Home

AIDA 1D Evaluation User's Manual (Windows)

1. 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 Scaling 1 antity Minimurn Maximum FE Logarithmic Automatic Sancel Integral Bkg AL Minimum M 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 Horizontal None C Big Tics C Small Tics Cancel Help None Big Tics C Small Tics 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 39 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 c
2. Intensity aL 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 24 AIDA ID Evaluation User s Manual Windows 2 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 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 respective
3. AIDA ID Evaluation for Windows User s Manual Band s lab Recalc Runlength TA C KE Lane s labels Part Number 2003 01 1 1 2003 raytest Isotopenmebger te GmbH Benzstr 4 D 75334 Straubenhardt Germany All rights reserved This product or document is protected by copyright and distributed under licenses restricting its use copying distribution and decompilation No part of this product or document may be reproduced in any form by any means without prior written authorization of raytest and its licensors if any RESTRICTED RIGHTS LEGEND Use duplication or disclosure by the government is subject to restrictions as set forth in subparagraph c 1 11 of the Rights in Technical Data and Computer Software clause at DFARS 252 227 7013 and FAR 52 227 19 The product described in this manual may be protected by one or more U S patents foreign patents or pending applications TRADEMARKS AIDA AIDA Image Analyzer AIDA Annotation AIDA AIDA Array Metrix AIDA Array Compare AIDA Multi Labeling AIDA 1D Evaluation AIDA 1D 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 rayt est in the 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
4. Preview button opens the Preview window directly since all layout setting are already made through the Print Protocol pane Aida 1D Evaluation Preview Ag test adf File Edit Evaluation Options View Window Help lt Close Page 1 of 3 1D Evaluation e Program Filesraytest amp ida Image Analyzer dev 3 10 028 Examples Ag test adf Parameter Thursday March 15 2001 11 14 34 AM Page 1 of 3 mage Tk esProgran FiksvayestAkia mage Arahzer cev3 10 025 Erampks ay estttt Digital resolutiva 256 Sbitper phe I Epos ure dae Tresday December 09 1997 8 1208 PM Image ste 752 582 376 2 91 mage resolttion 20ddpr ao0qp Commert Seketion 000 000 752 582 n Last Changed Thursday May 25 2000 1 38 17 PM Aida Development Version 3 10 028 Page 2 of 3 1D Evaluation eProgram Filesvraytestisida Image Analyzer dev 3 10 028 Examplesi4g test adf Profiles Thursday March 15 2001 11 16 20 AM Page 2 of 3 heast PY Position pixel hegral Brg WegrakBhy Sti ovaitty Recair Ovantty o py hal hal A AO a Ear ECO A E A IC E Tum E MO E sora E IC ETE fms oar amen ene 0 me rms MO EI sere em a prms fee as ere E E om Last Changed Thursday May 25 2000 1 38 17 PM Aida Development Version 3 10 028 Figure 2 73 Preview Example Part I 64 AIDA ID Evaluation User s Manual Windows DS SEE raytest Page 3 of 3 1D Evaluation Thursday M
5. The Direction drop down menu is used to select the lane type used for the calibration Vertical Lanes or Horizontal Lanes 56 AIDA ID Evaluation User s Manual Windows 2 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 below 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 M
6. Baseline Determination Edit Baseline Tool Remove Baseline Tool smoothed Opixel Auto Determine Baseline Tool search level T apply to lane determination Signal Smoothing Drop Down Menu Search Level Drop Down Menu Figure 2 59 Baseline Determination Pane Overview Edit Baseline Tool Clicking this button activates the Edit Baseline function If no baseline was determined yet a straight line is 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 can be set to various values reaching from 0 insensitive to 10 extremely sensitive 50 AIDA ID Evaluation User s Manual Windows 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 dl Peak Assignment Tool wam overlap Delete Peaks Button TEE E
7. Cancel Figure 2 84 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 T2 AIDA ID Evaluation User s Manual Windows 2 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 Al File name Jag test cal Files of type ADA Calibration Cancel Figure 2 85 Quantity Calibration Import Dialog Box Using 1D Evaluation 73 74 AIDA ID Evaluation User s 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 collected in a comprehensive printout With AIDA 1D Evaluation it is usually preferable to select all parameters that are necessary for an automatic detection o
8. 1 80 AIDA ID Evaluation User s 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 Peak Determination position by maximum si 122 warn overlap T k search level 2 smoothed 1 pixel g Figure 3 12 Enabling the Apply to Lane Determination Option Performing 1D Evaluations 81 11 Detect all lanes by opening a rectangle over each lane one by one in the search mode kr e Program Files raybest fide Image Analyzer dev 3 10 028Examples pools tf eee P a 5 cued AO O OF ki e Program Filesiraytest Aida Image Analyzer der 3 10 020 Examples pools 1 4 BETTE pH i ei e wee vend MAD A A oyo z HER uE 8 a1yOud 8 T Un Ml 6 O1NOid 36 ih _ E 1 reves IA 141 Ms HE 1 ouo AAAA AA A 7 Automatic Detection of All Lanes Figure 3 13 AIDA ID Evaluation User s Manual Windows 82 12 Delete peaks of no interest by opening a rectangle over them in the selection mode and pressing the DEL key Delete Selection pools1 tif xx 2 Delete the selected peaks or profiles Delete Profiles Can
