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p. 1
imageaide reference although all possible care has been taken in the preparation of this publication spectronics corporation accepts no liability for any inaccuracies that may be found spectronics corporation reserves the right to make changes without notice both to this publication and to the product that it describes no part of this publication may be reproduced stored in a retrieval system or transmitted in any form or by any means electronic mechanical photocopying recording or otherwise without the prior permission of the copyright owner © synoptics limited 20002004[close]
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spectronics corporation 959 brush hollow road p box .o westbury new york 11590 tel 1-800-274-8888 fax 1-800-491-6868 website www.spectroline.com[close]
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contents using imageaide for gel analysis 1 how to create and work with secure sample files 1-1 file formats 1-1 opening a secure sample file 1-2 creating a new secure sample file 1-10 using the browser 1-11 acquiring an image to create a secure sample file 1-13 importing settings from existing secure gel sample files 1-14 editing sample properties 1-20 saving a secure sample file 1-20 how to place position and work with tracks 1-21 overview 1-21 locating tracks automatically 1-23 locking tracks 1-24 placing individual tracks on a sample 1-25 deleting tracks 1-29 adjusting the overall position of the tracks on a sample 1-29 adjusting the track splay 1-31 moving and tilting a single track 1-33 adjusting track width 1-39 adjusting the rf start and end lines 1-41 enabling and disabling tracks 1-45 adding a description for a track 1-46 displaying track properties 1-47 how to set the background correction for tracks 1-50 setting the integration parameters 1-50 setting or changing a manual baseline 1-55 deleting a manual baseline 1-58 how to locate and edit peaks on a track 1-58 locating the peaks on a track automatically 1-59 imageaide user manual contents-1[close]
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contents adding a peak semi-automatically 1-60 adding a peak manually 1-62 selecting peaks 1-63 deleting peaks 1-66 adjusting the position of a peak 1-67 adjusting the position of a peak s bounds 1-69 displaying peak properties 1-70 how to create molecular weight calibrations 1-71 assigning molecular weights to a standard track 1-71 specifying how molecular weights are calculated from standard tracks 1-77 removing molecular weight assignments from peaks 1-79 reassigning molecular weights in a standard track 1-80 molecular weight configuration settings 1-82 the molecular weight calibration graph 1-83 viewing molecular weights calculated from the tracks 1-83 working with molecular weight libraries 1-85 how to create quantity calibrations 1-96 setting the quantity calibration method 1-97 assigning quantities to peaks 1-99 the quantity calibration graph 1-100 viewing quantities calculated from the tracks 1-101 how to compare peak profiles 1-102 changing the scale in the profile comparison window 1-106 printing reports profile comparisons 1-106 how to match peaks on different tracks 1-107 setting the tolerance and method used for peak matching 1-107 setting a matching standard 1-108 editing matches 1-109 viewing match results 1-110 dendrogram 1-113 how to print and export gel results 1-115 printing a report showing gel results 1-115 exporting results to excel 1-117 saving results in a comma separated values format file 1-118 creating a report in word 1-119 copying a picture of an image to the clipboard 1-120 contents-2 imageaide user manual[close]
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contents using imageaide for manual band quantification using imageaide for spot blot analysis 2 3 how to create and work with secure sample files 3-1 opening a secure sample file 3-2 creating a new secure sample file 3-6 using the browser 3-7 acquiring an image to create a secure sample file 3-9 importing settings from existing secure spot blot sample files 3-10 importing settings from a saved sample file 3-15 editing sample properties 3-16 saving a secure sample file 3-16 how to add and remove spot frames 3-17 adding spot frames automatically 3-17 adding new spot frames 3-18 removing spot frames 3-21 how to adjust the size and shape of spot frames 3-22 setting the size of all spot frames to the same size and shape 3-22 changing between circular and rectangular spot frames 3-22 adjusting the size of circular spot frames 3-23 adjusting the size and shape of rectangular spot frames 3-25 how to adjust the spot frame positions 3-26 adjusting the position of a single spot frame 3-26 adjusting the positions of all the spot frames in a grid 3-27 how to lock the spot frames on a sample 3-32 how to show or hide identification labels on the spot frames 3-33 how to set the quantity calibration method 3-33 how to assign quantities to spot frames 3-34 how to apply background correction to results 3-36 how to view spot blot results 3-38 how to define an incidence condition 3-39 how to print and export spot blot results 3-41 printing spot blot results 3-41 exporting spot blot results to excel 3-42 saving results in a comma separated values format file 3-43 copying a picture of an image to the clipboard 3-44 imageaide user manual contents-3[close]
