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AIDA 1D Evaluation+ for Windows User's Manual

1. Figure 2 29 Smooth Width Dialog Box Using 1D Evaluation 35 The Grid command opens the Grid dialog box which allows you to lay grids over both axis that are an extension of either the big or the small ticks on the axes Vertical None Big Tics C Small Tics Harizantal Mone C Big Tics C Small Tics Cancel Help Figure 2 30 Grid Dialog Box The Profile Overlays command opens a dialog box that allows you to select various labels for the peaks together with their font font size font style and rotation angle The display of peak Area and or Brackets indicating the bands borders can be selected To turn off the marking of the peaks on the graphics display click to deselect the Draw Peaks option Profile Overlays Pools adf k X Region Cancel v Draw peaks Mark peaks by v Area Brackets Figure 2 31 Profile Overlays Dialog Box Label Pane 36 AIDA ID Evaluation User s Manual Windows The location of the migration distance standards can be drawn into the profile by clicking the Draw Standard Lines option on the Runlength Calibration pane Further you can specify the font size of the labels by entering a value in the Label Size combo box Profile Overlays Pools adf X int Draw standard lines My E Cancel Label Size B Pt Came Figure 2 32 Profile Overlays Dialog Box Runlength Calibration Pane With the Change Profile Names com
2. Peak Determination pane Figure 2 53 Lane amp Peak Determination Toolbox Overview 52 AIDA 1D Evaluation User 5 Manual Windows To display the Lane amp Peak Determination toolbox e On the main menu click Evaluation and choose Lane amp Peak Determination Alternatively you can click the Lane amp Peak Determination button on the toolbar Lane Determination Pane The first step in the analysis of a 1D application is the determination of the lanes Lane Determination Manual Lane Marking Tool arbitrary T Delete Marked Lane Button Lane Marking Options Drop Down Menu ite A lane width Jimm Profile Width Determination Tool vertical T Lane Searching Parameter Drop Down Menu sensitivity medium E Search Sensitivity Drop Down Menu intensity summation pu Search Lane Tool Profile Type Selection Drop Down Menu Minaturized Profiles Window Figure 2 54 Lane Determination Pane Overview Manual Lane Marking Tool Select this tool to enter the manual lane marking mode The Lane Marking Options drop down menu next to the button offers the following lane marking options p Q arbitrary k w arbitrary horizontal vertical Fun direct expand to edges show prafile show drag Figure 2 55 Lane Marking Options Drop Down Menu Using 1D Evaluation 53 Da b 6 qm Pa QU aS E S EE raytest Densitometer windows c
3. Quantity Calibration Runlength Calibration Profile Options M Audit Trail all profiles i v M Use Colors Audit Trail Options include positive trail configuration v include complete trail configuration Figure 2 86 Print Dialog Box Using 1D Evaluation T 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 Footnote nam Iv file path Iv author did ril pm v page number v last change time ancel v current date v software version Figure 2 87 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 overlays the Image Profile Curves Profile Table Quantity Calibration and Runlength 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 9 o In the Profile Options pane you can specify to print the Curr
4. position hy maximum M i position by maximum E position by center Figure 2 62 Overlap Treatment Drop Down Menu The options Allow Overlap Avoid Overlap and Warn Overlap are available Selecting Avoid Overlap will turn off the overlapping of regions by either keeping peaks separate or by merging two strongly overlapping peaks after the region is confirmed Better results for quantification are obtained by using the manual peak assignment mode in the profile or image window Delete Peaks Button Click this button to delete all currently selected peaks Search Peaks Button Select this button after setting the Search Level and Smoothed parameters to search peaks automatically within the active profile s Although these parameters have the same names as the previously described ones setting the same values for the different functions will not give the same results All these parameters should be tested for a particular image type before they are applied to an actual image of the tested type The selected parameters can be saved under specific names using the Preferences command of the Options menu Clicking the Apply To Lane Determination box enables the immediate use of the selected parameters for the evaluation of the following densitometer windows Band Searching Tool Use this button to search for individual bands by clicking on them Bands that are not found with any set of parameters can be double clicked when this function
5. 1 Profile 1 Figure 3 3 Semi Automatic Lane Detection 88 AIDA ID Evaluation User 5 Manual Windows 4 Click the Profile Width Determination Tool and set the Profile Width to a value so that a little more than the band width 1s enclosed Figure 3 4 Setting Profile Width 5 Use the Search Peaks button to detect bands automatically Feak Determinatian position hy maximum xs i warn overlap rid search level 2 smoothed T pixel g Figure 3 5 Activating Automatic Band Detection Performing 1D Evaluations 89 If too many bands are found increase the Search Level If too many artifacts are detected as bands try to increase Smoothed Do not forget to save your selected parameters under a Preferences scheme so that you can use them as a default for the next analysis Once the parameters are selected appropriately the detection of lanes and bands should be fairly easy and straightforward Jni x Ee Program Files raytest Aida Image Analyzer dev 3 10 028 Exampl ce pools LH i 1 Profile 1 Figure 3 6 Automatic Band Detection Completed 6 Use the Auto Determine Baseline Tool to draw a baseline Baseline Determination search level 1 g smoothed 2 pixel apply to lane determination Figure 3 7 Activating Automatic Baseline Determination 90 AIDA ID Evaluation User 5 Manual Windows If the baseline is too flexible and enters into
6. Figure 2 5 Options Menu Opening the Image Overlays Dialog Box After choosing Image Overlays from the Options menu the Image Overlays dialog box opens allowing you to specify the labeling and marking of lanes and bands separately using the Lanes and Bands tab respectively In addition you can specify marking and labeling for Runlength Calibration 16 AIDA 1D Evaluation User 5 Manual Windows 2 For Lanes depending on the already performed evaluations various labels can be selected by clicking the Configure button in the Label pane of the Lanes tab Image Overlays Pools2 adf x Lanes Cancel Font Arial Size poo pti print B 7 B 2 z jut Mark by Middle line Border lines Figure 2 6 Image Overlays Dialog Box Lanes Tab This opens the Columns Selection dialog box which provides you with a list of possible labels e g lanes can be labeled by Number Profile name or not at all Columns Selection 7 x Show Columns Hide Columns B Bkg GL Integral GL Recale Quantity Integral Bkg GL Integral 95 Type Nama Integral Bkg 95 Raeralc Quantity 95 uiu Hidelegend Hide all Figure 2 7 Columns Selection Dialog Box Using 1D Evaluation 17 8 qe amp raytest After you have determined the type of labeling you can set the font font size and font style bold italic roman typeface on white background as well as the rotation 0 4
7. 0c eee 73 Print Options for Runlength Calibration llle 74 Print Options for Audit Trail 0 0 eee nes 75 PIs ais sce uc Erbe en bee eo rU oou caes aor iubeat BOS aS Ar teu 76 EXpor and IMPON euo ecar on ionis ordre abis qe d ke enc bona da mS c dre tad 78 Exporting Templates a ouais bt beo UR sdb SS Ed ee ce 78 Esporine er OCS t ee cuotas ees dchliot aec pci aret bottom tcu its 79 Exporting 1D Report lables so doceo So Tabor Eo ERE wired goles eee ES 80 Exporting Quantity Calibration 0 0 cece eee een ees 81 Exporting Runlength Calibration 000 eens 82 Exportino Audit Tal a d ce sce dessa edo ak ode anite Sean keds asit uq ore Fette quand 84 Importing Templates 4435s swe scent wih Eu RE RS E dens 84 Importing Quantity Calibration eser e ga 9 kaw e ede wae eure en 85 3 Performing 1D Evaluations 87 Assigning Lanes and Bands 0 0 0 ccc neenon erronee 87 Assigning Migration Distance Standards 0 0 0 96 Assigning Quantitative Calibration Standards naana anaua 99 Configuring the Evaluation Output 00 0 eens 102 iv AIDA 1D Evaluation User s Manual Windows Introduction 1 Overview The AIDA 1D Evaluation module combines the full functionality of the AIDA 1D Eval uation module with special features required for working under GLP Good Laboratory Practice conditions Most biochemical applications utilize only one dimension as for example SDS protein
8. AIDA ID Evaluation for Windows User s Manual Band s lal Recalc Runlength Epp E 5 9 Lane s labels Part Number 2004 04 1 1 2004raytest 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 ray test 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 ofthe 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 1D 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 ray test 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 name
9. Audit Trail Confirmation Dialog Box The desired modification is blocked until the user confirms the action by entering the intention for the change explicitly in the Audit Trail Confirmation dialog box im Audit Trail Confirma x m x System Administrator Administrator Wednesday April 28 2004 15 20 17 02 00 Lane 11 469 106 469 449 changed to Lane 11 468 77 469 gt 448y Aida 1D Evaluation GLP E xj e Please give a reason For Ehe changes Reason a Figure 2 75 GLP Error Message Box 68 AIDA ID Evaluation User s Manual Windows 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 Im Audit Trail Configuration Changes afterwards I 3 x Annotation Measurement Overlay Create M Modify M Delete M Template Import M Auto Export Parameter Lane Iv Create v Modify W Delete W Lane Name W Lane Number W Lane Int Mode Peak Region W Create v Modify W Delete Peak Name Background W Create Modify Delete Quantity Calibration I Std List M Equation Standard W Create Iw Modify Iv Delete Runlength Calibration M Change Method W Equation Standard W Create v Modify M Delete ca
10. Delete Selection pools1 tif q x 2 Delete the selected peaks ar profiles Delete Profiles Cancel Figure 3 14 Deleting Peaks 13 Check the Profiles window for peaks of interest 14 Save the search parameters under a name specific for the source of the image e g EtBr Gel using the Preferences command of the Options menu Preferences OK _Cancel_ __toad_ Saw Configuration Eter Gel Cancel Load Save Figure 3 15 Saving Search Parameters This allows you to simply reload them for the next image Performing 1D Evaluations 95 Assigning Migration Distance Standards In all 1D applications the migration distance of a sample can be correlated with a physical constant of the sample In the most widely used biochemical applications comparison 1s done according to size molecular weight fragment size or charge pl or a combination thereof RF It should be noted that the computer and software are unable to detect the migration distance as such They simply denote the absolute location of a band and compare it to the location of standard bands The evaluation is based on the image data in the lanes densitometers and evaluated utilizing the Runlength Calibration data Thus any tilt in the gel image and any smiling effect or distortion in the gel itself will hamper the evaluation Since these conditions are frequently encountered in gels blots or thin layer chromatography a m