9. 17 Intensity aL Intensity aL Intensity aL oJ ua 3 2 cay a AAA 2d 31 ao 35 FT Position mm Figure 2 14 Zooming X Axis in 3D Display Mode To zoom out again click the right mouse button when the b 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 18 AIDA ID Evaluation User s Manual Windows 2 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 SS SITITII ITA Ez 5555 std 3 5000 4000 3000 2000 1000 PSSS Visi ALLLLLLLL LLL A AL A o i ad a SER 26 98751123 28 5750119 30 16251 256 31 75001 322 33 33751388 34 92501 454 Figure 2 16 Zooming into Profiles Window Using 1D Evaluation 19 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 Te Apsua Position mm ha Apsua Position mm SON Ma Apsua Position mm Figure 2 17 Zooming into Profiles Window in 3D Display Mode AI
10. Menu of the Runlength Window 0 00005 43 Additional Toolboxes in 1D Evaluation 0 0 0 0 000 cece cee eee 46 Lane amp Peak Determination ToolboX o oooooooooo o ees 46 Lane Determination Pane 0 0 0 ccc eens 48 Baseline Determination Pane 0 00 eens 50 Peak Determination Pane as EOS hae RG dk oad OS ie oe Se 51 Quantity Calibration Toolbox 2 5 200 5 ook SS Oe be ak bean ba tak 52 Standard Pane sitio ra aaa a E a eae Re a a a Bh nds 53 Calibration Cuyo Pane aia ala darias 55 Migration Distance To0lboX o ooo oooooooooooorrrr eens S6 Publishing Layout Toolbox in 1D Evaluation 0 0 0 0 0 cece es 58 Print Options for Profiles ua ari acota aaa teed 59 Print Options for Quantity Calibration 0 0 0 0 00 ee eee 60 Print Options for Runlength Calibration 0 0 0 cee eee 61 DPE MUN A O EEE T DETS EET REPERE T 62 ili iv o A AA A e te es hy eo 63 Export and IMPOR cana adi 66 Exportine Templates nics is RNA 66 Exportine Prones ta Aa 67 Exporting 1D Report Table ooooooooooonoononoorornoo 68 Exporting Quantity Calibration 0 0 0 0 0 ce eens 69 Exporting Runlength Calibration 00 0 70 Importing Profile Templates iia 12 Importing Quantity Calibration 0 0 0 ro 73 Performing 1D Evaluations 75 Assigning Lanes and Bands sais it Gace a ae pi dead PA a 75 Assigning Migration Distance Standards
11. Point settings are the numerical representation of the change in 3D views as described above Data Scaling Runlength bp Minimum Maximum 12 318 M Logarithmic M Automatic _Cancel Position rm Minimum Mazximur M Logarithmic M Automatic 30 View Point A Position Y Position os OAs 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 Horizontal None Big Tics C Small Tics Vertical None C Big Tics C Small Tics Cancel Help 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 AIDA ID Evaluation User s Manual Windows 2 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 Profile ia Position Select Columns Std Aunlength Recalc Aunlength 11198 1 st 1 st Change Profile Mames radif 6 297377 7126 6863 Figure 2 50 Table Row Contextual Menu The following columns are available Table 2 4 Columns Available in the Runlength Window Columns Short Description Profile The
12. Profile 1 Scales v 3d 9000 i Hito Interpolated 8000 y f Profile Overlays v Extremal 3 ios A B Change Profile Names BESA 2 5000 dm HA v Show Baseline i 4000 L AA Hide Baseline 3000 ee Subtract Baseline my pA JA 1000 D 100 200 300 400 Position pixel Select Profiles lt gt BAE aa Toolbar Profile Peak Integral Bkg Integral Bkg Std Quantity Recalc Quantity OL OL QL ng ng 1 Profile 1 1 175938 65 45118 94 130819 71 100 00 100 6314 2 Profile 2 12477351 3993580 84837 71 50 00 48 8766 Result Table 53376 06 24891 95 5 Profile 5 1 4187664 27877 62 1399902 5 00 5 3633 E Profile E 1 182419 1442275 3801 44 2 00 ES Figure 2 11 Profiles Window Example In the graphics pane 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 RISE Figure 2 12 Select Tool on the Main Toolbar 16 AIDA ID 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 aL Intensity aL Intensity aL 26 98751123 28550119 3016251256 31 5001322 3539751500 34 92501454 Position mm Figure 2 13 Zooming X Axis 3 Release the mouse button The scaling will change accordingly Using 1D Evaluation
13. 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 Show Columns pe Abs Pos mm Os Std Runlenath bp Mm Recalc Runlength bp Recalc Quantity 9 Integral Bkg 9 Height AL Integral 9 Integral aL Bky EL Integral Bkg AL Std Quantity Recalc Quantity Figure 2 36 Columns Selection Dialog Box Using 1D Evaluation 35 In addition each row of the table has a contextual menu attached which includes commands for displaying special row types like Sum Total Reminder as well as for changing profile names Wi Profiles Pools2 adf i Ba Profile Peak Type Abs Pos mm Std Aunlength bp Recalc Aunle 1 std 1 22 1 EE IE E IESO IOMA tadi ajasa aol 0120 tadi af mstd zal 8162 E E G E Set to 100 M126 3 Bkg Region i 6108 1 1 4 FE Sseacotmms Y 5080 e Select Columns Select Rows 1 1 3 i LE Select Percent Reference 15 3054 i IES Change Profile Mames TET a eae a e ao 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 36 AIDA ID Evaluation Use
14. 0 cee eens 84 Assigning Quantitative Calibration Standards 0 0 0 cc eee ees 87 Configuring the Evaluation Output 0 0 0 0 0 ccc cee eee 90 AIDA ID Evaluation User s Manual Windows Introduction 1 Overview Most biochemical applications utilize only one dimension as for example SDS protein 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
