group Attributes Name Numbering Region Calibration M Change Method M Change Curve standard Create IY Modify M Delete mal Figure 2 72 Audit Trail Configuration Evaluation 2 Specify which user actions should be recorded This includes for example the modification deletion of annotations changing calibration method and or calibration curve or modifying deleting regions Using 2D TLC 63 If the working operator performs one of the actions checked in the Audit Trail Configuration dialog box a dialog box pops up prompting the user to give reasons for the desired change E Audit Trail Confirmation T ax Sunday April 18 2004 16 11 52 02 00 Region R in group 0 515 9296962 PSLY changed to Region R2 in group 0 515 9296962 PSLY Reason Figure 2 73 Audit Trail Confirmation Dialog Box The desired modification is blocked until the user confirms the action b
39. LE_UNIQUEID is reset to a new value To create or edit a master nametable 1 Make sure that no image is currently loaded 2 Select the GLP Supporting Tools toolbox 3 Click Master Nametable Region Mames apply Master Nametable Automatic Export Edit Setting Figure 3 41 Selecting Master Nametable The Master Name Table dialog box appears Performing 2D TLC Evaluations 109 4 Click Import to select an import table x Table Name Table ID OF Table Stamp 20030110 154255 7792 Import k Export Figure 3 42 Master Name Table Empty Master Name Table The Import Master Name Table dialog box open allowing you to select an import table file 5 Select an import table file and click Open to continue Import Master Name Table 2 x Look in 3 AIDA Examples e EF f Ed rat txt Clontech Hcancer Large Array tet onco Ext Clontech Hbroad Large Array txt master Ext Clontech Large Array txt Al master nametable2 txt demo txt bi Type Text Document Size 195 Byte File name master nametable txt Files of type Table ASCII File Cancel Figure 3 43 Import Master Table Dialog Box 110 AIDA 2D TLC User s Manual The import table is loaded and replaces the existing in this example empty master nametable All changes to the master name table are logged and displayed in the Changes in Master Nametable dialog box E Changes in Master Namet
40. Master Nametable2 Table ID Table Stamp 20040319 154805 2585 OK n0017 sample1l Import n l sampler nQ03 samples Figure 3 46 Exporting Master Nametable Step 1 The Export Master Name Table dialog box appears 112 AIDA 2D TLC User s Manual 3 Type a name and select a location and data format for the export file and click Save to complete the action Export Master Name Table i ajx Gave in ADA Examples e EF f Ed Clontech Hcancer Large Array txt Clontech Hbroad Large Array txt Clontech Large Array txt File name master nametable ts Save az Ippe Table ASCII File Cancel Figure 3 47 Exporting Master Nametable Step 2 In addition to the contents of the master nametable the internal master nametable identifier Table Stamp the Table Name and the Table ID are included in the export file Note On import the value for the Table Stamp TABLE UNIQUEID is not imported but set to a new value Performing 2D TLC Evaluations 113 Creating and Editing Work Nametables Work nametables are used to assign names to regions Any user can create a new or edit an existing work nametable Note that depending on whether a master nametable is applied or not the creation or editing procedure for work nametables is slightly different To create a new work nametable no master nametable applied 1 On the Region Determination toolbox click the Select Name List b
41. Publishing Layout TLC Calibration Region Determination Region Calibration w GLP Supporting Tools Figure 2 34 Mapping between Evaluation Menu Entries and Toolbar Buttons 2D TLC Calibration Toolbox The 2D TLC Calibration toolbox is used to set the TLC plate and the origin front overlays To display the 2D TLC Calibration toolbox e On the main menu click Evaluation and choose the 2D TLC Calibration entry Or e Click the 2D TLC Calibration button on the toolbar 38 AIDA 2D TLC User s Manual 20 TLC Calibration 1st Run Direction Bottom Top Run Direction Panel 2nd Run Direction Left Right sample Definition Oj El TLE Plate Definition A X pixel 968 Y pixel 0 i i TLC Plate Definition Panel Dimensions Distance A B pixel 968 Distance A D pixel 916 Angle I 270 F Origin Front Sample Definition Panel Distance A E pixel 112 Distance A G fpixel 116 o Z Origin Front Panel Dimensions Distance G H pixel 708 Distance E F pixel T59 Run Direction Panel From the ist Run Direction and 2nd Run Direction pull down menus you can select the run directions used to develop the TLC plate from which the image was obtained For ist Run Direction two vertical Top gt Bottom and Bottom gt Top and two horizontal options Right gt Left and Left gt Right are available whereas 2nd Run Direction has only the two horizontal options Right gt Left and Left
42. Region Calibration Figure 2 1 View Menu 13 ZAS ES Image Window in 2D TLC All data are evaluated from the Image window Although 1t might not always be on top it is always open The title bar of the Image window shows the full file path of the image Figure 2 2 Image Window Example You can copy the content of the Image window to the clipboard using the Copy Content command of the Edit menu See the Export Image section in the AIDA Image Analyzer Basic Concepts and Features User s Manual for further explanation 14 AIDA 2D TLC User s Manual Contextual Menu of the Image Window In 2D TLC the contextual menu of the Image window looks slightly different depending on the type of overlay you have chosen Single Selection If you have selected a single overlay region only the contextual menu that opens after right clicking the respective object provides you with the possible commands for this specific selection mode in addition to the standard editing functions like Cut Copy Paste Delete and Duplicate These include commands for labeling regions and setting integration limits F raytest Manuals a Mr ae AGAR Delete Duplicate Matrix Change name Change group name vi NameTable lg Move label Set as background Set as standard Set integration limits Figure 2 3 Contextual Menu Single Object Selected Multiple Selection If you have selected mul
43. VIDED AS IS WITHOUT WARRANTY OF ANY KIND EITHER EXPRESS OR IMPLIED INCLUDING BUT NOT LIMITED TO THE IMPLIED WARRANTIES OF MERCHANTABILITY FITNESS FOR A PARTICULAR PURPOSE OR NON INFRINGEMENT THIS PUBLICATION COULD INCLUDE TECHNICAL INACCURACIES OR TYPOGRAPHICAL ERRORS CHANGES ARE PERIODICALLY ADDED TO THE INFORMATION HEREIN THESE CHANGES WILL BE INCORPORATED IN NEW EDITIONS OF THE PUBLICATION raytest MAY MAKE IMPROVEMENTS AND OR CHANGES IN THE PRODUCT S AND OR THE PROGRAMS S DESCRIBED IN THIS PUBLICATION AT ANY TIME Contents 1 Introduction 5 ORE racer a an Das 5 Sitio 21 PLE taa ad dis oe delas de ide dd NE EETA eee TEN 6 Create User Resisiration o A a ee eee 7 2 Using 2D TLC 13 Data Windows HAD TLC a ase OS 13 maseWindow te 2 DIEC irura a erase a seca Maca A eet ion A kee eM 14 Contextual Menu of the Image Window 00 ccc eee eee 15 masias ita asp dpi podais dais 16 Region Report WindOW wire cd Sd A A AA ous 19 Data DESCAPIONid Dass he Wace Seed a es 19 Contextual Menu of the Region Report Window 24 Region Calibration WindoW 2 0 teen eens 30 Graphics Pane of the Regions Calibration Window 31 Contextual Menu of the Region Calibration Window 33 Audit Trail WIE OW no aria dean ew BR dle ele BS 37 Additional Toolboxes in 2D TLC aars aii eens 38 AD TEC Calibration Toolbox sia daran aA il 38 R n Direction Panel errre cate AS A
44. a with no signal activity using the Region Creation tools ojaa Create matrix Figure 3 25 Selecting Region Creation Tool y Region s labels o 5 i No 2 Figure 3 26 Selecting Image Area 100 AIDA 2D TLC User s Manual The newly created region is highlighted in the 2D Region Report window T Region Report 2dtlc adf i oj xj Area Grp Name AF 1 AF 2 Integral It pixel AAA m Figure 3 27 Highlighted Region in the 2D Region Report Window 2 To define the region as background region select the Background check box on the Integration panel of the Region Determination toolbox integration E Cocal kg Integration limits Figure 3 28 Backgound Checkbox 3 Review the changes in the 2D Region Report window Note that the value for Type of the region is changed to Bkg Area Grp Name pise MEN NT Bkg y 9972 Group O Sum 6990 0 T Group 0 Figure 3 29 Bkg Value in the 2D Region Report Window Performing 2D TLC Evaluations 101 To define one or more local background regions 1 Mark one or more image areas in the vicinity of spot for which the background calculation should be performed using the Region Creation tools of the Region Determination toolbox Region creation ojoje p Create matrix Figure 3 30 Selecting Region Creation Tool Figure 3 31 Selecting Image Areas The newly created region is highlighted in the 2D Region
