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Imaris 4.0

1. gt ppm Ready Zoom 1 00 Fig 95 ImarisColoc main screen Preview Window This area visualizes the data set together with a preview of the colocalized region It displays a Slice representation of the data set i e a 2D view from the top in the z axis direction A sliding bar with a handle allows inspecting and displaying the single slices in the data set along the z axis The preview window updates in real time the display of the colocalized region based on the actual settings of the colocalization parameters The original channels can be switched on or off using the channel visibility editor The user can zoom and pan the view with the middle and right mouse buttons respectively Page 130 Imaris 4 1 Operating Instructions BITPLANE AG Display Adjustment x Select all Channels Range Ch 2 name not specified 22 6863 Heset 135 1780 Auta Blend Opacity 27 040 Ch 2 Fig 96 Preview window with Channel visibility and Display adjustments Channel Histograms Channel Channel 1 namen v Threshold 36 00 This area allows the selection of the two channels for the colocalization analysis The intensity histograms are displayed for the currently selected channels With most data sets checking the Ignore border bin and Logarithmic options see Histogram options on page 137 allows a more representa tiv
2. 132 ja MN T SEE NHIEU 132 NE RN MEME 133 MM UU UI DAMM EM E NEC 134 BASIC OPERAT ON TER e ED EN pi UR E ERREUR 138 Nereus E cim 138 SELECTING THe CHANNELS epa 139 Page 4 Imaris 4 1 Operating Instructions SETTING THE THRESHOLDS COLOG DISPLAY E toS a aat COLOC VOLUME STATISTICS BUILD COLOC CHANNEL SAVE COLOC CHANNEL ADVANCED OPERATION POLYGON MODE inito tete ta ter otl eee rk te Edda MASK CHANNEL AND AUTOMATIC THRESHOLD TIME 5 056 077 7 000 4 EXAMPLES deiude iwi Eun VISUALIZE THE COLOC CHANNEL IN IMARIS VISUALIZE THE COLOC CHANNEL IN SURPASS REFERENCE GUIDE MAIN TOOLBAR THE EDIT MENU IMAGE PROPERTIES buiusmodi roo Uia tte ean OR GE OME CRV e T E et eae INI XCTI seman ee eae sete a dee FEIUMBNAID PARAMETERS ELE ADD 5 DELETE CHANNELS ADD RR Ex EXER 5 lt 2454 a Fexc
3. UR Kui OBJECT PROPERTIESAREA OBJECTS AREA 9 VIEWING AREA DRAW STYLE deca e aid SET CENTER e GROUPO raran lIGHTAOOUROE eeiam iea a a nace ein oun SUR AC E ORTHOSBIOBS ua sees CLIPPING PLANE sii eere TOPOGRAPHY EXTERNAL OBJECTS COMBINATIONSOF VISUALISATIONS COLOR AND MATERIAL 22 0 SPOT TRACKING THRESHOLD TRACKING MANUAL TRACKING TRACK EDITOR pU TRACK SETTINGS TRACK STATISTICS KEYFRAME ANIMATION SCENE FILES EXPORT OCENE 2 LOAD GCENE tete 5 MEASUREMENTPRO SLICE MEASUREMENT 2D SLICE MEASUREMENT 3D MEASUREMENT SURFACE AND VOLUME MEASUREMENTS PO SURFAGES ees SURFACE AND VOXEL STATISTICS SPOTS MEASUREMENTS 16 11 04 BITPLANE AG BITPLANE AG Operating Instructions n 94 POLS STAS NO A 94 95 IED IN
4. OK Cancel Fig 115 Geometry Box Type Shows the data set type Supported types are 8 bit 16 bit and 32 bit 12 bit data sets are stored as 16 bit Size Indicates the data set size in the x y z and t Resample Opens a dialog to change the dimensions of the data set i e to reduce the voxel density For a detailed description of the Change and Resam ple dialogs please see Resampling in this section Coordinates Indicates the data set position in the coordinate system Voxel Size Displays the voxel size Min Minimum coordinate value on the x y and z axis Max Maximum coordinate value on the x y and z axis All values may be altered When altering the Min or Max parameters the voxel size is recalculated When altering the voxel size the Max values are recalculated Unit Indicates the scale units Possible Values nm nanometers um micrometers u mm millimeters Page 156 Imaris 4 1 Operating Instructions BITPLANE AG m meters unknown Channels The channels section of the Image Properties menu is used to define the channels and layers used in the dataset Channels and layers can also be managed from the edit menu Image Properties P Channels Channel 1 Tou can add channels from another data set i Channel 2 x Thumbnail Add Channels i Parameters Delete channels from the data set Delete Channels Layers
5. Riokate Channel Shift Swap Z with T Image Smoothing Image Thresholding F Contrast Change AutaDeblur Huygens Essential Fig 6 Imaging Processing Menu 16 11 04 Page 15 BITPLANE AG Page 16 Operating Instructions The Help menu contains three important information items 1 License A box informs you about all currently found licenses for your IMARIS system 2 About Imaris In case of any problem requiring direct support from Bitplane you find here the exact Version number and date of your IMARIS system 3 Keys help Click Help Keys Help In addition the help menu provides access to some demos of Imaris func tionality Fie Edit View Image Processing Surpass Help License keys Help Demol Easy3D Demol Easy3D Info Demaoz Surpass Demo Surpass Info Demos Kevframe Demos Kevframe Info About Imaris Fig 7 Help Menu Imaris 4 1 Operating Instructions BITPLANE AG The use of IMARIS in the daily workflow comprises the following steps 1 Loading a data set Adjusting the image parameters Preparing the data set Exploring the data set using different display modes Creating the required output images Opening images Data sets can be loaded from various file formats Image file series If the data set consists of a whole series of images each stored as individ ual file select only one file to open and the system will automatically load the re
6. Surpass Slice Lin Camera oom Made C Default Meas View f Single sese y 0 23 at 36 14 55 24 32 00 Zoom 3 66 NUM Tools IF arid Fig 20 Slice mode Viewing is performed by Dragging the slider handle in the left control bar up and down or Clicking the slider and then using the up down Arrow keys 1 slice or the Page Up and Page Down keys on the keyboard Pressing the Begin or End key displays the first respectively last slice Click the arrow at the top of the slider to see a continuous display through all the slices in the stack A closer look at details can be achieved using the zoom function When moving the cursor over the image the current voxel intensity and the 3D position in the data set in brackets is displayed in the lower left corner of the view Time Bar If the user has a license for ImarisTime and a time series is loaded a time bar displays at the bottom of the main window It contains a slider show Page 34 Imaris 4 1 Operating Instructions BITPLANE AG ing all time points in the series a Time Settings button Start and Stop but ton e Record button pel For a full description of ImarisTime please see the ImarisTime section of this manual Section The Section view lets you inspect the environment of any given point within the data set by showing the cut through the point in the x y
7. 51 9 Surpass Scene C Ortho Frame Perspec IS Light Source 1 db TREE Full Featun box move Stereo Fig 49 Topography Coloring External Objects Surpass provides the ability to save and import external objects and display them concurrently with a Surpass object for comparison For example previously calculated IsoSurfaces which have been exported as selected objects can be imported to another data set Page 72 Imaris 4 1 Operating Instructions 16 11 04 BITPLANE AG All selected IsoSurface objects Contours and External Objects in the view are saved as an Inventor file with an iv extension and can then be loaded as external objects Exporting an Object In the objects tree highlight all objects to be exported To export all IsoSurfaces contours and external objects select the entire Surpass Scene folder Select Surpass Export Selected Objects The Save selection as external objects box is displayed Select the directory and enter the name for the file to be saved and click OK The objects are saved as an Inventor file Loading an Object P Click the External Object icon e in the objects toolbar or click eurpass Add New External Object An object entry is automatically created in the objects area Click Load in the object properties area The Load External Object File box is displayed Enter the path and the requested File name a
8. Fig 50 External Object File properties Position Scaling The following positional scaling options are available Translation Use these fields to move the external object in the X Y or Z direction Scaling Use this field to scale the external object in one of the dimensions or all together if fix ratio is checked Fix ratio is only valid with this method of scaling Hotation Use this field to rotate the external object around the X Y or Z axis Reset Size and Location Clicking this button sets the external object back to its original size and position before any translational scaling or rotational operations have been performed Combinationsof visualisations It can be useful to combine different visualizations of the same object in order to have a better view or possibility to compare Some examples are shown below Page 74 Imaris 4 1 Operating Instructions BITPLANE AG Fig 51 IsoSurface Volume Fig 52 IsoSurface and Ortho Slices combined 16 11 04 Page 75 BITPLANE AG Operating Instructions Fig 53 IsoSurfaces with different transparency Color Material Surpass offers the user numerous controls to adjust the colors of Isosurfaces external objects and contours 1 In the objects area highlight the object or group to which the color and material settings shall apply 2 Click the Color tab in the Properties area The Color Properties box is displayed Sur
9. i El MILQ Surpass Scene Pe Light Source 1 Frame 9149 volume Draw Style Ful Featured box move smooth lines Set Center Stereo Ready Zoom 100 2 Fig 45 Volume Display Adjustment and Blend Opacity The Display Adjustment and blend opacity can be adjusted for one or all channels Click Edit Display Adjustment Changes in Blend Opacity are seen in real time Changes in the Display adjustment will be calculated automatically See Figure 30 in the Visualization section of this manual Note The threshold setting is affected by the settings for Channel Visibility e and Display Adjustment If all channels are set visible and their display adjustment range is identical the threshold applies on all channels If the Display Adjustment range of the channels is different the threshold settings shown are only valid for the first visible channel For all other channels the applied threshold value is unknown The calculation of the volume may take a long time when the data set is large It is therefore possible to specify that only the xy textures are calculated and drawn Page 66 Imaris 4 1 Operating Instructions BITPLANE AG Go to Edit Preferences Display and check Use only xy textures for 3D preview then click OK See the Reference section of this manual for a complete description Orthoslices Ortho Slices represent an orthogonal plane orthogonal to the coord
10. Select File Open or click Open in the main toolbar The Open box appears Select Files of Type Leica LCS Left click the lei file to highlight it and click Settings The LeicaVista Image Selection box displays Select the required image stack on the left side of the dialog box The individual images that belong to that stack will display on the right side Click OK to open the image LeivaVista Image Selection B e X File contains 3 images Images Files belonging te image yeast 4 25 Senes 000 yeast phago 4 25 SenesU9 gt ch yeast phago 4 25 SenesU9 HIE gt ch yeast phago 4 25 09 100 001 chi yeast phago 4 25 SenesU9 gt 1 chu yeast phago 4 25 SenesU9 001 chu yeast phago 4 25 Senes 100 002 cht Fig 9 Leica LCS Settings Adjustable Tiff Series Reader Settings If the data set consists of a series of images individual images can be sorted according to various dimensions i e slices channels time points dimension sequence The selected sequence is shown in the File Ar rangement panel The reader can handle tiff series with single and multi ple running numbers Page 19 BITPLANE AG Operating Instructions o z p ERETTE S i mT ip Series Reading Sequence Documents and Settings Jaimer DeskkEop est serie a S inl x Apply Automatic File Filter Esp acuments and SettingssaimmersDresktep
11. Undo Image Properties Channel visibility Display Adjustment Add Time Points Delete Time Points Add Channels Delete Channels Add Layers Tine Crop 3D Resample Time Resample 3D Change Data Preferences Alt Backspace Ctrl I Fig 112 Edit Menu The undo function allows the user to undo the most recent image processing function Preferences Display The number of undo functions allowed is set in The copy function allows the user to copy the image on the screen and paste it into another application The Data Set entry in the Image Properties menu contains the basic infor mation and the log entries Data Set Name Description Log Data set name Valid description Log file Displays all previous actions on the data set Page 155 BITPLANE AG Operating Instructions Geometry The Geometry section of the Image Properties menu contains the ge ometical information about the data set Image Properties Geometry Data Set Channels Wide Channel 1 fe bit unsigned Thumbnail Parameters Size X Y 2 T Size 256 256 2 45 Resample 3D Coordinates Time Point VoxelSize 4 006274 4 006274 9 309678 f Date VY Y Y MM DD Min 0 000000 0 000000 0 000000 Max 1021 599976 1021 599976 288 600006 Time HH MM 55 10 30 00 Unit jum v All Equidistant
12. of material A above threshold colocalized Percentage of channel A material above threshold A that is colocalized SA iecoloc Y SA 10090 i ieobjectA of material B above threshold colocalized Percentage of channel B material above threshold B that is colocalized SB iecoloc 100 SB ieobjectB 96 of ROI colocalized Percentage colocalization of channel A and channel B volume inside the region of interest N cowe 100 mask of ROI material A colocalized Percentage colocalization of ROI channel A material SA iecoloc 10096 SA iemask of ROI material B colocalized Percentage colocalization of ROI channel B material SB iecoloc 100 SB iemask Channel correlation in colocalized volume Pearson correlation of channel A and channel B inside the colocalized region Note The correlation value ranges between 1 and 1 A value of 1 represents perfect correlation O no correlation and 1 perfect inverse correlation Page 143 BITPLANE AG Build Coloc Channel Page 144 Operating Instructions 7 core oc avg coloc avg coloc iecoloc coloc iecoloc iecoloc Channel correlation in data set volume Pearson correlation of channel A and channel B inside the entire data set volume gt USA SA BIS araser c dataset dataset dataset iecoloc Channel correlation in ROI volume Pe
13. 3 The data is exported to MS Excel and sorted in different sheets The data contains the Surface Statistics and Voxel Statistics for the group as well as for single objects Warning Area and volume of an IsoSurface depend on the chosen calculation parameters The Spots object is a computer generated representation of a point like structure It permits to automatically detect and visualize these structures See the Imaris Surpass User Manual for details on creating a Spots object Creating Statistics Spots statistics are automatically computed for each Spots object To each spot belongs a spatial position along x y and z as well as the intensity of the point it represents The number of spots per time point is also part of the standard statistics of a Spots object When a Tracking is done using the Spots object statistics such as track length track speed number of track branches and number of track fusions are made available See the Imaris Surpass Tracking User Manual for details on creating Spots tracking Radius Scale O00 Henderng Quality Settings Tracking Statistics Fig 69 Spots Settings box Imaris 4 1 Operating Instructions Contour Surfaces 16 11 04 Variable Value Unit gt Avg Track Speed 0 000 unknown Number of Branches 0 000 Number of Fusions 0 000 Position X 102 247 unknown 1 Posikian x 103 883 unknown i Position Y 98 157 unknown pp 4 Export Settings Spots Sta
14. If the x are missing in the first string then issue the following command chmod 755 bitplane to make it executable for the flexlm server Start FlexLM Server Verify if the server process is running by issuing the command ps ef grep Imgrd If the server runs the output should be something like root 1505 1 0 Feb 11 0 01 Imgrd c var flexlm license dat l var tmp Im log If you don t see an instance of Imgrd running start it manually with the Unix command Imgrd c var flexIm license dat var flexlm Imgrd log Then you can verify the server using the Imstat command Imstat c var flexlm license dat The output should read Imstat Copyright C 1989 2001 Globetrotter Software Inc Flexible License Manager status on Tue 2 12 2002 10 53 License server status 1700 sgi neurobio mydomain edu License file s on sgi neurobio mydomain edu var flexlm license dat sgi neurobio pitt edu license server UP MASTER v7 2 Vendor daemon status on sgi neurobio mydomain edu bitplane UP v7 0 If the server is not running the log file needs to be consulted for diagnostics information cat var flexiIm Imgrd log 1 Start the Imaris software on the computer on which you want to install licenses You will see the following dialog Page 189 BITPLANE AG Operating Instructions Imaris didn t Find any license E Would you like Ea specify your license configuration Click Yes to get to the license
15. L ancel 7 Fig 91 Add Time Points dialog box Select the required image from the Add Time Points dialog box and click Open Superfluous time points can be deleted Delete Time Points E E Fd E REC Cancel Fig 92 Delete Time Points dialog box 1 Select Edit Delete Time Points 2 Specify the time points to be deleted 3 Click OK 4 The selected time points are automatically deleted from the data set Page 127 BITPLANE AG Crop Time Resample Time Page 128 Operating Instructions The total number of time points can be reduced at the beginning or end of the series of images From Ta Cancel Fig 93 Crop Time Dialog Box Select Edit Crop Time Enter the time points to be included in the dataset Click OK All other time points are deleted from the dataset 5D I ox The total number of time points can be reduced to display the images at a faster speed Resample Time ENS x Mew number of time points Cancel Fig 94 Resampling Time 1 Select Edit Resample Time 2 Specify the number of time points needed 3 Click OK The process of resampling the time points takes a while When the process is finished the time bar displays the new number of time points Imaris 4 1 Operating Instructions Imaris Coloc What is Imaris Coloc Main screen 16 11 04 BITPLANE AG In fluorescent microscopy colocalization descri
16. 5 The threshold in the creation dialog is initially set to such a value that all detected spots are visible By increasing the lower threshold insignificant spots can be discarded The viewer immediately reacts to a change in threshold and displays only the remaining spots Select the threshold such that the result optimally fits the data It is helpful to view the data as a Volume and adjust the settings of the volume such that both the volume and the spots are visible Press Finish spots 1 Properties zoom 1 Radius Scale 1 500 1 fi 2 M Pointer Rendering gt Select EE v Navigate View Key Frame Animation Full Screen a r Camera Type Orthogonal z 9 Pers El zs Surpass Biens i H Mf Light Source 1 Draw T PU rra Frame Ful Feature cow W e Spots 1 14 Volume smooth lines Set Center Stereo hs Ortho Slicer 1 Hc Ie Ortho Slicer 2 ope Ortho Slicer 3 Fig 43 Spots Editing The properties window of a Spots object has an Edit tab When this tab is selected a slicer appears in the viewing area and spots can be deleted and added 1 To delete a spot switch the surpass viewer to select mode and shift left click onto the spot 2 Manual method To add a spot fully manually click on the Manual check box
17. Delete Channels 163 Delete Time Points 127 Diffuse 77 Diffuse color 78 Direction of the rotation 48 Directional Intensity 41 Disable subtexture 168 Display 167 Display Adjustment 19 31 32 46 66 Display Handling 73 Draw Style 53 Draw Styles 96 Drawing methods 146 Easy 3D 31 33 40 88 126 154 162 Edit Material 46 Emissive color 78 Equidistant Time Points 23 Export button 145 statistics 145 Export Scene 88 Exporting an Object 73 Imaris 4 1 Operating Instructions 16 11 04 BITPLANE AG Exporting Statistics 93 95 Exporting Statistics to Excel 93 95 Exporting Statistics to MS Excel 99 External Objects 72 100 Filament Tracer 105 Filter objects with less than nn triangles 61 Filtering 26 28 Fix ratio 73 Fix to Data Set 56 Fixed ratio 59 Flip 174 Frame 55 Frame rate 49 Frames 48 Free Measurement Points 102 Full 3D 31 33 42 45 65 88 126 154 Full Feature 53 Full screen 53 Gallery 33 38 154 Gamma Correction 178 180 Gaussian filter 59 Gaussian Filter 26 27 30 175 176 Geometry 23 70 90 156 Groups 51 56 Hidden lines 53 Hide Labels 101 Huygens Essential 181 Ignore border bins 137 Image Access 11 13 49 119 122 Image Parameters 22 Image Properties 155 ImageAccess 49 120 Imaris Coloc 129 Imaris MeasurementPro 89 Imaris Time 123 ImarisColoc 13 154 ImarisMeasurementPro 13 ImarisTime 13 23
18. Objects Area Properties Area Viewing Area Gecti Selected Properties 19 move Objects BE isis Area Bet Cerar Ecm 397 4 Fig 54 Surpass Window Page 50 Imaris 4 1 Operating Instructions Object Propertiesarea Objects Area 16 11 04 BITPLANE AG The Object Properties area displays all available functions and information for the selected object The content differs depending on the selected object type The properties of each object are described in the corresponding sections below This Objects area displays a tree list of all Surpass objects that the user has added to the viewing area Structure The tree list is automatically generated and updated when adding or deleting an object The first added object generates a group All following new objects are stored in this group A name is generated automatically for each tree item To change the name double click on the entry and enter a new name Move objects or groups from one group to another by dragging and dropping them with the left mouse button Display Each tree item includes a check box Check the box to make the object visible in the viewing area Uncheck the box to make the object invisible in the viewing area The currently active object in the viewing area is highlighted in the tree Groups The user can group objects into so called component groups
19. Operating Instructions 16 11 04 All time points Histogram options BITPLANE AG mode allows for faster browsing through the data set along the z axis The histograms are calculated and shown for an entire time data set i e for all time points within the 4D data set The histogram mode options are used to define the display of the 1D and 2D histograms Ignore border bins Logarithmic Color coded Coloc intensities Often a lot of voxels fall into the bin with the low est intensity In a frequency plot this causes a massive and unnecessary downscale of all other intensity bins which are of more interest The border bins can therefore be ignored when scal ing the display of the histogram Ignore is se lected as default The frequency plot is shown in a logarithmic scaling instead of a linear scaling Applies a false color look up table to the histo gram to improve the visibility of intensity differ ences The high frequencies bins in the 2D his togram into which a lot of voxels accumulate are shown in yellow to white bins with little voxels are shown in blue to black The Coloc intensities option defines the brightness of the calculated colocalization displayed in the Preview window Source channels Constant value Uses the intensities of the two channels for dis playing the intensity of a colocalized voxel as the square root of the product of the intensities of the original channels Display
20. Page 134 Colocalization Selection made Threshold C Polygon Automatic thresholding Histogram made C Single slice Single time point All time points Histogram options lanare border bins Logarithmic Color coded Coloc intensities Source channels C Constant value Build Coloc Channel hannel Statistics Fig 101 Control panel Selection mode Threshold The user can define for each of the channels separately an intensity value as a threshold Only voxels with an intensity value above the specified threshold will be considered for the colocalization analysis The two thresholds are visible and can be set in four different ways Imaris 4 1 Operating Instructions 16 11 04 BITPLANE AG As a hatched region with a bolded line on the 1D histograms As a hatched region with bolded lines on the 2D histogram The re gion always contains the top right part of the histogram The left and lower bolded lines define the intensity thresholds for the two channels As a value in the Threshold field of each channel As an active contour in the Preview window When using one of the methods to modify the threshold values the other indications as well as the Preview display are updated in real time Polygon The user can define a freely shaped polygonal region whose interior defines the intensity pairs considered as colocalized Page 135 BITP
21. vide the optimum amount of information from 2D or 3D still images time series and animations Once a data set has been loaded into IMARIS individual parameters such as channel colors geometrical settings or voxel sizes can be adjusted IMARIS has a variety of tools available such as cropping threshold cut ting and filters for processing the images to bring out the required details It provides 6 different viewing functions for the visualization and production of high quality images for presentation and storage gt A slice viewer gt Asection viewer for simultaneous viewing along three coordinate axes gt A gallery viewer for slice image overview and selection gt Asimple 3D viewer gt A full 3D and ray tracing viewer Allows 3D viewing from an arbitrary viewpoint and can also produce animations The Surpass viewer which offers numerous tools for data preparation presentation and manipulation of different types of data display as well as any combination of them the ability to define combine and group an arbitrary number of objects out of a set of viewing objects A Coloc viewer for colocalization computation Optional modules for colocalization measurements and to process time series are also available Page 8 Imaris 4 1 Operating Instructions BITPLANE AG It is easy to navigate within the IMARIS modules because the frequently used toolbars menus and inter active controls remain the same and
22. 0 ustom Bolme gt gt C aloriSalid Lum 20 Blue 0 Cancel Add to Custom Colors Fig 116 Base Color Box The base color selection uses the HSV Hue Saturation Value or RGB Red Green Blue method to define the basic channel color The currently active color is marked with a frame A Custom Color can be defined by clicking Define Custom Colors Color Solid Displays the current color Hue Color value Saturation Color saturation Lum Luminosity Red Green Blue Composition Use the left mouse button to drag the small cross on the color map in order to select the desired color directly from the display and the slider in the shading bar to define the color luminosity Or Enter the figures for Hue Saturation and Lum or RGB directly in the respective fields Click the Add to Custom Colors bar to store the new color in a Cus tom colors field When all colors are defined select the color by clicking on the re quested color field and click OK The channel appears in the new color Page 159 BITPLANE AG Page 160 Operating Instructions Mapped Color Color mapping is especially useful for single channel images by coloring the voxels differently according to their brightness value 7 Image Properties Hn Es Channel 1 Collagen vy Description Base color Mapped calor Selected Color Edit Copy Paste Interpolation Colorspace RG
23. 47 96 97 125 127 171 Topography 70 Track 62 Track Editor 82 Track Settings 85 Track Statistics 86 Trackball 73 Translation 74 Transparency 77 78 Triangle Reduction 61 Type 156 Undo 155 Use only xy textures 67 168 User Defined Settings 45 View 53 Viewing Area 50 Visualization 31 Volume 62 65 Voxel Size 22 156 Imaris 4 1 Operating Instructions BITPLANE AG Wireframe 53 Zoom 31 44 52 60 16 11 04 Page 7
24. Avg Track Speed If the track has no merges or splits and any vertex is connected to any other vertex via some path then the speed is calculated as the track length divided by the time between first and last object in the track The Keyframe animation feature in Surpass allows the user to create an animation from views keyframes selected by the user It interpolates frames between the selected views to create a smooth movie 1 Create an object isosurface volume etc from which to create the animation Click the Animation button on the main toolbar The keyframe window displays on the bottom of the screen Imaris 4 1 Operating Instructions BITPLANE AG retina ims Imaris 4 1 loj x File Edit View Image Processing Surpass Help NO in c 277 AVG 7 2 gt ES Open Snapshot Slice Section Gallery Easy 3D Surpass Animation Coloc Bitplane 4G Iso t28 c Properties Zoom Source Channel 2 Threshold 28 000 Curent Timepoint Time Index 1 Surface Triangles 311304 Reduce Number of Surfaces 3 Split Time Data Mask Channel Create Track Split All Settings Statistics Color Objects X o 2 1 sis Select Navigate View Full Screen i Camera Type Orthogonal Perspective Full Featun smooth lines Set Center Mr css