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contents using imageaide to count colonies windows 4 5 gel windows 5-2 adjusting the panes in a gel window 5-3 track label pane 5-3 description pane 5-4 image pane 5-4 profile pane 5-6 peak value pane 5-6 graphics pane 5-7 results pane 5-13 molecular weight library window 5-15 profile comparison window 5-16 colony counting window 5-20 spot blot/manual band quantification window 5-22 image pane 5-23 results/incidence/calibration graph pane 5-25 image controls 5-27 the histogram viewer 5-28 toolbars the standard toolbar open save help sample properties report toolbar report setup preview print export table to excel save table to csv file export to word save to clipboard track toolbar locate tracks 6 6-1 6-1 6-1 6-2 6-2 6-2 6-2 6-2 6-3 6-3 6-3 6-3 6-3 6-4 6-4 contents-4 imageaide user manual[close]
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contents lock all 6-4 position all 6-4 splay all 6-5 move/tilt 6-5 width 6-5 rf start position 6-5 rf end position 6-5 zoom toolbar 6-6 zoom in 6-6 zoom out 6-6 zoom reset 6-6 calibrations toolbar 6-6 integration parameters 6-7 quantity calibration 6-7 assign quantity 6-7 molecular weight calibration 6-7 mw standard 6-7 profile comparison window 6-8 peak matching toolbar 6-8 peak matching 6-8 matching standard 6-8 spot blot toolbar 6-9 all spots same size 6-9 position control points 6-9 locate 6-9 position any spot 6-9 background correction 6-10 lock position 6-10 spot incidence parameters 6-10 circle 6-10 rectangle 6-11 free hand 6-11 quantity calibration parameters 6-11 assign quantity 6-11 menus 7 file sample window 7-1 imageaide user manual contents-5[close]
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contents new import 7-2 open 7-7 browse 7-9 close 7-10 save 7-10 save as 7-11 select source twain 7-12 acquire twain 7-12 new library 7-13 open library 7-14 import method 7-14 export table to excel 7-16 save table to csv file 7-17 export to word 7-18 save to clipboard 7-19 printer setup 7-19 report setup 7-20 print preview 7-26 print 7-27 recently opened files 7-28 sample properties 7-28 exit 7-37 file molecular weight library window 7-38 unlock 7-38 lock 7-39 change password 7-40 properties 7-41 edit gel window 7-42 integration parameters 7-42 molecular weight calibration 7-48 quantity calibration 7-50 peak matching 7-52 edit spot blot/manual band quantification window 7-53 quantity calibration 7-53 edit colony counting window 7-54 edit molecular weight library window 7-54 new standard 7-54 edit standard 7-56 contents-6 imageaide user manual[close]
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contents delete standard rename standard edit profile comparison window view gel window profile comparison histogram zoom in zoom out zoom reset align banks of tracks display corrected profiles peak markers peak bounds tracks match lines peak numbers only molecular weight quantities peak matching maximize pane toolbars submenu toolbar buttons on menus status bar view spot blot/manual band quantification window spot numbers view colony counting window view profile comparison window view molecular weight library window track locate tracks width individual height individual lock all position all splay all move/tilt width rf start position 7-57 7-58 7-59 7-59 7-60 7-60 7-60 7-60 7-61 7-61 7-61 7-61 7-62 7-62 7-62 7-62 7-63 7-63 7-63 7-63 7-64 7-64 7-64 7-65 7-65 7-66 7-66 7-66 7-67 7-67 7-68 7-68 7-68 7-69 7-71 7-73 7-78 7-80 imageaide user manual contents-7[close]
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contents rf end position 7-80 invert profile 7-84 edit manual baseline 7-84 delete manual baseline 7-88 mw standard 7-88 matching standard 7-89 insert 7-90 delete 7-93 enable 7-93 disable 7-94 description 7-94 properties 7-95 peak 7-97 assign molecular weight 7-97 assign quantity 7-103 unassign molecular weight 7-105 unassign quantity 7-105 locate submenu 7-106 delete submenu 7-106 match submenu 7-107 properties 7-107 profile 7-108 reference 7-108 show 7-108 properties 7-109 spots 7-109 circle 7-110 rectangle 7-110 free hand 7-110 all spots same size 7-112 position control points 7-112 locate 7-113 position any spot 7-114 background correction 7-115 lock position 7-115 spot incidence parameters 7-116 delete 7-117 delete all 7-118 contents-8 imageaide user manual[close]
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contents assign quantity unassign quantity extras user name configuration save sample defaults load sample defaults window new window cascade tile arrange icons windows open in imageaide help help topics using help about imageaide image pane and profile context menus track label context menu active matching standard peak value pane context menu delete graphics pane context menu scale to fit save to clipboard gel window results pane context menu result table commands profile comparison window context menu spot blot/manual band quantification window spot blot/manual band quantification window result table commands table 1d/table 2d molecular weight library context menu image pane context menu results pane context menu 7-118 7-120 7-120 7-121 7-121 7-128 7-129 7-129 7-129 7-129 7-130 7-130 7-130 7-130 7-130 7-130 7-131 7-131 7-131 7-132 7-132 7-132 7-133 7-133 7-133 7-134 7-134 7-135 7-135 7-136 7-136 7-137 7-137 imageaide user manual contents-9[close]