11. gels DNA agarose gels thin layer chromatography and isoelectric focusing gels These applications result in lanes originating from different samples that can be evaluated on the basis of their migration distance and signal intensity The AIDA 1D Evaluation module facilitates the analysis of 1D applications using quantification and migrations distance evaluation For the lanes of a 1D application e g an agarose gel of ethidium bromide stained DNA rectangular densitometer windows can be defined to create a profile The profile is the signal intensity at each point plotted against the length of the densitometer The signal intensity can be integrated or averaged over the entire width of the densitometer It is important to enclose the lane each band entirely with the densitometer because the bands in many applications vary in their signal intensity over their entire width Encircling the entire band is especially important when using the integration option The integrated signal intensity can be compared between lanes relative comparison e g of expression rates or normalized by amount standards e g 10 20 50 100 ng of DNA that are also present in standard lanes in the same image or even in other images of the same series The profiles can also be used to evaluate the molecular weight fragment size pI or RF values of the samples depending on the employed separation method and sample To that end the position of the bands are detected and
12. intensity summation averaged intensity optical density Figure 2 58 Profile Type Selection Drop Down Menu The diagram shows the selected parameter y axis plotted against the distance unit selected in the Image Attributes dialog box of the Option menu intensity summation i mm Figure 2 59 Miniaturized Profiles Window Baseline Determination Pane This pane will be needed only for the quantification of the bands after standardization Baseline Determination Edit Baseline Tool Xx Remove Baseline Tool smoothed Opixel Auto Determine Baseline Tool search level apply to lane determination Signal Smoothing Drop Down Menu Search Level Drop Down Menu Figure 2 60 Baseline Determination Pane Overview Using 1D Evaluation 55 Edit Baseline Tool Clicking this button activates the Edit Baseline function If no baseline was determined yet a straight line 1s drawn from the leftmost intensity to the rightmost intensity of the profile This line can be edited in the Profiles window by moving the handles attached to the line Double clicking on any position of the baseline creates a new handle Remove Baseline Tool This button removes the baseline s of the currently selected profile s Auto Determine Baseline Tool This tool allows you to determined the baseline automatically The Smoothed parameter should be set to a value of 2 or higher to omit single pixel noise and the Search Level
13. profile or the Sum The later two are set via the following dialog box Percent Reference Pools2 adt Ts x Set 100 percent to Total profile C Peak summation OK Cancel Figure 2 39 Percent Reference Dialog Box The Change Profile Names command allows to re name the densitometer windows and resulting profiles according to your preferences The names will appear in all result tables and image menus Profile Name ae j x Set name af 1 profile to L ok Cancel Figure 2 40 Profile Name Dialog Box Using 1D Evaluation 41 raytest Quantity Calibration Window To open the Quantity Calibration window Choose Quantity Calibration from the View menu or Double click the miniaturized calibration curve display on the Quantity Calibration toolbox 4 Bl x Linear Regression y 13 893x 14 80 Correlation Coefficient r 0 9993 Intercept on x Axis 1 062 Integral Bkg QL ce c 0 10 20 30 40 50 60 70 60 390 100 120 14 Quantity ng Profile Peak Integral Bkg QL Quantity Std ng Quantity Recalc nq 1 Standa g 1402 9 100 0 99 66 11 Profili 1 494 6 34 44 Figure 2 41 Quantity Calibration Window Example The Quantity 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
14. quantity unit Recalc Quantity quantity unit Difference unit Rel Error 96 Deviation unit Rel Deviation 96 Columns Available in the Quantity Calibration Window Short Description The index and name of the profile The number of the peak in the profile The name for the peaks as given by the user The background corrected integral of the peak The quantity of the peak as entered for the calculation of the quantity standard curve A calculation of the peak quantity based on the current quantity calibration curve The difference without sign between the entered standard and the recalculated value The relative difference without sign of the entered standard and the recalculated value compared to the standard value 7 Difference Std Quantity The difference with sign between the entered standard and the recalculated value Recalc Quantity Std Quantity The relative difference with sign of the entered standard and the recalculated value compared to the standard value 7 Deviation 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 quantity unit and runlength unit unit of measurement specified by the user through quantity and runlength calibration respectively Using 1D Evaluation 45 way raytest Runlength Calibration W
15. 3 1D Evaluation eProgram Filesaytest 4ida Image Analyzer dev 3 10 028 ExamplesVAg test adf Parameter Thursday March 15 2001 11 14 34 AM Page 1 of 3 Image Tie eproqram FiksvayestAkia mage Axalzer deus I0 809 Examples wg te c ttf Digital resolto 255 8 bitpe r phe I Epose dae Tiesday December 09 1997 8 1206 PM mage st 752582 376290 mage resolto 209p 203p Commeit S kctior 000 000 052582 ae Last Changed Thursday May 25 2000 1 38 17 PM Aida Development Version 3 10 028 Page 2 of 3 1D Evaluation amp Program Files aytest 4ida Image Analyzer dev 3 10 028 ExamplesVAg test adf Profiles Thursday March 15 2001 11 16 20 AM Page 2 of 3 beastly pu Position pixel hegral Bhg hegasi StLOwaitfy Pecak Qatt py bu bu ng ng ej e e sew Er po ne ses ues we Eme cp omm me me 0 ew res ism mm umm mes mm sx Emme iem mae sme mx mm wow Exe pem eme zem cem e aan Emme em eee cma se a a Last Changed Thursday May 25 2000 1 38 17 PM Aida Development Version 3 10 028 Figure 2 88 Preview Example Part 1 Using 1D Evaluation 79 D e cu Lin Page 3 of 3 Thursday March 15 2001 11 16 20 AM Page 3 of 3 1D Evaluation amp Program Files aytest 4ida Image Analyzer dev 3 10 028 ExamplesVAg test adf Quantity Calibration Logarithmic Regression y 215403 4Tlog x 33 05 33 05 Correlation Coefficient r 0 333652 hegrabbkg
16. AIDA 1D Evaluation User 5 Manual Windows 1 Click the Evaluation button to set the recording options for changes during evaluation The Audit Trail Configuration Evaluation dialog box appears imi Audit Trail Configuration Evaluation E xj Annotation Overlay Create Iv Modify M Delete Measurement M Template Import Auto Export Parameter Lang Create v Modify Iv Delete Lane Name Lane Number Lane Int Mode Feak Region Create Iw Modit M Delete Peak Name Background f Create Iv Modify M Delete Quantity Calibration M Std List M Equation Standard Create Iw Modify Iv Delete Runlength Calibration M Change Method M Equation Standard Create Iv Modit Iv Delete cael Figure 2 73 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 1D Evaluation 67 If the working operator performs one of the actions checked in the Audit Trail Configuration dialog box a dialog box appears prompting the user to give reasons for the desired change I Audit Trail Confirmation E E x System Administrator Administrator wednesday April 28 2004 15 20 17 02 00 Lane 11 469 106 469 449 changed to Lane 11 468 77 468 gt 448y Reagan Figure 2 74
17. Horizontal Vertical Mone Mone C Big Tics C Big Tics Small Tics Small Tics Cancel Help Figure 2 49 Grid Dialog Box The table in the lower part of the Runlength window provides its own contextual menu The column headings are equipped with the standard contextual menus described in the Profiles Window section of this manual 48 AIDA ID Evaluation User s Manual Windows 2 PES If you right click on any of the rows in the table the row s contextual menu opens allowing you to Select Columns for displaying or Change Profile Names 1 st 1 st Profle Peak Position Std Hunlenath Hecale Hunlenath mm bp Select Columns Change Profile Mames radif 6 289737 7128 E968 Figure 2 50 Table Row Contextual Menu The following columns are available Table 2 4 Columns Available in the Runlength Window Columns Profile Peak Name Position ength unit Std Runlength runlength unit Recalc Runlength runlength unit Difference runlength unit Rel Error 96 Short Description The index and name of the profile The number of the peak in the profile The name for the peaks as given by the user The position of the peak in the profile The runlength standard value of the peak as entered Depending on the method for the runlength measurement this is the standard for the RF value Fragment Size Molecular Weight or
18. Linear Interpolation Linear Regression Logarithmic Regression Log Weighted Regression Linear Interpolation calculates lines to join each data point 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 Click to select the Origin option to force the curve through the origin The correlation coefficient Corr Coeff of the estimation 1s 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 Using 1D Evaluation 61 c aS SS abt raytest Double clicking on the graphics pane opens the Quantity Calibration window see the Quantity Calibration Window section of this manual for details Sometimes a warning will be displayed below the graphics pane stating bkg not dertermined to indicate that no background region has been assigned yet It can also indicate the lack of a local background for some regions Migration Distance T
19. 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 10 AIDA ID Evaluation User 5 Manual Windows 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 name Permissions Password Confirm password 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 operator 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 9E You can change the password of user doe Old password aerae Mew password Confirm password 3 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 Introduction 11 4 Click OK to apply the changes At the end of t
20. The variation of fluorescence of different DNA fragments is not larger than the error of the whole procedure itself less than 10 96 The profiles determined by the Lane amp Band Evaluation function can be visualized by activating the Profiles command of the View menu Any profile can be selected using the Select Profiles pop up menu or using the arrows under the Profiles window El x y SIS E ES gt z 20 f J im i qo Jt it J ANUN io N un N f ARNE NA A d i z An UNAN D Ao mi i j M A N N 1ANONPNA N DR A z INNON N HA N AN A N PHAM NN 8 Y NANYAN AAA NN MAN y TI NINANI BAN A AN WAN S DNNN We SN A 0 100 200 Pasition pixel Figure 3 18 Selecting Profiles In the absence of a run length evaluation you will not see the deduced fragment length If the bands can not be detected automatically the profiles can be set manually Performing 1D Evaluations 99 Q O 1y 87 BICE raytest After all peaks are properly determined the quantity standards can be assigned 1 Set the method for the intensity evaluation in the Lane amp Peak Determination toolbox to Intensity Summation Optical Density should be selected for color stained bands Intensity Summation for radioactive fluorescent stains and chemiluminescence applications and Averaged Intensity can be used for fluorescent stains and chemiluminescence applications intensity summation k v intensity summation averaged in