45. able added 1002 sample added 1003 sample3 Figure 3 44 Changes in the Master Nametable Dialog Box 6 Click OK to apply the changes or Cancel to discard them Activating Master Nametables To activate a master nametable e Click the Apply Master Nametable checkbox Region Mames Master Nametable aster Mametable Figure 3 45 Applying Master Nametable Performing 2D TLC Evaluations 111 Note Checking this option with GLP support enabled applies the master nametable to any new evaluation The Apply Master Nametable attribut is set on evaluation startup and cannot be changed for the currently running evaluation On startup the evaluation is associated with the current master nametable The association is logged in the audit trail If you open this evaluation with a version of AIDA which uses a different master nametable the master nametable of this version is used for any new name assignments made Names assigned using the previous master nametable are left unchanged Exporting Master Nametables You can save your current master nametable to a text file tab separated values for use as import table file using the Export option of the Master Nametable dialog box To export a master nametable 1 Click the Master Nametable button on the GLP Supporting Tools toolbar to open the Master Nametable dialog box 2 On the Master Nametable dialog box click Export ll Master Name Table EF x Table Name
46. ame of the region as defined in the general master nametable User ID The user ID Login Name of the user who recorded the region creation or last modification User Name The user name Full Name of the user who recorded the region creation or last modification Date and Time Date and time when the region was recorded creation or last modification The date and time format complies with the ISO 8601 specification yyy mm dd hh mm ss hh mm The following additional rows can be found or created exclusively in the Region Report Table 2 3 Additional Rows in the Region Report Sum Displays the scientifically correct sum for the selected column e g 2 Intensity Area means 2 Intensities Z Areas Different groups get individual sums Ttl Total Displays the values of the selected column of Ttl region in the group For group 0 the Ttl region is the entire image Remainder Ttl Sum Quantity results in italics indicate that they lay outside of the calibration curve values and therefore are prone to significant error You can select the number and type of columns you want to display using the contextual menu of any column in the window see below To change the column order 1 Click the heading of the column to be moved 2 Press the mouse button and drag to the desired position in the table 22 AIDA 2D TLC User s Manual 3 Release the mouse button ch Region Report 2D Calibrat Integral Bko DEERE
47. ating Work Nametable Master Nametable Applied Performing 2D TLC Evaluations 117 To modify a work nametable with master nametable applied 1 On the Region Determination toolbox click the Select Name List button The Region Nametables dialog box appears 2 Click Modify 3 On the Nametable Editor dialog box that appears select the entries you want to remove from the work nametable in the List of Names list box and click the right arrow button To add entries to the work nametable select the appropriate entries in the Master Nametable list box and click the left arrow button 4 Click Accept to confirm the changes Assigning Names to Regions To assign names to regions 1 Select a region Figure 3 52 Selecting a Region 118 AIDA 2D TLC User s Manual 2 On the Region Determination toolbox select the Region Name tab Region Label Region Name Apply at Region Creation Figure 3 53 Selecting Region Name Tab 3 Select the appropriate entry from the list to assign the name to the selected region The list shows the names defined in the current work nametable Region Label Region Name M Apply at Regi Select Nam AA a 4 Figure 3 54 Selecting a Name Performing 2D TLC Evaluations 119 To change a region name 1 Select the region whose name you want to change AA Figure 3 55 Selecting the Region 2 Click the appropriate name in the Regi
48. ays the Image Region Report and Region Calibration information can be selected in the left part of the Print dialog box In the right part of the dialog box Parameter and Image Options you can select Whole Image or Window Content from the first drop down menu Landscape or Portrait format can be selected from the second drop down menu The printing of Overlays Grayscales Scales and Overexposed can be selected The printout can be automatically fitted to the page or the relative size of the image representation can be selected in In the Profile Options pane you can specify to print the Current Selection or all profiles as well as the paper orientation Landscape or Portrait for the printout Finally using the Audit Trail Options pane you can specify whether to include information about the Data Trail settings at all and 1f yes to which extent configuration information should be printed Include Positive Trail Configuration Include Complete Trail Configuration T AIDA 2D TLC User s Manual Preview As for printing in AIDA there are two ways of previewing pages before they are actually printed e Using the Print Preview command from the File menu or e Using the Preview button on the Publishing Layout toolbox The Print Preview command of the File menu opens the Preview dialog box which allows you to specify the layout settings for the preview The options presented here are the same as in the Print dialog box and
49. ble applied 1 On the Region Determination toolbox click the Select Name List button The Region Nametables dialog box appears showing the current work nametable s list of names and a dropdown list with the names of all existing work nametables Only work nametables derived from the master nametable are display if no master nametable is applied only the custom work nametables are displayed 116 AIDA 2D TLC User s Manual 2 Click New to create a new work nametable derived from the current master nametable The Nametable Editor dialog box appears 3 Type a title for the work nametable in the Title text box 4 Select one or more entries from the Master Nametable list and click the left arrow button to copy the entries to the List of Names list of the work nametable The selected entries are inserted below the last entry in the List of Names list if no List of Names entries is highlighted If one or more List of Names entries are highlighted the selected names from the Master Nametable list are inserted above the first highlighted List of Names entry Note You can add multiple instances of one Master Nametable entry to the work nametable s List of Names list 5 To define a custom name type the name in the text box above the List of Names list and click Add to add it to the list Nametable Editor X Title work nametable List of Hames Master Nametable spinal cord Stomach wee Figure 3 51 Cre
50. cify the Range of the exported rows and columns Table Header All Rows Columns displayed in the Region Report window or only the Selected highlighted Rows Columns in the Region Report window as well as the List Delimiter and Decimal Delimiter in the respective panes of the dialog box Selecting the System Defaults button applies the Windows default settings to List Delimiter and Decimal Delimiter In addition the parameter settings for the evaluation can be exported by checking the Parameter option 3 Complete the action by clicking the OK button The data are saved to a simple ASCII text file In addition the calibration data can be exported to an AIDA Calibration file that can be imported into another image Using 2D TLC 79 Exporting Audit Trail To export Audit Trail data 1 On the File menu click the Export menu item and then choose Audit Trail from the submenu 2 In the Audit Trail Export dialog box that appears enter a name for the export file Audit Trail Export Rat adf Save irr E 20 5 ample da jt File name Est Cancel Save as type PICA Figure 2 94 Audit Trail Export Dialog Box 3 Complete the export action by clicking the Save button The data from the Audit Trail window are exported to an ASCII file Audit Trail Ascii File txt 80 AIDA 2D TLC User s Manual Automatic Export of the Region Reports Region Calibrations AIDA s Automatic Export option allows you to exp