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using imageaide for gel analysis this chapter contains detailed instructions for using imageaide for gel analysis it begins by describing how you can load sample files and images into imageaide it then shows you how to detect place and edit tracks on the image and then detect place and edit peaks on the tracks this is followed by instructions for calibrating the molecular weights represented by the peaks on a standard track including how you can use libraries of molecular weight standards there is then a section showing you how to calibrate the tracks so that you can measure quantities from the gel the chapter continues by showing you how to display a window comparing the profiles on different tracks including how to compare the profiles of tracks on different gels the next section describes how to use imageaide to match the peaks on different tracks to a standard track the final section in the chapter tells you how to print and export result reports how to create and work with secure sample files file formats imageaide stores data in a secure file format an .sgd file these files are also created by compatible acquisition programs such as imagesource the secure file format ensures that your data cannot be tampered with and that your results are both traceable and reproducible in keeping with good laboratory practice secure sample files may be analyzed files that have been previously saved in imageaide these will contain for a gel information about track positions peaks assigned standards results for molecular weights quantities and peak matching and so on imageaide user manual 1-1[close]
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using imageaide for gel analysis unanalyzed files that have been saved in an acquisition program but have not yet been loaded into and saved in imageaide opening a secure sample file below shows you how to open both unanalyzed and analyzed secure sample files however you can also load other image files created by non-secure sources such as tiff images generated by a scanner into imageaide for analysis when you do this imageaide copies the original image and saves it in a secure file any changes you then make to the original image will have no effect on the image in the secure file full details are given in creating a new secure sample file page 1-10 opening a secure sample file to open a secure sample file 1 choose open from the file menu to display a standard windows open dialog box 2 use the dialog box to select the required secure sample file 3 press open 1-2 imageaide user manual[close]
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how to create and work with secure sample files what happens next depends on whether you are opening an analyzed or unanalyzed secure sample and in the latter case on whether you have set auto import methods in the imageaide configuration dialog box if you are opening an analyzed secure sample file the sample will be opened in a gel window with all tracks and peaks displayed as they were when the file was last saved all previously analyzed results will be shown in the results tables if you are opening an unanalyzed secure sample file and you have selected auto import methods in the imageaide configuration dialog box the import method dialog box will open so that you can choose which settings to import see opening or creating a secure sample file with automatic import page 1-15 for further details if you are opening an unanalyzed secure sample file with auto import methods deselected the sample properties dialog box will open with the image loaded imageaide user manual 1-3[close]
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using imageaide for gel analysis the controls shown on the general page in the sample properties dialog box depend on the type of sample in the picture shown above the sample is an ethidium bromide stained dna gel to set the sample properties 1 if required select a different analysis type from the drop-down list box the default analysis type is set to gel when imageaide is first installed but you can change this using configuration in the extras menu see page 7-121 2 click on one of the direction radio buttons to set the electrophoresis direction the default is down 3 imageaide detects the image type automatically so you shouldn t need to change this setting however if you wish to change the setting a click on the fluorescence radio button for a fluorescence image bright bands on a dark background b click on the absorption radio button for an absorption image dark bands on a bright background 4 the red rectangle on the image preview defines the area of interest the area analyzed when imageaide performs an automatic track location if required for example if you are working with a multi-layer sample you can define a grid of independent areas of interest note each area of interest is nearly independent of all the other areas of interest defined on the gel molecular weight standard tracks must be assigned separately