21. ale o Pratacal Print I Page Layout Edges Top 20m 2 Bottom 20cm 24 Left 20m 2 Right 20cm 4 Header Iv File Path W Page Number M Current Date Footnote M Author M Last Change Time M Software Version Figure 2 81 Publishing Layout Toolbox Overview and Toolbar 74 AIDA ID Evaluation User 5 Manual Windows Print Options for Profiles Using the Profiles Options button you can configure layout setting for the Profiles window 1 Click the Profiles Options button The appearance of the Protocol Print pane changes Annotate Protocol Print Print Previews Profiles Format Portrait C Landscape Print Selection Only 4d Graphic Single C Multiple C 3p W Table C Single Tables Allin One Table To Graphic Figure 2 682 Print Options for the Profiles Window 2 Select the appropriate options In the Format pane select the paper orientation Portrait or Landscape by clicking the appropriate radio button In the Print pane the scope for the print output can be specified Click Selection Only to print only the densitometers that are currently selected Graphic selects the graphical representation of multiple selected profiles Single shows each densitometer curve in a separate graphic Multiple shows all selected curves on top of each other in a single graphic and 3D shows all selected curves in a 3 dimensional fashion within t
22. can be set to various values reaching from 0 insensitive to 10 extremely sensitive After optimizing all parameters for an example lane you can select the Apply To Lane Determination checkbox to use the settings for any other lane to be selected Peak Determination Pane This part of the toolbox is used for the determination of peaks Peak Determination k position by maximum hdi Peak Assignment Tool warn overlap 7 Delete Peaks Button M Bunan L Overlap Men Drop Down Menu eje Band Searching Tool search level 0 Ee Search Level Drop Down Menu smoothed Opixel E Signal Smoothing Drop Down Menu apply to lane determination Band Position Drop Down Menu Figure 2 61 Peak Determination Pane Overview Band Position drop down menu Use this drop down menu to mark the position of the band by Position by Maximum or Position by Center Peak Assignment Tool This button is used for manual peak assignment Click on the start of the peak in the image or in the profile window press the mouse button and drag the second mark to the end of the peak You can change the peak region by dragging one of the marks After the peak region is properly defined right click or press the ENTER key to confirm 56 AIDA 1D Evaluation User 5 Manual Windows Overlap Treatment Drop Down Menu The drop down menu next to the peak assignment tool is used to define AIDA s action if an overlap of peak regions is detected
23. dialog box that appears select the template to be imported and click Open Look in a 1D Samples amp t EX EJ Type OTF File Size 40 0 KB File name pools2 att Files of type ADA Template Format Cancel Figure 2 99 Import Template Dialog Box The imported profiles can be arranged to fit the image by moving the whole array using the AIDA standard overlay editing functions Using 1D Evaluation 87 Importing Quantity 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 Quantity Calibration Import dialog box appears allowing you to specify the calibration file cal Quantity Calibration Import Ag test adf Type CAL File Size 255 Byte Ju File name Jag test cal Files of type ADA Calibration L ancel Figure 2 100 Quantity Calibration Import Dialog Box 88 AIDA ID Evaluation User 5 Manual Windows Performing 1D Evaluations 3 Assigning Lanes and Bands The first step in the 1D evaluation is the detection marking of the lanes which is followed by the detection marking of the bands normalized by the setting of the migration distance standards and the setting of the quantity standards as applicable The result is a fully evaluated image where all the information can be
24. for setting several display options see below Bl nunlenath Pools2 adf ioj x s Multiple Display Tile Display w 3d Display Scales Position mm Profile Peak Position Std Runlength Recale Runlength mm bp bp 24 9944 10120 10284 8144 8017 radif ej 20737 mae 86 d 34 3558 Figure 2 47 Runlength Window With Contextual Menu Set the desired display mode using Multiple Display Tile Display and 3d Display explained in the Profiles Window section of this manual Using 1D Evaluation 47 The Scales command allows you to configure the graphical output The Minimum and Maximum value of the x Runlength and y axis Position can be selected together with a logarithmic representation for both axis In addition both axis can be set to Automatic scaling using the appropriate checkboxes The 3D View Point settings are the numerical representation of the change in 3D views as described above Data Scaling Runlenath bp Minirmurn Maxirmurn 12 318 M Logarithmic Iv Automatic _ Cancel Position rm Minimum haxirurm 21 5 71 2 as O M Logarithmic v Automatic 3D View Point A Position Y Position os E p Figure 2 48 Data Scaling Dialog Box Runlength Window The Grid command opens the Grid dialog box which allows you to lay grids over both axis that are an extension of either the big or the small ticks on the axes SS 5
25. given and recalculated standard values so you can easily evaluate their differences This can be useful to estimate the error for a certain amount calculated for an unknown band The graphical representation can be adapted using the contextual menu attached For a more detailed view of the plot you can activate the AIDA standard zoom functions described above The table can be exported as a text file or copied into the clipboard 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 42 AIDA 1D Evaluation User s Manual Windows Contextual Menu of the Quantity Calibration Window The contextual menu of the upper pane of the Quantity Calibration window contains two commands Scales and Grid With the Scales command you can 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 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 5caling i X iauantity hinirurm haxirurm DENN Logarithmic Automatic cancel Integral Bkg G
26. of the Display command you can specify the appropriate display mode Multiple Tile and 3d explained earlier as well as how the data should be displayed You can choose between the Discrete display of data points pixels in the profile the display of Extremal data representing the maximum value for all points between the determined data points or the display of Interpolated curves between data points Further you can specify to show or hide the baseline using the Show Baseline or Hide Baseline command respectively In addition you can choose to subtract the baseline from the data by selecting Subtract Baseline 34 AIDA ID Evaluation User 5 Manual Windows With the Data Scaling command you can configure the graphical output The Minimum and Maximum value of the x Position and y axis Intensity can be selected together with a logarithmic representation for y axis Both axis can be set to Automatic scaling using the appropriate checkboxes The 3D View Point settings are the numerical representation of the change in 3D views as described above Data 5caling X Position mrm Minimum Maximum T i Automatic Cancel Intensity GL Minimu haxirurm 754 7 UU M Logarithmic v Automatic 3D View Point A Position Y Position oo B E Be X Figure 2 28 Data Scaling Dialog Box The Data Smoothing function is used to smoothen the curve Smooth Width Pools1 adf Ea Set smooth width to mm Cancel
27. of the Profiles window the displayed range of both axis can be zoomed To select a display range on an axis 1 Choose the Select tool from the toolbar RSG Figure 2 12 Select Tool on the Main Toolbar 20 AIDA 1D Evaluation User s Manual Windows 2 Click at the smallest value of interest on the x axis and drag to the biggest value of interest on the same axis with the left mouse button pressed Intensity GL Intensity GL Intensity GL 26 98751123 28 5790119 30 16251256 31 75001322 33 33 81388 34 32501454 Position mm Figure 2 13 Zooming X Axis 3 Release the mouse button The scaling will change accordingly Using 1D Evaluation 21 Intensity GL Intensity GL Intensity GL ER e 3 2 roS M ap ed 3 33 35 Fo 33 4 Position mm Figure 2 14 Zooming X Axis in 3D Display Mode To zoom out again click the right mouse button when the 4 symbol is displayed The maximal zoom out factor is defined using the Data Scaling command see below The same procedure can be applied to the y axis 22 AIDA 1D Evaluation User s Manual Windows To zoom into the Profiles window using the Zoom tool 1 Click the Zoom tool on the toolbar 2 Draw a rectangle around the area of the plot you want to see in more detail Figure 2 15 Selecting Zoom Tool std 2 5000 4000 3000 2000 1000 Cass 2722222222
28. standards Linear Interpolation Linear Regression Logarithmic Interpolation Logarithmic Regression Hyperbolic Regression Hyperbolic Interpolation Using 1D Evaluation 63 Qe Dg 8 BICE raytest You can set your favorite calibration function as default by saving the current settings using the Preferences dialog box of the Options menu together with all other variables GLP Supporting Tools Toolbox The GLP Supporting Tools toolbox is used to specify all GLP relevant settings GLP GLP Supporting Tools Administration Iv apply GLP support proof PCA check sum Reason List Data Trail Settings for Evaluation Data Trail Settings Pane Changes Afterwards Finish Evaluation by v first save v first clase Iv explicit stop Administration Pane Finish Evaluation Pane Automatic Export Edit Setting Data Trailing Automatic Export Pane Data Trailing Pane view uit Trail Stop Evaluation Figure 2 68 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 Click the GLP Supporting Tools button on the toolbar 64 AIDA 1D Evaluation User 5 Manual Windows Note To configure the GLP support administrator rights are required Therefore make sure that you are logged in as system Administrator to change the GLP set
29. the File Name text box and complete the export action by clicking Save Export Template Pools adf i I Ed x Save im X 1D Samples frt Fae File name pools2 at Save as type alba Template Format Cancel Figure 2 91 Export Template Dialog Box Using 1D Evaluation 81 Exporting Profiles To export Profiles On the File menu click the Export menu item and then choose Profiles from the submenu 2 In the Profiles Export dialog box that appears type a name for the export file and specify the data format To export the graph in the Profiles window e Choose Windows Metafile The graph is saved to a separate file in the Windows Metafile format To export the profile data of the Profiles window 1 Select the Profile Ascii File data format 2 Click Save to confirm the export The Profile Export dialog box appears allowing you to set further option for the export of the result table Profile Export Ponls2 adf X Range i Header OK Cancel All Profiles C Selected Profiles Decimal Delimiter f Point List Delimiter Tabulator C Comma C Comma C Semicolon System Defaults Figure 2 92 Profile Export Dialog Box You can specify the Range of the exported rows Header only All Profiles or Selected Profiles only as well as the List Delimiter and Decimal Delimiter in the respective panes of the dialog box Selecting the System Defaults button applies the W