25. D uf nc stack bl xx O z 1 O06 4 nc stack bl xx O z 1 UDS uf nc stack bl xx O z 1 nc stack bl xx O z 1 nc stack bl xx 0 z 1 012 nc stack bl xx 0 z 1 Dimension Sequence C Z C T RR MEN 2 T C 2 Fig 88 Tiff Adjustable series sequence reader Resampling Open 4D images are usually quite large Imaris uses two mechanisms for efficiently dealing with large images e Cropping of images i e selecting a region of interest e Resampling of images Both mechanisms reduce the memory requirements for the currently loaded images and speed up the processing Cropping keeps the resolution whereas resampling keeps the perspective Imaris 4 1 Operating Instructions BITPLANE AG In the File Open dialog box highlight the image and click Resampling OpenThe Resampling dialog box displays Resampling File Open C Images wobbel ims wobbel_TOOO ims_ Original Size 6 000 iat a mM subsampling Factor 1 t m Crap Limits s C E zm Resampled Size 8 000 MB 1 gt Fig 89 Resampling Dialog box Enter the resampling factor or the crop limits and press Open Swap Z with T ImarisTime offers the ability to exchange the Z axis and the Time axis and re format the image in such a way that the time dimension is mapped onto z
26. Filter Width 4 7 4 4 Intensity Position Intensity Position Fig 137 Background Subtraction Connective Baseline The Connective Baseline works with two threshold values a lower and an upper threshold lt applies these thresholds to divide the image into regions and then processes each region with a Baseline Subtraction using either the lower or the upper threshold The three types of regions the method creates are the following A Connected region of voxels where the inensity of every voxel within the region is below the lower threshold B Connected region of voxels where the intensity of every voxel within the region is greater than the lower threshold and no voxel within the region has an intensity greater than the upper threshold C Connected region of voxels where the intensity of every voxel within the region is greater than the lower threshold and at least one voxel within the region has an intensity greater than the upper threshold The intensities of voxels from regions of type A are set to zero as are the intensities of voxels from regions of type B Only voxels from regions of type C retain a nonzero intensity namely the original intensity less the lower threshold value The following figure shows schematically how the Connective Baseline works Page 178 Imaris 4 1 BITPLANE AG Operating Instructions 100 upper Threshold 80 S lower Threshold 30 0 5 Pos
27. Full 3D Preview Section Preview Volume When this preview is selected the data set is calculated and rendered automatically with standard settings to produce a fully movable 3D image e Hold down the left mouse button and move the mouse to rotate the cube e Hold down the right mouse button and move the mouse to pan e Hold down the center mouse button and move the mouse to Zoom 16 11 04 Page 43 BITPLANE AG Operating Instructions th retina red ams Imaris 3 2 Bl File Edit View Image Processing Surpass Help of Full 3D yp man Qc som NM S i Surpass Open Save Snapshot ECH Slice Section Gallery Easy 3D Preview Cube Section Mode MIP Blend Light Edit Settings Edit Material Steren Off C Red Cyan Buffers Calculate Animation U reser a Fig 29 Full 3D Preview Volume e The Zoom buttons allow the user to quickly change the dimensions of the image Select a predefined camera position from the View list Select the Type Ortho parallel lines Perspec perspective display Stereo When working with special 3D glasses the Stereo Mode must be configured Red Cyan for red green glasses Buffers for crystal eye glasses image shuttering Select the Mode MIP Maximum Intensity Projection maximum intensity of all lay ers along the viewing
28. System Man and Cybernetics SMC 8 1978 630 632 Resample data set Check the Resample data set checkbox in order to reduce the amount of data before calculating the IsoSurface Bitplane recommends resampling for all data sets Resampling reduces the number of voxels in a grid but keeps the original relation between the voxels if specified by fixed ratio Select Standard Size or a reduced Quick Size setting Resample settings If Resample data set is not checked the respective fields in the Advanced Settings show the size of the loaded data set If Resample data set is selected the user can accept the defaults or customize the values for the voxel sizes Click the Advanced Settings button to customize the settings Enter the values for the x y or z dimension directly in the respective fields Check one of the Fixed ratio options to maintain the x y or x y z ratio of the data set when entering values directly The Gaussian filter width is calculated automatically from the voxel sizes oriented at the largest voxel dimension but can also be altered directly The value is given in image coordinates and not in fraction of voxels The Gaussian filter is isotropic with respect to the image coordinates Page 59 BITPLANE AG Page 60 Operating Instructions Smooth data set Check the Smooth data set option to smooth the structure of the object to obtain fewer triangles and therefore less data Smoothing applies a G
29. The edge preserving filter does not smooth the image in the direction of maximum intensity change in order to preserve edges Page 175 BITPLANE AG Operating Instructions Threshold Cutoff Threshold Cutoff compares the intensity of every voxel in the image to the threshold value If the intensity is greater than the threshold it remains unchanged lf a voxels has an intensity less than the threshold value it is assigned a new intensity value This value is 0 by default The following figure shows schematically how Threshold Cutoff works 80 2 30 2 0 Position 2 D New Value Position Fig 135 Threshold Cutoff Baseline Subtraction Baseline Subtraction subtracts the baseline value from the intensity of every voxel in the image If the result is positive this is the new intensity value if it is negative the voxels intensity is set to zero Page 176 Imaris 4 1 Operating Instructions BITPLANE AG 80 30 O Intensity Position 70 50 gt O c 9 0 Position Fig 136 Baseline Subtraction Background Subtrac tion Background Subtraction applies a Gaussian filter to define the background at each voxel and then performs a Baseline Subtraction of this variable background The filter width is the only parameter of the background subtraction method The following figure shows schematically how background subtraction works 16 11 04 Page 177 BITPLANE AG Operating Instructions
30. You can add layers from another data set Add Layers Fig 114 Channels box Channels Add Channels Opens a dialog to add one or several channels from another data set Delete Channels Opens a dialog to delete one or several channels from the data set Layers Add Layers Opens a dialog to add one or several layers from an other data set For a detailed description of the Channels and Layers dialogs please re fer to the Edit sections in this reference guide 16 11 04 Page 157 BITPLANE AG Channel 1 n Page 158 Operating Instructions The Channel entry in the Image Properties menu allows the complete color definition for each channel separately Image Properties Channel 1 Mame fee anne 1 Collagen I iL Channel 2 T Thumbnail Description i Parameters specified Base color Mapped color Cancel Fig 115 Channel Box Channel Name Channel name Description Informative description of the channel Base Color See separate description on the following pages Mapped Color See separate description on the following pages Edit Displays the color selection feature Imaris 4 1 Operating Instructions 16 11 04 BITPLANE AG Base Color Color Basic colors HI MIT EI rr EI See NIS BI NENNEN EENENININEN BER BH Custom colors 00 Hug Red 255 oe a ee Sat 240 Green
31. and z axes or a defined portion of the data set several slices together The illustration below shows the coordinates in the three different display areas of the section view Fig 21 Coordinates To select a point click with the left mouse in any of the three views hold the button down and drag the crosshairs to the requested point The crosshairs immediately snap to the cursor when clicking anywhere in the image The other views connected by one coordinate automatically follow the movements A closer look at details can be achieved using the zoom function 16 11 04 Page 35 BITPLANE AG Operating Instructions JOR of pm c NIE d gt D a AT Ni NS d Open Save 5 Snapshot Slice Section Gallery Easy 3D Full 3D Surpass Coloc Bitplane AG Section View oom Normal Extended Extende Fi Crosshair Normal M Extended Ready Zoom 296 z Fig 22 Section view Normal Checking View Normal selects a single cut through the data set You may eliminate the crosshair display on the image by unchecking the Crosshair Normal box It is then only visible at the edges Mode cannot be selected in this view The section view also displays additional information about any given point in the data set Click a location in the image display In the lower left corner of the view a display shows the voxel in
32. 0 2 0 Add as many measurement points as needed When adding a second measurement point it is automatically connected to the first point by a straight line Page 103 BITPLANE AG Operating Instructions Moving a Free Measurement Point The location of the measurement point can be changed either in the object properties area or in the viewing area Properties Position Distances X e 8 47 F1 F 121 3 um y F3 P4 740 um 64 00 cum g Entries 195 3 um ni 64 00 Faren Object none Fig 77 Measurement Properties e Inthe object properties area enter the x y and z coordinates of the new position e Inthe viewing area switch to Select mode left click the measurement point and move it along the required axis on the required plane Altering the Direction of the Handles The measurement point has one preferred axis which is marked with two handles When clicking on the point it moves in a plane perpendicular to the preferred axis 1 To enlarge the measurement point click the key on the number keypad 2 When the measurement point is large enough click the measurement point with the left mouse in Select mode Two handles pointing in the opposite direction are displayed To alter the direction of the handles move the mouse pointer onto the measurement point and press the Ctrl key The measurement point can now be dragged to the required axis and to the new position Page 104 I
33. 35 88 125 Installation 11 Interpolation 45 160 161 173 Invert 181 Isosurface 58 101 IsoSurface Setup 59 Key Frame Animation 173 Keyframe Animation 86 keys help 9 Keys help 16 Layers 157 164 Page 3 BITPLANE AG Page 4 Operating Instructions Leica LCS Settings 19 Licensing 13 Light 40 Light Direction 45 Light Source 52 55 56 Linear Stretch 179 Load Scene 88 Loading 169 Loading an Object 73 Log 155 Logarithmic 137 Main screen 129 Manipulator Active 69 Manual Tracking 82 Mapped Color 160 Mask Channel 133 Materials Palette 77 Maximum Number of Commands in History 171 Mean 37 Measurement color 169 Measurement Points 91 100 103 Median Filter 26 27 MIP 37 40 44 45 65 162 Mode 65 Movie settings 49 Naming conventions 52 Navigate 52 New Maximum 180 New Minimum 180 No Texture 53 Object Properties 51 Objection Creation Buttons 173 Objects Area 50 51 only xy textures 168 Opacity 47 Open 154 Opening images 17 Ortho 44 53 Orthoslices 67 Pan 31 Parameters 162 Persp 44 Perspec 53 Playback Modes 172 Pointer 52 Points 53 Polygon 135 Polygon Mode 146 Imaris 4 1 Operating Instructions 16 11 04 BITPLANE AG Position Above 40 Position Scaling 74 Preferences 166 Preview Cube 42 Preview Section 43 Preview Volume 43 Preview Window 130 Properties Area 50 Range Adjustment 166 Ray Cutoff
34. 78 shows the surpass viewer with the volume of the current data set A new filament object was created Filament 1 On the top left the properties to create the filament graph step by step are visible At the moment there is no 3D representation of the filament graph but only an orthogonal section Filament 1 Properties Show Modes Terminal Pts Style Line 1 pixel Cylinder 1 000 um Branch Pts C Done 1 000 scale M Root Point Rebuild Set Selection as Point Length 73 318 um Average Diameter 3 315 um Serial Resistance 12 041 1 Number of Branchings 59 Surpass Scene Light Source 1 s Ce Frame 8 Filament 1 Fig 79 shows the objects tree of the Surpass viewer The object tree allows users to hide or show objects change its properties and group various components Together with the scene view and the object property dialog it provides the user with a rich palette of interaction tools Page 108 Imaris 4 1 BITPLANE AG Operating Instructions Segmentation and Automatic Graph Compilation The automatic graph compilation creation is guided by a wizard like user interface and consists essentially of 3 steps 1 Select source channel and do some preprocessing 2 Segmentation to specify filamentous and non filamentous parts 3 Graph compilation 4 Finish The wizard allows to go forth and back until the optimal
35. Diffuse color Diff determines the basic color of the object e Specular color Spec e Emissive color Emis To specify a color manually 1 Check the Edit or Color checkbox of the requested effect Check Edit to apply the color selection to only one effect Check Color to apply on more than one effect concurrently and to apply the selected color to all checked effects When checking Edit or Color the Material Color box is displayed Material Diffuse Color 10 x Edit Sliders Options Fig 57 Material Color box 2 Select Sliders and your requested color mode if necessary Additional sliders are displayed 3 Drag the handle in the color disc until color and brightness of the ball in the material editor box are satisfactory Use additional sliders to adjust the settings if necessary The color settings are accepted for all checked effects in the material editor Close the material color box Adjust the brightness of any effect by means of the sliders in the material editor 6 If required adjust the Shininess of the IsoSurface object by means of the respective slider in the material editor 7 f required adjust the Transparency of the lsoSurface object by means of the respective slider in the material editor This may be useful if smaller objects are hidden inside a larger object and cannot be seen otherwise 8 Click Accept to apply the changes Page 78 Imaris 4 1 Operating Instructions BITP
36. Gr ON OU 96 CAECULATING NGONTODR SURFACE VE ho 98 SATIO TO nO centre 99 tus t AT 100 MEASUREMENT POINTS VERE N EU Us 100 MEASUREMENT POINT PROPERTIES 100 ATTACHED MEASUREMENT POINTS teal 101 FREE 102 FILAMENT TRACER 105 INPUT DA p ccr 105 SEGMENTA TION mercem 106 AUTOMATIC GRAPH COMPILATION 106 MANUAL GRAPH 107 SEMI AUTOMATIC GRAPH 107 USER INTERFACE 108 SEGMENTATION AND AUTOMATIC GRAPH COMPILATION 00000000000 0 00000000000000000000000 109 FILAMENT PROPERTIES Gin deductus MI ER M EE 112 TUME DATA CAD
37. Install Upgrade Product Licenses For this Computer IF this computer is connected to the Internet Imaris can directly download the license information from vw Press the Online button Online If this computer has na Interet access you can download the license information via another computer with Internet access Press the Offline button and follow the instructions Offline IF this computer applies license information from a floating license server ar if you would lke ta configure a license please enter the fle path ar the license server name L Fragram Files sBitplane sbirplanae lic or portiiservername ___ Mate will need to restart the application in order for the changes ta take effect Carosi 2 Enter port servername where servername is the name IP address of the computer running the license server and port is the port specified in the license file of the floating license server 3 Press OK and restart Imaris for the license information to take effect License Server Irix To use floating licenses you need to a install a floating license server and b configure Imaris to use the floating license server Both steps are described in this section Installation of License Server The license server is not part of the default installation of Irix Therefore it needs to be installed first We assume that the operator has experience installing
38. Median filter for a data set can have only one value This value can be e applied separately to any one channel or to all channels Structures that are smaller than the filter width will be removed In the menu bar select Image Processing Image Smoothing Me dian Filter The Median Filter box is displayed Select the channels to be filtered by marking the check box Check the requested Filter Size and click the Apply button The effect of the change can be seen in the image f necessary readjust the value When the results are satisfactory click the OK button The data set is modified Click Cancel to return to the original image Image Thresholding Whether or not it is necessary to apply a thresholding function to an image depends on its quality Inspect the image to see if any action is required For Image Thresholding a number of functions are available 1 Threshold Cutoff 2 Baseline Subtraction 3 Background Subtraction The following paragraphs describe the user interface to these filters For details on their function please refer to the Reference section in this manual Threshold Cutoff Threshold Cutoff 1 X Channel 1 name not specified Threshold value Ies Set values below to 0 Channel 1 name not specified cancel Fig 17 Threshold Cutoff box Page 28 Imaris 4 1 Operating Instructions S Baseline Subtraction Baselin
39. Pinhole Channel 1 es Color 2222 ColorMode 2225 DolorRange o c Bain 0 Mas c Offset o c Pinhole Image Do C 0 c EstMax 0 o EstMas2 0 c EstMinl o c EstMinl ii Delete Parameter Cancel Fig 119 Parameters Box Parameters A set of informational parameters is appended to the image file The value of the parameter currently selected is shown in the Parameters window Add Group Delete Group Add Parameter Opens a dialog to add a new group Deletes a group Opens a dialog to add a new parameter to the group Delete Parameter Deletes a parameter When images are imported from different file formats their parameters are saved in groups Imaris 4 1 Operating Instructions Add Channels Delete Channels 16 11 04 BITPLANE AG Adding channels is used to extend or complete the data set information if a microscope can only export one channel as a file at one time All channels of a selected data set are added The size of the data sets must be identical Add Channel Look in 3 wobbel ime 1 lt Swobbel_TOOO ims wobbel TOO6 ins wobbel TO12 ims wobbel TC wobbel TOOL ins ES wobbel_TOO ims wobbel TO13 ims wobbel TC wobbel TOO ims E wobbel_TOOS ims a wabbel TO14 ims wobbel TC wobbel TOO3 ims E2 wobbel TODO ims ES wobbel 015 inns wobbel_ TC wobbel TOO4 ims S wobbel 10 ims ums wobbel TO16 inns wobbel TC wobb
40. area The data is written into a text file csv file comma separated values Exporting Statistics to MS Excel 1 Highlight Surpass Scene or the required Group folder in the Objects area 2 Click Excel in the object properties area Choose whether you want to use pivot tables or not some configurations of MS Excel do not support them 3 MS Excel is then opened and the sheets are filled with the data Page 99 BITPLANE AG Operating Instructions External Objects Calculated contours can be saved as an External Object and Loaded into another data set See the External Objects section Measurement Points ImarisMeasurementPro allows the user to set Free Measurement Points as well as Attached Measurement Points and thus to perform measure ments in space Measurement Point Properties Measurement Point properties are specified on the Surpass Scene or Group Properties area e Highlight Surpass Scene or a Group folder in the Objects area e Click the Distances tab Surpass Scene Properties Line Pair AB CD Folkyqon AB BC CDI Origin AC AD F1 F2 hb 5 um Liraw Lines visible Points C all Points C no Points Hide Labels Hide Distance Labels Iv Time Depend Paints Export curtace Objects Distances Fig 74 MeasurementPoint Properties Box There are three different Line Modes for connecting Measurement Points e Pair Any two consecuti
41. be inverted by marking the check box on the left The effect of the change can be seen in the image When the results are satisfactory click the OK button The data set is modified Click Cancel to return to the original dataset AutoDeblur deconvolution software is integrated into Imaris It requires a separate license The pathway to AutoDeblur must be set in Edit Preferences Advanced Files AutoDeblurExeFileName When opening AutoDeblur the software will prompt for a destination for the file that is transferred to AutoDeblur and will automatically create an image in the ids ics format and open it in AutoDeblur e Click Image Processing AutoDeblur An error message appears if the program is not installed or the pathway is not set Huygens Essentials deconvolution software is integrated into Imaris It requires a separate license The pathway to Huygens Essentials must be set in Edit Preferences Advanced File HuygensExeFileName When opening Huygens Essential the loaded data set is automatically transferred to that program e Click Image Processing Huygens Essential An error message appears if the program is not installed or the pathway is not set Page 181 BITPLANE AG Operating Instructions License installation Guide Two different licensing schemes are available for the software node locked and floating Node locked licenses grant permisssion to run the licensed software on one computer node only Floatin
42. by means of the slider or a direct entry of the slice position given as the offset distance from the data set origin equals the absolute coordinates 3 Check show slice border to display a frame in the selection color around the active slice 4 The slice can be viewed and manipulated by all available functions in the viewing area The Clipping Plane cuts away objects on one side of the plane It can be freely moved and rotated in the scene and allows the user to look inside any object The Clipping Plane consists of a yellow frame with a white double rod in the middle Imaris 4 1 Operating Instructions 16 11 04 BITPLANE AG 1 To add a clipping plane click the Clipping Plane icon is on the objects toolbar or click Surpass Add New Clipping Plane from the main toolbar The Clipping Plane is displayed in the viewing area 2 To clip another object this object must be placed behind the clipping plane within the tree in the Objects Area that is it must be in the same group as the Clipping Plane and it must be placed below the Clipping Plane The figure on page 70 gives an example where the Clipping Plane cuts away part of a Volume and an Isosurface 3 The position of the Clipping Plane can be changed Choose Select mode highlight the Clipping Plane in the viewing area and left click on the bigger diameter rod It will turn yellow as it is selected move it to set the Clipping Plane to the required position Th
43. conditions that could result in problems such as a loss of data how to avoid them This symbol marks features of optional modules in imaris You may not have access to the described functionality without the appropriate module Bold text Bold text is used for e Menus and menu commands Example In the Edit menu select Delete e Buttons Example To confirm click Save Quotation marks Quotation marks are used for Internal cross references Example See the section xyz in this chapter Text displayed in a window or dialog box Example The status bar text reads Mean error 13 Italics Italics are used for References to other publications Example Refer to Manual xyz Menu path specifications Example Menu File Open Page 7 BITPLANE AG Operating Instructions Getting Familiar What is IMARIS This Chapter explains the basic IMARIS features and functions It also in troduces the most important terms used throughout the software and the manual and helps you to get started Today all optical microscopes commercially available can record several channels simultaneously to produce multi channel images IMARIS is an application designed to visualize such microscopic data IMARIS uses a special file format to store images with parameters and can incorporate image files for all major microscopes and image acquisition systems The images can be viewed in several different ways and processed to pro
44. create a spots object and perform spot detection as described in the section on Spots Then you can select the Tracking tab in the properties window of the Spots object Page 79 BITPLANE AG Operating Instructions Algorithm Brownian Motion Parameters Maximum Distance 1 500 Track Fig 59 Spots Tracking tab The Tracking tab provides several tracking algorithms to choose from e Brownian Motion This algorithm models the motion of each spot as a Brownian motion This model is appropriate if your spots actually perform a Brownian motion Autoregressive Motion This algorithm models the motion of each spot as an autoregressive process This model is apropriate if your spots perform any kind of continous motion The following figure shows the paths of a particle that moves according to a Brownian motion model and a particle that moves according to an autoregressive motion model respectively Brownian Motion AR 2 Motion Fig 60 Motion Models Page 80 Imaris 4 1 Operating Instructions Threshold Tracking 16 11 04 BITPLANE AG The motion models are used to predict the future positions of spots Based on these predicted positions the best matches to candidate spots can be determined The maximum distance parameter disallows connections between a spot and a candidate match if the distance between the predicted future position of the spot and the candidate position exceeds
45. defined Fig 83 shows 3 different representations of the same filament as lines as cylinders as cones Draw When the drawing tab is activated and the manual mode is checked an orthogonal section plane appears within the Surpass viewer Use the channel visibility and the display adjustments to optimize the section viewing In order to draw filaments onto the drawing plane switch the mode for the mouse pointer from Navigate to Select Surpass Camera tool bar 16 11 04 Page 113 BITPLANE AG Method Filament Diameter AutoPath unknown C AutoDepth C Manual Set as Starting Point Slice vz Plane Automatic Placement Cops Position 255329 unknowr f vy Plane Settings Dra Operating Instructions Select the drawing Method AutoPath mode AutoDepth mode or Manual mode Only in Manual mode Select the plane orientation of the drawing board xy xz yz Filament diameter Only in AutoPath mode Select a Starting Point in the picture a Volume object can be helpful by shift right click on the desired point Only in Manual mode The position of the drawing board can be altered in perpendicular direction by the Slice slider The same is achieved using the cursor keys of the keyboard up down Only in Manual mode Automatic Placement when activated the position is set automatically to the location of the highest data intensity In order to avoid unwanted hori
46. direction shows always a black background Blend blending all values along the viewing direction and includ ing their transparency shows the user defined background color Page 44 Imaris 4 1 Operating Instructions 16 11 04 BITPLANE AG With Light is selected the light direction and the corresponding shadow projection are shown The light direction can be modified Creating the final 3D Image Check the requested Mode Blend or MIP and Light if Blend is se lected Click the Edit Settings button The Edit Settings box is displayed Fig 31 f Blend mode was selected and Light enabled Enter the desired parameters for the Light DirectionYou may also set the light direction directly on the image in section or volume mode by pressing the Shift key and moving the white light direction indicator line with the left mouse button Intensity Enter a number between 1 5 to determine the light inten sity The upper numbers are unlimited but values gt 5 result in exces sive brightness of the image Check Shadow to create a shadow visible in the Full 3D view Check the requested Rendering Quality During standard processing a rendering quality from the available predefined list Low Medium High should produce a satisfactory im age If more specific effects are required check User Defined to en ter the Advanced Quality Settings dialog Advanced Quality Settings Interpolation Interpolation impro