15. 5 std 3 2 ASI Figure 2 26 Changing Column Width Using 1D Evaluation 29 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 Delete Peaks Create Peaks Delete Profiles Delete Baselines Display i Multiple Data Smoothing Tile Scales w 3d Grid Interpolated Profile Overlays w Extremal AI a Wiser Change Profile Mames Show Baseline Hide Baseline Subtract Baseline Figure 2 27 Contextual Menu of the Profiles Window s Graphics Pane From the submenu 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 30 AIDA ID Evaluation User s Manual Windows With the Data Scaling command you can configure t
16. Baron Ci Overlap quan Drop Down Menu Eee Band Searching Tool search level 0 H Search Level Drop Down Menu smoothed Opixel Ej Signal Smoothing Drop Down Menu apply to lane determination Band Position Drop Down Menu Figure 2 60 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 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 position by maximum KM EN w position by maximum a position by center Figure 2 61 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 1s confirmed Better results for quantification are obtained by using the manual peak assignment mode in the profile or image window Delete Peaks Button Click thi
17. DA ID Evaluation User s Manual Windows 20 2 To change your viewpoint in 3D display mode e Drag the axis origin with the mouse Intensity QL Intensity tL T a ir Cc di Intensity tL al 30 40 50 Position mrm Figure 2 18 Changing 3D Viewpoint Using 1D Evaluation 21 To edit peaks 1 Double click the peak whose borders or position you want to edit Vertical delimiter bars appear 2 Drag the vertical delimiter bar Y LN al Intensity QL Intensity QL T a ir Cc di E Position mm Figure 2 19 Editing Peaks 3 Right click to confirm the changes 22 AIDA ID Evaluation User s Manual Windows 2 To delete a peak 1 Right click on the hatched area indicating the peak s domain 2 Choose Delete Peaks from the contextual menu T a gt a E D E 00 600 500 400 amp 300 o En 200 reate Peaks 100 10 Delete Profiles Intensity aL Delete Baselines Select Profiles 4 Display ar Data Smoothing Scales Grid Profile Overlays 2 Lane 1 4 Figure 2 20 Selecting peak and Opening Contextual Menu A dialog box opens prompting you to confirm the command Using 1D Evaluation 23 3 Click OK to delete the selected peak W Profiles Pools2 adf Ioj x Delete Pools2 adf x oa Delete selected peaks caer e gt 2laner 7 laei sl
18. 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 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 Also the evaluation of radioactive binding e g in southern northern or dot blots yield accurate results It should a
19. Header Cancel f All Rows Selected Rows Cancel f AllColumns Selected Columns Decimal Delimiter f Point List Delimiter i Tabulator C Comma System Defaults Figure 2 81 Quantity Calibration Export Options Dialog Box C Comma 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 1 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 70 AIDA ID Evaluation User s Manual Windows File name Save as type Windows Metafile Cancel Windows Metafile Table ASCII File he Figure 2 82 Runlength Calibration Dialog Box Graph Export To export the result table of the Runlength Cal
20. abel pane of the Lanes tab Image Overlays Pools2 adf Cancel Font Size hoo pti print 2m Wark by FT 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 xl Show Columns Hide Columns a Akg L Integral iL Recale Quantity Integral Bkg AL Integral 9 Type Mame Integral Bkg 9 Recalc Quantity 9 slo FT Hide legend Hide all Figure 2 7 Columns Selection Dialog Box Using 1D Evaluation 13 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 45 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 i xj Lanes aa Font Arial Size bo reta print B Z B EIIE hark by IF Position FT 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 usi
21. aller 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 is shown below the curve A value of 1 identifies a perfect correlation a value of 0 indicates full independence of values Good standard curves should have value very near to 1 Using 1D Evaluation 55 Dy A e 5 C PS E N ZA 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 Toolbox 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 BRUKE 10120 Apply Button 0163 AutoAssign Button 8144 7126 Manual Assignment Button 6108 5090 Delete Assignment Button 4072 3054 a 2036 Edit Standard Button Calibration Curve Linear Interpolation pixel Figure 2 66 Migration Distance Toolbox Overview
22. ands will be assigned accordingly It should be checked that no band mentioned 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 86 AIDA ID 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 bro
23. antity Calibration Toolbox Overview 52 AIDA ID Evaluation User s Manual Windows 2 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 Standard Pane LA Quantity Calibration standard Standard Selection Drop Down Menu hre st List of Standard Values AutoAssign Tool Manual Assignment Tool ny Delete Standard Assignment Button HH Edit Standard Button Es Figure 2 63 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 specified 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 53 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 Figure 2 64 Quantity Standards Dialog Box In the Quantity Standards dialog box the Standard name and Values are entered and