30. the peaks you can increase the Smoothened value If too much baseline is detected as peak you can increase the Search Level If the baseline still does not fit your needs you can edit it manually by double clicking it in the Profiles Window 0 100 200 300 Profile Peak Type Abs Pos Std EN Hecalc P as pixel rig ng 1 Profile 1 LT o NN T RN RN I a Figure 3 8 Automatic Baseline Determination Completed All densitometer windows can be extended to the edges of the image choose Expand To Edges from the contextual menu of the Image window if necessary Multiple selections of profiles can be helpful for many manipulations and are achieved by opening a rectangle over all desired profiles or bands in the selection mode or clicking on each individual profile while holding down the CTRL key 7 Finally assign bands within profiles that were not automatically detected by activating the Band Searching Tool and then clicking directly on the middle of the band Performing 1D Evaluations 91 8 Images where the automatic lane detection does not work can be assigned manually Depending on the lane shape the Arbitrary Vertical or Horizontal densitometer function is activated Lane Determination arbitrary T vertical id search level 1 g smoothed pixel g Figure 3 9 Activating Manual Lane Detection a Activate the Manual Lane Marking Tool b Click in the middle of the lane to set the star
31. you with different display possibilities for the graphical output Multiple displays multiple curves for selected items in a single two dimensional co ordinate system using different colors for each 3D shows all curves for selected items in a single three dimensional co ordinate system Tile uses a separate 2D co ordinate system for each curve of a selected item The Normalize button is used to normalize all displayed profiles according to the determined migration distance The Show Baseline Hide Baseline Subtract Baseline buttons are used to display the determined baseline hide the baseline and subtract everything below the baseline respectively This function is only available if a baseline has been set in the Band amp Peak Determination toolbox The Smooth Width button opens a dialog box to enter the smooth width for the curve display A higher number will result in averaging over the given range of pixel mm inch where the unit is set using the Image Attributes commands of the Options menu Smooth Width Ponls2 adf l I X Smooth width 0 211 138 mm EI Figure 2 23 Smooth Width Dialog Box The lower part of the Profiles window contains a table henceforth Result Table listing the standards their given and recalculated values etc Using 1D Evaluation 29 You can change the display ratio between the graphics and the table pane by dragging the delimiter line located on the bottom of the toolbar EA Frofi
32. 1 2AT faut aa 23 7 tadi 3 rsd 250 10120 tadi 4 RStd 28a A NENNT E Set to 100 2b 5 0 Bkg Region 8 6108 i4 Select Percent Reference i x 2038 Change Profile Mames 5 m kan mmw mi we Si icd 1 5 7 Select Columns Select Rows 1 1 M DE Figure 2 37 Contextual Menu of a Result Table Row Set to 100 will set the value in the active row to 100 instead of using the Sum value as 100 and recalculate the other values accordingly To select the columns that should be displayed in the Result Table choose the Select Columns command see above 40 AIDA ID Evaluation User 5 Manual Windows Using the Select Rows command you can choose four special row types to be displayed in the Result Table In the dialog box that appears click to select or deselect the appropriate options Clicking Total Sum and or Remainder inserts the respective row s into the table Separator displays an extra separator row showing the name of the profile Select Rows Ponls2 adf X Show Profile Rows OK Separator Cancel v Total Gammel jv Sum Ps es eet Figure 2 38 Select Rows Dialog Box Select Percent Reference This command allows you to set the reference value for the percentage calculation This can be either a distinguished peak defined by the Set to 100 command of the table contextual menu see above the value for the total
33. 2 SNR std 3 5000 4000 3000 2000 1000 LLL ALLLLLLLLL LL Lae N e 1 aad ES 26 98751123 28 5750119 30 16251 256 31 75001322 33 33751388 34 92501 454 Figure 2 16 Zooming into Profiles Window Using 1D Evaluation 23 SAASSSS 4 Position mm Position mm 18 Apsua 18 Apsua 18 Apsua Position mm AIDA 1D Evaluation User s Manual Windows 3 Release the mouse button 4 Right click to zoom out This procedure is also applicable in the 3D display mode with the difference that you have to draw the rectangle on the back plane of the cuboid Figure 2 17 Zooming into Profiles Window in 3D Display Mode 24 To change your viewpoint in 3D display mode Drag the axis origin with the mouse Intensity aL Intensity aL T a ir c D E Intensity aL zn 3l 40 Position mm Figure 2 18 Changing 3D Viewpoint Using 1D Evaluation s 29 To edit peaks e Double click the peak whose borders or position you want to edit Vertical delimiter bars appear n Drag the vertical delimiter bar Intensity aL Intensity aL T a ir Cc D E Position mm Figure 2 19 Editing Peaks 3 Right click to confirm the changes 26 AIDA ID Evaluation User 5 Manual Windows To delete a peak 1 Right click on the hatched area indicating the peak s dom
34. 5 90 for the labels The detected lanes can be surrounded by a frame Border Lines and tracked by a Middle Line Similar to the lanes assigned peaks can be labeled using the Configure button of the Label pane on the Bands tab Image Overlays Pools2 adf X Lanes Bands Arial Size po Aptis print B B SFE Mark by Iv Position Border Figure 2 8 Image Overlays Dialog Box Bands Tab As for the lanes use the Font pane of the dialog box to set the font options font font size font style Specify the position of label relative to the band marks left center right using the controls next to the font style selection buttons To insert marks for the peaks on the image select the appropriate checkboxes in the Mark By part of the Image Overlays dialog box Position and or Border Besides the labeling for lanes and bands the Image Overlays dialog box allows you to configure the display of runlength calibration marks and labels Just click the second icon on the toolbar to switch to Runlength Calibration pane Figure 2 9 Image Overlays Dialog Box Toolbar 18 AIDA 1D Evaluation User 5 Manual Windows To turn on the drawing of standard lines on the image click to select the Draw Standard Lines option You can change the font size for the labels using the Label Size combo box Image Overlays Panls2 adf l x Ok M Draw standard lines Cancel Label Size Cancel Pt Figure 2 10
35. 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 76 AIDA ID Evaluation User 5 Manual Windows Print In AIDA there are two ways for printing your image and evaluation data e Choose Print from the File menu Or 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 reproducibility Choosing the Print 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 imi Print 1D Evaluation GLP Pools1 tif Printer Kyocera Mita F5 1010 KX at Megi OK Edges Left 2 0crn Tap 2 0cm cm Header Right 2 0cm Bottom 2 0cm v Parameter Parameter and Image Options Iv Image whole image I overlays v Profile Curves portrait v grayscale W scales Iv Profile Table automatic size i W overexposed
36. Image Overlays Dialog Box Runlength Calibration Tab Using 1D Evaluation 19 a raytest Profiles Window The Profiles window is bipartite the upper pane displays the change of intensity over the length of the densitometer window integrated or averaged as selected over the width of the densitometer window the lower one contains a table listing the standards their given and recalculated values etc m Profiles ag test adf Delete Peaks Create Peaks Delete Profiles Profile 3 Delete Baselines Profile 2 Display Multiple Data Smoothing Tile Profile 1 Scales v 3d 9000 i Gnd Interpolated 8000 A f Profile Overlays v Extremal a es ALY B Change Profile Names Discret 2 5000 Aa PE E LL v Show Baseline E 4000 p zw A JA Hide Baseline 3000 PA Subtract Baseline w AL ZA O 1000 0 100 200 300 400 Pasition pixel Profile Peak Integral Bkg Integral Bkg Std Quantity Recalc Quantity QL QL QL ng ng 1 Profile 1 1 175938 65 45118 34 130819 71 100 00 100 6314 84837 71 sooo 48 8766 53376 06 24891 95 5 Profie5 1 4187664 27877 62 1399902 5 00 5 3633 6 Profile amp 1 1822419 1442275 3801 44 2 00 E Figure 2 11 Profiles Window Example Shortcut Menu 2 Graphics Pane Toolbar Result Table In the graphics pane
37. L Minirmurn Logarithmic Automatic Figure 2 42 Data Scaling Dialog Box Quantity Calibration The Grid command opens the Grid dialog box which allows you to lay grids over both axis that are an extension of either the big or the small ticks on the axes SSS 05 Vertical Mone C Big Tics C Small Tics Harizantal Mone C Big Tics C Small Tics Cancel Help Figure 2 43 Grid Dialog Box The table in the lower part of the Quantity Calibration window provides its own contextual menu Using 1D Evaluation 43 The column headings are equipped with the standard contextual menus described in the Profiles 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 Integral Area Bkg Std Conc PSL mrr dpm cnr BD4 3 1107000 Select Columns Delete Standards Figure 2 44 Quantity Calibration Window Row Contextual Menu Columns Selection X Show Columns Hide Columns Profile Peak Rel Deviation Integral Bkg GL Deviation Std Quantity Difference Recale Quantity cancel Figure 2 45 Selecting Columns to be Displayed 44 AIDA 1D Evaluation User 5 Manual Windows The following columns are available Table 2 3 Columns Profile Peak Name Integral Bkg signal unit Std Quantity
38. PY 60 Quantity ng Last Changed Thursday May 25 2000 1 38 17 PM Aida Development Version 3 10 028 Page 5 of 6 Thursday March 15 2001 11 29 02 AM 1D Evaluation Page 5 of 6 amp Program Filesvaytest Aida Image Analyzer dev 3 10 028 Examples Pools2 adf Runlength Calibration 1000 2000 3000 4000 5000 6000 7000 8000 3000 10000 11000 12000 13000 Runlength bp Position Std Runlength Recalc er bp feast T siseas _evoe 5529 esr ef seasea soso saa a FERAE a a em Last Changed Thursday May 25 2000 1 35 19 PM Aida Development Version 3 10 028 Figure 2 89 Preview Example Part 2 80 AIDA ID Evaluation User 5 Manual Windows Export and Import In AIDA 1D Evaluation 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 button on the toolbar to open the Export Template dialog box Ear Figure 2 90 Main Toolbar Export Template Button 2 Type a name for the export file in
39. The percentage of the peak integral in comparison to the Sum or to the value in the row which is Set to 100 94 if defined The percentage of the peak integral reduced by the background intensity in comparison to the Sum or to the value in the row which is Set to 100 if defined 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 quantity unit and runlength unit unit of measurement specified by the user through quantity and runlength calibration respectively Using 1D Evaluation 31 Table 2 1 Columns Available in the Result Table of the Profiles Window Column Short Description Std Quantity quantity unit The quantity of the peak as entered for the calculation of the standard curve Recalc Quantity quantity unit A calculation of the peak quantity based on the current calibration curve Recalc Quantity 94 The percentage of the peak quantity in comparison to the Sum of all peak quantities or to the value in the row which is Set to 100 94 if defined Std Runlength runlength unit The runlength standard value of the peak as entered Depending on the method for the runlength measurement this is the standard for the RF value Fragment Size Molecular Weight or pI Value Recalc Runlength runlength unit A calculation of the peak runlength based on the current runlengt