47. in the properties window switch the surpass viewer to select mode move the slicer to the desired position and shift left click onto the slicer You can choose on which plane and in which position should the new spot be set It is easier to use a Volume object to add Spots Auto Depth method Click on the Auto Depth check box in the properties window switch Page 64 Imaris 4 1 Operating Instructions Volume 16 11 04 BITPLANE AG the surpass viewer to select mode and shift left click onto the de sired location The Auto Depth function will automatically set the spot depth where the object intensity is at a maximum depending on the current viewing angle e g if you are looking at the xy plane of your picture the spot z coordinate will be automatically computed Adding spots is easier when using a Volume object in MIP mode at the same time Manual Slice pz Plane xz Plane fy Plane Fig 44 Spots Edit box Settings The properties window of a Spots object has a Settings tab On this tab the rendering quality can be adjusted to produce faster performance of the display when the number of spots is very large The size of the spots can be changed via the Radius Scale box The Volume object is similar to the IMARIS Full 3D blend preview representation of the data set When a volume is added Surpass generates and displays the volume of the entire data set 1 Click the Volume icon in
48. installation dialog au can either specify a location with license information or download it via internet Install and Upgrade Product Licenses For this Computer IF this computer is connected to the Internet Imaris can directly download the license Information from ww biEplane com Press the Online button HE If this computer has na Internet access you can download the license information via another computer with Internet access Press the Offline button and follow the instructions Offline IF this computer applies license information fram a floating license server or if you would lke to configure a license please enter the fle path ar the license server name L Fragram Files sBitplane sbitplanae lic or servemname Mate au will need to restart the application in order Far the changes ta take effect 2 Enter port servername where servername is the name or IP address of the computer running the license server and port is the port specified in the license file of the floating license server 3 Press OK and restart Imaris for the license information to take effect Browse Files Page 190 Imaris 4 1 Operating Instructions BITPLANE AG 2D Histogram 132 2D slice measurement 89 2D Still Images 119 3D Measurement 91 Add Channels 163 Add Layers 164 Add Time Points 127 Adjustable Tiff Series 19 123 Advanced Quality Settings 45 Al
49. parameters are found Step 1 Preprocessing Select Source Channel Feature Preprocessing Enable Feature Preprocessing Appr Filament Diameter 0 441 um Preserve Edges lt Back Next gt Finish Create 16 11 04 Select a source Channel Specify if a preprocessing should be done Preprocessing is not necessary if the data is already deconvolved or if the noise has already been removed by a gaussian filter An approximate minimal Filament diameter has to be provided The user can also enable an edge preserving preprocessing to maintain filamentous structures Press Next to proceed with the next segmentation step The selected filter will be applied to the source channel and the result will be added as new Filament Working channel All future Filament segmentation will be done with this channel Press Finish to terminate the segmentation and build the filament graph manually the current step will not be executed Page 109 BITPLANE AG Operating Instructions Channel 2 Filament 1 Working Fig 80 shows the result from filtering with a new Filament Working channel using the Orthogonal Slicer Before continuing with setting up the parameters for this step the user can inspect the result of the previous step with any viewer of Imaris Slice Section Gallery Easy3D Isosurface etc Step 2 Segmentation Segmentation Connective Baseline 4 00
50. software on an SGI computer The command versions long grep flexim shows whether or not the server subsystem is installed If it is installed the output should read similar to the following sgi 24 versions long grep flexlm f 24676 1 license eoe sw flexlm server c etc config flexlm f 21297 1 license eoe sw flexlm server c etc config Imgrd options f 6955 4 license eoe sw flexlm server etc init d flexlm Page 188 Imaris 4 1 Operating Instructions Installation of Licenses Running the DAEMON Imaris configuration 16 11 04 BITPLANE AG O O license eoe sw flexilm server etc rcO d K30flexlm 10 0 license eoe sw flexlm server etc rc2 d S45flexlm f 22140 969 license eoe sw flexlm server usr etc Imgrd f 24318 1445 license eoe sw flexlm server usr etc sgifd If the software is not installed please load license eoe flexlIm server from your irix installation CDs please make sure to first load all the directories by inserting all CDs including overlays Bitplane will supply the licenses by email Please copy them into the file var flexlm license dat Make sure that the SERVER line is first and the VENDOR line is second The vendor daemon is an executable which is shipped by Bitplane It is called in usr local imaris3 bin bitplane Please make sure that this file is executable by root You can verify this by issueing the command Is l which should give the following output rwxr xr x 1 root sys 620224 Oct 30 20 09 bitplane
51. the series of images subsequently is much faster than playing the data set the first time Click the Time Settings button to display the Preferences box For a description see the Preferences section Click on the arrow eh next to the time slider to play or stop playing the 4D image The set of images can be played and viewed in all display modes available in Imaris Loading an Adjustable Tiff Series 1 Click File Open 2 Select Files of Type Tiff Adjustable file Series 3 Highlight one of the images in the series and click Settings The Series Reading Sequence dialog box opens The dialog s elements are as follows Apply Automatic File Filter This is activated automatically when the dialog is opened It has the same logics as the classic TIFF series reader of Imaris and will preselect the first series detected in the directory Be aware that you may not see all files in the directory 16 11 04 Page 123 BITPLANE AG nc stack xx DU 01 0 nc stack x 0 201_001 4 nc stack xx O 201 002 tif nc stack 61 x O z01_003 tif nc stack 61 xx D 01 0044 nc stack xx 0 01 nc stack x 0 z 1 nc stack xx O 01 nc stack OU z 1 nc stack 61 xx D 201 0094 nc stack bl xx _0_z01_010 tif nc stack 61 x O _z01_011 tif nc stack xx O 01 0124 nc stack bl xx O z 1 Page 124 Series Reading Sequence GiImages
52. the object contours on 2D slices The user can employ this method whenever simple thresholding does not yield individual structures Page 95 BITPLANE AG Operating Instructions e g touching cells in confocal microscopy complex tissue recorded by transmission microscopy Building Contours It is recommended to uncheck any other objects in the object tree before drawing the contour so that only the contour is visible in the view area Aag e 1 Click the Contour Surfaces icon in the objects toolbar or click Surpass Add New Contour in the toolbar A drawing plane is displayed in the viewing area The z direction is suggested by default The object properties area shows the position of the plane relative to the data set Camera Zoom Fit Pointer C Select Navigate View Key Frame Animation Full Screen Camera Type C Orthogonal Perspective Draw Style Fu Featured box move Contour Surface 1 Properties Draw Board Size Cyz x Cx fie xy Drawing Mode C Click Time 200 ms C Dist 35 7414 Slice 22 Paste Calculate Contour Surface Settings Statistics Color Objects Delete ME Surpass Scene ES Light Source 1 MB Frame smooth lines Set Center Stereo Fig 71 Contour Surface Window Draw Styles The contours can be dr
53. top of the slice bar in Slice view In addition the data set can also be saved to the Image Access database Check the box Data Output Add to ImageAccess database After the file is saved the dialog for ImageAccess starts Follow the instructions to save the data set to the Image Access database Page 122 Imaris 4 1 Operating Instructions BITPLANE AG Imaris Time ImarisTime is an optional task within the IMARIS program and is not available as a standalone program The user must have a license for Imaris and a license for ImarisTime ImarisTime extends the image processing and viewing functions of Imaris to time resolved 2D or 3D images so called 4D images and is fully integrated into all views It offers the user the ability to view a series of images that have been taken at successive times Storage Imaris interprets loads file series of various 3D file formats as a time series 4D In the case of a few formats Imaris can directly recognize 4D images and read them from a single file Storing 4D images in the Imaris3 file format is recommended as this format ensures faster loading Loading a Time Series To view a time series proceed as follows 1 Click File Open or by click the Open button in the main toolbar Depending on the number and the size of the images the loading procedure may take a while Once the loading process is finished the data is stored in cache Owing to the caching function playing
54. when the grid contains a lot of Segment Length avg 1 667 values Note Because of lack of space some values Segment Resistance avg 0 376 iiun are displayed as average value avg When exporting 4 it to Excel all individual values are available This um TF allowes to calculate sums medians or other statistical Export _ classification numbers Settings Draw E dit Statistics e The two buttons on the bottom of the tab allow data LM Each point of a filament line has its individual measured diameter The Diameter is measured as shortest distance from the center line to the Iso Surface defined by the lower threshold automatic creation Branch Point Diameter The Diameter at a branch point Full Depth Each point of the graph has a depth depending on the root point The depth of a point is defined by the number of branch point of the shortest path to the root point Full depth is the depth of the point with the largest number of branchings Full Length The sum of all lines within the filament No Branch Points The number of branch points No Edges The filament graph is composed of a lot of small linear components called edges The size depends on the voxel size of the original data set No Segments A segment is the path between branch points or end points This value counts also the number of end segments Page 116 Imaris 4 1 Operating Instructions BITPLANE AG No Unconnected Filaments
55. which the number of triangles is to be reduced Either select the appropriate percentage or move the slider to the required number of triangles 4 Click Apply Reduction The fewer triangles an object contains the bigger the triangles are To view triangles choose Hidden Lines from the Draw Style list in the right camera bar The Surface object cannot be created directly Surface Objects are generated as a result from the split operation on an isosurface when exported from a contour surface or when using automatic threshold tracking in a track object The Surface object posses a time point index It is only visible at this time point and invisible at all other time points For a description of the Contour Surface object y refer to the chapter MeasurementPro further down The Track object T can be created directly as a starting point for a tracking session or out of the isosurface properties window when only objects in a isosurface should be tracked Refer to the Tracking description further down for a complete description on how to use the Track object The Spots object is available to model point like structures in the data It provides a procedure to automatically detect point like structures an editor to manually correct detection errors a viewer to visualize the point like structures as spheres and together with Measurement Pro Statistics output Creation 1 Click the Spots icon in the objects area
56. your selected color thus clearly indicating the Contour Surface currently in use If more than one Contour Surface has been created in the objects tree the Contour Surfaces not currently in use have a gray border The user can draw as many contours on a plane as required All contours drawn on a plane will become part of the same object Use the slider in the properties area to select the slice for the next contour or move the drawing plane in the viewing area with the mouse pointer in the select mode Draw contours on as many slices as required for the contour surface In the object properties area the user can choose Visibility options in order to display contours previously drawn None contour currently being drawn is made visible e Next The contour s next to the contour currently drawn is are shown e All All contours are shown Moving Inserting and Deleting Vertices If the user is in the Time draw style vertices can only be modified when returning to the slice on which the vertex is drawn Moving vertices Vertices can be moved by clicking on them while holding down the Shift key and simultaneously dragging the left mouse button Inserting vertices Insert vertices by holding down the Shift key and clicking or double clicking with the left mouse button on the line between two vertices Page 97 BITPLANE AG Operating Instructions Deleting vertices Delete vertices by holding down the Ctrl key and dou
57. 0 56 000 Fill Cavities fully enclosed volumes Back M est gt Finish Create Page 110 Key in two values for the Connective Baseline double Threshold or click into the histogram left mouse button for lower right mouse button for higher threshold The figure above shows the preprocessed data segmented into 3 regions The black region below the lower threshold is background The gray region above the lower threshold is part of the filament but only under the condition that a white region upper threshold is included by this gray region all the gray regions which do not contain a white region will be background Check Fill Cavities to avoid hollow regions These regions would be enclosed by a filament mesh which is not desired in most of the cases Press Next to proceed to the Automatic Graph Compilation Press Back to refine the preprocessing Press Finish to terminate the segmentation and build the filament graph manually the current step will not be executed Imaris 4 1 Operating Instructions BITPLANE AG Step 3 Graph compilation Graph Compilation Minimal Ratio of Branch 30 Length at Trunk A adius ue Iw Find Root Point lt Back gt Finish Create Enter a minimal ratio of branch length to trunc radius see figure below Branches shorter than this value will be removed A root point has to be specified for specific Statis
58. 45 Ray Increment 45 Read only one time point 18 Rebuild 60 Red Cyan 44 Rendering Mode 40 Rendering Quality 45 Resample 3D 164 Resample data set 59 Resample settings 59 Resample Time 128 Resampling 59 124 156 164 Resampling Open 18 21 Reset Size and Location 74 Reverse left and right views 54 Rotate 31 175 Rotation 48 74 Save 154 Save Animation 48 Save Coloc Channel 145 Saving 26 Saving a 3D data set 121 Scale Bar 169 Scaling 74 Scene Files 88 Section 35 Select 52 Select all 32 Selected Color 160 Selection color 168 Selection mode 134 Set Center 53 Settings 19 Shadow 45 47 Shadow Projection 41 Shininess 78 show slice border 68 Single slice 136 Single time point 136 Page 5 BITPLANE AG Page 6 Operating Instructions Size 156 Slice 33 Smooth data set 60 Smooth lines 53 Snapshot 119 120 154 Source channels 137 Specular color 78 Splitting Objects 60 Spots 94 Spots Measurements 94 Spots Statistics 94 Statistics 92 99 Stereo 44 53 54 Stereo Preferences 53 Supported file formats 11 Surface 62 Surface and Volume Measurements 92 Surface and Voxel Statistics 92 Surpass 50 154 172 Swap Z with T 125 System 166 Take colors from 169 Terminology 10 Texture cache limit 168 Threshold 59 65 Threshold Cutoff 28 176 177 Threshold Tracking 79 81 105 Thumbnail 161 Tile size 168 Time 10 21 35 41
59. B interpolate Color T able File Import Esport EIE ETES EZ E ER ERR EET UE yy EERE EERE Fig 117 Mapped Color Box Selected Color Edit Opens the Color selection dialog Same as base color Copy Copies the selected color field or area Paste Pastes the copied field or area Interpolation Colorspace Select the Color mode HSV Hue Saturation Value RGB Red Green Blue Interpolate Interpolates the color between two selected fields or areas Direct color selection Use the left mouse button to click on a field or mark an area in the color map Click the Edit button The Base Color box is displayed Select the requested color from it as described in the Base Color sec tion and click OK selected color is copied into the marked field or area in the color map Repeat as desired for other fields or areas Imaris 4 1 Operating Instructions Thumbnail 16 11 04 BITPLANE AG Interpolation Use the left mouse button to click on a field or mark an area in the color map Click the Edit button and select the requested color from the color selection box Click OK Click on a second field or mark a second area the color map Select the second color from the Base Color box and click OK Select the interpolation mode from Colorspace HSV or RGB In the Base Color map RGB interpolates in straight direction between the two selection
60. BITPLANE AG Operating Instructions 1 2 NIE Esc EB Slice Section Gallery Slice Section Gallery Vee E Slice Section Gallery JORE nA ees Gallery Full 3L Surpass Full 3D Ful 30 Surpass Ful Full 3D p CIE NN CIE Ont NY Slice Section nie u 3b Surpass Manual Version 1 9 February 2004 EL m image revolution starts here Operating Instructions BITPLANE AG This manual provides basic knowledge and step by step procedures for using the IMARIS Suite Release 4 0 software in the workflow In addition it contains a reference section with a description of all screens functions and parameters and an installation guide Information in this book is subject to change without notice and does not represent a commitment on the part of Bitplane AG Bitplane AG is not li able for errors contained in this book or for incidental or consequential damages in connection with the use of this software Copyright This document contains proprietary information protected by copyright No part of this document may be reproduced translated or transmitted with out the express written permission of Bitplane AG Zurich Switzerland 4 please contact us Telephone FAX Support WWW Bitplane AG Badenerstrasse 682 CH 8048 Z rich O 2003 Bitplane AG Z rich All rig
61. D HOSTID INTERNET 136 142 DUP_GROUP UH ISSUER Bitplane AG FEATURE NeuronTracer bitplane 1 0 1 jan 00 2 4C665C29D5825DE6F161 HOSTID INTERNET 136 142 DUP_GROUP UH ISSUER Bitplane AG The licenses above only can be served by a computer with hostid 69139cc4 The hostid can be obtained from the Imaris license dialog by clicking the Offline button The count red indicates how many instances of the program can be started concurrently The last entry on the SERVER line 1700 is the port number of the server that is necessary for the Imaris configuration A different port number requires a different Imaris configuration The HOSTID field in the licenses above indicates that these licenses may only be used by clients with IP addresses in the indicated range While the HOSTID line is compulsory for node locked licenses it is optional for floating licenses If it is missing every client on the internet can use the server provided that it has a copy of the SERVER and all the FEATURE lines and it can reach the server 1 Start the Imaris software on the computer on which you want to install licenses You will see the following dialog 2 j Imaris didn t Find any license Would vau like Ea specify your license configuration Yes Mo Click Yes to get to the license installation dialog Page 187 BITPLANE AG Operating Instructions You can either specify a location with license information or download it via internet
62. EE cs EREnE SYSTEM SURPASS pb MEC 2 0 0 00 0001 5 EDGE PRESERVING FILTER THRESHOLD CUTOFF BASELINE SUBTRACTION BACKGROUND SUBTRACTION CONNECTIVE BASELINE LINEAR STRETCH 16 11 04 BITPLANE AG BITPLANE AG Operating Instructions HUYGENS ESSENTIA E C 181 LICENSE INSTALLATION GUIDE 182 IMARIS B virine 182 IMARIS ol cB zn oon ee Rear eee ei 184 LICENSE SERVER WINDOW S a E Eaa 186 LICENSE SERVER IRIX conna r EEEREN 188 INDEX 191 Page 6 Imaris 4 1 Operating Instructions BITPLANE AG Introduction Before you begin Conventions v A 16 11 04 This manual assumes that you are familiar with Microsoft Windows and the process of working with microscopic data in general Before first using the IMARIS application e Read your Windows User documentation to become familiar with the working environment e Make sure that you have the necessary working environment and the data transfer equipment needed to import the data from the acquisition stations This symbol marks non critical information about the IMARIS application such as user requirements computer messages suggestions and short cuts This symbol marks actions or
63. Filaments over time but a filament graph for each time point When a huge time data set is loaded the graph generation maybe takes some time the user has to crop the data set himself To change Settings or Color the parent group from the Surpass tree can be selected The filament settings are available as tab named Filaments A filament graph is only visible if the current Time point is the same as the time point where the filament creation passes the graph compilation Create Step 3 16 11 04 Page 117 BITPLANE AG Operating Instructions The selection within the graph remains if the global Imaris time point changes Storing output data e Save statistic values as CSV File statistics tab filament graph and all its properties can be saved as Imaris Scene file File Export Scene as e Export the currently selected Filament object as Inventor file Surpass Export Selected Objects e Export the filament graph as Neuron file Edit Tab e Store the temporary Filament Working channel together with the original dataset in various file formats Imaris3 Tiff series ICS etc Save as Page 118 Imaris 4 1 Operating Instructions BITPLANE AG Creating Output This chapter explains how to create output from the images prepared in the visualization process Images can be stored as a File or added to a database record if Image Access is installed ImageAccess TestBase iaa
64. File Edit Archive Image Acquisition Options View Window Help DO Image 44 EI 725 L2 TestBase iaa O Title retinaFulll 6bit Site Leader Bitplane Development sgi m eR esampled not sp Eretina_red ims Imaris 3 2 2 at 2 Gl x Resampled2 not sp File Edit View Image Processing Surpass Help descr SR ings cu en_16bit autofl d Mm be id ran zi soo Md obit nol sp Open Save Snapshot Slice Section Gallery Easy 3D Full 3D Surpass not sp Ppammum c ampled not sp Section Hesampled2 not sp Crosshair Save Desktop 6 amp 3 c EJ Normal My Computer goof y ims wetter Extended SE My Network Places quat wobbel ims _Jradar NI grid bpIAnImaris test b tif FractalHypercomplex ims top secret NI c tif Ld FractalQuaternion ims topo view View Zoom fame not kr Normal Extended Save Snapshot As B 2 xl e Fi pollen_16bit File 002 tif Save as type Tiff file format tif Cancel Data Dutput Save to File Copy to Clipboard Ready Zoom 4 28 NUM A Fig 85 Saving images to a database 2D Still Images Saving 2D still images is supported by the Snapshotoption e Still images can be stored in TIFF Format or as database records in Image Access in order to manage them m
65. Functions applied to the component group apply to all of its members This facilitates the application of colors or the deletion of objects Note If a group folder is checked invisible all items in the folder are invisible Multiple selection You may select more than one listed item at a time for an operation The selection functions in Surpass correspond to the Windows functions gt Consecutive Press and hold the Shift key down and select the first then the last entry to be selected from the list All entries in between the two are also selected gt Selective Press and hold the Ctrl key down and select any required entries from the list All selected entries are highlighted and commands or operations apply to all of them Delete To Delete an object highlight the object and click the Delete bution Page 51 BITPLANE AG Viewing area Viewing Functions Zoom Pointer Page 52 Operating Instructions Naming conventions Objects are created by clicking on the respective icon in the Objects toolbar Delete Fig 35 Objects Toolbar Objects are automatically named by Surpass as follows e Group Group n n is a consecutive number e Light Sources Light Source n e Clipping Planes Clipping Plane n Iso txxx cy n txxx is the current threshold limit cy is the channel number and n is a consecutive number e Volume Volume only one volume can be created e Ortho Slices
66. IQ Stereo m T Vy Surpass Scene 9 Light Source 1 MEB Frame v Q Iso 148 ci MI Iso 128 c2 Key Frame Animation Add Ofod X Del 100 Frames Auto Distribute Settings LI Ready Zoom NUM A Fig 65 Surpass with Keyframe Animation Window Open 3 In the animation field enter the number of frames to be included in the movie 4 Click the Settings button and enter the number of frames per second and the compression factor 5 With the pointer in Navigate mode position the image or the time slider to create the view for the first keyframe 6 Click the Add button to add the keyframe 7 Position the image to the desired view for the second keyframe and click the Add button to place the second keyframe 8 Repeat as desired Thick blue lines represent user defined keyframes and thin blue lines represent interpolated keyframes White lines represent the active keyframe User defined keyframes can be deleted by clicking the X Del button and moved by dragging the keyframe line while holding down the left mouse button The first and last keyframes cannot be moved Large spaces between the user defined keyframes thick blue lines will cause that section of the movie to play more slowly Placing the lines close together will cause that section of the movie to play more quickly The Auto Distribute function will distribute th
67. LANE AG Given a time data set that describes several biological objects at several time points the term tracking refers to a procedure that links objects of consecutive time points and results in a description like Object 3 at time 1 develops into object 7 at time 2 then into object 7 at time 3 then splits into objects 6 and 13 at time 4 In ImarisSurpass the objects that can be used for tracking are the Surface objects and Spots The result of tracking is a set of Tracks symbolized by T in Imaris Surpass each of which should describe a biological object if tracking was successful This has two important consequences The different Tracks can be visualized separately or with different colors and statistics can be produced separately for each Track The following figure displays the result of threshold tracking visualizing different biological objects in different colors for three time points Fig 58 Objects colored by threshold tracking at 3 time points Spot Tracking 16 11 04 In ImarisSurpass tracking can be performed in several ways Automatic tracking possibilities exist for Spots and SurfaceObjects For Spots several algorithms are provided that create tracks without splits or merges For IsoSurfaces a threshold tracking algorithm is available that generally produces tracks with splits and merges Furthermore a track editor allows to modify or create tracks manually To track spots it is necessary to
68. LANE AG Page 136 Operating Instructions Automatic thresholding This button performs an automatic threshold run on both channels based on an algorithm developed by Costes and Lockett at the National Institute of Health NCI SAIC which is based on the exclusion of intensity pairs that exhibit no correlation Pearson s correlation below zero The automatic threshold search is done using the 2D histogram Starting with the highest intensity value the algorithm reduces the threshold value step by step along a line shown in Figure 8 and computes the correlation coefficient of the image using only voxels with intensities below the threshold The algorithm continues reducing the thresholds until the corre lation reaches 0 thus defining the automatic threshold Channel A line along which algorithm proceeds Correlation 0 on both Channel Fig 102 Automatic Threshold Detection For more information please refer to the following publication S Costes E Cho M Catalfamo T Karpova J McNally P Henkart S Lockett Proceedings Microsc Microanal 8 Suppl 2 1040 41CD 2002 Histogram mode The histogram mode options are used to define the calculation basis for the colocalization within the data set Single slice The histograms are calculated and displayed for the current slice only Single time point The histograms are calculated and shown for the entire 3D stack of the current time point This Imaris 4 1