24. arch 15 2001 11 16 20 AM eProgram Filesaytest Aida Image Analyzer dev 3 10 023 Examples 4g test adt Page 3 of 3 Quantity Calibration Logarithmic Regression y 215403 47 log x 33 05 33 05 Correlation Coefficient r 0 999882 hegrabbkg PY 60 Quantity ng WegrakBhg saai Recak Quantty py ng BOC Era a eer a as s A sone AE ser er as oa ECON A sa sms E Last Changed Thursday May 25 2000 1 38 17 PM Aida Development Version 3 10 028 Page 5 of 6 1D Evaluation Thursday March 15 2001 11 29 02 AM eProgram FilesvraytestiSida Image Analyzer dev 3 10 028 Examples Pools2 adf Page 5 of 6 Runlength Calibration 1000 2000 3000 4000 5000 6000 7000 8000 3000 10000 11000 12000 13000 Runlength bp Position mm Last Changed Thursday May 25 2000 1 35 19 PM Aida Development Version 3 10 028 Figure 2 74 Preview Example Part 2 Using 1D Evaluation 65 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 c
25. cel 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 As OK _Cancel_ __toad_ Save 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 83 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 is done according to size molecular weight fragment size or charge pI 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 more elaborate standardization method should be employed It is suggested to include at least two standar
26. cking it in the Profiles Window JE Profiles pools1 tif loj x l 2 N y IR z UN i R S i N Y 0 100 200 300 Position SE Era ED a a Profile Peak Type Abs Pos Std eee Recalc sae pixel ng ng 1 Profile 1 E 2 E E a 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 E and then clicking directly on the middle of the band Performing 1D Evaluations 79 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 vertical t search level 1 a smoothed 5pixel 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 start point and drag the densitometer to the end of the lane Figure 3 10 Manual Lane Marking
27. d 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 EP MBEN L gt kk AAT T USE FT p El III FE pa 3 HH W i el pisi HEE ip cle E 7 Figure 3 16 Migration Distance Example 84 AIDA ID Evaluation User s Manual Windows When all bands have been assigned the Migration Distance toolbox can be used to assign the standards H Migration Distance Direction 71126 Apply Butt PPS 2 6108 AutoAssign Button 2 5090 Manual Assignment Button 4072 2 3054 Delete Assignment Button 2036 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 85 All other b
28. 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 42 AIDA ID Evaluation User s Manual Windows 2 Contextual Menu of the Runlength Window Right clicking on the Runlength window s graphics pane opens a contextual menu providing you with commands for setting several display options see below BN Runlength Pools2 adf Ds Multiple Display Tile Display w 3d Display Scales Position mm Profile Peak Position Std Runlengih Recalc Runlenath mm bp bp 24 9944 10120 10284 8144 8017 radif ef 28 73771 mel 6963 radif 7 31 84231 e 564 T 5 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 43 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
29. dth Determination Tool and set the Profile Width to a value so that a little more than the band width is enclosed Figure 3 4 Setting Profile Width 5 Use the Search Peaks button to detect bands automatically Peak Determination position by maximum Bl 122 warn overlap 7 Ea N search level 2 g smoothed 1 pixel g Figure 3 5 Activating Automatic Band Detection Performing 1D Evaluations TT 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 ae e Program Files raybest fide Image Analyzer der 10 078 Exam Profile 1 Figure 3 6 Automatic Band Detection Completed 6 Use the Auto Determine Baseline Tool to draw a baseline Baseline Determination fe search level 1 smoothed pixel T apply to lane determination Figure 3 7 Activating Automatic Baseline Determination 78 AIDA ID Evaluation User s Manual Windows If the baseline is too flexible and enters into 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 cli
30. ect the Draw Peaks option Profile Overlays Pools2 adf Region Cancel IY Draw peaks Mark peaks by M Area Brackets Figure 2 31 Profile Overlays Dialog Box Label Pane 32 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 Pools2 adf int Draw standard lines an Label Size js l Figure 2 32 Profile Overlays Dialog Box Runlength Calibration Pane With the Change Profile Names command 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 TE O e Figure 2 33 Profile Name Dialog Box Using 1D Evaluation 33 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 Ee A Profile Peak Type Abs Pos mm Std Aunlength bp Recalc Aunle Sort ascending by Abs Pos Sort descending by Abs Pos Select Abs Pos 1 std 1 4 AStd Hide bs Pos Settings of 46s Pos tsd 6 Astd Two lined Header Se
31. elected 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 28 AIDA ID Evaluation User s 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 MM Profiles Pools2 adf Wi Profiles Pools adf JE Profiles Pools2 adF Profiles Pools2 adf SelectProfiles 4 Select Profiles Select Profiles NIKE Select Profiles Elo Profile Select Profiles Type Profile Ty II 2 A5td 5 43 Astd 2 std 3 AStd 5 std 3 2 Asta 5 std3 2 Rstd 5 43 2 Asta 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 ma Profiles P00ls2 profiles Pools2 select Profiles select Profiles select Profiles 5sd3 2 5sd3 2 5sd3 2