51. e region was recorded creation or last modification The date and time format complies with the ISO 8601 specification yyy mm dd hh mm ss hh mm Audit Trail Window In the Audit Trail window you can view all GLP relevant changes to the currently active evaluation as defined via the Data Trail Settings for controls of the GLP Supporting Tools toolbox including timestamp user id evaluation status and reasons for evaluation changes indicated by the current user Audit Trail c Program Files yraytest Aida Image Analyzer d a Oj x 1 Friday March 19 2004 13 37 21 01 00 System Administrator Administrator al 2D Thin Layer Chromatography GLF evaluation started Master Name Table 20040319 125552 9153 ts applied Figure 2 33 Audit Trail Window To open the Audit Trail window either choose the Audit Trail command from the View menu or alternatively click the View Audit Trail button in the Data Trailing pane of the GLP Supporting Tools toolbox Using 2D TLC 37 Additional Toolboxes in 2D TLC In addition to the standard AIDA toolboxes Display Control and Publishing Layout the following toolboxes become available after selecting the 2D TLC mode TLC Calibration e Region Determination e Region Calibration e GLP Supporting Tools You can access these additional toolboxes from the Evaluation menu or by clicking the appropriate button on the toolbar Evaluation Options View Display Control
52. e type of the imaging device used to obtain the image and the options set in the Image Attributes dialog box concentration unit and quantity unit unit of measurement specified by the user through calibration unit quantity or concentration unit of measurement depending on the argument Using 2D TLC 35 Table 2 4 Columns Available in the Region Calibration Window Columns Std Quantity quantity unit Short Description The quantity of the region as entered for the calculation of Recalc Quantity quantity unit Concentration Calibration Integral Area Bkg signal unit length unit Std Conc concentration unit Recalc Conc concentration unit Difference unit Rel Deviation Deviation unit Rel Error the standard curve With the Quantity Standards the background corrected Intensity values of the regions are calibrated A calculation of the quantity in the region based on the current calibration curve The background intensity 1s subtracted from the calculated intensity of the region and this signal intensity is divided by the area The concentration of the region as entered for the calculation of the standard curve With the Concentration Standards the background corrected Intensity per Area values of the regions are calibrated A calculation of the concentration in the region based on the current calibration curve The difference without sign between the
53. ect or deselect the Included in Export option to include exclude the respective column Depending on the column you selected you can specify additional settings in this example the Decimal Digits for the exported values 3 Select the appropriate options and click OK to complete the action Using 2D TLC 83 Saving Automatic Export Settings 1 On the Automatic Export Setting dialog box click Save Setting The Save Automatic Export Setting dialog box opens ll Save Automatic Export Setting E x Name 2d tle2 ID Cancel 20040319 142049 5802 Delete 2d tle Figure 2 97 Save Automatic Export Setting Dialog Box 2 Type a unique name and an ID for the settings and click Save to confirm the action Selecting Automatic Export Settings 1 On the Automatic Export Setting dialog box click Select Setting The Select Automatic Export Setting dialog box appears ll Select Automatic Export Setting 3 X K Select Cancel 20040319 142049 5802 Delete 2d tle Figure 2 98 Select Automatic Eport Setting Dialog Box 2 Select a settings set and click Select to complete the action 84 AIDA 2D TLC User s Manual To delete a settings set 1 Select the set you want to delete in the list of the Select Automatic Export Setting dialog box 2 Click Delete Note If GLP support is turned on only user with administrator privileges can edit save or delete automatic export settings Import
54. entered standard and the recalculated value The relative difference with sign of the entered standard and the recalculated value compared to the standard value Deviation Std Conc Std Quantity The difference with sign between the entered standard and the recalculated value Recalc Conc Recalc Quantity Std Conc Std Quantity The relative difference without sign of the entered standard and the recalculated value compared to the standard value Difference Std Conc Std Quantity signal unit and length unit units of measurement depending on the type of the imaging device used to obtain the image and the options set in the Image Attributes dialog box concentration unit and quantity unit unit of measurement specified by the user through calibration unit quantity or concentration unit of measurement depending on the argument 36 AIDA 2D TLC User s Manual If GLP support is turned on the following additional columns are available Table 2 5 Additional Columns in the Region Calibration Window GLP Support Enabled Column Short Description Name ID The unique identifier for the Name of the region as defined in the general master nametable User ID The user ID Login Name of the user who recorded the region creation or last modification User Name The user name Full Name of the user who recorded the region creation or last modification Date and Time Date and time when th
55. f the Region Report s Type Cell Single Selection 26 AIDA 2D TLC User s Manual If multiple rows are selected the contextual menu of the Type cells contains the additional command Average Regions which inserts an additional row for the average values of regions The three above mentioned menu items for changing the region type are unavailable in this context and therefore displayed in gray T Region Report 2D Calibratio Grp Name Type Area Integral Group Regions Ungroup Regions Group standards Hide standards Average Regions Setas normal Sebas background Set as total Figure 2 17 Contextual Menu of the Region Reports Type Cell Multiple Selection The contextual menu of the Name cells contains the additional command Change Name for renaming the selected region ai Region Report 2D Calibration adf Type Area Group Regions Ungroup Regions Group standards Hide standards Change name Hide sum Show remainder Show total Show header Figure 2 18 Contextual Menu of the Region Reports Name Cell Using 2D TLC 2l The additional menu item Set Standard of the Std row cells opens the Region Calibration dialog box allowing you to change the standard value and unit for the selected region Wi Region Report 2D Calibration ad Area Std Conc Grp Name Integral dem er Group Regions Ungroup Regions Group standards Hide standards Set standard Hide sum Sh
56. fer to the Creating and Editing Work Nametables section for instructions 54 AIDA 2D TLC User s Manual Actual Region Pane The Actual Region pane provides information about the selected region Actual Region Configure Intensity 4625 0131 Recalc Figure 2 59 Actual Region Pane Use the Configure button to add or delete categories from the list shown below the Configure button This opens the Column Selection dialog box described in the Region Report section of this manual Region Calibration Toolbox The controls of the Region Calibration toolbox allow you to perform the calibration of the 2D densitometer For the calibration reference regions for the creation of standard curves are necessary on the same image l Region Calibration Method Concentration Method Pane Standard Test Source Array high 1 Standard Pane Calibration Curve Log weighted Regression FP linear fit Calibration Curve Pane FSL demic m Corr Coeff r 0 999865 Figure 2 60 Region Calibration Toolbox Overview Using 2D TLC 55 To display the Region Calibration toolbox e On the main menu click Evaluation and then click Region Calibration Or e Select the Region Calibration button on the toolbar Method Pane The drop down menu of the Method control pane is used to specify the calibration method Quantity or Concentration l Region Calibration Method Figure 2 6