69. Open Topo File box is displayed Page 70 Imaris 4 1 Operating Instructions 16 11 04 BITPLANE AG 4 Select the requested File and click Open The new Topography is displayed 5 If necessary scale the height of the topographic representation Automatic Clicking the Auto button rescales the heights to a default value relative to the file s 2D extensions Manually The scaling fields in Translation and Scaling of Z allow manual scaling to the individual requirements Maximum height calibrates the geometric height distance in z direction between the lowest and the highest gray value in the input slice 6 Ifthe topography source is different from the loaded data set in Imaris e g when visualizing simultaneously topographic and volume data The topography can be moved in z direction relative to the data set Bycursor Check the Select field of the camera toolbar and then move the topography manually within the data set Byscale Use the translation field in Translation and Scaling of Z to specify the required value 7 By clicking Cut Above Cut Below in the Mask Volume panel the volume above below the topo line can be cut A texture can be applied to the relief from different sources Click on the Coloring tab 8 To apply a color to the relief check Base Color tab and proceed as described in the Color and Material section of this manual The color is overlaid on the Topo View relief 9 Checking the Data Set sele
70. Ortho Slicer n e External Object External Object n e Topography Topography n separate license required e Contour Surface Contour n separate license required Measurementpoint Pn separate license required This area displays all objects that are added to the tree list in the object area and are checked visible The viewing area includes a number of tools for data preparation presentation and manipulation most of which are displayed as buttons in the right frame bar Some can be called up with short keys gt 1x Displays the scene from a top view gt Fit Fits the entire scene into the view gt Select The cursor becomes an arrow Select a single object gt Navigate The cursor becomes a hand View rotate and or zoom an object in the view The keyboard Esc button can also be used to change pointer modes Imaris 4 1 Operating Instructions View Camera Type Draw Style Set Center Stereo 16 11 04 Full screen Ortho Perspec BITPLANE AG Displays the viewing window as a full screen To return to the standard window click on the screen then press the spacebar Orthogonal view Perspective view Select the draw style of the object from the pull down list Full Feature gt Wireframe gt Hidden lines Shows all objects as they are Draws isosurfaces as wireframe models Draws isosurfaces as wireframe models and hides all background lines Note Set Volume and orthoslicer obj
71. Parts of the filament graph without any connection When splitting the filament edit tab the same number of fila ment objects will be created Each new filament will con sist of one connected piece No Vertices A vertex is a point within the filament graph which are connected through edges Some special vertices are end points branch points or the root point defined there is also a corresponding depth available The sum of length of all edges which compose a segment If a root point is defined there is also a corresponding depth available Segment Resistance This value is proportional to the length and indirect proportional to the cross section area A variable diameter is considered Multiply this value with an electrical material constant to obtain an electrical resistor composed from small cones Segment Volume The sum of volume of all edges cones which compose a segment If a root point is defined there is also a corresponding depth available Terminal Point Diameter The Diameter of an end point Color e Specifies the color of the branches refer to the Imaris Diffusion manual for detailed operating instructions nc Specular n Emission Transp 04 Falette 0 000 1 000 0 000 Settings Draw Edit Statistics Color Time Data 4D All segmentation steps and graph compilation is applied with the same set of parameters to each time point There is not one track of
72. Resample dataset Standard Size f Quick Size Viorel size Urita E 514 E 5 0485 m Mg E 13 4 8452 2 580 Gaussian filter width Close objects at border Smooth dataset Split objects Create no more than 10 objects Ede EN Filerabjects with less than tangles Cancel Fig 109 Iso Surface settings for colocalized channel 5 Inthe Object area click the Add New Volume button Imaris 4 1 Operating Instructions BITPLANE AG 6 In the IMARIS menu bar select Edit Channel Visibility In the Channel Visibility window uncheck the coloc channel The Surpass Volume object displays only the two initial channels 8 In the IMARIS menu bar select Edit Display Adjustment In the Display Adjustment window select the two initial channels and adjust their settings e g decrease the blend opacity retina ims Imaris 3 3 _ File Edit View Image Processing Surpass Help m NAREN Colac ECA Open Save Snapshot Slice Section Gallery Easy 3D 3D Iso t14 c3 Properties Channel isibility x C Diffusion I Fi Surpass Specular Emission t E Pointer A Select Transp 0 Navigate Material 1 Palette 0 389 fo 389 0 389 Surface Statistics Voxel Statistics Color Objects Full Screen gt Delete Camera Typ
73. S program and is not available as a standalone program When selected in the IMARIS menu bar the ImarisColoc main screen is displayed The screen consists of the following areas Page 129 BITPLANE AG Operating Instructions retina Imaris 3 3 File Edit View Image Processing Surpass Help s a m NE B Open Saveas Snapshot Slice Section Galley Easy3D Full3D RE 4 Surpass Coloc Channel 2D Histogram Channel 1 name n n Selection mode 2D Histogram 500 00 Polygon 11 20 of data selected Automatic thresholding Threshold Threshold 4 Channel Histograms Channel B _ B Histogram mode Channel 2 name n Single slice Threshold 8 Single time point T 52 00 n All time points Histogram options 5 10 of data selected Ignore border bins of channel amp volume colocalized 479 of cha ocha Volume Statistics i Preview Window of cha channel correlation in colocalized volume 0 0686 Coloc color MASA Gramm Edit 459 Mask dataset Channel 1 name ax Fit NRA Z nf data selected T c Coloc Volume Statistics Quantit Value number of colocalized voxels 21333 of dataset colocalized 0 54 z Build Coloc Channel Hane Statistics
74. Status Available Amount of internal memory Select the viewing properties and the basic colors for the backgrounds and selection in the gallery o RES ES Display Display Advanced Iv Interpalate Disable subtexture Use only xy textures for 3D preview Texture cache limit ME fou Background Background color select Checkered for blending Tile size 20 000 Linear color progress for blending Selection Selection color DE Select Scale Bar Show Date P r hM DD Background color 2 Show Time 55 Measurement Measurement color 21 Select 10 0090 Cancel Fig 126 Display Box Display Interpolate If checked the images are automatically interpolated for a smoother display Page 167 BITPLANE AG Page 168 Disable subtexture Operating Instructions Some drivers for Open GL graphics cards cause ir regularities in the display of the images If this oc curs check Disable subtextures to prevent possi ble problems Use only xy textures in 3D preview Since rendering is quite time consuming the processing of images in full 3D volume preview can be accelerated by checking this box The resulting volume image preview still produces a fine display when the dataset is viewed in the xy direction or is tilted The missing z texture information becomes a limitation when looking at the data set in the xz or yz direction It is es
75. age 16 11 04 Page 21 BITPLANE AG Operating Instructions Adjusting the Im age Parameters When opening a data set the following parameters should be checked or modified gt Name and Description gt Voxel Sizes gt Channel Colors Name and Descrip tion Image Properties B E 1 E CIS Channels Name pe Channel 1 bpgw u3 people pandolfa retina ims Channel 2 Thumbnail Parameters Description not specified Log Cancel Fig 9 Image Properties box Data Set Inthe main toolbar select Edit Image Properties Display The Data Set box is displayed Type a Name and short Description in the respective fields Voxel Size The voxel sizes directly influence the views because they control the e height of the image relative to its width Page 22 Imaris 4 1 Operating Instructions BITPLANE AG Image Properties en Geometry m Channels d Channel 1 8 hit unsigned Thumbnail Parameters Resample 30 Vowel Size 4 008274 4 008274 2 302678 Date v Y MSM D Min 0 000000 0 000008 0 000000 2003 03 01 1021 599976 1021 599976 288 600006 Time HH MM 55 10 30 00 All Equidistant Fig 10 Image Properties box Geometry Click Geometry in the index of the Image Properties box The geometrical settings are displayed Check the parameters and adjust the Voxel Size and or other settings if necessary Enter the collecti
76. ain to calculate the result th retina Imaris 3 3 i xl File Edit View Image Processing tem Help a NNNM SE Select all Channels N m Gallery v 3D Coloc Open Saveas Snapshot Rendering Range Ch 2 name not specified Min o 0000 Reset deme ERE m Blend Opacity 21 160 Ch 2 Channel Visibility O Che name eS e e T Image calculated Zoom 283 Fig 106 Easy 5D representation of the Coloc channel with one original channel 16 11 04 Page 151 BITPLANE AG Visualize the Coloc Channel in SURPASS Page 152 Operating Instructions The colocalization channel can be visualized like any other channel in Surpass Surpass is an optional module of IMARIS and may be not available Example 2 Create an Iso Surface of the Coloc channel add the two initial channels as volume rendering 1 After building the Coloc channel click on the Surpass button in the IMARIS menu bar In the Object area click the Add New Iso Surface button In the Iso Surface Setup window check and select the Coloc channel Select the desired parameters In this example we set the threshold just below the lowest intensity values in the histogram Iso Surface Setup E x Channel 3 Colocalzation Aesult Channel 3 Colocalization Result Threshold 12 000 Advanced Settings
77. alog box Both options reduce the size of the data set decreasing the time needed to read the file and speeding up any operations on the data This can be particularly important when reading large datasets over a network The revision applies to all views in Imaris and in Surpass ini xi Fig 8 Resampling File Open Page 18 Select File Open or click on the Open button in the main toolbar The Open box appears Left click a file to highlight it and click on Resampling The Resampling File Open box is displayed The user has the option to specify the Subsampling Factor i e the fraction of data points to be retained The subsampling factor can be specified for the x y z directions the channels and the time points The Crop Limits Min Max can also be specified Imaris 4 1 Operating Instructions Settings 16 11 04 BITPLANE AG 3 The view on the right side displays a single time point of the data set Left click in the view and move the mouse pointer to the right to increase brightness Move the pointer upwards to increase contrast This fills the same function as the Display Adjustment function of the main screen 4 Click Open The data set is cropped and resampled while loading Allows the user to specify options for reading certain file formats Leica LCS Settings A LeicaVista data set consists of a number of image stacks or experi ments A dialog box can be opened to select a specific image stack
78. ame Animation Full Screen Finish Camera Type C Orthogonal Perspective Delete Draw Style Ful Feature box move smooth lines Set Center Stereo Zoom 1 00 NUM Fig 82 The result of the Graph Compilation step 3 is immediately visible in the Surpass viewer e The Filament Working channel can be kept for Build Filaments for all Time Paints other use Principally it is not needed any more Delete Working Channel Create Filament Properties Page 112 when finishing the filament graph e When the data set contains multiple time points the Filament creation wizard can create Filament objects for each time point with the same set of parameters They will be visible within the Surpass tree However the 3D filament graph is only visible according to its time point Finish e Press Back to refine parameters of previous steps e Press Finish to terminate the segmentation When finishing the graph can be edited manually When the creation process is finished the Create Tab disappears while some other property tabs appear They allow the manipulation of the filament graph Settings Change Filament appearance and inspect selection statistics Draw Manually draw filaments and connections Edit Select automatically filament parts and delete or smooth the
79. and the z dimension is mapped onto t The section viewer now shows x t and y t views 1 Select mage Processing Swap Z with T The Z and the T axes are exchanged This allows the user to play the images over Z and T 2 Click Start to play the series of images along the z axis The time bar is visible in all display modes Slice Section Gallery 3D Preview and Surpass It allows the user to view a particular time point or to automatically play 4D images The views change when selecting a new time point 16 11 04 Page 125 BITPLANE AG Operating Instructions retina red ims Imaris 3 2 E uu 0 x File Edit wiew Image Processing Surpass Help x m B Nx 1 amp NX S ELE Open Save Snapshot of Full 3D Pi nai nr F Surpass Slice Section Gallery Easy 3D Mode Default Meas f Single Multi Style Hair C Line C Star C Paint Paints Tools Ir Brid 9 outside at 134 07 121 50 32 00 Zoom 3 55 Fig 90 Main screen with time bar Easy 3D and Full 3D Imaris renders the image when the Calculate button is pressed If the time point is subsequently changed Imaris will render the new image automatically If Calculate is not pressed in Full 3D Imaris remains in Preview mode In this case the time point can be changed without the time consuming renderi
80. arson correlation of channel A and channel B inside the region of interest 2 SA SA mask SB SB vg iemask IR usas iemask iemask Calculation and building After its build the Coloc channel is added to the current data set The new channel is then available in all IMARIS views most interestingly in Surpass 1 Click the Build Coloc Channel button The colocalization channel is calculated while a box is displayed showing the process Viewing statistics The calculated statistics for each colocalization channel can be viewed in the Image properties 1 Click the Channel Statistics button or in the menu bar select Edit Image Properties The Image Properties window is displayed 2 Click the required channel in the menu tree The Name of the channel the Description of the source channels and the selection method are indicated and the Coloc Statistics are displayed Imaris 4 1 Operating Instructions Save Coloc Channel 16 11 04 BITPLANE AG Channel 3 Name Colocalization Result Description Source Channels 0 amp 1 Selection Method Threshold Threshold 52 00 Threshold B 36 00 Base color Mapped color Coloc Statistics time Frame 1 of dataset colocalized 0 57 5 of ROI colocalized 8 46 SOF volume above threshold colocalized 5 46 of volume B above threshold colocalized 100 00 Sb of material above threshold colocalized 5 09 af material B above threshol
81. at for each channel if applicable Click Apply The effect of the changes can be seen in the image f necessary readjust the value When the results are satisfactory click the OK button The data set is modified Click Cancel to return to the original image Background Subtraction The Background Subtraction filter has the same user interface as the Gaussian Filter Page 30 Imaris 4 1 Operating Instructions BITPLANE AG Visualization The process of visualization is probably the most valuable part of your ap plication because its goal is to evaluate the essence of the image and to communicate it to others IMARIS provides a wide range of display modes and functions to create in formative images that illustrate the relevant details of the data set Viewing Aids Zoomin Out The display modes provide several aids to enhance the evaluation proc ess The processes involved can be performed in different ways Zooming can be performed by Clicking the 1x original size and Fit to window button in the right control bar In the slicer section gallery Easy 3D and Full 3D hold down the middle mouse button click on the image and drag the mouse up and down or press and hold the shift key and the right mouse button and drag the mouse Surpass With the pointer in navigate mode hold down the Control key and the center mouse button and drag the mouse Pan To pan move the image within the view in Imaris
82. aussian filter to the dataset and requires a great deal of memory Uncheck Smoothing if your system runs out of memory Smoothing can result in the loss of small objects so should not be used if the dataset contains very small objects Close objects at border If structures on the border of a data set are cut open the volume is not calculated Check Close objects at border to close these structures before calculating the IsoSurface object Note If a new IsoSurface object is oversized and does not fit into the viewing area select Zoom Fit in the frame bar on the right hand side Modifications Once the IsoSurface is displayed it can be viewed and manipulated by all available functions in the viewing area Other objects can be combined with the IsoSurface in the display Check the objects desired All modifications apply to the object highlighted in the Objects area Single objects groups or the entire Surpass scene can be selected Rebuild Rebuild allows the user to re specify the settings for the creation of an IsoSurface object The main reason for performing a rebuild is when testing for the best threshold value the calculated IsoSurface can be quickly replaced with a rebuild Splitting Objects The number of unconnected surfaces is displayed in the Properties area If there is more than one unconnected surface the Split button in the IsoSurface object properties is active 1 To split the IsoSurface click the S
83. aw Edit Statistics Calor BITPLANE the nucleus The automatic computation of all the possible paths is then completed Hold down the shift key and place the mouse cursor at the ends of the structure The computed paths are displayed Still holding down the shift key left click and the displayed path is definitely drawn If some false paths appear you can change the Starting Point at any time by repeating the procedure or switch to AutoDepth Manual mode to make corrections It is very useful to use a Volume object to help drawing the desired filaments Drawing in AutoDepth mode Select AutoDepth in the Draw box and shift and hold down the left mouse button to draw a filament The depth is automatically computed by comparing local intensities Drawing in AutoDepth mode is then similar to drawing in Manual mode In order to fully use the potential of the AutoDepth mode the pictures should not be too noisy A pre filtering would be then necessary Key Mouse Button Action Cursor Down Move plane position down left back Edit e Mouse Selects Specify how to select parts of the filament by a single mouse click onto the filament graph Edge selects only the position near to the mouse pointer Segment selects the filament between two branch points Branches selects the filament starting from the clicked point to all connected end points a root point should be defined lows individual coloring and individual statisti
84. awn by means of the following Draw Styles Select the draw style in the object properties area Click Hold down the Shift key and left click the mouse to insert point a so called vertex on the contour e Time Hold down the Shift key click once on the contour and then drag the mouse along the contour The number of vertices depends on the time interval ms specified in the object properties area The interval can be changed by manually specifying the required value Page 96 Imaris 4 1 Operating Instructions 16 11 04 5 This draw style allows the user to draw a structure that contains fine details Dist Hold down the Shift key click once on the contour and then simply drag the mouse along the contour The number of vertices depends on the distance interval um specified in the object properties area To change the interval specify the required value in the object properties area This draw style is convenient if the structure is smooth Select the direction of the Contour Surface by specifying the required direction x y z in the object properties area Choose the slice for the first contour by moving the slider in the properties area or by entering the position of the plane The position of the drawing plane can also be changed in the viewing area Switch to Select mode and move the drawing plane by means of the mouse pointer The border of the active drawing plane is shown in
85. been generated Follow the instructions to save the license file and then copy the file to the computer for which you generated licenses It is suggested that you copy the file to the directory where the Bitplane software is installed typically C Program Files Bitplane and that you name the file bitplaneW lic Restart Imaris for the new licenses to take effect To use floating licenses you need to a install a floating license server and b configure Imaris to use the floating license server Both steps are described in this section NM O 10 Click the button Save Service and answer yes to the question to save 11 12 13 Create a folder called flexim on the C drive of the server Unzip the Win32bit Fl Lic attachment and place Imgrd exe Imgrd dl Imutil Imtools and bitplane exe in the flexlm folder Install the license file in the same folder Open Imtools Click the Service License File tab Click Configuration using Services Click the Configure Services tab Type Bitplane as the service name Path to the Imgrd exe file Use the browse button to find the Imgrd exe file in the folder flexlm Path to the license file Use the browse button to find the license file in the folder flexim Change Files of type to all files to find the file Path to the debug log file Use the browse button to find the Imgrd dl file in the folder flexim Change Files of type to all files to find the file It w
86. bes the presence of two fluorochromes at the same physical location ImarisColoc provides extended functionality for the analysis and visualization of colocalization in multi channel data sets It provides an automated selection of colocalization based on a method introduced by Costes amp Lockett at the NIH NCI SAIC ImarisColoc processes 2D 3D and in conjunction with ImarisTime also 4D data sets It operates on two channels simultaneously and measures the degree of overlap of the two channels With Coloc the user can determine and display locations within the data set with common information from all channels e g to illustrate where all types of proteins can be found simultaneously or to detect low density tissues in all channels The desired range for signal analysis can be limited by defining intensity thresholds or sets of intensity values for each of the involved channels All voxels falling inside every of these channel specific intensity limits represent a colocalization They are emphasized in the view and statistics are calculated and displayed When the selection is changed the numeric and visual feedback happens in real time The result of the colocalization as a whole can be saved as a separate channel which can then be processed and viewed as any originally acquired channel in IMARIS Multiple colocalization images are easily combined into a single view in ImarisSurpass ImarisColoc is an optional module within the IMARI
87. between two vertices left click and drag the line around to the desired location The polygon changes its shape Moving the polygon Point somewhere beside of the polygon edge left click and drag the polygon around to the desired location Deleting points Point on one of the vertices Press the DEL key to delete the vertex Deleting the polygon Point somewhere beside of the polygon edge and press the DEL key to delete the entire polygon Fore more information select Help Keys Help in the menu bar Page 147 BITPLANE AG Operating Instructions 3 24 of data selected Fig 106 Typical polygon selection for channel bleed through Mask Channel and Automatic Threshold Time Settings Page 148 Automatic thresholding produces only relevant results if an adequate mask is defined please refer to the publication by Costes and Lockett mentioned on page 136 1 In the Mask channel area select the channel to be used from the pull down list In the Mask channel area check the Mask dataset field The 1D histogram of the mask channel is displayed and the Threshold field is activated Point with the cursor into the histogram and click to set the threshold A bolded line marks the threshold in the histogram and the corresponding value is indicated in the threshold field The masked volume of the data set appears hatched in the Preview window Drag the cursor in the histogram to modify the threshold Eff
88. ble clicking with the left mouse button Size of Vertices and Lines In the drawing mode the size of the vertices as well as the connecting lines can be increased or decreased by repeatedly pressing the key or key on the numerical keypad The process of drawing the contour can be interrupted and continued later To change the position of the object while drawing the contour switch the pointer to Navigate mode move to the required location and switch the pointer back to Select Contour Sunace 2 Properties I Bie oe ees Oe Rene og M EIE x MM CMM d Direction E yf oy Paste M un mimm mmm mnn naim amm nm TEE 7 2 Dum AdvSelect C Time po Dist 141712 um S EERE EAS ve Navigate DIERUM E pum z Objects 126 lt 2 re x Camera Type NI E mE AT C Ortho LIE Frame Rune c f Perspec Light Source 1 ai g E uar Era Surface 2 4 Full Featun move E B 4 Stereo Fig 72 Contour Surface with Vertices Calculating a Contour Surface When all contours on different planes have been drawn calculate the surface by clicking on Calculate Surface in the object properties area A surface based on the contours is generated and can be viewed in the viewing area Note The program uses a
89. box is displayed 2 Select any of the colors and click OK The indication bar shows the selected color and the colocalization areas in the Preview window are colored accordingly If required click Color definition and use the colorwash to create and add your own colors for selection Page 141 BITPLANE AG Operating Instructions Coloc Volume Statistics The values are updated in real time and refer to the entire volume data of the current time point regardless of the settings for the Histogram mode The values are calculated by the following formulas Definitions Regions Data set all data set voxels Coloc colocalized voxels Object A Voxels with channel A intensity above threshold A Object B Voxels with channel B intensity above threshold B Indicators N Number of voxels SA Channel A intensity of i voxel SBi Channel B intensity of i voxel SAavg coloc Average channel A intensity in the coloc region Number of colocalized voxels Total count of colocalized voxels of dataset colocalized Percentage of total data set voxels colocalized 100 dataset 2e of volume A above threshold colocalized Percentage of channel A voxels above threshold A colocalized 19091 objectA of volume B above threshold colocalized Percentage of channel B voxels above threshold B colocalized N clo 100 objectB Page 142 Imaris 4 1 Operating Instructions 16 11 04 BITPLANE AG
90. can all be operated with the mouse buttons A Keys help menu is always available by clicking Help Keys Help 16 11 04 Page 9 BITPLANE AG Operating Instructions Terminology Term Description Voxels A volume data set is composed of volume elements called voxels The voxels are the smallest units within the image about which we have distinct information in the form of a measured intensity The dimensions of an individual voxel are specified in most image file formats supported by Imaris Channel A channel is a particular piece of information that has been recorded for all voxels Most of the time in light microscopy the different channels are intensities that have been measured at different wavelengths For instance a two channel data set con tains two intensities for each voxel one intensity measured in channel 1 e g green light and one intensity in channel 2 e g red light Ideally the intensity values pre sent in different channels are independent of one another Time Point A time point is a single 3D image containing one or many channels The term is used regardless of what data is actually stored in the channels In particular the channels could contain data taken at different points in time with a changing object Rendering A technique that visualizes a multi dimensional data set as a two dimensional image that can be displayed on a computer screen or printed on a laser printer Volume Ren A rendering technique which