32. f 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 Pools2 adf i x Image Interpretation Cancel E mission 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 i 1 Activate the Lane Searching function Lane amp Peak Determination Lane Determination LO arbitrary pf All lane width 5 72mm J vertical de sensitivity medium g 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 a i EL jis El Figure 3 3 Semi Automatic Lane Detection 76 AIDA ID Evaluation User s Manual Windows 4 Click the Profile Wi
33. he 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 Scaling Position rm Minimum Maximum W Automatic Cancel Intensity CL Minimum haximurn 764 007 M Logarithmic M Automatic 30 View Point A Position Y Position os fe Hs X Figure 2 28 Data Scaling Dialog Box The Data Smoothing function is used to smoothen the curve Smooth Width Pools1 adf Set smooth width to mm Cancel Figure 2 29 Smooth Width Dialog Box Using ID Evaluation 31 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 i None Big Tics C Small Tics Cancel Help Horizontal None C Big Tics C Small Tics 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 desel
34. he layout setting for the Runlength window in the Protocol Print pane Protocol Print AD Print Preview Format Portrait C Landscape Print I Graphic C Single C multiple 3D Y Table C Single Tables Allin One To Graphic Figure 2 70 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 61 Print In AIDA there are two ways for printing your image and evaluation data e Choose Print from the File menu Or e Use the Protocol Print pane on the Publishing Layout toolbox The Print command of the File menu allows you to print your data on the fly without permanently storing the layout printing settings whereas the Protocol Print pane provides you with a complete set of printing features w
35. hich 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 Print 1D Evaluation Pools2 adf Printer EPSON EPL 5700L Advanced at LPT1 Cancel A 20cm E Jom Y Header Printer tight 2 0cm E bottom 200m W Parameter Parameter and Image Options M Image whole image Y overlays M Profile Curves portrait Y grayscale iv scales iv Profile Table automatic size M overexposed Quantity Calibration M Runlength Calibration Profile Options all profiles de portrait ig Figure 2 71 Print Dialog Box 62 AIDA ID Evaluation User s Manual Windows 2 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 OK M file path IP author IV page number IV last change time Cancel IV current date IV software version Figure 2 72 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 Calibrat
36. ibration window 1 Select the Table Ascii File data format 2 Click Save to confirm the export The Runlength Calibration Export Options dialog box appears allowing you to set further option for the export of the result table Runlength Calibration Export Options Range US x M Header Cancel f All Rows Selected Rows f AllColumns Selected Columns Decimal Delimiter f Point List Delimiter i Tabulator C Comma System Defaults Figure 2 83 Runlength Calibration Export Option Dialog Box C Comma 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 Using 1D Evaluation 71 3 Complete the action by clicking OK The data are saved to a simple ASCII text file Importing Profile Templates To import previously saved profile 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 dialog box that appears select the template to be imported and click Open Import Pools adF Look in E 10 Samples dm Type ATF File Size 40 0 KB File name pools2 atf Files of type ADA Template Format
37. igration 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 e None this option will shut off the background calibration e g if this function is too time consuming during editing of the standards e Linear Interpolation e Linear Regression e Logarithmic Interpolation e Logarithmic Regression e Hyperbolic Regression e Hyperbolic Interpolation Using 1D Evaluation 37 PS SS abt 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 Publishing Layout Toolbox in 1D Evaluation In AIDA 1D Evaluation the standard AIDA Publishing Layout toolbox is expanded by three additional buttons f
38. index and name of the profile Peak The number of the peak in the profile Name The name for the peaks as given by the user Position length unit The position of the peak in the profile 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 runlength curve Depending on the method for the runlength measurement this is the standard for the RF value Fragment Size Molecular Weight or pI Value Difference runlength unit The difference without sign between the entered standard and the recalculated value Rel Error The relative difference without sign of the entered standard and the recalculated value compared to the standard value 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 45 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 e Lane amp Peak Determinatio
39. ion You can access these data windows by choosing the appropriate entry Profiles Quantity Calibration and Runlenght Calibration respectively from the View menu view window Help Image Histogram Profiles Quantity Calibration Runlength Calibration 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 lt en Program Files raytest Aida Image Analyzer dev 3 20 017 Examples Pools2 adf k e Oy ni o Hpg umm 5707 ERE 3744 126 5108 5090 4072 5054 2056 B35 018 Band s lak Recalc Runlength f FE i Lane s labels y Figure 2 2 Image Window Example You can copy the content of the Image window to the clipboard using the Copy Content command of the Edit menu See the Export Image section in the AIDA Image Analyzer Basic Concepts and Features User s Manual for further explanation 10 AIDA ID Evaluation User s Manual Windows 2 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 th