within each area and band matching is carried out between the tracks in the same area and not with any tracks in other areas separate quantity calibrations can be used for each area though quantity calibrations can be shared between areas a single set of integration parameters is used for all areas but molecular weight calibration quantity calibration and peak matching parameters can be chosen independently for each area of interest tracks can be located adjusted and locked independently within each area of interest rf start and end lines can be adjusted independently for each area of interest the results and graphics panes show details for the selected area of interest only to define a grid of independent areas of interest on the gel image a enter the number of columns in the grid 1-4 imageaide user manual[close]
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how to create and work with secure sample files b enter the number of rows in the grid leave the numbers of columns and rows set to 1 if you just want a single area of interest as you enter the numbers a grid of red rectangles appears on the image for example imageaide user manual 1-5[close]
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using imageaide for gel analysis if required as in this example you can adjust the shape and position of the areas of interest see the following steps 5 to adjust the shape of an area of interest a click in the area of interest that you want to adjust to select it blue drag handles appear at the corners and in the middle of the sides of the area of interest showing that it is selected b move the pointer over the drag handle for the corner or side that you want to adjust the shape of the pointer will change to a two-headed arrow showing that you can drag the handle c press and drag the corner or side as required the outline of the area of interest will be redrawn as you drag the handle 1-6 imageaide user manual[close]
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how to create and work with secure sample files d when the corner or side is in the required position release the mouse button 6 to move an area of interest a move the pointer over the area of interest you want to move b press and drag the area of interest the area of interest will become selected the pointer will change to a four-headed arrow and the outline of the area of interest will be redrawn to show its new position as you drag it c when the area of interest is in the required position release the mouse button imageaide user manual 1-7[close]
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using imageaide for gel analysis 7 the next step is to choose between automatic and manual track detection note automatic track detection is the recommended way of working with imageaide you can use configuration in the extras menu see page 7-121 to set imageaide to detect the tracks automatically by default to choose automatic track detection for an area of interest you can choose the setting separately for each area of interest a click in the area of interest to select it this step is unnecessary if there is only one area of interest b press locate tracks automatically note generally this is all you have to do as in nearly all cases the imageaide automatic track location will be able to locate the tracks on the gel correctly and position track markers over them accurately without further assistance however very occasionally you may have a gel that causes problems and the following step allows you to help out the track detection software by giving an indication of the number of tracks you will only need to use this step if the fully automatic track detection has failed c if automatic track location has failed to identify the tracks correctly i check hint ii enter the approximate number of tracks in the area of interest to choose manually how many tracks you want to place in each area of interest on the gel a click in the area of interest to select it this step is unnecessary if there is only one area of interest b press the create radio button to enable the tracks for manual positioning spin box c enter the number of tracks you want to place in that area of interest the tracks will be placed at default positions and you will need to position them manually later 1-8 imageaide user manual[close]
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how to create and work with secure sample files note the leave the tracks unchanged radio button is only enabled when the sample properties dialog box is displayed for an existing analyzed sample see the entry for sample properties in the file gel section in the menus chapter page 7-28 8 click on the analysis notes tab and enter any notes you want to save with the secure sample file the other two pages give information saved with the file in the acquisition program see the entry for sample properties in the file gel section in the menus chapter page 7-28 for details 9 press ok to close the dialog box and display the sample in a gel window if you have set automatic track location the tracks will be shown on the image and locked if you have also set automatic peak location the peaks will be marked on the tracks note the recommended way of working with imageaide is to use automatic peak location use configuration in the extras menu see page 7-121 to set imageaide to locate peaks automatically if you have not set automatic track location and have specified a number of tracks the tracks will be unlocked ready for you to position them accurately on the image see how to place position and work with tracks page 1-21 for details once you have opened an unanalyzed file you should save it in order to save the track peak and other settings see saving a secure sample file page 1-20 for details imageaide user manual 1-9[close]