91. ce of the direction and the angle of rotation Click the Animation button in the 3D View The Animation dialog box displays Page 47 BITPLANE AG Page 48 Operating Instructions Animation Preview Rotation Full rotation Half rotation C Quarter rotation Cancel Fig 32 Animation box Direction Horizontal Select the Direction of the rotation horizontal or vertical axis The rotating cube shows the selected movement direction Select the angle of Rotation 360 180 90 or user defined The rotating cube shows the selected rotation path Specify the number of Frames to be produced The quality of the animation depends directly on the number of frames pre sented in the rotation More frames result in a slower and more fluid movement of the object Rendering is a time consuming process and requires about 1 minute per frame depending on your computer and the complexity of the image We recommend checking how long it takes to render a few images before specifying a large number of frames Click OK The Save Animation As box is displayed Imaris 4 1 Operating Instructions BITPLANE AG Saving an Animation animation as Save Desktop a Computer zu Network Neighborhood Filter S Mu Briefcase Lj ScreenShots surpass snapshots File name retina float 1 Save as type Microsoft videa 1 4 Can
92. cecauaeeecececeuaateeeseceeeas 18 SETTING nsec TEN 19 ADJUSTING THE IMAGE PARAMETERS errem nnn nun nene 22 NAIVIE AND DESCRIPTION RR E 22 VOEL OZE 22 CANINE LC OOS RCM EUER ur uM EFE 24 PREPARATIONS FOR VISUALIZATION nnne nennen nnn nnn 24 ITHE CROP DIALOG 25 SAVING IN IMARIS FILE FORMAT scccccccccccseececcecccccsseeecccccccasseecccccecaasseeeececccuaaeteecccceeeausteeececeeeaaaeess 26 cee 26 URE SO Bp CERRO ra E ea 28 VISUALIZATION 31 irae M 31 RH 31 FAN 31 mee IM eM 31 DISPLAY ADJUSTMENT BLEND OPACITY AND CHANNEL VISIBILITY eere 31 DISPLAY eb 33 EIN T RE 33 SECTION M E 35 A 37 2 Imaris 4 1 Operating Instructions SURPASS MAIN SCREEN 25
93. cel Movie Settings Compression 15 Low compression High compression High quality Low quality Frame rate 10 frames per second Database Add to ImageAccess database Fig 33 Save Animation As Adjust the Movie settings Compression Use the slider to set the compression factor be tween 0 and 100 A lower factor results a lower compression and therefore a better quality but also in a larger movie file Frame rate Enter the requested frames per second Image Access The animation can be saved to the Image Access database if the user has purchased Image Access Check the box Add to ImageAccess database After the animation is saved the dialog for ImageAccess starts Follow the instructions to save the animation in the Image Access Select the directory and enter the name for the file to be saved and click Save The animation file is saved as an avi movie 16 11 04 Page 49 BITPLANE AG Operating Instructions Surpass provides extended functions for visualization including gt A large viewing area with numerous tools for data preparation presentation and manipulation A selection of different types of data display as well as any combination of them gt The possibility of loading additional external objects for comparison gt Grouping functions When selected in the IMARIS menu bar the empty Surpass main screen is displayed The screen consists of three different areas
94. created IsoSurfaces are listed in a tree list in the objects area Click the Statistics tab in the object properties area to view and compare the voxels statistics for each surface Iso 120 cl Properties variable Value Unit F Area 52 945 urn volume 15 224 3 Center of Mass X 12 411 um Center of Mass Y 14 280 um Center of Mass Z 3 316 ur Number of Triangles 1616 000 4 H Export Excel settings Statistics Fig 68 Statistics Box Exporting Statistics The values can be exported in tabulated form 1 Highlight the Group containing the required object s or select the entire Surpass Scene Click Export Statistics in the object properties area The Export Statistics box is displayed Select the directory and enter the name for the file to be saved and click OK The data is written into a text file csv file comma separated values The data contains the Surface Statistics and Voxel Statistics for the group as well as for single objects Exporting Statistics to MS Excel The values can be directly imported into MS Excel Highlight the Group containing the required object s or select the entire Surpass Scene Page 93 BITPLANE AG A Spots Measure Spots Spots Statistics Page 94 Operating Instructions 2 Click Excel in the object properties area You are asked whether or not you want to use pivot tables with MS Excel some configurations do not support this feature
95. cs On the contrary separated filaments can be merged us ing the Merge button Smooth removes roughness of the filament path It affects the position and the di Page 115 BITPLANE AG Operating Instructions ameter of all segments except the branch points and end points e Global Selection There are various methods to make automatic selections on the whole filament grap Rel Branch selects short parts of the filament defined by the ratio of the branch length to trunk radius B R see figure above Abs Branch selects all branches shorter than an absolute length B Loops finds circular closed filaments limited by a maximum length Invert selects all unselected parts of the graph and deselects all selected parts Path requires at least two selected points from the filament graph The shortest path between these two selections will be calculated Clear removes the selection Process selection lets you delete or smooth the current selection e Export The whole filament graph can be stored in the Neuron file format The Neuron application is freeware and is able to do electrical simulations Statistics variable value Unit 4 The tabular grid allows selection for copy paste flo Seaments 547 00 Select some cells with the mouse and press ctrl c to copy the selection Unconnected Filamen 2 000 vertices 3563 000 export as CSV file or immediate launching of Microsoft Lid us Excel Use Excel
96. ction overlays per default the image of the first slice in the loaded data set on the Topography relief If you want to change the slice and channel within the loaded data set select Slice and Channel or check Select All in the Data Set amp File Options panel of the Coloring tab Checking the File selection allows you to select a specific 2D input file possibly a 2D color file to overlay on the Topography relief Page 71 BITPLANE AG Operating Instructions Topography 2 Properties ry moam Data set 1 File EE Data Set amp File Options Mask Valume A Fit Channel fi Cut Above Pointer Slice 17 Below f Select Translation and scaling C amp dvSelect 0 000329 Jum 0 106988 umvstep C Navigate Jum Auto Scale Z 27241 83 Tm Store Recall Geometry Objects MEN ae ESL ASI HADE Delete Camera Type E E Surpass Scene Ortho 7 Frame Perspec ES Light Source 1 opography 2 Draw Style Full Faatun box move Fig 48 Topography Geometry Topography Properties moam f Base Color Color 5 Material 1 fe DataSet Fit C File Data Set amp File Options Pointer Channel H I Select al Select Hz Slice 17 AdyvSelect Navigate 100 gt O Diffusion D E mission View Geomety Coloring Store Recall Camera
97. ctions 8i retina Imaris 3 3 e x File Edit View Image Processing Surpass Help E Display Adjustment s m NEMO 9 See Select all Channels Open Save Snapshot Slice Section Gallery Easy 30 Full 3D Surpass Coloc Rendering Light Left C Bight Range Ch 3 Colocalization Result Min 0 0000 Reset Max 255 0000 Auto Blend Opacity 100 000 Ch 3 Settings Calculate ol eee Image calculated Zom 203 Fig 107 Easy 3D representation of all three channels Page 150 Imaris 4 1 Operating Instructions BITPLANE AG As the colocalized region always lays inside the initial channels making the initial channels invisible or more transparent allows a better view of the colocalized region 3 Inthe IMARIS menu bar select Edit Channel Visibility 4 In the Channel Visibility window uncheck one of the two original channels then click the Calculate button again The 3D image of the original and the Coloc channel is calculated and displayed In the IMARIS menu bar select Edit Display Adjustment In the Display Adjustment window select the Coloc channel and adjust its settings 7 n the Display Adjustment window select the remaining original channel and adjust its settings In this example the channel is set as partly opaque 8 After each modification click the Calculate button ag
98. d channel Check box to switch the channel visibility e Click on the Channel name to access the channel properties Display Modes Display Modes are used to create still images or animations to illustrate and save a certain state or detail of a microscopic data set There are three types of 2D Display Modes Slice general overview by slice Section front view top view and side view at any given point in the data set or as a portion of the data set Gallery display of all available slices or a user defined selection of the slices There are three types of 3D Display Modes Easy 3D fixed view with standardized parameters Full 3D freely movable with extended parameter defini tions Surpass provides numerous tools for data presentation Animations can be created from the Slice and 3D modes or with the Key frame animator in Surpass Slice The slice view mode is the view in which an image is displayed when load ing a data set into IMARIS The Slice representation shows a 2D view from the top in the z axis It shows all images in the data set along the z axis so the user can in spect any slice along the z axis for a general overview of the data set 16 11 04 Page 33 BITPLANE AG Operating Instructions Eretina red ims Imaris 3 2 p y RE inl xj File Edit View Image Processing Surpass Help y N e NIE ba Lar lc Full 3D Open Save Snapshot EE Slice Section Gallery Easy 3D
99. d colocalized 100 00 oF ROI material colocalized 8 09 of ROI material B colocalized 52 55 channel correlation in dataset volume 0 0172 channel correlation in ROT volume 0 0392 channel correlation in colocalized volume 0 0951 ba Export statistics The statistics can be exported 1 In the Image Properties window click the Export button The Export Coloc Statistics window is displayed Select the directory and enter the name for the file to be saved The statistics are saved as csv Excel file and can be opened directly with Microsoft Excel Before the channel is added permanently to the data set it must be saved 1 In the menu bar select File Save As or click the Save as button in the toolbar The Save As box is displayed Select the directory and enter the name for the file to be saved or confirm the suggestion Select the requested file format and click OK The data set is saved Page 145 BITPLANE AG Operating Instructions mu 0 MES Save in E Images amp wabbel ims pollen inns retina ims File name retina red Save as type Bitplane mars 3 uncompressed 1 Cancel Data Output 2 Fig 105 Save As dialog Advanced Opera tion Polygon Mode Instead of using the basic threshold mode for defining the area where a colocalization will be determined the user can define a freely shaped polygonal region The polygon can be drawn
100. d drag the entire ROI around Shape the ROI Click on a handle with the left mouse button hold it down and reshape the ROI Side handles affect one direction corner handles two directions The modifications apply to all slices and all time points of the image Click OK when finished The data set is cut down to the marked ROI The rest is erased 16 11 04 Page 25 BITPLANE AG Saving in IMARIS file format Image Smoothing Page 26 Operating Instructions Saving in Imaris3 format is recommended whenever the data set is cropped or the parameters changed Saving a data set in IMARIS file format provides the advantage of a faster loading process and the possibility of using thumbnails In addition most parameters are saved with the images Save in E images amp t wobbel ims pollen ims retina ims File name retina red Save as type Bitplane Imaris 3 uncompressed ims Cancel Data Output 2 Fig 14 Save As box n the menu bar select File Save As or click the Save button in the toolbar The Save As box is displayed Select the directory and enter the name for the file to be saved or con firm the suggestion Select the requested file format and click OK The data set is saved Whether or not images need to be smoothed depends on their quality Inspect the image to see if any smoothing is required For Image Smoothing several different filters are available 1 Ga
101. d8010 The webbrowser will prompt you to save the license information into a File fon Floppy disk As next come back bo this computer and let Imaris read Ehe license information Fram this File Continue with reading the license information From File 3 Enter the Host ID of the computer on which you want to install licenses and the Order ID into the specified fields on the web page and click Submit Identification The web page will then be updated to display the list of products corresponding to your Order ID Your identification Your computer s Host LD Your OrderLD Done This new order tiem has been registered io the Host ID you specified Products mstalled on computer 00055 4448010 Imaris 4 0 for Windows upgradeable until 2004 04 21 Imaris Measurement Pro Win ready for installation on this computer Imaris Time Win ready for installation on this computer ImarisColoc ready for installation on this computer Page 185 BITPLANE AG License Server Windows Installation of License Server Page 186 6 Operating Instructions From the list of products carefully select those that you want to install on the computer you are currently using Note that it is not possible to repeat the license installation on another computer unless you have purchased several licenses Press Install Upgrade selected Product Licenses The web page will be updated to tell you that a license file has
102. djusted directly by entering values in the Size fields or by means of their up and down arrows Uncheck the Crosshair Normal and or Crosshair Extended boxes to eliminate the crosshair and or the focus range lines display on the image When View Extended is selected you may choose the best display mode for the slice portion MIP Calculated maximum over all slices in the range Mean Calculated mean value over all slices in the range Blend Calculated upon the transparency of the slices Gallery The Gallery displays all available slice images of the data set 16 11 04 Page 37 BITPLANE AG Operating Instructions In the Gallery view you can adapt the display to your needs to gain more information from the slice series fle Edit View Image Processing Surpass Help E X ad 6 x 5 HU Y WE he e Open Save as Snapshot Slice Section Galery Easy 3D Ful 3D Surpass Coloc Bitplane AG Slice 4 r Page Columns 4 Skip 34 Display 1 Lest 6 E H Selection oom Fit Page 1 2 30 slices selected Zoom 296 p Fig 24 Gallery view When the Gallery view is selected all slices are displayed Click on images to select or deselect them Selected images are marked with a yellow frame Holding the Shift button lets you select a continuous series of images Hold Ctrl button to select vari
103. e C Orthogonal Perspective View 21 28 Surpass Scene be 9 Light Source 1 Draw Style Full Feature box move smooth lines Set Center Stereo Display Adjustment Select all Channels Range Ch 2 name not specified Min 22 6363 Reset Max 136 1780 Auto Blend Opacity 27 040 Ch 2 Ready foom 1 823 Fig 110 Iso Surface of colocalized region and volume rendering of initial data set 16 11 04 Page 153 BITPLANE AG Operating Instructions Reference Guide Main Toolbar The following buttons are located in the main toolbar of ON SS he ies ER Open Save as Snapshot Slice Section Gallery Easy 3D Full 3D Surpass Colac Fig 111 Main toolbar gt Open Opens a new data set gt Save Saves a data set gt Snapshot Saves a 2D image as a TIFF file and stores in a database record gt Slice selects the slice view gt Section selects the section view gt Gallery Selects the gallery view gt Easy 3D Selects the Easy 3D view gt Full 3D Selects the Full 3D view gt Surpass Selects the Surpass view gt ImarisColoc Opens ImarisColoc Separate license for ImarisColoc required Page 154 Imaris 4 1 Operating Instructions BITPLANE AG The Edit Menu Copy Image Properties Data Set 16 11 04 File Edit view Image Processing Surpass Help
104. e Clipping Plane can be rotated around the x y and the z axes In Select mode left click the smaller diameter rod It will turn yellow as it is selected move it to rotate the Clipping Plane Manipulator Active To make the frame of the Clipping Plane visible check the Manipulator Active box Configurations Store Recall After changing the position and or orientation of the Clipping Plane the new configuration can be stored by clicking on the Configuration Store button in the object properties area The position previously stored can be recalled by selecting Configuration Recall in the object properties area Orthogonal View The camera is set to a position which is perpendicular to the Clipping Plane Recenter The Clipping Plane center is moved to the dataset center Orientation The Clipping Plane is oriented parallel to either the xy Plane the xz Plane or the yz Plane Perpendicular movement By pressing Ctrl and the left mouse button on the bigger diameter rod the Clipping Plane be moved in the directions parallel to the plane The pointer must be in select mode Page 69 BITPLANE AG Operating Instructions retina ims Imaris 4 1 File Edit View Image Processing Surpass Help o EEN ECA Open Snapshot Slice Section Gallery Easy 3D C Coloc Bitplane AG Surpass Animation Clipping Plane 1 Properties Position Configuration Recente
105. e Subtraction 16 11 04 BITPLANE AG The Threshold Cutoff values for the channels may differ In the menu bar select Image Processing Image Thresholding Threshold Cutoff The Threshold Cutoff box is displayed showing a histogram of the voxels in the dataset Select the channels to be cut by marking the check box to the left of the channel bar Highlight a channel A colored bar in the histogram shows which channel has been se lected Enter the requested values for that channel in the Threshold field or drag the bar in the histogram Repeat for each channel if applicable Click Apply The effect of the changes can be seen in the image If necessary readjust the value When the results are satisfactory click the OK button The data set is modified Click Cancel to return to the original image omen Fig 18 Baseline Subtractionbox The Baseline Subtraction values for the channels may differ In the menu bar select mage Processing Image Thresholding Baseline Subtraction The Baseline Subtraction box is displayed showing a histogram of the voxels in the dataset Select the channels to be cut by marking the check box to the left of the channel bar Highlight a channel A colored bar in the histogram shows which channel has been se lected Enter the requested value for that channel in the Baseline field or drag the bar in the histogram Page 29 BITPLANE AG Operating Instructions Repe
106. e and click the Color tab Click into the color wheel to select the desired color for the light source Check Fix to Data Set on the Settings tab in the properties area to fix the Light Source to the object When the box is not checked the light source is fixed to a particular position in space and remains in place when the object moves or rotates retina ims Imaris 3 3 E ml x view Image Processing Surpass Help JOR ot gm c NYY 4 6 2 a AT NS Open Save as Snapshot Slice Section Gallery Easy 3D Ful3D Surpass Coloc Bitplane AG ight Source 1 Properties Zoom 1x Eit Pointer Select C Navigate EF 3 0983 0988 100 View Key Frame Animation Settings Color Full Screen Camera f Orthogonal Perspective 2 Delete v L3 Surpass Scene MES Light Source 1 MEB Frame ivi 9 Iso 120 ci Iso 124 c2 Draw Style Full Featured box move smooth lines Stereo Ready Zoom 2 96 Fig 38 Light Source 16 11 04 Page 57 BITPLANE AG Operating Instructions Fig 39 Two Light Sources blue light from the left yellow from the right white object Isosurface The IsoSurface visualization is a computer generated representation of a specified gray value range in the data set It creates an artificial solid object in order to visua
107. e file name and click OK The tree objects are saved as Imaris Scene file Any Scene file with the file extension imx can be loaded into Surpass for further viewing 1 Select File Load Scene in the IMARIS menu bar The Load Surpass Scene window is displayed 2 Select the directory and requested file to be loaded and click OK The scene file is loaded for viewing Isosurfaces included in the scene must be recalculated after the scene is loaded Imaris 4 1 Operating Instructions BITPLANE AG Imaris Slice Measure ment 2D slice measure ment 16 11 04 ImarisMeasurementPro is an optional module within the IMARIS program and is not available as a standalone program The user must have a license for Imaris and a license for ImarisMeasurementPro ImarisMeasurementPro adds geometric and intensity measurement capabilities to the volume and surface rendering features and the object slicing functionality of Imaris It allows users to Measure distances in 2D or 3D Measure intensities gt Manually segment an object Export statistical data to a text file that can be imported into a spread sheet ImarisMeasurementPro allows users to perform a number measurements in the slice view The user can measure distances within a particular slice 2D or between two different slices 3D 1 Load a data set into Imaris and select the Slice view 2 Select the mode Meas o
108. e histogram display The threshold for each channel can then be defined by dragging the yellow line directly in the histogram or by entering the value in the threshold field 15 55 of data selected Channel B Channel 2 namen 7 Threshold 76 00 1 7 X of data selected 16 11 04 Fig 97 Channel selection and intensity Histograms Page 131 BITPLANE AG Operating Instructions 2D Histogram This area displays a two dimensional intensity histogram of the selected channels It reflects the distribution of pairs of voxel intensities occurring in the two selected channels The range of intensity pairs considered as colocalized can be defined on the histogram e either as channel thresholds visible in form of a rectangle if selected as an intensity area in form of a free configurable polygon 20 Histogram 0 10 of data selected Fig 98 2D Histogram Volume Statistics This area displays statistics about the resulting colocalized volume The display adapts instantly to all modifications in the selection Definitions of the displayed values are given in chapter coloc volume statistics in the next chapter Coloc Volume Statistics Quantity Value number of colocalized yorels 40529 of ROI colocalized 1 02 x valume 4 above threshold colocalized 8 33 Volume B above threshold colocalized 12 96 4 gt material amp above threshold colocaliz
109. e keyframes to equalize the speed of the movie throughout its entirety 9 To preview the movie click the arrow in the animation field 10 When you are satisfied with the movie click the red dot in the animation field to record 16 11 04 Page 87 BITPLANE AG Export Scene Load Scene Page 88 Operating Instructions Users with ImarisTime only Time settings Affect playback only Arrow plays through time Red dot Automatically records an avi movie of the selected object moving through time No settings are possible with this automated option A Scene File stores a configuration of Imaris and Surpass and allows the user to restore the work at any time by loading the Scene File The file stores the camera position of the Full 3D and Surpass views the rendering modes of the Easy 3D and Full 3D views and information about all Surpass objects It is thus possible to create several objects in Surpass save a scene file and later load this scene into the software in order to recreate the object tree from the time of writing A scene comprises the tree of objects in the objects area and the camera settings This scene can be saved as an Imaris Scene file with an imx extension The scene can be loaded again to the same data set or to another data set The Scene File can be written as follows Select File Export Scene as in the IMARIS toolbar The Save Imaris Scene box is displayed Select the directory and enter th
110. ear voxel represen tation Select the Advanced button Click the requested channel to select it or check Select all Channels to apply the settings to all channels Drag the upper or lower handle of the adjustment line to adjust the min and max limits or enter direct values in the Min and Max fields The effect of the change can be seen on the channels channels ap pear brighter or darker Click the Reset button to set the image back to the original values When clicking the Auto button the system detects the real high and low values e g 10 150 and sets the max and min limits automati cally to these values Page 32 Imaris 4 1 Operating Instructions BITPLANE AG The Blend Opacity adjustment allows the user to change the opacity in real time in Blend projections in Section Full 3D and Surpass Volume views e Inthe menu bar select Edit Display Adjustment e Select the Advanced button e Click the requested channel to select it or check Select all Channels to apply the settings to all channels e Drag the Blend Opacity slider bar to adjust the blend opacity e The effect of the change can be seen on the channels channels appear more or less transparent The Channel Visibility adjustment allows the user to switch the individual channels on or off and to change channel parameters such as name color and description e In the menu bar select Edit Display Adjustment Check or uncheck the require
111. ections are drawn as truncated cones with radii proportional to the volume of the Surface objects at their end points The slider allows adjusting the width of lines cylinders or cones The following figure displays a Track with connections drawn as cylinders The connections are visualized for all time points while the Surface objects are only visualized for one time point Playing through time would have the effect that Surface objects would move along their tracks Page 85 BITPLANE AG Track Statistics Keyframe Anima tion Page 86 Operating Instructions Fig 64 A Track with Surface objects and cylinders Selecting Time Color Map draws lines cylinders or cones in colors with a range from blue to white and dark to bright corresponding to the range of time points Alternatively it is possible to assign a single color via the color editor that can be opened by pressing the Edit Color button The Track object has a Statistics Tab that displays some statistics values of its children as well as several Track specific statistics values Number Of Branches A Branch occurs when a Surface object is connected to several other Surface objects of a future time point Number of Fusions A Fusion occurs when a Surface object is connected to several Surface objects of a past time point Track Length The length of a Track is computed as the sum of the lengths of all connections between Surface objects of a Track
112. ects can be selected by left clicking onto the line corresponding to the connection in the Track Editor Window Using Shift left click it is possible to perform multiple selection Imaris 4 1 Operating Instructions 16 11 04 BITPLANE AG Track 2 Editor j euUrfacenl295 T2 m I ha Surfaces T3 E m ll E Surface0g0_T4 m I en Surtacel 13 T5 Surfacel46 Surfaces T7 m Il Time 9 ime 8 uUrfacez 8 T3 Time 100 Surfacez33 Add Connect Select entire Branch Merge Connect Select Branch Points Split Select longest Path Delete Connections Delete Objects Fig 62 The Track Editor Window Add Connect of a single Surface object To add a new Surface object to a track select this object either within the Viewing Area or within the Objects Area Then press the Add Connect button the Track Editor Window As a result the selected Surface object appears in the Track Editor Window and it is moved from its original location in the Object Area tree to become a child of the Track object to which the Track Editor Window belongs Add Connect of multiple Surface objects Several Surface objects can be added to the track at once To do so use multiple selection to select several Surface objects Then press the Add Connect button in the Track Editor Window All selected S
113. ects invisible before selecting hidden lines gt Points Y No Texture Y Bounding box boxmove Smooth lines Draws isosurfaces as a point model Draws objects without textures Only relevant for Topography objects Shows only the boxes surrounding the objects Displays only the surrounding box when moving the object Freezes the object in the view area and prevents movement Select a new center of rotation Click into the Surpass scene to define the new center on which the camera zooms in Note Rotation centers be set on IsoSurfaces Contour Surface Ortho Slices External objects and Topographies Calls up the Stereo Preferences dialog box to specify the requested stereo viewing mode and adjust the views Page 53 BITPLANE AG Keys Page 54 Operating Instructions Stereo Preferences Use Stereo Flav Stereo 0 Stereo Adjustment Reverse left and right views ero paralla balance Cameras offset A Fig 36 Stereo Preferences dialog box Select one of the following modes Raw Stereo Open GL e SSDI Stereo e Horizontal Interlaced Fast e Vertical Interlaced Fast e Horizontal Interlaced Best e Vertical Interlaced Best The modes listed above require special viewing equipment e Horizontal Half filled screen not recommended as it distorts the object e Horizontal Half screen not recommended as it distorts the object e Ve