57. gion Calibration Options Button Audit Trail Options Button Header M File Path M Page Number i Current Date Footnote M Author M Last Change Time M Software Version Figure 2 81 Publishing Layout Toolbox Overview and Toolbar 68 AIDA 2D TLC User s Manual Print Options for Region Report With the first one you can configure layout setting for the Region Report window in the Protocol Print pane Protocol Print FE Print Preview ES Region Report Formal Portrait C Landscape Print M Table Figure 2 82 Print Options for the Region Report Window Here you can select the paper orientation Portrait or Landscape by clicking the appropriate radio button in the Format pane In the Print pane the scope for the print output can be specified To turn on printing of the Region Report table click the Table box Print Options for Region Calibration The second additional button of the Publishing Layout toolbox is used to configure the layout setting for the Region Calibration window Protocol Print FE ES Print Preview E Region Calibration Format Portrait C Landscape W Graphic W Table Figure 2 83 Print Option for the Region Calibration Window Using 2D TLC 69 You can select the paper orientation Portrait or Landscape by clicking the appropriate radio button in the Format pane The options in the Print pane enable or disable the printing of the calibration cur
58. graphics pane opens the Region Calibration window see the Region Calibration Window section of this manual for details Sometimes a warning will be displayed below the graphics pane stating bkg not determined to indicate that no background region has been assigned yet It can also indicate the lack of a local background for some regions Using 2D TLC 59 GLP Supporting Tools Toolbox The GLP Supporting Tools toolbox is used to specify all GLP relevant settings GLP GLF Supporting Tools Administration M apply GLP support proof PCE check sum Administration Pane Reason List Data Trail Settings for Evaluation Data Trail Settings Pane Changes Afterwards Finish Evaluation by M first save M first clase 4 explicit stop Finish Evaluation Pane Region Names apply Master Nametable Master Marnetable Automatic Export Edit Setting Region Names Pane Automatic Export Pane lo Je Data Trailing view Audit Trail Data Trailing Pane stop Evaluation Figure 2 67 GLP Supporting Tools Toolbox Overview To display the GLP Supporting Tools toolbox e On the main menu click Evaluation and then click GLP Supporting Tools Or e Select the GLP Supporting Tools button on the toolbar 60 AIDA 2D TLC User s Manual Note To configure the GLP support administrator rights are required Therefore make sure that you are logged in as system
59. ing Region Calibration 1 To import the calibration curve from another image e g taken at the same time with the same BAS screen or from a second gel handled at the same time and captured under identical conditions use the Import command of the File menu The Region Calibration Import dialog box appears allowing you to specify the calibration file cal Region Calibration Import 2D Calibration adf Look in E 20 5 ample d E 2D Calibration cal File name 2D Calibration cal Files of type ADA Calibration Cancel Figure 2 99 Region Calibration Import Dialog Box Using 2D TLC 85 86 AIDA 2D TLC User s Manual GY y Performing 2D TLC Evaluations 3 Setting the TLC Plate and 2D Origin Fron Overlay The first thing to do when doing a 2D TLC evaluation is to set the TLC plate with the origin and front lines Consider the following image oe ST hee so EAN i Ae Figure 3 1 TLC Image Example Here you can see the 2D TLC overlays The outer rectangle represents the TLC plate the inner rectangle is used for the calibration of the two TLC runs 87 The TLC plate overlay is used for the calculation of the Total value shown in the 2D Region Report The little cross axes indicates the two run directions The corner of the inner rectangle where the little cross axes are drawn has the RF value 0 for the first and second run direction The opposite corner has the RF value 1 for
60. ion Using 2D TLC 19 Table 2 1 Columns Available in the Region Report Window Column Type Int Limits Position ength unit Position X length unit Position Y length unit Area length unit Area Integral signal unit Integral Integral Bkg signal unit Integral Bkg Integral Area signal unit length unit Integral Area Integral Area Bkg signal unit length unit Short Description The type of the region e g bkg background Integration limits for the region The position of the region as determined by x and y coordinates in the image The position of the region as determined by x coordinate in the image The position of the region as determined by y coordinate in the image The calculated area of the region The percentage of the region area compared to the reference row in the group of the region e g the sum of all evaluated areas in a group The calculated intensity of the region The percentage of the intensity of the region compared to the reference row in the group of the region The background intensity is subtracted from the calculated intensity of the region to result in the signal intensity of the region For each group of regions the background is determined in the following order the local background area around the region or the local background defined for the group or the general background defined in group 0
61. ion of the development is set for both steps Then the TLC plate is defined on the image and the origin and front line are defined All these steps can be performed using the mouse However these positions can also be defined using the preferences settings or by entering the appropriate values with the keyboard which can be helpful for reproducible evaluations or to fasten the procedure for similar samples Following this procedure regions are defined around the signals from the various compounds which have been separated by the chromatography The software provides several region types like rectangle ellipsis or polygon You can also draw a line by hand using the mouse to mark a region or get the region marked automatically by the software Regions at suitable positions are defined as background If an absolute calibration is needed standards have to be measured for calculation of a calibration curve The results can be documented by a result report which includes RF values for both directions and activities if a calibration of the measured image has been performed Additionally all parts of the measured data and the evaluation can be printed out It is also possible to export the images and the result report to other software packages for further data reduction Starting 2D TLC To select the 2D TLC mode 1 Click the Evaluation button in the toolbar A dialog box appears offering you a list of available evaluation modes E
62. ither the big or the small ticks on the axes SS Vertical None C Big Tics C Small Tics Horizontal None Big Tics C Small Tics Cancel Help Figure 2 31 Grid Dialog Box 34 AIDA 2D TLC User s Manual The table in the lower part of the Region Calibration window provides its own contextual menu Select Columns Delete Standards Figure 2 32 Contextual Menu of the Region Calibration Window Table Pane The column headings are equipped with the standard contextual menus described in the Region Report Window section of this manual If you right click on any of the table rows the row s contextual menu opens allowing you to Select Columns for displaying or Delete Standards from the table The following columns are available Table 2 4 Columns Available in the Region Calibration Window Columns Short Description No The number of the region Name The name of the region as it was assigned by the user Quantity Calibration Integral Bkg signal unit The background intensity is subtracted from the calculated intensity of the region to result in the signal intensity of the region For each group of regions the background is determined in the following order the local background area around the region or the local background defined for the group or the general background defined in group 0 whatever is available signal unit and length unit units of measurement depending on th