40. ain 2 Choose Delete Peaks from the contextual menu T e a n E Wl Profiles Pools2 adf B x rog 600 500 400 k 300 o a 200 reate Peaks 100 10 Delete Profiles Intensity GL Delete Baselines Select Profiles 4 Display ar Data Smoothing Scales Grid Profile Overlays 4 Figure 2 20 Selecting peak and Opening Contextual Menu A dialog box opens prompting you to confirm the command Using 1D Evaluation 21 3 Click OK to delete the selected peak m Profiles Pools2 adf E B x Delete Pools adf x Delete selected peaks Cancel Intensity GL Figure 2 21 Confirming Deletion of Peak Alternatively you can delete peaks by first selecting the peaks in the Image window and pressing the DEL key on your keyboard 28 AIDA 1D Evaluation User 5 Manual Windows Toolbar of the Profiles Window Select Profiles Back Button Smooth Width Button Forward Button Subtract Baseline Button Multiple Button Hide Baseline Button Tile Display Button Show Baseline Button 3D Display Button Normalize Button Figure 2 22 Profiles Window Toolbar Using the Select Profiles button you can select any profile by its name With the arrow buttons next to the Select Profiles button you can browse forward or backwards through the available profiles The three buttons in the middle provides
41. allowing you to set further option for the export of the result table Runlength Calibration Export Options bs X Range Ok M Header Cancel i All Rows Selected Rows il AllColumns Selected Columns Decimal Delimiter f Point List Delimiter Tabulator C Comma System Defaults Figure 2 98 Runlength Calibration Export Option Dialog Box C Comma C Semicolon You can specify the Range of the exported rows Header only All Profiles or Selected Profiles only 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 86 AIDA ID Evaluation User 5 Manual Windows 3 Complete the action by clicking OK The data are saved to a simple ASCII text file Exporting Audit Trail To export Audit Trail data 1 On the File menu choose 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 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 Importing Templates To import previously saved templates to another equivalent image 1 On the File menu click the Import menu item and then choose Templates from the submenu 2 In the Import
42. an be drawn Arbitrary in any angle Horizontal or Vertical Expand Lanes to Edges allows the densitometer windows to go from top to bottom of the image Show Profile opens the Profiles window directly after the densitometer window has been drawn If the Show Drag option is turned on the profile of the currently dragged densitometer window is continuously updated in the miniaturized profiles window of the toolbox Delete Marked Lane Button Use this button to delete a previously marked lane Profile Width Determination Tool This tool is used to enter the width of the densitometer window in the currently selected unit of measurement pixels cm or inch The width should be selected to enclose the entire lane without too much extra space Figure 2 56 Determinating Profile Width Search Lane Tool Clicking this button activates the lane search mode The above mentioned options Horizontal Vertical and Expand to Edges can be selected from the adjacent Lane Searching Parameter drop down menu f Hf vertical k horizontal wv vertical expand to edges Figure 2 57 Lane Searching Parameter Drop Down Menu 54 AIDA 1D Evaluation User 5 Manual Windows The miniaturized profiles window allows you to display the Intensity Summation the Averaged Intensity and the Optical Density for a selected lane To choose the desired graph use the Profile Type Selection drop down menu on top of the diagram intensity summation Lu
43. ation Export Options Dialog Box C Comma C Semicolon You can specify the Range of the exported rows Header only All Profiles or Selected Profiles only 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 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 Exporting Runlength Calibration To export Runlength Calibration Data On the File menu click the Export menu item and then choose Runlength Calibration from the submenu 2 In the Runlength Calibration Export dialog box that appears type a name for the export file and specify the data format To export the graph of the Runlength Calibration window e Choose Windows Metafile Using 1D Evaluation 85 Runlength Calibration Export Pools2 adF ajx Save in Examples 4 r File name Save as type Windows Metafile Cancel Windows Metafile Table ASCII File cu Figure 2 97 Runlength Calibration Dialog Box Graph Export To export the result table of the Runlength Calibration window 1 Select the Table Ascii File data format 2 Click Save to confirm the export The Runlength Calibration Export Options dialog box appears
44. collected in a comprehensive printout With AIDA 1D Evaluation it is usually preferable to select all parameters that are necessary for an automatic detection of lanes background and peaks first with one example lane When all parameters are well chosen further lanes can be automatically selected and assigned The detected bands can than be normalized by entering the values for the standard lanes quantification and migration distance standards To begin with the evaluation of lanes it is important to set the Image Attributes via the Options menu first see AIDA Image Analyzer Basic Concepts and Features User s Manual for details If these settings are not correct further image evaluation will not result in meaningful results Image Attributes Ponls2 adf X Image Interpretatian Emission x Cancel Dark signal on white background Figure 3 1 Image Attributes Dialog Box When you use the Lane amp Peak Determination toolbox for an application the first time various parameters should be set 87 1 Activate the Lane Searching function A Lane amp Peak Determination Lane Determination U arbitrary b Bi lane width 572mm FF Ki erc ibi sensitivity medium B Figure 3 2 Activating Lane Searching 2 Click to the upper left corner of your lane hold down the mouse button and drag a rectangle to the lower right of the lane 3 Release the mouse button AIDA will search for the lane
45. ecified standard values are displayed in the List of Standard Values pane below the drop down menu Delete Standard Assignment Button Using this button you can delete selected standard assignment s Using 1D Evaluation 59 Edit Standard Button Use this button to enter and edit predefined standards Clicking this button opens the Quantity Standards dialog box DNA ladder DNA ladder Unit Precision Default Add Modify Delete OK Cancel Figure 2 65 Quantity Standards Dialog Box In the Quantity Standards dialog box the Standard name and Values are entered and the dimension of the amounts can be selected The Add Modify and Delete buttons are used to alter the list of standards All standards defined 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 60 AIDA 1D Evaluation User 5 Manual Windows Calibration Curve Pane In the Calibration Curve pane of the Quantity 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 Logarithmic Regression Iv 0 0 origin C ng Corr Coeff f 0 999882 Warning bkg not determined Figure 2 66 Calibration Curve Pane The Calibration Curve drop down menu provides the following functions
46. ected Figure 2 4 The Delete Profiles and Delete Peaks commands are only executed for the selected lane s and selected peaks respectively Using 1D Evaluation 15 With the Duplicate Profile commands you can insert another profile identical to the currently selected one Edit Profile allows you to reshape the currently selected profile The currently selected peak area band can be re defined with the Edit Peak command The Image Overlays command opens the Image Overlays dialog box described below Change Profile Name is used to change the name of the currently selected lane Set Profile Width is used to adapt the width of the densitometer windows drawn around the lanes Flip Profiles changes the densitometer orientation upside down and Expand To Edges expands the densitometer window beyond its narrow sides over the entire length or width of the image If no overlay 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 Image Overlays You can specify the label type and font you want to apply to the overlays lanes and bands using the Image Overlays command of the Options menu Options View Window Hi Image Attributes Image Overlays Preferences Automatic Export Configuration Path Image Search Path Aubosaving Overlay Colors User Registration
47. el _ Figure 2 76 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 v first save v first clase Iv explicit stop Figure 2 77 Finish Evaluation Pane If you click First Save the settings of the Changes 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 1D Evaluation 69 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 Profiles Quantity Calibration Runlength 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 Data Trailing Pane After starting an evaluation the controls of the Data Trailing pane are enabled Data Trailing View Audit Trail Stop Evaluation FCB check sum not proofed Evaluation is in p
48. ent 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 if yes to which extent configuration information should be printed Include Positive Trail Configuration Include Complete Trail Configuration Preview As for printing in AIDA there are two ways of previewing pages before they are actually printed Using the Print Preview option of the File menu or Using the Preview button on the Publishing Layout toolbox The Print Preview option 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 those selectable from the various panes of the Publishing Layout toolbox However they are not stored with the data file 78 AIDA ID Evaluation User s Manual Windows 5 SSA r iced 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 since all layout setting are already made through the Print Protocol pane EE Aida 1D Evaluation Preview Ag test adf File Edit Evaluation Options View Window Help gt Close Page 1 of
49. entioned in the Values was unassigned in the image so that the following band would get a wrong value 3 The procedure should be repeated for all other standard lanes After all standard lanes are assigned the normalization lines show the distortion effect in the current application The fragment size of all assigned bands will be automatically deduced and receive a label indicating the result The exact representation in the image depends on the Image Overlays settings see the Contextual Menu of the Image Window section above You can print the image as well as the standard curve that was used to evaluate the run length data a hyperbolic regression in this case using the Publishing Layout toolbox controls as described in the respective section above The evaluation of other physical parameters by the run length of samples in SDS polyacrylamide gels kDa isoelectric focusing gels pI and thin layer chromatography RF values can be performed analogously 98 AIDA 1D Evaluation User s Manual Windows Assigning Quantitative Calibration Standards As detailed in the introduction to this manual the scientific relevance of the following quantification process has to be validated to obtain meaningful results The example presented below is the fluorescence of ethidium bromide stained DNA It has been established that the fluorescence resulting from ethidium bromide staining 1s proportional to the DNA amount within certain ranges