114. ects of the modification are instantly visible in the threshold field as well as in the Preview window When the mask is set as required click the Automatic thresholding buttons to calculate the thresholds Build the colocalization channel by clicking the Build Coloc Channel button For a detailed description of the Time option please refer to the IMARIS manual ImarisTime is an optional module of IMARIS and may be not available Imaris 4 1 Operating Instructions Visualize the Coloc Channel in IMARIS 16 11 04 BITPLANE AG There are no time dependent parameters for the colocalization analysis The Preview window always displays the currently selected time point of the data set The values in the Statistics window always refer to the vol ume of the current time point However when the Build coloc channel but ton is pressed the coloc channel and statistical values for all time points are calculated The colocalization channel can be visualized like any other channel in IMARIS In the following examples a coloc channel was built on the retina data set Example 1 Visualize and adjust the Coloc channel in IMARIS Easy 3D 1 After building the Coloc channel click on the Easy 3D button in the IMARIS menu bar 2 Check the desired Rendering mode parameters and adjust the Settings if necessary then click the Calculate button The 3D image of channels is calculated and displayed Page 149 BITPLANE AG Operating Instru
115. ed 8135 material B above threshold colocalized 10 94 of AU material colocalized 5 25 x of ROI maternal B colocalized LSF channel correlation in colocalized volume 0 0790 Fig 99 Volume Statistics Page 132 Imaris 4 1 Operating Instructions ROI Definition 16 11 04 BITPLANE AG This feature allows the user to define a region of interest ROI for the en tire analysis A third channel can be selected as a masking area for the en tire analysis A masking channel is e g a third microscope channel acquired with settings that allows defining a region of interest by simple intensity thresholding All voxels outside of the region of interest defined by the mask channel are ignored for the colocalization analysis The volume excluded by the mask appears hatched in the Preview window The mask channel is used in conjunction with the Automatic Threshold function For this function the selection of an appropriate region of interest is essential Defne Region of Interest iw Mask dataset Channel 1 Call Threshold oF 6 69 of data selected Fig 100 ROI Definition and Mask Channel Page 133 BITPLANE AG Control Panel Operating Instructions The Control panel contains several sections with all possible selection and definition tools and settings for the histograms the thresholding and the display as well as the buttons for the color definition and the build of the coloc channel
116. el TOOS ins ES wobbel_TO1 1 ims RE wobbel TO17 ims wobbel TC File name wobbel TOC ims Files of type AI Formats L ancel Fig 120 Add Channels dialog box 7 Select the requested file from the Open box and click Open All channels from this data set are added automatically If the size of the data set does not match a warning message is issued Deleting channels is used to erase unwanted information in a data set Delete Channels Select Channel to Delete Channel 1 Collagen 1 Cancel Fig 121 Delete Channels dialog box Select the channel to be deleted in the Delete Channels box and click OK The effect is immediately visible on the image Page 163 BITPLANE AG Operating Instructions Add Layers Adding layers may become necessary if through manipulation or during file export or formatting the data set consists of less than the originally ac quired number of slices Add Layers Look ir 3 images T pollen ins retina ime File name D SWIMMNT sPrafilesslaimersD esktap sretina ims Bitplane maris Classic ing ICS file 168 ids eis LOM570 sm eles LS5MATOLLSM3TU Leica TCS NT Leica Series Leica LES lei Bio Rad 1024 600 Fig 122 Add Layers dialog box Select the requested file from the Add Layers box and click O
117. er 117 STORING OUTPUT DATA 2 118 CREATING OUTPUT 119 2D SHBLIMAGES ae A eae ee a SUN ME 119 SANOA NEF IMAGE 120 SAVING ADATABASE BECORD 7 neutre deb do Ed ead ous 120 COPYING TO THE CLIPBOARDS 55 Dod 120 M VIE DA DUE 121 IMARIS TIME 123 LOADING A TIME SERIE 123 SWAP Z WR dietis 125 PADD Mies Fe ONS er ce 177 DELETE tT 127 128 RE AMPLE EL Seu LE 128 IMARIS COLOC 129 WHAT IS IMARBISCOLEOQG 2 Din MNA CE FED PAID 129 MANGO REE ee tries rt d ioter colo E oc MERE eee 129 OV 130 GHANNEEJFISTOGRAM S tado es 131 2DF FO GPA
118. eshold in the control panel Select Histogram mode Check e Single Slice for calculating the histogram for current slice only Page 139 BITPLANE AG Page 140 Operating Instructions One Time Point to calculate the histogram for the entire 3D time point All Time Points for the entire 4D image Threshold setting methods There are basically four different methods how the thresholds can be defined On the 1D Channel Histogram 4 Point with the cursor on the histogram The cursor turns into a cross Click on the histogram to determine a threshold The bolded left line of the overlaid hatched area indicating the selected voxel intensity range for colocalization jumps on the marked spot Click on the line and drag it to another position in the histogram Effects of the modification are instantly visible in the other indications as well as in the Preview window Perform for both channels until the settings are as desired By entering a value Click in the Threshold field of one of the channels and enter a value then press Enter The bolded left line of the overlaid hatched area indicating the selected voxel intensity range for colocalization jumps on the marked spot Effects of the modification are instantly visible in the other indications as well as in the Preview display 2 Perform for both channels until the settings are as desired On the 2D Histogram 1 Point with the cursor on the 2D histogram T
119. essary change the settings When the results are satisfactory click the OK button The data set is modified Click Cancel to return to the original image Imaris 4 1 Operating Instructions Channel Shift Gaussian Filter Median Filter Edge Preserving Fil ler 16 11 04 BITPLANE AG The Channel Shift function allows to move channels relative to one another In the channel selector you can select the channels that should be moved relative to the remaining channels The translation is specified in terms of integer numbers of voxels to move along each of the X Y and Z directions Channel Shift X Pixel shift For all selected channels B Channel 1 name not specified Direction Y z W Channel 3 fname not specified Channel 4 fname nat specified voxels 5 m H Apply Cancel Fig 134 Channel Shift The Gaussian filter smooths the image lt has some nice properties 1 Total image intensity is preserved 2 Noise is suppressed homogeneously 3 No artefacts are introduced The Median Filter replaces the intensity of each voxel with the statistical median of the intensities of neighbouring voxels There are several choices for the neighbouring voxels to be included in the median calculation The median filter is useful when the image data are corrupted by salt and pepper noise The edge preserving filter smooths the image and has all the nice properties of a Gaussian filter
120. g 2 File Menu The Edit menu allows you to open different dialogs to adjust images edit channels layers and time points and modify the data type and general pa rameters you can also crop images File Edit view Image Processing Surpass Help Copy Image Properties Ctrl I Channel visibility Display Adjustment Ctrl D Add Time Points Delete Time Points Add Channels Delete Channels Add Layers Crop Time Crop 30 Resample Time Resample 3D Change Data Preferences Fig 3 Edit Menu Imaris 4 1 Operating Instructions BITPLANE AG The View menu allows the selection between the different viewing modes File Edit jew Image Processing Surpass Help Slice View Section View Gallery View Easy 3D View Full 3D view Surpass View Toolbars b Refresh Ctrl F Fig 4 View Menu The Surpass menu contains the functions for adding new Surpass objects for exporting selected objects in a viewer file and for deleting selected objects Fie Edit wiew Image Processing Surpass Help Add Mew Group Frame Export Selected Objects Light Source Delete Selected Objects Clipping Plane External Object Iso Surface Contour Surface Volume Ortho Slice Topography Measurement Point Fig 5 Surpass menu The Image Processing menu contains the image processing operations File Edit View Image Processing Surpass Help Flip
121. g licenses grant permission to run the licensed software on several computers One floating license can activate only one instance of the program at any given time Node locked licenses are the easiest way to license the software The installation of node locked licenses is guided by the Imaris software itself Floating licenses require the installation of a separate license server that can run either on a Windows or on an Irix computer The server installation requires more technical knowhow The following procedure describes how install node locked licenses for Imaris if the computer on which you want to run is connected to the internet If your computer is not connected to the internet follow the instructions in the section Imaris Offline 1 Start the Imaris software on the computer on which you want to install licenses You will see the following dialog 9 Imaris didn t find any license Would vau like bo specify your license configuration Yes Click Yes to get to the license installation dialog Page 182 Imaris 4 1 Operating Instructions BITPLANE AG fou can either specify a location with license information or download it via internet Install Upgrade Product Licenses For this Computer IF this computer is connected to the Internet Imaris can directly download the license information from wea biEplane com Press the Online button On
122. g the point and e drag it to the new position Deleting Measurement Points Measurement points can be deleted one by one i e the point set last will be deleted first by pressing and holding down DELETE on the keyboard All measurement points are simultaneously deleted when clicking on the Delete button in the right frame bar It is also possible to select and delete an arbitrary measurement point i e not necessarily the last one by moving the mouse pointer directly over it thus highlighting it and pressing DELETE All measurement points are automatically deleted when a new data set is loaded Color The color for the measurement points and lines can be specified Select Edit Preferences from the toolbar In the Preferences box select Display from the list and choose the Measurement color Page 91 BITPLANE AG Surface and Vol ume Measure ments Isosurfaces Surface and Voxel Statistics Page 92 Operating Instructions The IsoSurface visualization is a computer generated representation of a specified gray value range in the data set It creates an artificial solid object in order to visualize the range of interest of a real volume object See the Imaris Surpass User Manual for details of creating an isosurface Creating Statistics Surface and Voxel Statistics are calculated automatically for each IsoSurface object The surface is made up of connected triangles The surface statistics describe the su
123. gh quality Object Roatation Center optimizing default user interactions High Low quality howe Up Move Down Cancel Fig 130 Surpass Preferences Box Objection Creation Buttons Select the icons to be displayed on the Objects toolbar in Surpass Highlight a button and click Move Up or Move Down to define the order of the icons in the Surpass Objects toolbar Imaris 4 1 Operating Instructions Advanced Image Processing 16 11 04 BITPLANE AG Key Frame Interpolation Select Camera Rotation Center for fly through animation Select Object Rotation Center for all other animations Key Frame Animation Select the playback mode Play one time or Repeat forever Select the playback frame rate and compression factor Low Compres sion High quality creates a larger file and requires more disk space Displays a window similar to the Parameters window It shows internal settings and parameters in groups and parameter lists Changing the Advanced parameter settings may endanger your system and lead to erroneous shutdowns The Image Processing menu provides a range of functions to change the image data Frequently used functions are Baseline Subtraction and Thresdhold Cutoff from the Image Thresholding submenu or the Gaussian Filter from the Image Smoothing submenu f a time series is loaded clicking Apply applies the image processing function to the current time point displayed on t
124. he cursor turns into a cross 2 Click on the histogram to determine the thresholds The edge of the overlaid hatched area indicating the selected voxel intensity range for colocalization jumps on the marked spot 3 Click on the edge and drag it to any another position in the histogram Effects of the modification are instantly visible in the other indications as well as in the Preview display On the Preview Window Imaris 4 1 Operating Instructions Coloc Display 16 11 04 BITPLANE AG 1 Click with the cursor on the image in the Preview window An active contour is displayed outlining image elements brighter than the location clicked 2 Dragthe line on the image to define the threshold of channel A The selection works best when starting with brighter spots and dragging towards darker areas 3 Press and hold the SHIFT key click with the cursor on the image then drag the line to determine the threshold of channel B Colocalization occurs where the voxels are above threshold in channel A and channel B ax Fig 104 Threshold selection on the Preview window The user may determine the intensity for the colocalized voxels to be shown Coloc intensities Check e Source channel for using the intensities of the two channels for displaying the intensity Constant value for displaying the intensity of a colocalized voxel defined by the specified value Coloc Color 1 Click the Edit button The color
125. he screen Clicking OK applies the image processing function to all time points File Edit View Image Processing Surpass Help Flip Rotae Channel Swap Z with T Image Smoothing k Image Threshalding Contrast Change k AutaDeblur Huygens Essential Fig 131 Image Processing menu Page 173 BITPLANE AG Flip Rotate Page 174 Operating Instructions The channels can be flipped together or separately Ix All Channaelis Direction Fig 132 Flip Dialog Box Inthe menu bar select Image Processing Flip The Flip box is displayed Select the channels to be flipped by marking the check box on the left Check the requested Direction box and click the Apply button The effect of the change can be seen in the image f necessary change the direction When the results are satisfactory click the OK button The data set is modified Click Cancel to return to the original image All Channel s Axis x Orientation Counter clockwise Clockwise cem Fig 133 Rotate Dialog Box Rotate direction applies to all channels together Single channel appli cation is not possible Inthe menu bar select Image Processing Rotate The Rotate box is displayed Enter the requested Axis and rotation direction Orientation then click the Apply button The effect result can be seen in the image f nec
126. hold down the right mouse button click in the image and drag the mouse To pan in Surpass with the pointer in navigate mode hold down the cen ter mouse button and drag the mouse Rotate To rotate hold down the left mouse button and drag the mouse In Surpass the pointer must be in navigate mode Click the Set Center button to set the center of the rotation Display Adjustment Blend Opacity and Channel Visibility The Display Adjustment function lets you choose the channel visibility as well as improve the image display by concentrating on a limited color con trast range of voxels Usually the color contrast values of the voxels stretch over a wide range e g 0 255 The Display Adjustment function lets you set an upper limit for maximum color and a lower limit for minimum color i e black The range between these two limits is then extrapolated in a linear mode to the full data set range and the new voxel values are calculated 16 11 04 Page 31 BITPLANE AG Operating Instructions Display Adjustment X ER Collagenty FITCH v Advanced Select all Channels Range Collagen TxAed 8 000 Reset 194 321 Auto Blend Opacity 16 300 Ch 1 Fig I9 Display Adjustment Blend Opacity and Channel Visibility box Inthe menu bar select Edit Display Adjustment The Display Setting box is displayed showing a lin
127. hts reserved Printed in Switzerland 16 11 04 If you find any information missing in this book or need additional support 41 1 430 11 00 41 1 430 11 01 support bitplane com http www bitplane com Page 1 BITPLANE AG Operating Instructions Contents PREFACE 1 NOTICE assist bM Lov OL Eu SM 1 DIDA EI E a RUP 1 CONTENTS 2 INTRODUCTION 7 BEFORE YOU BEGIN 7 CONVENT ONO m 7 GETTING FAMILIAR 8 WHAT IS IMAHBIS e Coss oleae qua cues Ei tao eese cire sudo e us 8 TERMINOLOGY ECCE 10 GETTING STAHTE D 11 Mee 11 SUPPORTED FILE FORMA LS 5 o rnoeo excu EreEEoERO codes ouster 11 STARTING IMABHBIS poser euet 13 Bie zie tC DT 13 d asc 14 USING IMARIS 17 Dove vus e E Duces cec D 17 READ ONLY ONE TIVE POINT sroine 1 0 5 22 50 50 Dodo Ens PN EAE Sands 18 FRRESAMPLINGOPEN ccsseececcccccccsececccccccacsseccccccccaseeeeccccccuacsseeeecccccaueseeecececsauuseeccc
128. i channel multiple colors but only one channel will be traced at a time The following input images are acceptable ideal input is a fluorescently labeled confocal image Such images generally have good contrast selective labeling and good resolution in all three spatial dimensions e 3D images can also be acquired using a standard microscope non confocal equipped with a CCD camera and a stepper motor Non confocal optical arrangements do not provide resolution along the optical axis which is why every single plane consists of in focus components and out of focus components These images require deconvolution prior to tracing The technique of deconvolution uses information about the imaging process the point spread function to remove the out of focus components from every section e Finally there are 3D images taken in transmission light microscopy Staining is usually less specific than in the fluorescent case and it will be more difficult to automatically segment the filament Deconvolution can also be applied to transmission images although theoretically its performance is weaker than in the fluorescent case 16 11 04 Page 105 BITPLANE AG Segmentation Automatic Graph Compilation Page 106 Operating Instructions The segmentation process will filter the original images in such a way that noise is suppressed as good as possible and the image becomes optimally suited for the next step the aut
129. ick fixed view 3D image with a few pa rameters for material rendering and light setting This lets you explore the data set in the 3D mode before doing any further processing AB Edit View Image Processing Surpass Help E NM a s m had E 3 NS S Open Save as Snapshot Slice Section Gallery Easy 3D Ful3D Surpass Coloc Bitplane AG Renderinc Mode m Zoom C ME Blend Fit Light Left Position C Right Above C Below Calculate Image calculated Zoom 2 98 Fig 25 Easy 3Dview When the Easy 3Dview is selected the screen is empty Select the requested Rendering Mode from MIP Maximum Intensity Projection shows the maximum intensity of all layers along the viewing direction Blend shows the color obtained by blending all values along the viewing direction and including their transparency If Blend is selected check Light on or off There are two predefined light setting parameters in Easy 3D to choose from Left light from the left shadow to the right Right light from the right shadow to the left Select the direction of the camera on the data set by clicking Position Above or Below Page 40 Imaris 4 1 Operating Instructions 16 11 04 BITPLANE AG Click the Calculate button The image is calculated and displayed A closer look at details can be achieved using the zoom function Shadow Projec
130. ill have a small notepad next to it Enable Start Server at Power Up and Use Services the Bitplane service Click the Start Stop Reread tab Bitplane should be highlighted in the window Flexlm license services installed on this computer Click Start Server The operations line at the bottom of the Imtools screen should report Server starting Click the Server Status tab and the Perform Status Enquiry button The enquiry should report that the license manager on the server and Imaris 4 1 Operating Instructions License File Imaris configuration 16 11 04 BITPLANE AG the bitplane vendor daemon are UP and the licenses are available for check out 14 Accidentally clicking the Start server button when the server is already running may cause multiple processes to run on the machine and use all of its resources If this happens click Control gt Alt gt Delete Select Task Manager and click the Processes tab End all the bitplane exe and Imgrd exe processes Reopen Imtools and continue A floating license contains three components a SERVER line a VENDOR line and one or several FEATURE lines An example license file may look as follows SERVER sgi neurobio mydomain edu 69139cc4 1700 VENDOR bitplane usr local imaris3 bin bitplane FEATURE ImarisFull3D bitplane 3 1 1 jan 00 1 5CA86299C288DAF81EO0F HOSTID INTERNET 136 142 DUP_GROUP UH ISSUER Bitplane AG FEATURE ImarisSurpass bitplane 3 1 1 jan 00 1 5C2892E92D16B16CA38
131. image before the calculation Preview Cube When selecting Full 3D a view with an empty cube structure is presented first Imaris 3 3 5 x File Edit View ImageProcessing Surpass Help SH MJ ies a 29 NI a 6 AZ EB 14 Open Save as Snapshot Slice Section Gallery Easy 3D Ful 3D Surpass Coloc Bitplane AG Renderin Preview p Zoom Cube 1 C Section Fit C Volume LEE Mode View MIP Selected C Blend i Light Type Ortho Edit Settings C Perspec Edit Material Stereo Off Calculate C Red Cyan Animation Buffers No image calculated Zoom 1 00 Fig 27 Full 3D Preview Cube Preview Section This preview shows a sectional cut through the center point of the data set to illustrate the position without having to wait for calculation Page 42 Imaris 4 1 Operating Instructions BITPLANE AG 6 retina_red ims Imaris 3 2 BD XX B File Edit View Image Processing Surpass Help s u NS SS he ud Open Save Snapshot Slice Section Gallery Easy 3D Full 30 Surpass Camera oom Preview Valume C MIP Selecter Blend Light Type C Ortho Perspec Stereo Off C Red Cyan Buffers E A E ge zm c d si Prise pa 2 22 4 image calculated Fig 26
132. imation Full Screen Surface Objects Distances Obiects Camera Type aa 25 C Orthogonal Perspective Draw Style Full Featured box move YJB Frame v Iso 120 cl Iso t20 ce MO smooth lines Z Ivi P2 B eS P3 go P4 Set Center P5 Stereo PE WS P v P8 VB v PIO Ready Zoom 1 J 2 Fig 75 Measurement Points on IsoSurface Free Measurement Points Free measurement points are freely movable in space The user can set as many free measurement points in space as needed Page 102 Imaris 4 1 BITPLANE AG Operating Instructions Fig 76 Free Measurement Points Adding Free Measurement Points AutoDepth Method 1 2 Create a Volume object Visualize only the channel for which you want to set Measurement Points Shift Left Click onto the position you want to set a Measurement Point The depth is automatically computed at the position of maximum intensity along the line of view e g if the picture is displayed on its xy plane z coordinate of the Measurement Point is computed Adding Free Measurement Points Manual method 1 16 11 04 Click the New Measurement Point icon e in the objects toolbar or click Surpass Add New Measurement Point in the main toolbar The new measurement point is displayed It is located in the zero origin of the coordinate system of the data set x20 y
133. inate axis of the data set that can be moved within the data set When adding slices Surpass suggests per default the directions Z X Y as sequence for the first three and all subsequent slices 16 11 04 Page 67 BITPLANE AG Operating Instructions Edit View Image Processing Surpass Help gt B Open Save as Snapshot Ortho Slicer Properties Direction le E show slice border Slice Objects 3 Surpass Scene Pe Light Source 1 MEB Frame Ortho Slicer 1 Ortho Slicer 2 Ortho Slicer 3 fi 42 000 4 um r View d Key Frame i Animation Full Screen RON ON a n 5 had T 3 2 NS ND he m 5 Slice Section Gallery Easy 3D Full 3D Surpass Coloc Bitplane AG Navigate m Draw Style Full Featured box move Camera Type Delete Orthogonal Perspective smooth lines Set Center Stereo I 7224 Ready Clipping Plane Page 68 Fig 46 Orthoslice window 1 Click the Ortho Slice icon in the objects area or click Surpass Add New Orthoslice in the main toolbar The slice is displayed in the viewing area and the object properties area shows the suggested parameters for it 2 Change the Direction if required and choose the relative position in the data set
134. iple selection to select several Surface objects Then press the Merge Connect button in the Track Editor Window All selected Surface objects will be merged into the track together with the Surface objects to which they were previously connected in another track if any In addition connections are created between all selected Surface objects in the order in which they were selected Split A track contains Surface objects and connections between these objects both of which are visualized in the Track Editor Window It may happen that a Track can be split into two or more Tracks with exactly the same connections between Surface objects without having to delete any connections If this is the case the operation is performed by pressing the Split button If it is not the case pressing the Split button has no effect Deleting Connections A connection between two Surface objects can be selected by left clicking onto the line depicting the connection within the Track Editor Window Pressing the Delete Connections button in the Track Editor Window will delete all selected connections Deleting Objects Pressing the Delete Objects button in the Track Editor Window will remove all selected Surface objects of that Track The Surface objects are not actually deleted but they are made invisible and moved into a group called Trash in the tree of the Object Area Selecting Branches If a Surface object or a connectio
135. ition Intensity Position Fig 136 Connective Baseline Linear Stretch Linear stretching may be necessary whenever 12 bit data is read in the 16 bit mode Then the contrast can then be extended to the new limits Picture data is changed when stretching Linear stretching results in a picture displaying dense data in a wider contrast range Linear Stretch Channel 1 name not specified Parameters Current minimurn 0 Few minimum Current maximum 55 Few maximum cancel Fig 139 Linear Stretch Dialog Box In the menu bar select Image Processing Contrast Change Linear Stretch The Linear Stretch box is displayed Select the channels to be stretched by marking the check box 16 11 04 Page 179 BITPLANE AG Gamma Correction Page 180 Channel 1 name not specified Operating Instructions Enter the requested values in the parameter fields New Maximum and New Minimum and click the Apply button The effect of the change can be seen in the image If necessary readjust the value When the results are satisfactory click the OK button The data set is modified Click Cancel to return to the original image The Gamma correction function lets you improve the image display by inten sifying the gray value of a specific range of voxels Usually the color contrast values of the voxels stretch linear over a wide range e g 0 255 The Gamma correction function lets y
136. l time points 137 Ambient color 78 Ambient Intensity 41 Animations 33 42 47 Apply Automatic File Filter 20 123 Attached Measurement Points 101 Auto 32 Auto Distribute 87 AutoDeblur 181 Automatic File Format Detection Sequence 170 Automatic thresholding 136 Background 168 Base Color 24 159 Baseline Subtraction 28 29 176 1777 Blend 37 65 Blend Opacity 33 66 Bounding box 53 Box handle 73 box move 53 Buffers 44 Calculate 41 Calculating a Contour Surface 98 Calculation 170 Camera Type 53 Cameras offset 54 Change Data Type 165 Channel Colors 22 24 Channel Opacity 41 Channel Visibility 33 66 Channels 59 157 Checkered 168 clipboard 120 Clipping Plane 52 68 Close objects at border 60 Color 56 Color and Material 71 76 100 Color Calculation 46 16 11 04 Page 191 BITPLANE AG Page 2 Operating Instructions Color coded 137 Color Properties 76 Color Table File 161 Colorspace 161 Combinations of visualisations 74 Compression 49 173 Constant value 137 Contour Surface 62 Contour Surfaces 95 Control Panel 134 Converting csv format to MS Excel 101 Coordinates 156 Copy 155 Create no more than nn objects 61 Creating 47 Creating Statistics 94 Crop Time 128 Cropping 25 124 Crosshair 36 cut high 65 cut low 65 Data Cache 171 Data Set 155 database record 120 Default colors 170 Define Custom Colors 159