63. ity Choosing the Print command from the File menu opens the Print dialog box allowing you to configure the printout to contain only the overlays results and graphs that are currently needed Click the checkboxes for the various items to turn them on or off Select the number of copies you want to print E Print 2D Thin Layer Chromatography GLP 2D Tlc pcb E E X Printer Kyocera Mita FS 1010 KX at Megi OK Edges Left 2 0cm Top 2 0cm cm Header Right 2 0cm Bottom 2 0cm W Parameter Parameter and Image Options I Image whole image overlays M Region Report portrait Y grayscale Rate A Ai aan e scales Region Calibration a Audit Trail 100 M overexposed Region Report Options portrait Audit Trail Options include positive trail configuration iv include complete trail configuration Figure 2 85 Print Dialog Box Using 2D TLC 71 raytest The margins of the printout can be selected in the Edges part of the dialog box The Header button displays the following dialog box Page Header and Footer x Header r Footnote i LAN IV file path M author ol lod Gena IV page number IV last change time Cancel IV current date IV software version Figure 2 86 Page Header and Footer Dialog Box Here the Header and Footer of the report can be configured to contain various image and evaluation specific data The Parameter data overl
64. n process Exposure time of the plate entered during the scan process Image and evaluation comments combined Digital resolution of image Width and height of image Spatial resolution of image Operator of the scan process 76 AIDA 2D TLC User s Manual Table 2 6 Exported Parameter Information Parameter Desciption Evaluation File Parameters Evaluation file Evaluation file path adf Evaluation date Date and time of evaluation Evaluation operator Operator of evaluation The actual image file parameters depend on the data file format and version Aida 2D TLC GLP 2D Region Report Export setting 20021104 110236 3896 Till Default Setting 04 11 2002 Master nametable 20021104 142426 7666 Master Name Table 2002 08 16 35 Parameter file i Aida Demo 2d tlc inf Image data file i Aida Demo 2d tlc img Original file 2d tlc Scan date Tuesday April 20 1993 4 02 43 AM Imaging plate type 20 40 Scanner Fuji BAS 1000 Sensitivity 10000 Latitude 4 Digital resolution 256 8 bit per pixel Image size 348 940 69 60mm 188 00mm Pixel size 200um 200um Evaluation file i Aida Demo 2d tlc adf Evaluation date Monday November 04 2002 3 22 42 PM Evaluation operator System Administrator Administrator Ho od dE dE E HHH HHH HH E E E E E E E E E Figure 2 91 Example of Exported Parameter Information 4 Complete the action by clicking Ok The data are saved to a simple ASCII text file U
65. nd drag it to the desired lower right end of the shape 3 Release the mouse button The area can be reshaped by moving the handles m Clicking within the area changes the handles to this symbol now the object can be rotated around its center Clicking within the area again changes the handles back When the overlay has received its final shape and tilt right click or press the ENTER key to save the object and go on with the next action Polygon Tool This Tool allows you to draw an enclosure by clicking at multiple subsequent points surrounding the object and finalizing the encircling with a double click Freehand Drawing Tool Click at a start point and drawing the outside shape of the object while holding down the mouse button On release the object is closed automatically The areas can be changed in size by moving the handles around The corner handles are used for total size change the side handles are used for size changes in the direction of the handle Clicking in the middle of the object switches to the turning mode as outlined above A third click will switch to the re shape modus where all edges of the object are shown with an individual handle Using 2D TLC 43 raytest The AutoContour Tool connects pixels of similar intensity to encircle an object automatically 1 After activating the function click on the border area of a region within the image that you would like to encircle and drag the mouse pointer
66. nt 0 999865 a Integrall rea Bk q PSL mm 100000 Conc deme Figure 2 28 Zoomed Curve Segment Contextual Menu of the Region Calibration Window The contextual menu of the upper pane of the Region Calibration window contains two commands Scales and Grid JE Region Calibration 2D Calibration adf JO xj Log weighted Regression log y 0 9691 9 log 3 06108 Correlation Coefficient 0 999865 k a L Integrall rea Bk q PSL mm 10000 100000 1000000 Conc demem l l l Figure 2 29 Contextual menu of the Region Calibration Window Graphics Pane The Scales command allows you to configure the graphical output After clicking the Scales command the Data Scaling dialog box opens Here the Minimum and Maximum value of the x Concentration and y axis Intensity can be selected In addition you can Using 2D TLC 33 turn on logarithmic representation and automatic scaling of either axis separately by clicking the appropriate option Logarithmic and Automatic below the Minimum and Maximum spin boxes Data scalino x Conc dprnicm dl Minimum Maximum 3 555 1 217 700 W Logarithmic W Automatic _Cancel Integralifrea Bkg PS Limrn oa r E ut Figure 2 30 Data Scaling Dialog Box Region Calibration Window The Grid command opens the Grid dialog box which allows you to lay grids over both axis that are an extension of e
67. on Name list Region Label Region Name Apply at Region Creat Select Name List Figure 3 56 Changing Name 120 AIDA 2D TLC User s Manual To assign names automatically upon creation of a region 1 On the Region Determination toolbox select the Region Name tab Apply at Region Creation Select Name List Figure 3 57 Selecting Region Name Tab 2 Click Apply at Region Creation Figure 3 58 Checking Apply at Region Creation 3 Select a tool from the Region Creation pane and mark regions sucessively Region Creation ojoje Create Mati le Figure 3 59 Selecting Region Creation Tool Performing 2D TLC Evaluations 121 y E CE The names in the Region Name list are assigned one after another a bl i M Apply at Region Creation Heart p Figure 3 60 Assigning Names sucessively 122 AIDA 2D TLC User s Manual Audit Trail Logging If a master nametable is applied to an evaluation the master nametable s name ID and table stamp are logged in the audit trail report y Audit Trail c Program Files raytest Aida Image Analyze dev JO x 1 Friday March 19 2004 13 37 21 01 00 System Administrator Administrator al 2D Thin Layer Chromatography GLF evaluation started Master Name Table 20040319 125552 9153 ts applied Figure 3 61 Audit Trail Logging Muster Nametable Applied If you change the master nametable modify or
68. ons 91 Assigning Regions To define regions for signal intensity evaluation it is important to set the Image Attributes in the Options menu first see the AIDA Image Analyzer Basic Concepts and Features User s Manual If these settings are not correct further image evaluation will not result in meaningful results Image Attributes L RTEST PCE k X Image Interpretation Image specific Fired interpretation defined by image format Cancel Length Unit Figure 3 9 Setting Image Attributes To enable signal intensity evaluation the regions of interest must be marked by an enclosure of the appropriate shape In the beginning you might find it difficult to exactly encircle a region of interest Some experience is needed to get this done on the first attempt To select regions of interest 1 Click the appropriate button here circle in the Region Creation pane of the Region Determination toolbox to select a shape alternatively click the circle symbol on the toolbar 92 Region Determination Region Creation olole Matrix Figure 3 10 Selecting Circle Tool 92 AIDA 2D TLC User s Manual 2 Position the mouse pointer over the top left edge of the region in the image press the mouse button and drag the mouse to the lower right edge of the region to be assigned Figure 3 11 Marking Region 3 Release the mouse button 4 Drag on the handles