40. ion 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 In the Profile Options pane you can specify to print the Current Selection or all profiles as well as the paper orientation Landscape or Portrait for the printout 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 Using 1D Evaluation 63 raytest 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
41. is 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 hd Figure 2 55 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 p vertical IN horizontal w vertical expand to edges Figure 2 56 Lane Searching Parameter Drop Down Menu 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 hae intensity summation averaged intensity optical density Figure 2 57 Profile Type Selection Drop Down Menu Using 1D Evaluation 49 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 bi mm Figure 2 58 Miniaturized Profiles Window Baseline Determination Pane This pane will be needed only for the quantification of the bands after standardization
42. is 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 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 if you select multiple bands the peak editing command of the contextual menu is turned off I Delete Profile Duplicate Profile Edit Profile Delete Peaks Edit Peak Image Overlays Change Profile Mame Set Profile width Flip Profile Expand To Egdes Figure 2 4 Contextual Menu Band Selected The Delete Profiles and Delete Peaks commands are only executed for the selected lane s and selected peaks respectively Using 1D Evaluation 11 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 Na
43. ity Calibration from the View menu or e Double click the miniaturized calibration curve display on the Quantity Calibration toolbox 5 x Linear Regression y 13 93 x 14 80 Correlation Coefficient r 0 9993 Intercept on x Axis 1 062 Integral Bkg QL o O o 0 10 20 30 40 50 60 70 60 390 100 120 140 Quantity ng Profile Peak Integral Bkg QL Quantity Std ng Quantity Recalc ng 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 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 38 AIDA ID Evaluation User s Manual Windows 2
44. l Bkg signal unit Integral Integral Bkg 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 The percentage of the peak integral in comparison to the Sum or to the value in the row which is Set to 100 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 27 Table 2 1 C
45. lect Columns Aadi std 1 g Std Ad 0 5090 tsat af rst 374 4072 4 Figure 2 34 Contextual Menu of the Profiles Windows 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 columns using the Select Columns command see below 34 AIDA ID Evaluation User s 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 Decimal digits 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
46. lso 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 samples will also make quantitative estimations safer Starting 1D Evaluation To select the 1D Evaluation mode 1 Click the Evaluation button in the toolbar A dialog box appears offering you a list of available evaluation modes Change Evaluation Mode xj Available Evaluation Modes 1D Thin Laver Yiromatography 20 Densitometry C 20 Thin Layer Chromatography sues Annotation Array Metrix Crystallography Material Science Multi Channel Display Whole Body Autoradiography SLP Figure 1 1 Changing Evaluation Mode The list entries reflect the installed AIDA application modules 2 Select the 1D Evaluation list item und click OK 6 AIDA ID Evaluation User s Manual Windows Note This evaluation mode is only available if you purchased the AIDA 1D Evaluation module Introduction 7 8 AIDA ID Evaluation User s 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 e Profiles e Quantity Calibration Runlenght Calibrat
47. lue Recalc Quantity Std Quantity The relative difference with sign of the entered standard and the recalculated value compared to the standard value 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 41 uy raytest Runlength Calibration Window 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 BN Runlength Pools2 adf i lo x std 3 std 2 std 1 E 5 E ou 0 2000 5000 8000 11000 D Aunlength bp Profile Peak Position Std Runlengih Recalc Runlenath mm bp bp 230731 aa IE asu ma one radif a 23548 482 127 radif ef 28 73771 mel 886 radif 7 31 84231 e 5364 EEE IO ELE E son Figure 2 46 Runlength Window Example Again the graph and adjacent table can be adapted by using the contextual menu For a more
48. ly 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 Pools2 adf smooth width 0 277 138 mm _ Cancel 0 mim Cancel 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 25 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 Profile Peak Type Abs Pos m 2 FE Profiles Pools2 adf Fr m Std A at Intensity aL Hel F Figure 2 24 Changing the Display Ratio Between Graphics Pane And Result Table 26 AIDA ID Evaluation User s Manual Windows 2 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 Po Integra