137. le Format Detection Sequence Allows the user to specify the order in which the file format readers are called when loading images Move the most frequently used file format readers to the top of the list to hasten file loading 1 Highlight a file reader 2 Click Move up or Move Down to arrange the format readers in the desired sequence Calculation Preferences System Calculation Display Loading Calculation Time surpass Data Cache Image Processing History Cancel Fig 126 Calculation Box Calculation opecify the number of processors used in calculations See maximum number of available processors in the System box Page 170 Imaris 4 1 Operating Instructions Time 16 11 04 BITPLANE AG Data Cache Imaris uses a data caching mechanism that allows the user to process im ages that are significantly larger than the physical memory RAM installed in the computer system This mechanism writes image data blocks to the disk and reads them back into the physical memory when they are needed The value of data cache limit controls the amount of data blocks Imaris will keep in memory at any time Enter a value slightly less than the amount of physical memory installed on the machine but not higher than 1 5 GB Maximum Number of Commands in History Defines the maximum number of image processing operations that can be undone Each level of image processing requires an additio
138. licer plane an IsoSurface a Contour Surface or a Topography These points can be moved across the object they are attached to but they cannot be moved away from that object When the object is moved the measurement point moves correspondingly Setting and Moving a Measurement Point To set and move a measurement point 1 Create an Orthoslice Isosurface Contour surface or Topography See the Imaris Surpass User Manual for details Page 101 BITPLANE AG Operating Instructions 2 Switch the cursor to Select mode and move the cursor to the required position on the object in the view area 3 Hold down the Shift key and left click with the mouse The measurement point is displayed 4 To change the position switch to select mode click on the measurement point and drag it to the new location Remember that the measurement point is anchored to the object or the particular OrthoSlicer plane and cannot be moved to a location outside the object or plane Camera Zoom Fit Pointer C Select Navigate View Surpass Scene Properties Line Mode Pair CD P1 P2 64 0 um P3 P4 34 5 um P5 P6 104 1 um Polygon 0 pe w Origin Entries 2 AD Sum 5 Entries 288 3 um Draw Lines to visible Points C all Points Hide Labels Hide Distance Labels Time Depend Paints C no Points Export jw n
139. line If this computer has na Internet access you can download the license information via another computer with Internet access Press the Offline button and follow the instructions Offline IF this computer applies license information from a floating license server ar if you would like to configure a license file please enter the file path ar the license server name Browse C Program FilessBitplane sbitplane lic or portt servername Files Mate au will need to restart the application in order Far the changes ta take effect 2 f your computer is connected to the internet click the Online button on the license installation dialog You will then be asked to enter the Order ID from your purchase order Given the Order ID the software will try to fetch the corresponting list of purchased products The following figure shows a list with four products The checked product licenses below will be installed an this computer Do not check products which you Would like to install on another computer run the Online Install Upgrade Products there Gray colored product licenses are allready installed on this computer and can t be unchecked have ordered additional new products vou should have recereed an identification number In order to install this additional product licenses press the Enter Order ID button below Imaris 4 0 for Windows ready for installation on this compu
140. lize the range of interest of a volume object Iso Surface Setup X Channel 1 sacb gfp Channel 2 concnavilin amp 533 lll Channel 3 mitatracker red Threshald Advanced Settings Viorel size Units E 1 1938 um qe 1 one Fired ratio 2 Fixed ratio nipe 250 Gaussian filter width Close objects at border Resample dataset 7 Standard Size Quick Size Smooth dataset Advanced Settings gt gt Fig 40 IsoSurface Setup dialog box Page 58 Imaris 4 1 Operating Instructions 16 11 04 BITPLANE AG 1 To add an lsosurface click the isosurface icon a in the Objects toolbar or click Surpass Add New sosurface in the main toolbar The IsoSurface Setup window is displayed 2 Define the parameters for the IsoSurface Channel Select the requested channel s to be included by checking the corresponding checkboxes You may select either one channel or multiple channels together Select the channel to apply the settings by clicking on the colored channel bar active channel becomes bold Threshold Define the requested threshold for each channel individually by clicking into the histogram or by entering the value directly in the Threshold field The initial threshold value is automatically computed using an algorithm based on T W Ridler and S Calvard Picture thresholding using an iterative selection method IEEE Trans
141. m Statistics Inspect all statistic values Color Change the color appearance material of the filament Imaris 4 1 Operating Instructions Settings Show Hodes Terminal Pts Branch Pts Style f Line 1 pixel C Cylinder 1 000 uum C Cone 1 000 scale Length 73 318 um Average Diameter 3 315 urn Serial Resistance 12 041 1 um Number of Branchings 53 Settings Statistics m 1 zi a N aliens a 3 E w sn 9 BITPLANE The visualization of the graph is done as lines with a specified thickness pixel as cylinders with a defined diameter or cones using the measured graph diameter Special nodes are highlighted with color coded spheres green terminal points red normal branch points blue branch points with 4 or more joining segments and the root point cyan Rebuild starts a new filament creation with the previous used parameters The current filament graph will be deleted The root point anchor node defines the order of other nodes within the graph Report and measurement of the currently selected segment s Length Average Diameter Volume Serial Resistance Depth Number of Branchings Number of End Points etc See the Statistics Tab for more information If multiple parts of the filament graph are selected the measurements are summarized or averaged the depth may appears as not
142. mage or a file to the IMARIS program icon maris be started directly from the Image Access database The software opens with the main screen fretina_red ims Imaris 3 2 BEP a B x File Edit View Image Processing Surpass Help T i H oh wi Mei SYN of EH ES Open Save Snapshot Slice Section Gallery Easy 3D Full Surpass c i m oom m Controls Controls Mode C Default gu View f Single Multi Style Eh os a Ear t Point Paints Tools Grid p outside at 134 07 121 50 32 00 Zoom 3 66 2 Fig 1 Imaris main Status Bar Time Bar To run the IMARIS system the appropriate licenses for the required mod ules such as the IMARIS base including Surpass ImarisTime Imaris Coloc ImarisMeasurementPro or Topography Without licenses the IMARIS can only be run in a restricted mode In case of any license problems please refer to the support information on our website www bitplane com for detailed instructions 16 11 04 Page 13 BITPLANE AG Page 14 Operating Instructions The File menu contains the basic image operations File Edit View Image Processing Surpass Help Open Revert to File Ctrl F Save as Chrl 5 Load Scene Export Scene as Snapshot Ctrl T 1 retina red ims Exit Fi
143. maris 4 1 Operating Instructions BITPLANE AG Filament Tracer The Imaris FilamentTracer offers features to segment track edit draw display and measure filaments While the precursor software NeuronTracer was dedicated to analyzing dendritic trees of labeled neurons the FilamentTracer is designed to operate on a wider range of filamentous structures and uses corresponding language The FilamentTracer processes one channel color at a time and extract objects that qualify as filaments e g length and average thickness comply with criteria After an automatic filament extraction the user can launch an editor operation to delete filaments connect filaments or draw new filaments The user may also skip the automatic filament generation and process the data with semi automatic or manual segmentation and filament generation Generated filaments objects will be visualized in the context of Surpass This will allow multiple viewing combinations in which the filaments are displayed individually or together with other cellular components that may have been imaged in other channels Properties and measures of entire filaments or selected segments will be generated automatically and can be exported in tabular format or directly to MS Excel Input Data The FilamentTracer is designed to operate on 3D images which provides sufficient resolution to resolve the filaments to be studied in all three spatial directions These images can be mult
144. mputer is connected to the Internet Imaris can directly download the license information from www biEplane com Press the Online button Online If this computer has na Interet access you can download the license information via another computer with Internet access Press the Offline button and follow the instructions Offline IF this computer applies license information from a floating license server or if you would like ta configure a license please enter the fle path ar the license server name L Fragram Files sBitplane sbitplane lic or portiiservername E ____ Mate rau will need to restart the application in order for the changes ta take effect 184 Imaris 4 1 Operating Instructions 16 11 04 BITPLANE AG 2 If your computer is not connected to the internet click the Offline button on the license installation dialog You will then be asked to go to another computer that is connected to the internet and to open a specified web page You need to take with you the Host ID of the computer on which you want to install licenses You find the Host ID in the dialog a Please start any webbrowser on another computer where you pl have Internet access and Follow this link f wwiw biEplane comg activakian acti offline ident cfm You will be asked For the Host ID of this computer and For the Crder ID which you should have received by mail Host ID 0005Sdd
145. n algorithm that can create forks and merges and can thus deal with complicated objects Page 98 Imaris 4 1 Operating Instructions Contour Surface 2 Praperties Color Material Surface Statistics Ara 9754 5 ume Triangles 3737345 uma Center 165 5 159 3 58 3 Em Heedit Objects Surpass Scene n IE Frame vp Light Source 1 po amp Contour Surface 3 v ws Ortho Slicer 2 Statistics 16 11 04 EAEE uma S Delete BITPLANE AG Camera Pointer Select AdvSelect Navigate JF me m c EN Camera Type 1 B IE A Ortho j RN Tis 1 E ua z gt A 22 Perspec I Pal fe zr Draw Style Featun box move Set Genter Steren Fig 73 Calculated Surface The contour can be modified and recalculated if necessary Click Reedit in the Properties area Click Reduce in the Properties area to reduce the number of triangle Color and material properties can be changed on the Color tab in the object properties area See the Color section for details Click the Statistics tab in the object properties area to view the surface and voxels statistics for the calculated contour To export the statistics 1 Highlight Surpass Scene or the required Group folder in the Objects area 2 Click Export Statistics in the object properties
146. n is selected and you press the Select entire Branch button all other Surface objects and connections of the track will also be selected unless there is a branch between them and the originally selected Surface objects or connections Imaris 4 1 Operating Instructions Track Settings 16 11 04 BITPLANE AG Selecting Branch Points By pressing the button Select Branch Points it is possible to select and thus highlight all Surface objects at that have more than two connections The Settings tab of a Track object is shown in the following figure Track 2 Properties T Edit Track Show Object Shape Path Connections Off E dit Color C Line Time Color Cylinder gt Volume o or go 1 Track invisible Settings statistics Color Fig 63 The Track Settings Tab The Edit Track button displays or hides the Track Editor Window that has been described above When Show Object Shape is ticked the Surface objects of the Track are visible in the Viewing Area Otherwise they are invisible The box Path Connections has settings that determine the display of connections between the Surface objects of a track When Off is selected no connections are drawn and the other settings within the box have no effect When Line or Cylinder is selected the connections are dawn as lines or cylinders When Volume is selected the conn
147. n the right frame bar 3 Select the drawing Style best suited to your needs pair line star point on the right frame bar e Pair Consecutive pairs of measurement points are connected by lines This is the default mode and is the active draw style when starting ImarisMeasurementPro e Line All measurement points are consecutively connected by lines The displayed distance is the sum of the distances between the points e Star All measurement points are connected with the first point set This is very useful for measuring various distances from the same starting point e Point The measurement points are displayed without connecting lines The coordinates of the points are displayed in the status bar Page 89 BITPLANE AG Operating Instructions 4 Set the measurement point by left clicking with the mouse on the desired spot on the image A small cross appears marking the selected point 5 Set the second and subsequent points in the same manner The points are connected by a solid line according to the draw style selected The coordinates x y z of the last point set last as well as the distance between them are displayed in the status bar in the following format Distance 8 20 um The distance is displayed in the same unit of measurement specified in Image Properties Edit Image Properties Geometry D retina red ims Imaris 3 2 RN File Edit View Image Proce
148. nal copy of the full image in memory If your machine runs out of memory set Maximum number of commands in history to 1 Allows the user to specify the playback mode Preferences Display 2 Loading Calculation Play one time Repeat forever i Advanced C Swing back and forth forever Frame rate f 00 frames per second Fig 129 Time Preferences Box Page 171 BITPLANE AG Surpass Preferences System Display Loading Calculation Time Surpass Advanced Page 172 Playback Modes Operating Instructions Play one time all time points of the dataset are shown one time The playback stops when the last time point is reached e Repeat forever once the playback has reached the last time point it starts at the first time point again never ending e Swing back and forth when the last time point is reached the time sequence is shown in reverse until the first time point is reached The user can further specify the frame rate i e the number of frames per second Surpass Object Creation Buttons Mlso Surface Contour Surface wiOrtha Slicer i Topography wiExtemal Object Framae Measurement Point Key Frame Interpolation Camera Rotation Center optimizing fly trough animation Key Frame Animation Play one time Repeat forever Frame rate 5 000 frames per second Movie compression factor 35 Low comp Hi
149. nd or File type for the object to be loaded and click Open The selected file is loaded and displayed as an external object Display Handling The object properties area displays a number of parameters for the positioning and display of the external object The following display handling options can be checked Fixratio If this option is checked and if scaling is performed on the external object by entering a value the same scaling factor is applied to all three dimensions Boxhandle The object shows a surrounding box with handles These handles allow to transform distort the object in the X Y and Z direction Fix Ratio not possible gt Trackball The object shows surrounding circular lines These lines allow rotation of the external object in any direction Fix Ratio not possible Page 73 BITPLANE AG Operating Instructions External Object Properties Camera f Box Handle C Trackball ponr E Documents and 5ettingsslaimersD esktopsf Reset Size and Location Translation Hotatian Scaling Iv n zd m Ee 5 1 0 28588 ET 0 00000 EY 1 000 T 7 353084 771 0 00000 1 0000 3 Z Fointer Select AdvSelect C Navigate View Store Recall 0 l sel i Delete Camera Ortho 2 e Perspec NES Li A E EEstemal Object Draw Style Full Featun box move Set Fenter Stered
150. ne AG Zoom Low Fit Pointer C Select Navigate View Key Frame Animation Full Screen Camera Type Orthogonal C Perspective E NS SS he a Section Gallery Easy 3D Full 3D Draw Style Full Featured box move smooth lines Stereo Fig 37 Frame with Grid The Frame serves to outline the object in the viewing area The Frame offers a number of functions that can be selected in the object properties area Frame Settings Highlight Frame in the object area and click the Settings tab The Frame settings display in the Properties area Check Box for the frame to display Uncheck Box to make the frame invisible Check Tickmarks to display tickmarks on the edges of the frame Check Grid to display a grid The grid serves as a scale bar in 3D perspective views Page 55 BITPLANE AG Groups Light Source Page 56 Operating Instructions Specify the grid and tickmark spacing in the Grid and Tickmark Settings box Check Coordinate Axes to display the x y and z axes to clearly show the orientation of the object Check Fix to Data Set to fix the x y and z axes to the same side when navigating the object Color To change the color of the frame highlight Frame in the object area and click the Color tab The Color editor displays in the properties area Click the desired frame color in the color wheel Groups are folders in the Surpass tree
151. nes visible in the image Slice View Surpass Allows the user to select the color assignment method used when loading datasets and to define the default colors Images in Imaris format will display in the colors defined in the image file How should colors be assigned when loading from other than Imaris 3 data Take Colors from Default colors Lookup table if available from file otherwise take default colors Emission wavelength if available fram file otherwise take default colors Detault Colors First channel Second channel Third channel Other channels Automatic File Format Detection Sequence Bitolane Imaris 3 series Bitolane Imaris Classic Olympus Fluoview TIFF ICS file feiss LSM5BTU Leica series Leica LCS Bio Rad MRC 1024 600 Tiff series Move Up Move Down Fig 127 Loading Box Take colors from Default colors Use the default color selection to display the loaded data set Lookup table from file Use the original color definition of the loaded data set usually stored in a lookup table Emission wavelength Use the color according to the emitted wavelength from the file corresponds to the appearance under the microscope Page 169 BITPLANE AG Operating Instructions Please note that not all file formats support lookup tables and emission wavelength Default colors Define the color selection for the available number of channels Automatic Fi
152. ng of each new image Surpass Selecting a new time point affects immediately Isosurfaces Volume Ortho Slice and Topography e Time does not affect Group Frame Light Source Clipping Plane Contour Surface External Object e When choosing a new time point in IsoSurface a new surface is automatically calculated unless the Split or the Triangle Reduction functions have been activated Page 126 Imaris 4 1 Operating Instructions Add Time Points Delete Time 16 11 04 BITPLANE AG Time related Operations ImarisTime contains additional time related operations in the Edit menu The new features are applicable to all views offered by the program Additional time points can be added to the data set The time points to be added must contain the same number of channels and be of the same format as the current data set A warning message appears if the formatting does not match Add Time Points Look in Cyl wobbel ims t p lt Swobbel_TOOO ims wobbel TOO ins wobbel TO12 ims wobbel TC wobbel TOOL ins 22 wobbel_TOO ims wobbel TO13 ims wobbel TC wobbel TOO ins ES wobbel_TOOS ims Emm wabbel TO14 ims wobbel TC wobbel TOO3 ims wobbel TODO ims ums wobbel 015 inns wobbel_ TC wobbel TOO4 ims S wobbel 10 ims wobbel TO16 inns wobbel TC wobbel TOOS ins E wobbel_TO1 1 ims UE wobbel TO17 ims wobbel TC p File name wabbel T O00 ims Files of type All Formats
153. oSurface After pressing OK the track algorithm begins calculation Tracks can be created or modified manually from existing Surface objects These Surface objects can come from either the IsoSurface or the Contour or a Track object To create a Track manually press the Create Manually button on the Create tab of a new Track object Next press the Edit Track button This will display a Track Editor window that allows to modify the set of Surface objects contained in a track as well as their interconnections The Track Editor Window displays an abstract view of all the Surface objects contained in a Track as well as their interconnections It also allows to modify both the set of Surface objects as well as the connections When the Track Editor Window is displayed for a new Track that is to be created manually it is initially empty The following figure displays a Track Editor Window for a track that contains one object per time point of the data set The Track Editor Window allows the following operations on a Track Selecting Surface objects A Surface object can be selected in the Irack Editor Window by left clicking onto the corresponding box displaying its name Using Shift left click it is possible to perform multiple selection The Surface objects selected in the Track Editor Window are also selected in the Surpass Viewing Area and the Object Area Selecting Connections A connection between two Surface obj
154. om www bitplane com The CD includes a folder containing the necessary manuals or the manuals can be downloaded Minimum hardware software requirements are gt Windows NT 4 0 or a more recent version Windows 2000 or XP CD ROM Graphics card with 3D accelerator Network facilities for image import from the microscope 512 MB RAM 1 GB recommended VV ON Y Bitplane also recommends gt database for storing images e g Image Access To install the software please proceed as follows Insert your IMARIS CD Rom the computer Follow the instructions on the screen installation is completed automatically The following file formats are supported by IMARIS e maris e maris Classic e ICS file e Zeiss LSM510 Zeiss LSM410 LSM310 e Zeiss Axiovision e Leica TCS NT e Leica Series e Leica LCS e MRC 1024 600 e Till TILLvisiON e Universal Imaging Metamorph STK e Delta Vision e Olympus FluoView e Olympus Cell R e Olympus OIF e Olympus OIB e Open Microscopy Environment XML OME Scanalytics IPLab Page 11 BITPLANE AG Operating Instructions e series e adjustable series e BMP series Page 12 Imaris 4 1 Operating Instructions BITPLANE AG Starting IMARIS IMARIS can be started by one of the following methods Double click on the IMARIS icon we recommend copying the icon to the desktop OR Drag the icon of an i
155. omatic graph compilation Gaussian Filtering The staining the illumination and the photon detection have strong influence on the amount of noise present in the image A gaussian filter is provided in the segmentation interface to reduce that noise The gaussian filter is quite a fast operation taking only a few seconds on a 512x512x40 image Images that have been deconvolved prior to tracing do not require any additional filter Edge Preserving Filter An anisotropic filler is used to preserve filamentous structures An approximate thickness of structures of interest must be provided This preprocessing method offers some advantages over the gaussian filter because noise can be suppressed without deleting the filamentous structure Connective Thresholding Simple thresholding methods classify voxels purely based on a single intensity value This approach often does not lead to good results because the staining the illumination and the detection operate within a precision range The connective thresholding scheme takes care of this and searches for object voxels using an intensity range rather than a single intensity The user must provide two threshold values The lower threshold is the intensity below which a voxel is recognized as part of the background the upper threshold is the minimal intensity for a voxel to be part of the filament object Voxels with an intensity value between the lower and the upper threshold are treated as pa
156. on data and time if desired Click OK when finished or select another heading for further adjust ments Users with an ImarisTime license If the dataset is a time series enter the date time for each time point or click All Equidistant to open the Set Equidistant Time Points dialog box Set Equidistant Time Points E Teina Start Time HH MM SS 10 30 00 Time Interval 00 00 00 Cancel Fig 11 Set Equidistant Time Points Dialog Box Enter the Start Date and Time and the Time Interval Click OK when finished Imaris will calculate the time for each time point in the series 16 11 04 Page 23 BITPLANE AG Operating Instructions The dataset must be saved to retain the changes Channel Colors 3 PUTAS SET ie 2 NUM Image Properties ES ir Geometry Channel 1 Data Set Channels Name Channel 1 Collagen Channel 2 lt lt Thumbnail Description Parameters pe specified Basic colors Base color Mapped color Custom colors JB JO S B B B Hue p Red 255 JB JO S BJ J B mg Define Custom Colors gt gt ColorlS olid Lum 120 Blue o Cancel Add to Custom Colors OK Cancel Fig 12 Image Properties box Channel 1 n the Index of the Image Properties box click the Channel entry Channel 1 Channel 2 etc to select the required channel Click the Base Color tab if necessary Click the Edit b
157. on the 2D histogram when the Selection mode Polygon is checked This method is especially useful for eliminating channel bleed through regions along the border of the lower intensity bins Drawing methods After checking the Polygon mode the overlaid threshold rectangle in the 2D histogram is converted into a polygon with four corner points The user can now start to modify this polygon or to draw a completely new one The initial point of the polygon is visible as black filled Setting points Press and hold the SHIFT key The cursor turns into a cross with a little sign Click on the 2D histogram with the left mouse button to place a point vertex Any additional point connects with lines to the initial point and the last point inserted Page 146 Imaris 4 1 Operating Instructions 16 11 04 BITPLANE AG Inserting points in lines Press and hold the SHIFT key The cursor turns into a cross with a little sign Point on the line between two vertices and click on the line with the left mouse button to add a point Automatic point setting Press and hold the CTRL key The cursor turns into a cross with a little sign and a stack symbol Drag the cursor around New vertices are added automatically at each change of cursor position Moving points Point on one of the vertices left click and drag the vertex around to the desired location The polygon changes its shape Moving lines Point on a line
158. operates directly on the voxel data The input image dering consisting of a collection of voxels arranged in a regular grid is converted directly into the 2D output image Resultant An image that has been computed using Imaris Image Original A 3D data set that cannot be derived from any other data set using Bitplane software data set Usually Original data sets have been acquired using an image sensor and are loaded into Imaris Coordinates Any position in the data set can be described either in the voxel grid e g voxel no 5 in x direction or by a coordinate which corresponds to a distance relative to a de fined origin in the real world e g 2 The latter requires the correct calibration of the data set i e that the voxel size is defined appropriately Generally in Imaris the origin of the data set is in the left bottom corner If the data set was cropped from a larger data set the bottom left corner may have minimum coordinates that are not equal to 0 0 0 Threshold A threshold in Imaris is a gray value which acts as a limit All gray values above or below are assigned a particular functionality Image stack A 3D data set is sometimes called an image stack consisting of 2D images stacked on each other Page 10 Imaris 4 1 Operating Instructions Getting Started Installation Supported file formats 16 11 04 BITPLANE AG The software is delivered on a standard CD or downloaded fr