50. gs to List Delimiter and Decimal Delimiter 4 Complete the action by clicking OK The data are saved to a simple ASCII text file Exporting Quantity Calibration To export Quantity Calibration Data On the File menu click the Export menu item and then choose Quantity Calibration from the submenu 2 In the Quantity Calibration Export dialog box that appears type a name for the export file and specify the data format Quantity Calibration Export Ag Eesk adf Save in a Examples e t File name Ag test cal Save as type ADA Calibration Cancel Windows Metafile Table ASCII File AIDA Calibration k Figure 2 95 Quantity Calibration Export Dialog Box To export the graph in the Quantity 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 Quantity Calibration window 1 Select the Table Ascii File data format 2 Click Save to confirm the export 84 AIDA 1D Evaluation User 5 Manual Windows The Quantity Calibration Export Options dialog box appears allowing you to set further option for the export of the result table Quantity Calibration Export Mans X Range Header Cancel 6 All Rows Selected Rows Cancel amp All Columns Selected Columns Decimal Delimiter f Point List Delimiter Tabulator C Comma System Defaults Figure 2 96 Quantity Calibr
51. h curve Depending on the method for the runlength measurement this is the standard for the RF value Fragment Size Molecular Weight or pI Value 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 quantity unit and runlength unit unit of measurement specified by the user through quantity and runlength calibration respectively The following additional rows can be found or created exclusively in the Region Report Table 2 2 Additional Columns in the Region Report Sum Displays the scientifically correct sum for the selected column Ttl Total sum over the entire profile Remainder Ttl Sum Numbers in italics indicate that the value was interpolated over the end of the defined standard curve and is therefore only representing a tendency not a result When a field is not relevant for a profile a dash is displayed The numbers can be formatted as outlined below 32 AIDA 1D Evaluation User 5 Manual Windows 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 3 Release the mouse button Wl Profiles Pools2 adf Wl Profiles Pools2 adf Bib Profiles Pools2 adF Profile
52. he same graphic Using 1D Evaluation 73 To turn on printing of the result tables for the quantification and or evaluation of the migration data click to select the Table box Single Tables prints a separate table for each 1D densitometer All in one prints all results combined in a single table To Graphic prints the tables together with its associated densitometer graphic on a single page Print Options for Quantity Calibration The second additional button of the Publishing Layout toolbox is used to configure the layout setting for the Quantity Calibration window Fratacol Print Frint Preview Quantity Calibration Format Portrait Landscape Print W Graphic W Table Figure 2 683 Print Option for the Quantity Calibration Window As for the Profiles 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 curve Graphic and or the calibration table Table 74 AIDA 1D Evaluation User 5 Manual Windows Print Options for Runlength Calibration The last button of the Publishing Layout toolbox is used to configure the layout setting for the Runlength window in the Protocol Print pane Protocal Print Print Preview Format Portrait C Landscape Frint Iv Graphic C Single C Multiple i 3D Iv Table C Single Tables Allin One Tab
53. he session you can choose to log on as a different user instead of exiting from the program Close all and registrate with another name C Ewitthe Aida program Cancel Figure 1 8 Exit Aida Dialog Box 12 AIDA ID Evaluation User 5 Manual Windows Using 1D Evaluation 2 Data Windows in 1D Evaluation In addition to the AIDA standard data windows Image and Histogram the following data windows are available in the 1D Evaluation mode Profiles Quantity Calibration e Runlength Calibration Audit Trail You can access these data windows by choosing the appropriate entry Profiles Quantity Calibration Runlength Calibration and Audit Trail respectively from the View menu Mie Window Help Le image Histogram Profiles Quantity Calibration Runlength Calibration Audit Trail Figure 2 1 View Menu Image Window in 1D Evaluation All data are evaluated from the Image window Although it 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 EZ e Program Files raytest Aida Image Analyzer dev 3 20 01 7ExamplessPoolsz adf B xj LER i E ad 126 5105 San 4072 3054 O36 B35 ma Band s lab Recalc Runlength TERI A Lane s labels an 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 Expor
54. hould be very close to 1 for a good standard curve The calibration curve can be selected on the basis of theoretical considerations or simply to fit the observed situation To evaluate the accuracy of the fit of the curve you can check the difference between the quantity standard value and the recalculated standard value 5 x Linear Regression y 1 521x Correlation Coefficient r 1 0000 100 Integral Bkg a u 0 10 20 30 40 50 60 70 80 90 100 110 120 Quantity nq Figure 3 22 Quantity Calibration Curve Example Performing 1D Evaluations 101 Configuring the Evaluation Output Following the calibration process outlined above the entire image is calibrated and the results will be shown in the table pane of the Profiles window One or more profiles can be displayed at the same time as outlined in the Profiles Window part of this manual It is important to use the Migration Distance Normalization function ge to get peaks of identical size at comparable positions ial x P ANNAE Profile 2 pn S AI ue Ie uu M rrr LE p gt fa j LOW FRDR BX Figure 3 23 Migration Distance Normalization Example 102 AIDA 1D Evaluation User 5 Manual Windows
55. indow 1 0 0 0 0 ee 42 Contextual Menu of the Quantity Calibration Window 43 Runlength Calibration Window 0 0 ccc eee eens 46 Contextual Menu of the Runlength Window 0 000000 47 ZA dit Trak MWIndoo 4 ea vo eto doeet Dare eh ear Cue eta tre VERS 50 Additional Toolboxes in 1D Evaluation 0 0 0 0 00 51 Lane amp Peak Determination Toolbox 1 02 6 ee eee 51 Lane Determination Pane 244 vo34 43 8S CREE REP EEIex amp Xo Rs 53 Baseline D termination Pane iusserat e Ea n e RN E Rs 55 Peak Determination Pane uu hee ho ee PRR UU RRS E RR aS SA 56 Ouantity Calibration Toolbox 2er REG U E HERE VES 58 Standard Pane 9 23 td but odo EIS dS aul M aeu ede Pa deba 58 Calibration C UEVO Pale ous d Soy erm aos so Rate alat RR dade Sa 60 Migration Distance Toolbox llllssssslelleeee e 61 iii GLP Supporting Tools Toolbox ssllllleeeeee eens 63 Admita non Panes o cse eben E Oe uae ua Hn Neo ein a ee 64 Pata brat Sewines Patieisus cad Etha d ON te ELA ERA aped taces 65 LanishoEvaluatton Pane o bsc roca b rae Is RR HEAR CHE AS CU pen 68 A tomatic Export Pane aoo eet e wl b Ew Ve v ud 69 Data Traine Palieussoseossw setate ptis Corp ea oa hg Dainese uw dit 69 Publishing Layout Toolbox in 1D Evaluation 0 eee eee 71 Print Options Tor Profiles 244 9s Exe Ac R ENS RR ta eee See UP 72 Print Options for Quantity Calibration 0 0 0
56. indow To open the Runlength window choose Runlength Calibration from the View menu Just as the Quantity Calibration window the Runlength window is bipartite the upper pane displays the curve of the calibration function for the different lanes The graphical representation of the calibration function is shown for every lane The lower part of the window contains the Result Table of the run length calibration function Blnunlenath Pools2 adf Ioj x std 3 std 2 std 1 Position mm 2000 5000 8000 11000 I RHunlength bp Profile Peak Position Std Runlength Recale Hunlength mm bp bp 23 073 raf 3 asu ma one Tadif a 263048 9162 S127 radif ej 280737 mab 886 radif z aeza eoj 6 MTE NE NE 37 3375 Figure 2 46 Runlength Window Example Again the graph and adjacent table can be adapted by using the contextual menu For a more detailed view of the plot you can activate the AIDA standard zoom functions described above You can change the display ration between the graphics and the table pane by dragging the delimiter bar The standard curves of non standard lanes are interpolated from the evaluated standard lanes This requires multiple standard lanes on the gel that are correctly assigned 46 AIDA ID Evaluation User s Manual Windows Contextual Menu of the Runlength Window Right clicking on the Runlength window s graphics pane opens a contextual menu providing you with commands
57. indows default settings to List Delimiter and Decimal Delimiter 3 Complete the action by clicking Ok The data are saved to a simple ASCII text file 82 AIDA 1D Evaluation User 5 Manual Windows Exporting 1D Report Table To export the 1D Report Table 1 On the File menu click the Export menu item and then choose 1D Report Table from the submenu 2 In the 1D Report Table Export dialog box that appears type a name for the export file 1D Report Table Export Panls2 adf i A x Save in C Examples e ct Be ww r Save az type Table ASCII File Cancel Figure 2 93 1D Report Table Export Dialog Box 3 Click Save to confirm the export The Profiles Export Options dialog box appears allowing you to set further option for the export of the result table Profiles Export Options K X Range M Header Cancel 6 All Rows C Selected Rows AllColumns Selected Columns Decimal Delimiter C Point List Delimiter Tabulator Comma System Detaults Figure 2 94 Profiles Export Options Dialog Box 1D Report Table Export C Comma C Semicolon Using 1D Evaluation 83 Qe You can specify the Range of the exported rows Header only All Profiles or Selected Profiles only 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 settin
58. is activated to obtain migration distance analysis for them Using 1D Evaluation 57 Quantity Calibration Toolbox The controls of the Quantity Calibration toolbox allow you to perform quantity calibrations of 1D densitometers by assigning amount standards to the bands L7 Quantity Calibration Standard Juser defined Standard pane Calibration Curve Logarithmic Regression IY 0 0 origin Calibration Curve pane Di Apo ng Carr Coeff r2 0 999882 Warning bkg nat determined Figure 2 63 Quantity Calibration Toolbox Overview To display the Quantity Calibration toolbox e On the main menu click Evaluation and then click Quantity Calibration Alternatively you can select the Quantity Calibration button on the toolbar 58 AIDA ID Evaluation User 5 Manual Windows Standard Pane L7 Quantity Calibration standard Standard Selection Drop Down Menu he st a 100 List of Standard Values AutoAssign Tool Manual Assignment Tool Delete Standard Assignment Button Edit Standard Button Figure 2 64 Standard Pane Overview Manual Assignment Tool This button allows you to assign amount standards manually to the bands by entering appropriate values via a dialog box AutoAssign Tool Use this button to semi automatically apply Standard values to bands one by one using a predefined list The predefined list can be selected from the Standard Selection drop down menu The sp