49. me 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 He Image Attributes be Image Overlays Preferences Configuration Path Image Search Path Autosa ing Overlay Drag Mode Overlay Colors User Registration 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 12 AIDA ID Evaluation User s Manual Windows 2 For Lanes depending on the already performed evaluations various labels can be selected by clicking the Configure button in the L
50. mide staining 1s proportional to the DNA amount within certain ranges The variation of fluorescence of different DNA fragments is not larger than the error of the whole procedure itself less than 10 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 Ol x ESERE E E 20 ER fon fito fin ie 4 KN Aron N Ao R f MENAN AN A A M i NANA ON RR AEN A N AN A 3 NONA N A N AN g N Er VE B 1 NAVYRYWINYGA NN AN WY gt INIA N WANN K 0 100 200 Position 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 87 oo lo EN 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 w intensity summation a
51. n Quantity Calibration Migration Distance 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 Figure 2 51 Mapping between Evaluation Menu Entries and Toolbar Buttons 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 46 AIDA ID Evaluation User s Manual Windows 2 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 amp Peak Determination Lane Determination LO arbitrary be All ane width 16mm G P vertical hal sensitivity medium g intensity summation Lane Determination pane Baseline Determination e search level i g Baseline Determination pane smoothed Opixel F apply to lane determination Peak Determination position by maximum E LEE Jlanow overlap Peak Determination pane search level 0 g smoothed pixel g apply to lane determination Figure 2 52 Lane amp Peak Determination Toolbox Overview Using 1D Evaluation 47 Lane Determinatio
52. n Pane The first step in the analysis of a 1D application is the determination of the lanes Lane Determination Manual Lane Marking Tool arbitrary Delete Marked Lane Button _ ee Lane Marking Options Drop Down Menu S Al lane width erm Profile Width Determination Tool ve tical Lane Searching Parameter Drop Down Menu sensitivity medium EI Search Sensitivity Drop Down Menu intensity summation inf Search Lane Tool Profile Type Selection Drop Down Menu Minaturized Profiles Window Figure 2 53 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 E Q arbitrary DS w arbitrary horizontal vertical fun direct expand to edges show profile show drag Figure 2 54 Lane Marking Options Drop Down Menu Densitometer windows can 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 48 AIDA ID Evaluation User s Manual Windows 2 Delete Marked Lane Button Use th
53. ng 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 14 AIDA ID Evaluation User s 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 Pools2 adf Figure 2 10 Image Overlays Dialog Box Runlength Calibration Tab Using ID Evaluation 15 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 a Profiles ag test adf Delete Peaks Shortcut Menu Create Peaks Delete Profiles G o Graphics Pane Profile 3 Delete Baselines P Profile 2 Display Multiple Data Smoothing Tile
54. olumns 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 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 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 pl Value Recalc Runlength runlength unit A calculation of the peak runlength based on the current runlength 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 s
55. onfirm the action by clicking Save or 1 Click the Export Template button on the toolbar to open the Export Template dialog box Ela Aj eje Figure 2 75 Main Toolbar Export Template Button 2 Type a name for the export file in the File Name text box and complete the export action by clicking Save Export Template PoolsZ adf Save in a 10 Samplez dm File name pools2 atf Save as type alba Template Format Cancel Figure 2 76 Export Template Dialog Box 66 AIDA ID Evaluation User s Manual Windows 2 Exporting Profiles To export Profiles 1 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 Pools2 adf JE Range IF Header lx f All Profiles _ Cancel Selected Profiles Decimal Delimiter f Point List Delimiter Tabulator C Comma C Comma C Semicolon System Defaults Figure 2 77 Profile Export Dialog Bo
56. ontextual menu opens allowing you to Select Columns for displaying or Delete Standards from the table Integral Srea Bkg Std Cone F5 Lmt dem em 604 91 1107000 Select Columns Delete Standards Columns Selection Show Columns Hide Columns Rel Deviation Deviation Difference Recalc Quantity cancel Figure 2 45 Selecting Columns to be Displayed 40 AIDA ID Evaluation User s Manual Windows 2 The following columns are available Table 2 3 Columns Profile Peak Name Integral Bkg signal unit Std Quantity quantity unit Recalc Quantity quantity unit Difference unit Rel Error Deviation unit Rel Deviation 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 Difference Std Quantity The difference with sign between the entered standard and the recalculated va
57. or specifying the print options for the Profiles Quantity Calibration and Runlength Calibration alla 12 41 Publishing Layout Annotate LIMA Protocol Print A Print Preview E Page Layout Edges w p E Profiles Options Button STUDE 20m pal Quantity Calibration Options Button FR u ight y om cm Runlength Calibration Options Button Header e file path page number M current date Footnote author M last change time M software version Figure 2 67 Publishing Layout Toolbox Overview and Toolbar 58 AIDA ID Evaluation User s Manual Windows 2 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 Protocol Print A Print Preview Profiles Format Portrait C Landscape Selection Only ol Graphic Single multiple 3D M Table Single Tables Allin One To Graphic Figure 2 68 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 repre