69. ontal distance mid to mid between objects in the matrix 44 AIDA 2D TLC User s Manual Numbering Options The regions in the matrix can be numbered By Lines from left to right or By Column from top to bottom Clicking the Alternating option results in numbering the first line from left to right the second line from right to left and so on The Alternating numbering is also available for columns Automatic Region Grouping The resulting regions can also be grouped together By Lines or By Columns if they represent multiple independent analysis points for example Integration Pane The Integration pane of the Region Determination toolbox is used to define the background calculation for the integration of regions Any region that is marked with the Background option turned on is taken as an example for the background signal Multiple background areas can be assigned and will be averaged Integration Background Integration limits Local bka Figure 2 41 Integration Pane To assign a frame around the active region as a local background select the checkbox next to the Local Bkg button The general background calculated by the Background function is ignored for regions that have a local background assigned to them This function 1s not advised for blurred spots of usual 2D gel applications because part of the actual sample might be mistaken for local background Clicking the Local Bkg button opens the Local Bkg Se
70. ort 2D Region Reports and or Region Calibrations automatically when you save an evaluation You can specify preferences for the range of the table columns to be automatically exported as well as export settings which are independent from the current work configurations of the table s The naming of the export files follows the scheme Evaluation Name Region Report txt or Evaluation Name Region Calibration txt 1 e if the name of the evaluation file is example adf the Region Report export file has the name example Region Report txt and the Region Calibration export file is named example Region Calibration txt Existing export files are overwritten without prompting the user However a backup file of the previous export file version is created Automatic Export always exports the entire result table Restricting the export to selected rows is not possible The enabling of the automatic export option as well as its settings are evaluation specific attributes which are valid until they are explicitly changed using the Preferences or Automatic Export options of the Options menu or by importing an AIDA template If you create a new evaluation the current default configuration for Automatic Export is applied and will be saved with the evaluation data in the adf file The configuration remains valid unless it is explicitly changed by the user If GLP support is enabled only user with administrator privileges can create or change a
71. ow remainder Show total Show header Figure 2 19 Contextual Menu of the Region Reports Std Row Cell Region Calibration k i xx Region no 1 Unit ng Figure 2 20 Region Calibration Dialog Box 28 AIDA 2D TLC User s Manual A E Y The contextual menu of cells containing percentages provides an additional Set to 100 item used to set the value of the selected cell to 100 and recalculating the percentages of all other rows in this column accordingly Hi Region Report 2D Calibratio IntegralBkg Integral A amp rea Bkg 5 PS Lm Group Regions Ungroup Regions Group standards Hide standards Set to 100 Hide sum Show remainder Show total Show header Figure 2 21 Contextual Menu of the Region Reports Cells Using 2D TLC 29 s2 raytest Region Calibration Window To open the Region Calibration window e Choose Quantity Calibration from the View menu or e Double click the miniaturized calibration curve display on the Region Calibration toolbox JE Region Calibration 2D Calibration adf Ioj x Log weighted Regression log y 0 969 9 logis 3 06708 Correlation Coefficient 0 999865 Cc Integral 4rea Bk g PSL mm 10000 100000 1000000 Conc dormcrr No Integrall rea Bkg Std Conc Recalc Conc PSL rom dem or dprncrr 604 3 110000 0 106 870 1 255 4 424000 0 438707 8 63 21 112000 0 114080 0 25 41500 0 41100
72. rovides the following functions Linear Interpolation e Linear Regression Logarithmic Regression e Log Weighted Regression Calibration Curve Log weighted Regression Linear Interpolation Linear Regression Logarithmic Regression Log weighted Regression Figure 2 66 Selecting Calibration Curve Type Linear Interpolation calculates lines to join each data point 58 AIDA 2D TLC User s Manual Linear Regression optimizes a linear graph so that the deviation of the actual points from the curve is minimized Logarithmic Regression is used to determine the standard curve with logarithmic characteristics which is found in many saturating effects e g absorption Log Weighted Regression can be described as linear regression using a double logarithmic scaling The result is a better optimization of the curve fit for the smaller values instead of a better fit for the higher values Linear Regression If you choose Linear Interpolation Linear Regression or Logorithmic Regression you can select the 0 0 Origin option to force the curve through the origin For Log Weighted Regression select the Linear fit option to force the curve to a linear function The correlation coefficient Corr Coeff of the estimation is shown below the curve A value of 1 identifies a perfect correlation a value of 0 indicates full independence of values Good standard curves should have value very near to 1 Double clicking on the
73. ser management Be sure not to forget this password Password ae an Confirm password Eas Cancel Figure 1 3 Entering Administrator Password 3 Click OK to apply the changes Now the user registration is activated and only registered operators have access to the program When the user registration is activated and the operator has administrator privileges the User Registration command is selectable from the Options menu On choosing this command the AIDA Registration User Management dialog box appears allowing you to add new users or to modify or delete existing user accounts AIDA Registration User Management X Add Modify Delete User informations Full name System Administrator Pemigsion Administrator Comment Fixed account for the administration of the user database Figure 1 4 AIDA Registration User Management Dialog Box 8 AIDA 2D TLC User s Manual To add new users 1 On the AIDA Registration User Management dialog box click Add to open the Add User dialog box x User name idoe Full name John Doe Permissions User Password J xxxxxxxx Figure 1 5 Adding Users 2 Type a user name full name and password for the new user and confirm the password by retyping it in the Confirm password field A User name can consist of up to 31 characters and must be unique in the registration database The Full name is used whenever the program has to display the name of the
74. sing 2D TLC 77 Exporting Region Calibration To export Region Calibration Data 1 On the File menu click the Export menu item and then choose Region Calibration from the submenu 2 In the Region Calibration Export dialog box that appears type a name for the export file and specify the data format Region Calibration Export 2D Calibration adf ajx Save in E 2D Sample amp cf EE a 2D Calibration cal File name 2D Calibration cal Save as type AIDA Calibration bh Cancel Windows Metafile Table ASCII File AIDA Calibration Figure 2 92 Region Calibration Export Dialog Box To export the graph in the Region Calibration window e Choose Windows Metafile The graph is saved to a separate file in the Windows Metafile format To export the result table of the Region Calibration window 1 Select the Table Ascii File data format 2 Click Save to confirm the export 78 AIDA 2D TLC User s Manual The Quantity Calibration Export Options dialog box appears allowing you to set further option for the export of the result table Region Calibration Export Options xX Fange l Parameter W Table Header Cancel i All Rows Selected Rows _ Cancel f All Columns O Selected Columns List Delimiter Decimal Delimiter i Tabulator f Point C Comma System Detaults C Comma Semicolon Figure 2 93 Region Calibration Export Options Dialog Box You can spe
75. t f 0 999865 Correlation Coefficient f 0 999865 E E E E J al in in a 10 a 10 Gi an 5 5 T T m uk z E e 1 D D E E ar 10000 100000 1000000 10000 100000 1000000 Conc dprn ore Conc dprn ore Figure 2 24 Clicking and Dragging the X Axis Using 2D TLC 31 3 Release the button Region Calibration 2D Calibration adf Ioj x Log weighted Regression log y 0 9691 9 log s 3 06108 Correlation Coefficient 0 999865 Integrall rea Bk q PSL mm 10000 Conc demem l l l Figure 2 25 Zoomed Graph To zoom a curve segment 1 Click the Zoom button on the toolbar Window Help RRE Figure 2 26 Zoom Button Main Toolbar 2 Draw a rectangle around the area of the plot you want to see in more detail Region Calibration 2D Calibration adf i Region Calibration 2D Calibration adf ioj xj Log weighted Regression log y 0 96919 logis 3 06108 Log weighted Regression log y 0 96979 log 3 06108 Correlation Coefficient f 0 999865 Correlation Coefficient f 0 999865 E E E E im T a 10 a 10 mu ET Gi 5 5 T T I I Eo l E D D E E 10000 100000 1000000 10000 100000 1000000 Conc dprn ore Conc dprn ore Figure 2 27 Specifying the Area to be Zoomed 32 AIDA 2D TLC User s Manual 3 Release the button JE Region Calibration 2D Calibration adf Ioj xj Log weighted Regression logly 0 9691 9 log s 3 06108 Correlation Coefficie