59. istrator Comment Fited account for the administration of the user database Figure 1 4 AIDA Registration User Management Dialog Box Introduction 9 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 Jahn Dae 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 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
60. le Tao Graphic Figure 2 64 Print Options for the Runlength Calibration Window As for the Profiles 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 runlength calibration curve s Graphic checkbox Clicking Single shows each runlength calibration curve in a separate graphic Multiple shows all curves on top of each other in a single graphic and 3D shows all curves in a 3 dimensional fashion within the same graphic To turn on the printing of the runlength calibration data click to select the Table box Single Tables prints a separate table for each calibration data set All in One prints all results combined in a single table Using 1D Evaluation 13 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 Protocal Print Print Preview Audit Trail Format Portrait C Landscape Frint W Configuration complete lists C positive lists W Data Trailing Figure 2 85 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
61. le Peak Tine Abs Pos mm Std A Intensity GL 0 0 20 30 40 50 F Position mm Select Profiles EE Figure 2 24 Changing the Display Ratio Between Graphics Pane And Result Table 30 AIDA ID Evaluation User 5 Manual Windows Data Description Available Columns in the Result Table Table 2 1 Columns Available in the Result Table of the Profiles Window Column Profile Peak Type Name Rel Pos length unit Abs Pos length unit Start Pos length unit End Pos length unit Width length unit Height signal unit Integral signal unit Bkg signal unit Bkg 76 Integral Bkg signal unit Integral 9 6 Integral Bkg 96 Short Description The index and name of the profile The number of the peak The type of the peaks e g QStd for Quantity Standards RStd for Runlength Standard The name of the peak as given by the user The position of the peak in the profile The position of the band in the image measured from the edges of the image The start position of the peak in the profile The end position of the peak in the profile The width of the peak The maximum height of the peak The integrated peak intensity The integrated background intensity The percentage of the peak background in comparison to the Sum of all peak backgrounds or to the value in the row which is Set to 100 if defined The background corrected integral of the peak
62. mand you can re name the densitometer windows and the resulting profiles according to your preferences The names will appear in all result tables and image menus Profile Name Pools1 adf X Set name of 4 profile to EM Figure 2 33 Profile Name Dialog Box Using 1D Evaluation 37 Contextual Menu of the Profiles Window Result Table Each column in the Result Table has a contextual menu attached To open the contextual menu right click on the column heading gray area ET Es A Profile Peak Type Abs Pos mm Std Aunlength bn Recalc Runle Sort ascending by amp bs Pos sx Sort descending by Abs Pos Select Abs Pos 1 std 1 4 R amp 5td Hide Abs Pos Settings of Abs Pos tsd B RStd Twa lined Header Select Columns Aadi std 1 8 RStd 4 UD tsat f sf std 34 4072 4 Figure 2 34 Contextual Menu of the Profiles Window s Result Table With the first two commands of the contextual menu you can sort the table of the Result Table 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 to export The column heading of selected columns are highlighted To hide a column choose Hide command from its contextual menu You can recover hidden col
63. oolbox This toolbox is used to enter the values for the bands in the standard lanes The resulting calibration curves are subsequently used for the determination of unknown bands SH Migration Distance Direction Vertical Lanes Method Fragment Size Values BRL KB 10120 Apply Button 4169 AutoAssign Button 8144 7126 Manual Assignment Button 6108 5090 Delete Assignment Button 3e 4072 3054 3 2036 Edit Standard Button Calibration Curre Linear Interpolation pixel Figure 2 67 Migration Distance Toolbox Overview The Direction drop down menu is used to select the lane type used for the calibration Vertical Lanes or Horizontal Lanes 62 AIDA ID Evaluation User 5 Manual Windows The Method drop down menu is used to select the Migration Distance determination method RF Value Fragment Size Molecular Weight or PI Value Choosing one of this methods will call predefined standard lists and calibration curve functions associated with the selected method The predefined standard is selected from the Values drop down list The buttons on the left side of the standard value window are used to assign the underlined value in the list to an assigned band in the image or in the profile by clicking on it Apply Button This button applies the current value to a band AutoApply Button This button assigns the currently underlined value to the clicked band and all values above and bel
64. ore elaborate standardization method should be employed It is suggested to include at least two standard lanes one on each side of the sample and preferably a third one in the middle of the gel This way the distortion of migration distance can be taken into account see horizontal equimigration lines and the most accurate results can be produced Figure 3 16 Migration Distance Example 96 AIDA ID Evaluation User 5 Manual Windows When all bands have been assigned the Migration Distance toolbox can be used to assign the standards mi Migration Distance Direction Apply Button AutoAssign Button Manual Assignment Button Delete Assignment Button Edit Standard Button Calibration Curve Logarithmic Interpolation bp 1 Select the Method and Values of the standard Figure 3 17 Migration Distance Toolbox All methods are automatically linked to the most suited evaluation function for the Calibration Curve However different functions can be selected to obtain a better fit of the calibration curve to the actual data You can automatically assign all values from the List of Standard Values new standard lists can be easily edited to the assigned bands 2 Activate the AutoAssign tool and click on the band corresponding to the highlighted value on the list Performing 1D Evaluations 97 All other bands will be assigned accordingly It should be checked that no band m
65. ow to the bands that are marked above and below of this band resulting in an automatic assignment of all standard bands at ones Manual Assignment Button This button is used to manually assign a value that is not part of a predefined standard to a clicked band Delete Assignment Button This button deletes all selected standard assignments Edit Standard Button This button is used to edit predefined standards After clicking this button a dialog box appears allowing you to add modify or delete standards Only standards for the currently selected Method in the Migration Distance toolbox can be set in the Standard pane Click the Modify button to get access to the currently selected scheme Click Delete to erase the current standard value list or Add to enter a new list of standard values The Standard name Values Unit Precision digits after the comma and Default Value the selected default value in the list can be entered The Default Value should be set to the value of the band that is easiest to spot in the standard lane In the lower part of the Migration Distance toolbox the Calibration Curve function is selected The method of standard curve determination can be choosen from the predefined list Usually the default function works best The following functions are available depending on the selected Method None this option will shut off the background calibration e g if this function 1s too time consuming during editing of the
66. pI Value A calculation of the peak runlength based on the current runlength curve Depending on the method for the runlength measurement this 1s the standard for the RF value Fragment Size Molecular Weight or pI Value The difference without sign between the entered standard and the recalculated value The relative difference without sign of the entered standard and the recalculated value compared to the standard value 7 Difference Std Runlength length unit unit 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 runlength unit unit of measurement specified by the user through runlength calibration Using 1D Evaluation 49 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 zi Audit Trail c Program FilessraytestAida Image Analyz ar d ev m 1 Friday March 2b 2004 12 24 25 01 00 System Administrator Administrator al 10 Evaluation GLF evaluation started Figure 2 51 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 on the Da
67. r 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 8 AIDA 1D Evaluation User 5 Manual Windows 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 actrzate the user management of this program From now on the program will require a user login an each start Please enter an administrator password for the user management Be sure nat to Forget this password Password us Ium Confirm password Cancel Figure 1 3 Entering Administrator Password 3 Click OK to apply the changes Now the user registration 1s 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 Modi Delete User informations Full name System Administrator Pemmigsion Admin
68. rocess 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 70 AIDA ID Evaluation User s Manual Windows 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 ker Evaluation FCB check sum not proofed Evaluation is finished by explicit stop Figure 2 80 Data Trailing Pane Evaluation Aborted by User Using 1D Evaluation 71 Publishing Layout Toolbox in 1D Evaluation In AIDA 1D Evaluation the standard AIDA Publishing Layout toolbox is expanded by four additional buttons for specifying the print options for the Profiles Quantity Calibration Runlength Calibration and Audit Trail Profiles Options Button Quantity Calibration Options Button Runlength Calibration Options Button Audit Trail Options Button Publishing Layout Annotate Z ai
69. s Ponls2 adf SelectProfiles 4 Select Profiles Select Profiles 0 Select Profiles Gaerne te Profile Select Profiles Type Profile Ty 2 AStd sd3 Red 2 std 3 Fistd 5 std 3 2 RStd 5 43 2 Rsu 5 43 2 Reu std 3 Figure 2 25 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 EX Profiles Pools2 El ProrilesPoolsz ll WI Profiles Pools2 Select Profiles Select Profiles select Profiles Pe Profile Peak Typ ps3 2 2 ps3 2 2 xsd3 a 5 std 3 2 RSI Figure 2 26 Changing Column Width Using 1D Evaluation 33 Contextual Menu of the Profiles Window Graphics Pane Right clicking on the Profiles window s graphics pane opens a contextual menu providing you with commands for deleting creating peaks deleting profiles baselines as well as several display settings see below i a Delete Peaks Create Peaks Delete Profiles Delete Baselines Display k Multiple Data Smoothing Tile Scales wv 3d rid os Ter prolate Profile Overlays wv Extremal INE MEN Ins Change Profile Mames wv Show Baseline Hide Baseline Subtract Baseline Figure 2 27 Contextual Menu of the Profiles Window s Graphics Pane From the submenu