58. r s 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 Pools adf Show Profile Rows Separator Cancel IF Total e Sum PA Remainder 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 profile or the Sum The later two are set via the following dialog box Percent Reference Pools2 adf h E Set 100 percent to 0 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 Pools acf as Set name of 1 profile to Cancel Figure 2 40 Profile Name Dialog Box Using ID Evaluation 37 e e raytest Quantity Calibration Window To open the Quantity Calibration window e Choose Quant
59. 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 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 1 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 4g test adf Save itr E Examples da t File name Ag test cal Save as type alba Calibration Cancel Windows Metafile Table 45Cll File AIDA Calibration k Figure 2 80 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 Using 1D Evaluation 69 Ta uy 2 E The Quantity Calibration Export Options dialog box appears allowing you to set further option for the export of the result table Quantity Calibration Export ore Range il OK M
60. s button to delete all currently selected peaks Using 1D Evaluation 51 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 is activated to obtain migration distance analysis for them 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 l Quantity Calibration Standard Juser defined Standard pane Calibration Curve Logarithmic Regression iv 0 05 origin Calibration Curve pane x ng Corr Coeff F 0 999582 warning bkg not determined Figure 2 62 Qu
61. 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 MAKE IMPROVEMENTS AND OR CHANGES IN THE PRODUCT S AND OR THE PROGRAMS S DESCRIBED IN THIS PUBLICATION AT ANY TIME Contents 1 Introduction 5 DU ta ASE EE EEE T EE E EE E E peed asa 5 Sarine ID Eya alon retarda AS aaa 6 2 Using 1D Evaluation 9 Data Windows in 1D Evaluation 0 c lt e0re0cdseeead oe4d sex wa seach ewa wes 9 Image Window in 1D Evaluation 0 0 0 0 000 cc ee eens 10 Contextual Menu of the Image Window 0 nennen 11 Miage Overlays ds ia 12 Prones WIN asar a 16 Toolbar of the Profiles Window o o o ooooooooooonnn o 25 Data CSC HOI sd cna eos ae a aetna en id 2 Contextual Menu of the Profiles Window Graphics Pane 30 Contextual Menu of the Profiles Window Result Table 34 Quantity Calibration Window 0 0 0 ete eens 38 Contextual Menu of the Quantity Calibration Window 39 Runlength Calibration Window 0 0 0 0 eee ee eens 42 Contextual
62. sentation 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 the same graphic Using 1D Evaluation 59 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 Protocol Print FE A Frint Preview Quantity Calibration Format Portrait C Landscape I Graphic e Table Figure 2 69 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 und or the calibration table Table 60 AIDA ID Evaluation User s Manual Windows 2 Print Options for Runlength Calibration The last button of the Publishing Layout toolbox is used to configure t
63. should 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 ALO x Linear Regression y 1 621 x 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 89 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 g to get peaks of identical size at comparable positions loj x If Wa sti SE AA SS Figure 3 23 Migration Distance Normalization Example 90 AIDA ID Evaluation User s Manual Windows
64. 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 54 AIDA ID Evaluation User s Manual Windows 2 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 09 origin Gi ng Corr Coeff f 0 999882 Warning bkg not determined Figure 2 65 Calibration Curve Pane The Calibration Curve drop down menu provides the following functions Linear Interpolation e Linear Regression Logarithmic Regression e 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 sm
65. veraged intensity 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 l Quantity Calibration Standard Test 0 100 0 50 Manual Assignment Tool ny Figure 3 20 Manual Assigment Tool Quantity Calibration Toolbox 88 AIDA ID Evaluation User s Manual Windows 3 Enter the value and the dimension in the Quantity Calibration dialog box that appears Set standard of 11 peaks rer 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
66. x 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 Ok The data are saved to a simple ASCII text file Using ID Evaluation 67 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 Pools2 adf ajx Save in E Examples e amp cf En wm E Save az hype Table ASCII File Cancel Figure 2 78 ID 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 xj M Header Cancel f All Rows Selected Rows f AllColumns Selected Columns Range Decimal Delimiter C Point List Delimiter F Tabulator f Comma System Detaults Figure 2 79 Profiles Export Options Dialog Box 1D Report Table Export C Comma Semicolon 68 AIDA ID Evaluation User s Manual Windows You can specify the Range of the exported

AIDA 1D Evaluation User's Manual (Windows)

Contents

Download Pdf Manuals

Related Search

Related Contents