76. the edges of the image If your TLC plate is not identical to the whole image 1 Double click near an edge of the image This will activate the Editing mode for the TLC plate overlay 2 Move rotate and stretch this overlay until it is congruent with the TLC plate Performing 2D TLC Evaluations 89 ER gt 3 Press the Return key to confirm the changes Figure 3 6 Editing TLC Plate Overlay Adjusting Origin and Start Lines 1 Now double click near an edge of the inner rectangle which represents the origin and front lines 2 Move rotate and stretch this overlay until it is congruent with the origin and start lines Figure 3 7 Adjusting Origin and Start Lines You can use the various combo boxes to type in directly the dimensions of your plate and your TLC runs 1f you know them 90 AIDA 2D TLC User s Manual Changing Run Direction of Existing Overlays If you want to change the run directions after the overlays have been set you have to delete the already set overlays first by clicking the Delete Overlay button and then redefine them Figure 3 8 Delete Overlay Button Sample Definition Panel Now you have completed the setting of the TLC plate and the 2D origin front overlay You may proceed now with the usual 2D densitometry evaluation Select the Region Determination toolbox to set regions and then use the Region Calibration toolbox to calibrate your measurement Performing 2D TLC Evaluati
77. tiple overlays regions the resulting contextual menu is considerably reduced compared with the single selection contextual menu showing only those commands available in this selection mode The provided menu items form a subset of the functions available in the single selection mode st Manuals ee ae Bie ied Delete Duplicate Matrix Change name Change group name Set as background Set as standard Figure 2 4 Contextual Menu Multiple Objects Selected Using 2D TLC 15 If no region is selected only the basic editing functions of the standard contextual menu described in the AIDA Image Analyzer Basic Concepts and Features User s Manual will be accessible Note All of the functions in these contextual menus are also accessible through different means menus buttons toolboxes windows They will be explained in the respective sections of this manual However it is often more convenient to activate the function through the readily accessible background menu This will make repetitive and routine tasks faster and more convenient Image Overlays You can specify the label type and font you want to apply to the region overlays lanes and bands using the Image Overlays command of the Options menu Options View Window Hi Triage Attributes Image Overlays Preferences Automatic Export Configuration Path Image Search Path Autosav ing Overlay Colors
78. to export The column heading of selected columns are highlighted To hide a column select the Hide command from its contextual menu You can recover hidden columns using the Select Columns command see below To specify the decimal digits for the value output in a column choose Settings of from the contextual menu and enter the value for the decimal digits in the dialog box displayed Column Integral Bkg Lx Decimal digits E OK Cancel Figure 2 12 Setting Output Format for Columns Use the One lined Header Two lined Header command to reduce or expand the column heading by one line the second line indicates the units of measurement To select deselect the columns that should be displayed in the Region Report 1 Choose Select Columns A dialog box appears displaying two lists Show Columns Hide Columns 24 AIDA 2D TLC User s Manual 2 2 Select the columns you want to show or hide and click the appropriate arrow symbol to transfer the items between the two lists 3 To deselect a particular item just click it again Columns Selection x Show Columns TA Integral L Integral Bkg EL Integral Bkg 9 IntegrallArea Bkg ALm Recalc Quantity na Std Quantity ng Hide Columns Area Position mmm Int Limits Position x mrm Integral Position Y mm Integral Area QLimm IntegralfArea IntegrallArea Bkg Recalc Quantity Std Quantity 9 cancel
79. ton or alternatively by pressing the ENTER key on your keyboard Figure 2 50 Label Repositioning Confirmed You can select the Font type and Size of the characters as well as their font style bold B italic I and roman typeface on white background using the Font button The Choose Font dialog box is displayed allowing you to specify the font options for the label Choose Font xj o A Font Size 10 0 Pt 1 1 print BIZIA Figure 2 51 Choosing Font Properties 50 AIDA 2D TLC User s Manual The regions can be renumbered if necessary by activating the Renumber function This function opens the Renumber Regions dialog box The numbers are displayed subsequently in a combo box and can be assigned to the desired regions by simply clicking on them Renumber Regions k X Nest region number to be assigned 1 E Click on a region to assign the number Choose Done to end renumbering Figure 2 52 Renumber Regions Dialog Box The Region Name tab controls are used to assign names to the various regions To assign names to regions 1 Select a region on the image 2 Click the Region Name tab Region Label Region Name Apply at Region Creation Figure 2 53 Region Name Tab Common names can be entered via a text box into a list that can facilitate the designation of redundant names Negative Control Positive Control Standard Background etc 3 Type a name in the text box or select one
80. tor has user permission the Change Password command instead of the User Registration command is selectable from the Options menu To change your password 1 Choose Change Password from the Options menu The Change AIDA Registration Password dialog box appears Change AIDA Registration Password X You can change the password of user doe Old password deco FE New password saca EH Confirm password acens 4 H Cancel Figure 1 7 Changing Password 2 Type your current password in the Old password text box 3 Type your new password in the New password text field and confirm it by retyping it in the Confirm password field 10 AIDA 2D TLC User s Manual 4 Click OK to apply the changes At the end of the session you can choose to log on as a different user instead of exiting from the program Close all and registrate with another name Exitihe Aida program Cancel Figure 1 8 Exit Aida Dialog Box Introduction 11 12 AIDA 2D TLC User s Manual Using 2D TLC Data Windows in 2D TLC In addition to the AIDA standard data windows Image and Histogram the following data windows are available in the 2D TLC mode Region Report e Region Calibration e Audit Trail You can access these data windows by choosing the appropriate entry 2D Region Report Region Calibration or Audit Trail respectively from the View menu view window Help Image Histogram 2D Region Report
81. ttings dialog box where a shape and size in pixels can be selected for the local background frame Using 2D TLC 45 local bkg Settings Frame shape C None C Rectangle Figure 2 42 Local Bkg Settings Dialog Box The Integration Limits option of the Integration pane allows you to integrate only those pixels that lie within a defined intensity range Thus a region that has some noise dirt on it This is useful for eliminating cavities from the surrounding tissue Since the level of intensity is different the surrounding region can be integrated without the unwanted signal originating from the cavity The intensity borders can be assigned via the Change Integration Limits dialog box A Change Integration Limits X i lower limit upper limit Full Range Uze colors for integrated area C norrintegrated area Figure 2 43 Change Integration Limits Dialog Box 46 AIDA 2D TLC User s Manual The intensity histogram is used to set the lower left and upper right intensity levels that will be used for the integration This is done by dragging the vertical black lines from the right and left end of the histogram to the desired position or by entering the appropriate values in the designated fields Lower Limit Upper Limit As a consequence the pixels that have a lower intensity or a higher intensity than the designated integration area peaks on the left or right of the margins shown in the example