70. s by their respective companies THIS PUBLICATION IS PROVIDED 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 MAKEIMPROVEMENTS AND OR CHANGES IN THE PRODUCT S AND OR THE PROGRAMS S DESCRIBED IN THIS PUBLICATION AT ANY TIME Contents 1 Introduction 5 Dad ASE EEE TEE T EE EEE E E E EOE E EEE 5 Starine ID ByaliallOf ic ooa dO S dere Roche e i eS tma 7 Create User Reci stano ao bates dain das ed eue d eara bu a V EAE A T7 2 Using 1D Evaluation 13 Data Windows in ID Evaluation llleleeeeeeeeee e 13 Image Window in 1D Evaluationt 0 0 000 000 cc eee eee 14 Contextual Menu of the Image Window 0 0 c eee eee eee 15 Miage Overlay sabe as pt ae Gh edes dex dete m dotada d bere up etd 16 Probes M INdO Wor ss See oS 28 h45 SRE PRATER SASS ADORE EASE e onn i s 20 Toolbar of the Profiles Window us ves ER CER GS 29 Data Descnpuon 4 3654462 EORR REQUE sone bie eee Gee ds 31 Contextual Menu of the Profiles Window Graphics Pane 34 Contextual Menu of the Profiles Window Result Table 38 Quantity Calibration W
71. samples will also make quantitative estimations safer To comply to GPL standards AIDA 1D Evaluation provides record keeping features in addition to its 1D Evaluation features All changes to a previously performed evaluation can be recorded and are saved to a AIDA Lock File alf The system administrator can fully configure the behavior of the GLP supporting tools requiring for example to prompt the user to explicitly account for all modification of existing evaluations 6 AIDA ID Evaluation User 5 Manual Windows Starting 1D Evaluation To select the 1D Evaluation mode 1 On the toolbar click the Evaluation button A dialog box appears offering you a list of available evaluation modes le Change Evaluation Mode E X Available Evaluation Modes 1D Densitometry 1D Densitometr 1D Evaluation PRESE MMC 4 Cancel 1D Multi Labeling 1D Runlength Determination 1D Runlength Determinatian 1D Thin Layer Chromatography 20 Densitometry 20 Densitometr GLFP 20 Multi Labeling 20 Thin Layer Chromatography Annotation Array Matrix Crystallagraphwy Figure l 1 Changing Evaluation Mode The list entries reflect the installed AIDA application modules 2 Select the 1D Evaluation list item und click OK Note This evaluation mode is only available if you purchased the AIDA 1D Evaluation module Note further that there are two possible 1D Evaluation evaluation modes in AIDA 1D E
72. t Image section in the AIDA Image Analyzer Basic Concepts and Features User s Manual for further explanation 14 AIDA 1D Evaluation User 5 Manual Windows Contextual Menu of the Image Window In 1D Evaluation the contextual menu of the Image window looks slightly different depending on the type of overlay you have chosen Lane Selection If you have selected one or more lanes the contextual menu that opens after right clicking the respective object provides you with the possible commands for this specific selection These include commands for editing and labeling profiles lanes Duplicate Profile Edit Profile Delete Peaks Edit Peak Image Overlays Change Profile Mame Set Profile width Flip Profile Expand To Egdes Contextual Menu Lane Selected Figure 2 3 Band Selection If you have selected a single band the contextual menu that opens after right clicking the respective object provides you with the possible commands for this type of selection These include commands for editing and labeling profiles lanes as well as commands for editing peaks bands Note that 1f you select multiple bands the peak editing command of the contextual menu is turned off Ti Delete Profile Duplicate Profile Edit Profile Delete Peaks Edit Peak Image Overlays Change Profile Mame Set Profile Width Flip Profile E Expand To Egdes j Contextual Menu Band Sel
73. t point and drag the densitometer to the end of the lane m f tt rnm Figure 3 10 Manual Lane Marking 1 92 AIDA ID Evaluation User 5 Manual Windows c Double click on a position in the frame to add a handle which can be moved independently enabling curved densitometer windows Figure 3 11 Adding Handles 9 Optimize search parameters for lanes background and peaks with a representative lane 10 Activate the Apply To Lane Determination function for all parameters to turn on automatic detection during the next lane assignment Feak Determinatian position hy maximum E search level Z g smoothed 1 pixel g Figure 3 12 Enabling the Apply to Lane Determination Option Performing 1D Evaluations 93 11 Detect all lanes by opening a rectangle over each lane one by one in the search mode i Er e Promqeam Files sraytest Aida Image Analyzer dewv 3 LU D2B Examples pools lif Lu JIBIEE Er e Program Files sraytest Aida Image Analyzer dewv 3 LU 2B Examples pools ttf cono c TE suas NI ELTIF JH ml v oid Wied t E Ug 080 eris LL es Yd LI Mai Pom aosd 1 quuni dod dn Automatic Detection of All Lanes Figure 3 13 AIDA ID Evaluation User s Manual Windows 94 12 Delete peaks of no interest by opening a rectangle over them in the selection mode and pressing the DEL key
74. ta Trailing panel of the GLP Supporting Tools toolbox 50 AIDA ID Evaluation User 5 Manual Windows Additional Toolboxes in 1D Evaluation In addition to the standard AIDA toolboxes Display Control and Publishing Layout the following toolboxes become available after selecting the 1D Evaluation mode Lane amp Peak Determination Quantity Calibration Migration Distance e GLP Supporting Tool You can access these additional toolboxes from the Evaluation menu or by clicking the appropriate button on the toolbar Evaluation Options View Wine Display Control Publishing Layout Lane amp Peak Determination Quantity Calibration Migration Distance w GLP Supporting Tools Figure 2 52 Mapping between Evaluation Menu Entries and Toolbar Buttons Using 1D Evaluation 5 Lane amp Peak Determination Toolbox The controls of the Lane amp Peak Determination toolbox allow you to define lanes profiles and peaks on the image for signal intensity evaluation J Lane amp Peak Determination Lane Determination U arbitrary bj Bi iane width 16mm G vertical hi sensitivity medium B intensity summation hd Lane Determination pane Baseline Determinatian SEED LETS 0 Baseline Determination pane smoothed Opixel g apply to lane determination Peak Determination position hy maximum E br allow overlap vi search level g smoothed Opixel E apply to lane determination
75. tensity optical density Figure 3 19 Setting the Intensity Evaluation Method It should be noted that integration over the entire densitometer window should be done in sufficiently large densitometer windows to accommodate the entire band Averaging can be performed best with densitometer windows that cover only the majority of the bands and omit empty space around them 2 Assign bands representing a known quantity as Standards For Calibration By Selecting The Manual Assigment Tool from the Standard controls of the Quantity Calibration toolbox and click on the appropriate band in the image or the peak in the profile L7 Quantity Calibration Standard Test 0 100 0 50 1 25 Manual Assignment Tool 1 410 Figure 3 20 Manual Assigment Tool Quantity Calibration Toolbox 100 AIDA 1D Evaluation User 5 Manual Windows 3 Enter the value and the dimension in the Quantity Calibration dialog box that appears Set standard of 11 peaks in 1 profile Profile 1 Cancel Standard Clear Figure 3 21 Quantity Calibration Dialog Box 4 After all standards are entered select the appropriate Calibration Curve type that fits the data and application 5 For most applications with properly assigned background it might be advisable to select the 0 0 Origin option so that the standard curve goes through the origin 6 Check the fit of the standard curve The r value gives a clue about the fit it s
76. the migration distance of the samples is compared to that of standards on the same image It is very important to understand and acknowledge the function of these evaluations to prevent pitfalls that will lead to results probably not reflecting the actual situation Thus some suggestions for practical considerations for the sample preparation and image capturing will be given at the various evaluation section In general it should be noted that all comparisons of samples to standards should be validated Thus it is for example not a good idea to evaluate the amount of protein A by using a standard of protein B because in general various proteins stain different in coomassie and silver stains This is also true for western blots of different proteins However the amount of the same protein in different lanes can be evaluated On the other hand the evaluation of the fluorescence signal of ethidium bromide stained DNA samples can be compared between any sample with acceptable variations around 5 9o Also the evaluation of radioactive binding e g in southern northern or dot blots yield accurate results It should also be kept in mind that the evaluations get more accurate with more standard values Standards should be well scattered around the intensities or migration positions that are going to be examined When in doubt it might be a good idea to check the reproducibility of known results Including and evaluating two fold dilutions of
77. tings If you are logged in as normal user the GLP Supporting Tools toolbox is considerably reduced providing you with the Data Trailing controls only HJA A 1 je GLP GLP Supporting Tools Data Trailing View Audit Trail Stop Evaluation Figure 2 69 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 Iv apply GLP support proof PCA check sum Reason List Figure 2 70 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 Select Apply GLP Support 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 1D Evaluation 65 The Reason List button opens the Audit Trail Reason List dialog box for predefining evaluation change reasons x Modity it Delete eu Figure 2 71 Audit Trail Reason List Dialog Box Data Trail Settings Pane The behavior of the GLP recording function 1s configured using the Data Trail Settings for controls Data Trail Settings for Evaluation Changes Afterwards Figure 2 72 Data Trail Settings Pane 66
78. umns using the Select Columns command see below 38 AIDA ID Evaluation User 5 Manual Windows 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 Settings Abs Pos X Decimal digits i i E OK Cancel Figure 2 35 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 Result Table 1 Choose Select Columns A dialog box appears displaying two lists Show Columns Hide Columns 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 1t again Columns Selection k X Show Columns Hide Columns A Std Runiength bp Recale Runlength bp Recale Quantity 95 Integral Bkg 9 5 Height GL Figure 2 36 Columns Selection Dialog Box Using 1D Evaluation 39 In addition each row of the table has a contextual menu attached which includes commands for displaying special row types like Sum Total Remainder as well as for changing profile names iol EXITUS 3 i Frofile Peak Type Abs Pos mm Std Hunlenath bp Hecalc Rune 1 std
79. valuation one with GLP features indicated by the acronym GLP in parentheses and the other without those functionality The 1D Evaluation GLP evaluation mode is only available after establishing access control by applying the user registration see the Create User Registration section below Introduction 7 Q O 1y 87 BICE raytest 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 1s a fixed account in AIDA which cannot be changed or deleted When no 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 xj Please enter user name and password User name Administrata Password Canes Figure 1 2 AIDA Registration Login Dialog Box To activate the user registration in AIDA the first time you must enter a password fo

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