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using imageaide for gel analysis creating a new secure sample file to create a new secure sample file for example from a .tif or .bmp image file 1 choose new import from the file menu to display a standard windows open dialog box 2 choose the type of image file from the files of type drop-down list box 3 use the look in drop-down list box to select the folder containing the image file 4 click on the file in the list box to select it the name of the file will appear in the file name box 5 press open to close the dialog box what happens next depends on whether you have selected auto import methods in the imageaide configuration dialog box if you have selected auto import methods in the imageaide configuration dialog box the import method dialog box will open so that you can choose which settings to import see opening or creating a secure sample file with automatic import page 1-15 for further details 1-10 imageaide user manual[close]
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how to create and work with secure sample files if auto import methods is not selected the sample properties dialog box will open so that you can set the type of image and make other settings see the picture on page 1-3 and the instructions following it using the browser as an alternative to using the open command to open an existing secure sample file see steps 13 in opening a secure sample file page 1-2 or the new import command to create a new secure sample file for example from a non-secure .tif or .bmp image file see the previous section you can use the imageaide built-in browser to preview and open image files to open an existing secure sample file or create a new secure sample file using the browser 1 choose browse from the file menu to display the browser imageaide user manual 1-11[close]
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using imageaide for gel analysis 2 use the left-hand pane in the browser in exactly the same way as you use the left-hand pane in windows explorer to select the folder containing the required image the right-hand pane in the browser will show a preview of all the images in the selected folder 3 double-click on the image that you want to open what happens next depends on whether you are opening an analyzed or unanalyzed secure sample or creating a new secure sample file if you are opening an analyzed secure sample file the sample will be opened in a gel window with all tracks and peaks displayed as they were when the file was last saved all previously analyzed results will be shown in the results tables if you are opening an unanalyzed secure sample file or creating a new secure sample file if you have selected auto import methods in the imageaide configuration dialog box the import method dialog box will open so that you can choose which settings to import see opening or creating a secure sample file with automatic import page 1-15 for further details if auto import methods is deselected the sample properties dialog box will open so that you can set the type of image and make other settings see the picture on page 1-3 and the instructions following it 1-12 imageaide user manual[close]
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how to create and work with secure sample files acquiring an image to create a secure sample file imageaide allows you to acquire an image from a twain source such as a scanner or a digital camera and create a secure sample file from it to select a twain source so that you can acquire an image from it 1 choose select source twain from the file menu to display the select source dialog box 2 click on the required source to select it 3 press select to confirm the selection and close the dialog box to acquire an image from the selected twain source and create a secure sample file 1 choose acquire twain from the file menu a dialog box will be displayed for the selected source so that you can select acquisition options and acquire the image 2 use the dialog box to acquire the image what happens next depends on whether you have selected auto import methods in the imageaide configuration dialog box if you have selected auto import methods in the imageaide configuration dialog box the import method dialog box will open so that you can choose which settings to import see opening or creating a secure sample file with automatic import page 1-15 for further details if auto import methods is not selected the sample properties dialog box will open so that you can set the type of image and make other settings see the picture on page 1-3 and the instructions following it imageaide user manual 1-13[close]
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