82. utomatic export settings If the Auto Export Parameter options is enabled via the Data Trail Settings for pane any changes to the automatic export settings are logged in the audit trail report The automatic export settings are timestamped internally In addition you can add a name and an ID which are included in the Parameter pane of the export file together with the internal time stamp The parameter settings for the automatic export in AIDA 2D TLC are saved to separate set files named Parameter Name 2D Thin Layer Chromatography Automatic Export set in the AIDA Config directory where Parameter Name is the name you used in the Save Automatic Export Setting dialog box see below Using 2D TLC 81 To create automatic export settings 1 From the Options menu choose Automatic Export or click the Edit Settings button on the Automatic Export pane of the GLP Supporting Tools toolbar The Automatic Export Setting dialog box appears E Automatic Export Setting E X Setting Mame 2d tle Select Setting Setting ID Edit Setting Setting Stamp 20040319 142049 6802 Save Setting 2D Region Report Cancel Export on Save prompt for Parameter Included YES Table Header Included yes List Delimiter tabulator Decimal Delimiter comma Setting for Report Columns Ho Included in Export YES Decimal Digits 3 Figure 2 95 Automatic Export Setting Dialog Box The top pane of
83. utton Region Label Region Name Move Label Font None C Number C Name C Free Choice Configure Figure 3 48 Region Determination Toolbox Select Name List Button 114 AIDA 2D TLC User s Manual raytest The Region Nametables dialog box appears showing the current work nametable s list of names and a dropdown list with the names of all existing work nametables x o New Modify List of Names Remove Remove a Figure 3 49 Region Nametables Dialog Box 2 Click New The Nametable Editor dialog box appears Performing 2D TLC Evaluations 115 Q O lo EN ee raytest 3 Type a name for the work nametable in the Title text box and a list of names in the List of Names text box Mametable Editor xX Title work nametablel List of Names Cancel Type in your list of names in the edit box on the right each name on a line Type in a tithe for this list in the edit box above Figure 3 50 Nametable Editor Dialog Box 4 Click Accept to confirm the changes To modify a work nametable no master nametable applied 1 On the Region Determination toolbox click the Select Name List button The Region Nametables dialog box appears 2 Click Modify The Nametable Editor dialog box appears 3 Edit the entries in the in the List of Names list box 4 Click Accept to confirm the changes To create a new work nametable with master nameta
84. value compared to the reference row in the group of the region A calculation of the quantity in the region based on the current calibration curve The percentage of the quantity for the region determined on the basis of the calibration curve compared to the reference row in the group of the region The concentration of the region as entered for the calculation of the standard curve With the Concentration Standards the background corrected Intensity per Area values of the regions are calibrated The percentage of the concentration standard value compared to the reference row in the group of the region A calculation of the concentration in the region based on the current calibration curve The percentage of the quantity for the region determined on the basis of the calibration curve compared to the reference row in the group of the region The retention factor 1 run The retention factor 2 run signal unit and length unit units of measurement depending on the type of the imaging device used to obtain the image and the options set in the Image Attributes dialog box concentration unit and quantity unit unit of measurement specified by the user through calibration Using 2D TLC 21 If GLP support is turned on the following additional columns are available Table 2 2 Additional Columns in the Region Report GLP Support Enabled Column Short Description Name ID The unique identifier for the N
85. ve Graphic und or the calibration table Table Print Options for Audit Trail The last button of the Publishing Layout toolbox is used to configure the layout setting for the Audit Trail window in the Protocol Print pane Protocol Print FE ES Print Preview Audit Trail Format Portrait C Landscape Print W Configuration complete lists positive lists M Data Trailing Figure 2 84 Protocol Print Audit Trail Pane As for the Region Report you can select the paper orientation Portrait or Landscape by clicking the appropriate radio button in the Format pane The options in the Print pane enable or disable the printing of the Data Trail settings Configuration and or the recorded data from the Audit Trail window Data Trailing In addition if the Configuration option is checked you can choose to print the Complete Lists of settings or just the Positive Lists the checked options only 70 AIDA 2D TLC User s Manual Print In AIDA there are two ways for printing your image and evaluation data e Choose Print from the File menu or e Use the Protocol Print pane on the Publishing Layout toolbox The Print command of the File menu allows you to print your data on the fly without permanently storing the layout printing settings whereas the Protocol Print pane provides you with a complete set of printing features which includes saving the setting with the data file for reasons of reproducibil
86. y entering the intention for the change explicitly in the Audit Trail Confirmation dialog box Sunday April 18 2004 16 11 52 02 00 Region R2 in group 0 1515 9296962 PSL changed to Region R2 in group 0 515 9296962 PSLY Figure 2 74 GLP Error Message Box 64 AIDA 2D TLC User s Manual As for changes during evaluation you can also set recording options for changes afterwards Just click the Changes Afterwards button on the Data Trail Settings for pane The Audit Trail Configuration Changes Afterwards dialog box appears allowing you to select the actions to be watched by the GLP system al Audit Trail Configuration Changes afterwards Measurement M Template Import iv Auto Export Parameter Region W Create h Modify iv Delete M Background M Integration Limits M Group Attributes M Name M Numbering TL Calibration iv Create je je Region Calibration M Change Method M Change Curve Create Modify Delete Modify Delete iv Create h Modify iv Delete Figure 2 75 Audit Trail Configuration Changes Afterwards Dialog Box Finish Evaluation Pane The checkboxes of the Finish Evaluation by pane allow you to specify the user action which indicates the end of an evaluation Finish Evaluation by M first save iv first close W explicit stop Figure 2 76 Finish Evaluation Pane If you click First Save the settings of the Changes
87. y near to 1 Performing 2D TLC Evaluations 107 Using Nametables Nametables are used to name regions for reference purpose To standardize the naming you can use a master nametable which contains all region names in the correct predefined spelling This is especially useful for postprocessing of evaluation data using a database Using predefined names ensures that the mapping between regions and names is consistent across different evaluations For easy referencing every name in the master nametable has a unique identifier associated with it The predefined names as well as the associated identifiers are strings so identifiers are not required to be of the number type Names and identifiers must not exceed 1024 characters On the Region Name tab only about 20 characters are displayed In addition to the list of predefined names and associated IDs the master nametable contains a Table Name Table ID and Table Stamp a unique internal master nametable ID which consists of date time and a random number The master nametable is saved to a file named 2d densitometry name glp in the AIDA Config folder To use the table on another computer just copy the file to the Config folder of the AIDA program on the respective machine or alternatively use the Export Import option of the Master Name Table dialog box see the Exporting Master Nametables section below After applying a master nametable you can assign the names listed

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