159. or click Surpass Add New Spots in the main toolbar The new spots object appears in the object area The properties area displays the first window of a creation dialog Imaris 4 1 Operating Instructions BITPLANE AG 2 The properties area displays the first window of a creation dialog Select Source Channel Spot Detection Spot Diameter 1 1 568 unknowr Background Object Subtraction Object Diameter 1 558 Lil ner Back Finish Create 3 Fill in the appropriate values 1 Source channel channel from which spots are to be computed 2 Appr Spot Diameter spots with diameter smaller than this are not detected 3 Background Object Subtraction if checked background will be removed prior to spot detection 4 Object Diameter Objects with diameter greater than this diameter will be considered as background This diameter must be larger than the Spot Diameter Technically background subtraction smoothes the image with a Gaussian filter sigma Object Diameter 2 and subtracts this filtered image from the original Press Next If you want to manually point out all the spots you can also do that by clicking on the Finish button 4 The properties area displays the second window of the creation dialog and the viewing area displays spots as spheres of the specified diameter Threshold Threshold low 50 611 high 86 000 16 11 04 Page 63 BITPLANE AG Operating Instructions
160. ore efficiently The process of creating a snapshot in these formats has been semi automated in IMARIS When an image is ready to be stored make sure that it is fully visible on the screen and not obstructed by any other windows or displays To save the image IMARIS reads from the internal buffer so other objects on the screen would appear superimposed on the image 16 11 04 Page 119 BITPLANE AG Saving a TIFF image Saving a database record Copying to the clip board Page 120 Save Snapshot As Operating Instructions Click the Snapshot button in the main toolbar or select File Snap shot The Save Snapshot box is displayed 4 goofy inns time 00 dim top secret grid 2 ImageBase 2 topo view grid ori J wobbel ims grid l bpIAnImaris radar retir Copy of wobbel ims D 2 tif File name retina red 001 type rit file format Cancel Data Output Save to File Add to Image ccess database Copy to Clipboard Fig 66 Save Snapshot Dialog Box Select the requested directory and accept the automatic naming suggestion or enter a new name for the image Click Save The image is saved as TIFF file Make sure that the Database ImageAccess is started Check Add to ImageAccess database Select the requested directory and enter a name for the image Click Save The image is sa
161. ou set a new gamma value according to which the gamma curve for the voxels is calculated automatically Thus the brightness of the voxels can be intensified or weakened in certain areas Gamma Correction B x Channel 1 name not specified Gamma value 1 0 cancel Fig 140 Gamma CorrectionDialog Box In the menu bar select mage Processing Contrast Change Gamma Correction The Gamma Correction box is displayed with the default Gamma Value set to 1 therefore showing a straight line as a linear voxel representation Select the requested channels by clicking marking the check box Enter the requested value in the Gamma value field or click directly in the gray Gamma Curve area to set the new gamma value point The curve and the field value are set accordingly and the effect of the change can be seen on the channels channels appear differently Press Apply If necessary readjust the value When the results are satisfactory click the OK button The data set is modified Click Cancel to return to the original image Imaris 4 1 Operating Instructions Invert AutoDeblur Huygens Essential 16 11 04 Invert BITPLANE AG The channels can be inverted together or separately Channel 1 name not specified Fig Invert Dialog Box Inthe menu bar select Image Processing Invert The Invert box is displayed Select the channels to
162. ous images spread over the gallery In addition the following display and selection controls are available Slice By clicking on the arrows you move up and down the images in the gallery one by one This is especially useful whenever the display on the screen does not include all the slices Page Page 38 Imaris 4 1 BITPLANE AG Operating Instructions If there are more images than can be shown in the display move up and down the pages by clicking on the arrows or go directly to the first or last page by using the respective buttons Columns opecify the number of columns to be currently displayed on the screen by clicking on the up down arrows or by entering the requested number Skip Select every second image from the data set by selecting 1 image to skip or every third image by selecting 2 images to skip or any other skip inter val analogously by clicking on the up down arrows or entering the appro priate number directly Display Specify inclusion of all available images or of selected images only Selection in the display on the screen The slice number is displayed beneath each slice The status bar indicates which of how many available pages is currently being displayed and how many slices have been selected Changes in the gallery view only affect the current display and do not crop the dataset 16 11 04 Page 39 BITPLANE AG Operating Instructions Easy 3D The Easy 3D view provides a qu
163. pass Scene Properties Diffusion Specular C Emission Transp 0 O Material Palette f O00 0 500 0 008 curtace Objects Distances Color Fig 54 Color Properties Dialog Box Page 76 Imaris 4 1 Operating Instructions BITPLANE AG Click into the color wheel to select a color 4 Dragthe Transparencyslider to adjust the transparency For further controls of the color click the Material Palette box The Material Palette box is displayed Edit Palette Options Palette Name Autumn Maternal Name Autumn3b Fig 55 Material Palette box 6 On the Materials Palette toolbar click Palette and select a color scheme from the list The Palette scheme displays in the box 7 Click the desired ball to select the color The objects in the view area display in the chosen color 8 To further adjust the color click Edit Material on the Materials Palette toolbar The template ball appears in the material editor box with the selected color and brightness settings Material Editor Edit Options Edit Calar oo Accept Fig 56 Material Editor Dialog Box 9 Inthe material editor box click on the slider handle in the Diffuse Diff row to take over the effects on the IsoSurface object 16 11 04 Page 77 BITPLANE AG Operating Instructions The following effects in the Material Editor can be set separately or together e Ambient color Amb e
164. pecially apparent if an animation is created Texture cache limit MB box Background Background color Background color 2 Checkered for blending Tile size Linear color progress for blending Selection Selection color Scale Bar Before displaying any image data Imaris converts the data into a configuration called textures that is optimized by the graphics hardware The value of the Texture Cache Limit determines how many tex tures can be stored in RAM Set the value to slightly less that the amount of RAM installed but not higher than 2 GB Normal background if an image does not completely fill a view Background color for Surpass Background marking the original position if an image is moved When using the blending mode a checkered back ground displays in Full 3D blend and in Surpass Allows definition of the tile size for a checkered background The background displays a color gradient in blend projections and in Surpass Color of selection frame and drawing lines in con tour surfaces Select to display date or time on screen in slider section or gallery view Imaris 4 1 Operating Instructions Loading 16 11 04 Preferences System Display Loading Calculation Time Surpass Advanced poem 47 Loading formats BITPLANE AG Measurement Measurement color Color of measurements points and li
165. pen All layers from this data set are added automatically The x and y values of the two data sets must be identical and the same number of channels is required Resample3D Resampling reduces the voxel density in a data set in order quicken its processing Reducing the data size also deteriorates the resolution Resampling reduces the number of voxels in a grid but keeps the original relationship between the voxels Page 164 Imaris 4 1 Operating Instructions BITPLANE AG Hesample Mew Size Aspect Fixed ratio Fined ratio x y z Cancel Fig 123 Resampling Box The fields display the current x y and z values The requested values can be directly entered in the fields The Aspect Ratio of the data set s dimensions can be kept by checking the respective Fixed ratio options Clicking the OK button resamples the data set to the entered values e Use a Gaussian filter as low pass before sampling down an image Change Data Type Change Data Type Range Adjustment None Source range to target range Data range to target range Fig 124 Change Data Type Box Type 16 11 04 Page 165 BITPLANE AG From To Supported types Range Adjustment none Source Range gt Target Range Data Range gt Target Range Preferences System Preferences System Display Loading Calculation Time SUrpass Advanced P
166. plit button The Split box is displayed Imaris 4 1 Operating Instructions 16 11 04 BITPLANE AG Create no more than 10 objects Filter objects with less than triangles Cancel Fig 41 IsoSurface Split 1 Check Create no more than nn objects to specify the number of independent objects created The objects are created beginning with the object that contains the greatest number of triangles depending on the shape it is possible that an object consisting of more triangles has a smaller volume than another one with less triangles Remaining objects are grouped together in the final object 2 Check Filter objects with less than nn triangles to eliminate very small objects which may not be relevant 3 Click OK Surface objects are created and displayed according to the setup parameters Triangle Reduction Triangle Reduction ae X APES Triangles in Object Reduce to 23076 Triangles E 104 2 PO Apply Reduction Cancel Fig 42 Triangle Reduction box To reduce memory requirements and increase speed the number of triangles can be reduced 1 Select an IsoSurface from the tree list in the object properties area Page 61 BITPLANE AG Surface Contour Surface Track Spots Page 62 Operating Instructions 2 Click on the Reduce button in the object properties area The Triangle Reduction box is displayed 3 Specify the percentage by
167. points HSV follows the colors along the outline of the map Click on the Interpolate button The system automatically interpolates the colors between the two fields or areas When the result on the color map is satisfactory click OK The effect is visible on the image Color Table File A selected color palette can be imported or exported to another picture by clicking Import or Export in the Color Table File panel The Thumbnail function allows the user to define a small 2D or 3D image to be used as an icon for the data set in order to illustrate its subject Image Properties Thumbnail Type C None 7 Middle slice 1 Blend Channel 1 Channel 2 io Parameters Fig 118 Thumbnail Box Type Page 161 BITPLANE AG Parameters Page 162 None Middle slice MIP Blend Operating Instructions No image is displayed The middle slice is displayed The image displayed is the same as Easy 3D with MIP settings The image displayed is the same as Easy 3D with Blend and Light settings The Parameters entry in Image Properties menu lets you build and main tain the environmental information for the data set Image Properties Parameters Generic 2 3 Add Group Add Parameter Delete Group l Channels gt Channel 1 Channel 2 Thumbnail El Channel 0 5 Dolor 2225 ColorMode v DolorRange c Bain 0 Mas c Offset
168. r Store Recall Orientation Manipulator Plane Active Settings oxPlne Camera Orthogonal View Objects yz Plane e T Delete E M Surpass Scene ES Light Source 1 3 1j Zoom e Pointer Select C Navigate View Full Screen Camera Orthogonal Perspective ii Draw Style Full Featun smooth lines Set Center Stereo Zoom ZA Fig 47 Clipping Plane Topography Topography is a separate module within the IMARIS program and requires a separate license Topography is relief representation created from a 2D input file which contains intensity coded height data This input file can be an ordinary TIFF file or a selected slice of a 3D data set Topography allows the user to overlay an independent texture on the relief image to colorize the representation 1 Click Surpass Add New Topography on the main toolbar An absolute topography of the file or of the slice 1 of channel 1 default of the loaded data set is created and displayed in the viewing area The object properties area shows the suggested parameters for it 2 Select the requested Channel and Slice in the Data Set amp File Options panel of the Geometry tab The new Topography is displayed To open another file for Topography check the File field The
169. rface mesh number of triangles surface area enclosed volume The volume is made up of rectangular voxels The voxel statistics describe the voxels enclosed within the surface number of voxels sum mean standard deviation min max and median of voxel intensities The voxel statistics are based on the voxels that are contained inside the surface represented If the center of mass of a voxel is inside the triangle surface then the value of that entire voxel is counted as inside the surface Iso 120 Properties Source Channel 1 Rebuild Threshold 20 000 __Febuild_ Curent Timeport Time Indes 1 Surface Triangles 14288 Reduce Number of Surfaces 1 Split Time Data Lreate Track Split All Settings Statistics Color Fig 67 Isosurface settings box IsoSurfaces that consist of two or more unconnected surfaces can be split into objects This procedure allows you to view and compare the voxels statistics for each unconnected object Imaris 4 1 Operating Instructions 16 11 04 BITPLANE AG Create an IsoSurface object and highlight the object in the tree list See the Isosurfaces section for details Click the Statistics tab in the object properties area to see the measurements Click the Settings tab in the object properties area and click on the Split button The Split dialog box appears Specify the required number of unconnected surfaces to be created Click OK Newly
170. rocessor Number of processors 1 Fracessor architecture Processor speed Graphics OpenGL renderer OpenGL version OpenGL extensions Operating System Version build Service pack Memory Status Physical memory installed Physical memory available Fig 125 System Box Processor Page 166 Operating Instructions Displays the current data set type Pull down list to select the requested data set type from unsigned 8 bit for the range 0 255 unsigned 16 bit 0 65535 32 bit float Check field to determine how the data values are translated during the change Data values are imported in the new type Maximum data values are scaled to the new range e g 0 255 to 0 65535 Actual data range values are interpolated to the new range e g 0 150 to 0 65535 Displays the basic system parameters of your Windows computer ie Intel Pentium 1700 GeFarce3 PCI SSE 2 1 4 0 3L depth texture GL imaging ARE multisample GL multitexture iL paint parameters Ea OS Widows 2000 2185 Service Pack 3 1023 MB b34 MB Cancel Imaris 4 1 Operating Instructions BITPLANE AG Display 16 11 04 Information about the number of Processors Processor Speed and Type Graphics Information about the OpenGL Version and the Graphics board Operation System Installed Version of the Operating System Memory
171. rs further controls for setting the opacity of the image Click Edit Display Adjustment and drag the Blend opacity slider to ad just the opacity in real time for one or all channels See Fig 20 Click the Edit Material button The Edit Material box displays allowing the user to specify the opacity and the shadow of the material for each channel Adjustments made in the Edit Material box are used in the final calculation Page 46 Imaris 4 1 Operating Instructions Creating Animations 16 11 04 BITPLANE AG E dit Material Channel 1 Collagen IV Cancel Fig 31 Edit Material box Select the requested channel Use the Opacity slider to specify the opacity for the materials 0 00 fully transparent 1 00 fully opaque Use the Shadow slider to specify the shadow intensity of the struc tures 0 00 no shadow 1 00 maximum shadow intensity It is recommended to start with equal values for the opacity and the shadow and then adjust the shadow to your liking Click the OK button when finished Click the Calculate button The image is calculated and displayed Time If the slider of the time bar is moved after having calculated an image at a certain point in time a new image is automatically calculated When re turning to the previous image it is immediately displayed without being recalculated Animations show a display rotating around the horizontal or vertical axis allowing a choi
172. rt of the filament object if and only if they are connected to a voxel within intensity above the upper threshold double thresholding with feature AND The result of the segmentation process described above is a clean image where the non zero voxels are describing the objects filaments and some non filamentous structures Because such a description of the data is not suitable for morphometric analysis the structures are converted Graph compilation is a process involving several image processing operations such as skeletonization erosion dilation and the creation of a vector definition for the filaments The result of the process is a database kept in memory describing the filaments as a set of segments that may contain branches Imaris 4 1 Operating Instructions Manual Graph Construction Semi Automatic Graph Construc tion 16 11 04 BITPLANE AG The manual segmentation feature is used to edit automatically traced filaments or to build the filaments manually from the image template Essentially the user draws the filaments onto the image which serves as a visual reference template Drawing is done in the xy plane For z positioning in the image the user can choose to change planes manually arrow keys or mouse or to have the system change the planes automatically in such a way that the center of the filament is kept in focus The semi automatic segmentation facilitates manual segmentation by providing t
173. rtical Half Screen side by side stereo Blue Yellow Stereo Red Cyan Stereo requires color glasses requires color glasses Green Magenta Stereo requires color glasses The modes listed above are interesting for microscopic data If required check the Reverse left and right views box for exchanging the views If necessary and or the Cameras offsetby means of the sliders When the settings are satisfactory close the Stereo Preferences dialog box Click Help Keys Help for a list of all keyboard shortcuts Imaris 4 1 Operating Instructions Adding Objects teretina ims Imaris B Open Save as Snapshot Frame Properties Box Tickmarks Grid Coordinate Axes Grid and Tickmark Settings Spacing fi 1 280 um Fixto Data Set Settings Objects Surpass Scene mS Light Source 1 MA Frame Iso 120 ci Iso 124 c2 Ready Frame 16 11 04 File Edit View ImageProcessing Surpass Help 55 Slice a e els eja Delete BITPLANE AG From a data set different objects can be created and added to the Surpass scene either by selecting Surpass Add New from the toolbar menu or by clicking on the icon in the Objects area A Surpass Scene folder a Frame box surrounding the data set and a Light Source are automatically created with the first object added ml x 5 e Y me e Surpass Coloc Bitpla
174. s the intensity of a colocalized voxel de fined by the specified value No distinction is made whether high intensity or low intensity voxels colocalize as long as they are within the selection Page 137 BITPLANE AG Basic Operation Loading a data set Page 138 Operating Instructions Coloc color Coloc color allows the user to specify his personal color settings for the colocalization display in the Preview window Edit Clicking on the Edit button opens the Color window The user can either select one of the predefined colors to apply on the colocalization areas or define add and apply his own color definitions Build Coloc Channel Clicking this button calculates the colocalization channel according to the selected settings and adds it to the data set The channel can then be saved and is part of the data set Channel Statistics After building the coloc channel its statistics become available in the Image Properties The Channel Statistics button displays directly the Image Properties window with the statistics for the calculated channel The basic use of ImarisColoc in the daily workflow comprises the following steps 6 Loading a data set 7 Selecting the channels 8 Setting the thresholds 9 Checking the statistics 10 Building the Coloc channel 11 Saving the Coloc channel Any 2D 3D or 4D multi channel and or time data set of the all types supported by IMARIS can be loaded and used for colocali
175. slim 60 1 nc stack60 1 cropped nc 2a Apply Automatic File Filter File List Operating Instructions Apply This button will use the regular expression to the left and select all files in the current directory that match the criterion Example tif selects all files with ending tif Example2 myfile tif selects all files that start with followed by any kind of letters or digits followed by Example 3 myfile tif selects all files starting with myfile followed by exactly two letters or digits followed by the ending tif Note Depending on the filter you may not see all files in the directory Note the selection may take a while because each file is opened and only files with identical xy dimension are selected Dimensions Enter the number of slices channels and time points e Dimension Sequence Defines how the individual images which are sorted in alphabetical manner are to be assigned to slices z channels c and time points t x meu CAlmages Nimsbl nc stackYbl cropped nc zaYnc stack bl xx D 201 0004 Apply Dimensions 14 Slices 2 Channels C Time Points T 14 of 14 files used File Arrangement 1 nc stack bl xx D z 1 0 nc stack bl xx DU 201_001 4 nc stack bl xx D z 1 UD2 uf nc stack 61 xx O z 1 D3 f nc stack bl xx D z 1 nc stack xx D z 1 O05 tf nc stack bl xx O z 1 nc stack xx O z 1
176. ssing Surpass Help TA i A gt Ne NS SS Ew EH Open Save Snapshot Slice Section Gallery Easy 3D Camera oom Slice Ld 1k Measure Mode Default Meas Single C Multi Style Pair 33 Time Settings Oat 83 36 74 99 32 00 P31 at 131 50 88 78 32 00 20 yum oom 4 26 NUM 27 Fig 66 Slice view with different drawing styles Note An incorrect Z voxel size can prevent measurement points from displaying Page 90 Imaris 4 1 Operating Instructions 3D Measurement 16 11 04 BITPLANE AG ImarisMeasurementPro also allows the user to measure distances in 3D 1 Load a set and select the mode Meas and the draw Style as directed for 2D slice measurement Select Multi view in the right frame bar Left click with the mouse to set the first measurement point on the selected slice 4 Drag the slider in the left control bar up or down to reach the required slice 5 Left click with the mouse to set another measurement point The measuring points are connected by a dashed line to show that they have been set on different slices The distance between the points is displayed on the status bar Moving Measurement Points The position of a measurement point already set can be changed as follows e move the mouse pointer over the measurement point e press the LEFT mouse button thus highlightin
177. st of the images that belong to the data set Look in E wobbel ims amp tj wobbel TOO ims wobbel TOO6 ins wobbel TO12 ims wobbel TC wobbel TOOL ins ES wobbel_TOO ims wobbel TO13 ims wobbel TC wobbel TOO ims S wobbel TOD amp ims Rs wobbel TO014 ims wobbel TC wobbel TOO3 ims 22 wobbel_TO09 ims is wobbel TO15 ims wobbel TC wobbel TOO ims E wobbel TO 10 ims ES wobbel TO16 ins ums wobbel TC wobbel TOOS ims 22 wobbel_TO1 1 ims wobbel TOL7 ims wobbel TC Cancel gt File name wobbel T OOD inm Files af type auto for tection Bitplane maris 2 Header Configuration Readanly one Time Point settings Resampling Open 2 Fig 7 Open box Inthe main toolbar select File Open The Open box is displayed Select file type from the Files of Type pull down menu Select a file name from the list and click Open or double click on the requested file entry The file is loaded 16 11 04 Page 17 BITPLANE AG Read only one time point ResamplingOpen Original Size 8 000000 MB Subsampling m Operating Instructions Loads a single time point of a time series The Resampling Open dialog box can be used as preview before loading a data set and allows the user to change the resolution of the data subsampling and to select only a part of the data set cropping by specifying parameters in the Resampling Open di
178. stestsserie imald ul tit hif Apply File List Dimensions 4 Slices File Arrangement imgDU ti imgDU3 ti ing Cle ti img005 tif 2 Channels C 2 Time Points T 16 of 16 files used imt tf imgQ tif 002 img tf 004 img005 tif ima tf img tif img 1 tif imat12 tif 1 int tif Dimension Sequence ingQ06 tif ing 4 tif ingly tit i s imt tit img OD8 ti imqL09 tif v2 ima Ll tif ima 1 tif ing 2 tit ing 3 tit ing 4 tit ing 5 tit Fig 6 Adjustable Tiff Series Reader To activate the Adjustable Tiff Series Reader dialog box Inthe main toolbar select File Open or click on the Open button The Open box is displayed Select Files of Type Tiff adjustable file series and not Tiff series from the pull down list Open the folder containing the series Click Settings which is grayed out if the file type selector is on automatic or if the current directory does not contain a series The Adjustable Tiff SeriesReader is displayed Use Apply Automatic File Filter Apply Dimensions Dimension Sequence described as follows to define the series Apply Automatic File Filter This is activated automatically when the dialog is opened It has the same logics as the classic TIFF series reader of Imaris and will pre se lect the first series detected in the directory Be a
179. tensity values for the channel and the relative 3D position in the data set in brackets Checking View Extended allows you to select a portion of the data set in the x y and z direction to be displayed On the left side of the screen select the mode In the main screen separate the two cross hairs The planes between the cross hairs are rendered in the views showing the other axis This allows you to see only certain interior structures without both the top and the bottom Page 36 Imaris 4 1 Operating Instructions BITPLANE AG ES lt JOR of p c iy 25 3 2 gt AS Ni sey 4 Slice Section Gallery Easy 3D Full 3D Surpass Coloc Bitplane AG Open Save as Snapshot Zoom View C Normal Extended Crosshair Iv Normal Extended Size x 3 000 y 3 000 z 3 000 4 Mode MIP C Mean C Blend Zoom 2 96 Fig 23 Section view Extended The image shows the white crosshair line together with yellow focus range lines The focus range can be set in any of the three directions as follows Move the cursor on a yellow line The cursor changes its form to a straight arrow with a bar Drag the line in the respective direction The range is extended or reduced Its depth is reflected in the x y and z fields in the Size display The Size values are indicated in the data set s dimensions e g in um It can also be a
180. ter 0 Measurement Pro Win ready for installation on this computer Time Win ready for installation on this computer IImarisCalac ready For installation an this computer Enter Order ID Install Upgrade seleced Product Licenses Cancel 16 11 04 Page 183 BITPLANE AG Operating Instructions 3 From the list of products carefully select those that you want to install on the computer you are currently using Note that it is not possible to repeat the license installation on another computer unless you have purchased several licenses 4 Press Install Upgrade selected Product Licenses to install licenses for the selected products 5 Restart Imaris for the new licenses to take effect Imaris Offline The following procedure describes how to install node locked licenses for Imaris if the computer on which you want Imaris to run is not connected to the internet If your computer is connected to the internet follow the instructions in the section Imaris Online 1 Start the Imaris software on the computer on which you want to install licenses You will see the following dialog j Imaris didn t Find any license Would vau like to specify your license configuration Yes Mo Click Yes to get to the license installation dialog You can either specify a location with license information or download it via internet Install and Upgrade Product Licenses For this Computer IF this co
181. that contain other Surpass objects They are visible in the objects area and allow the user to create a treelike hierarchy of Surpass objects When the first Surpass object is created it is automatically placed in a Group called Surpass Scene Additional Groups can be created in order to sort objects of the scene 1 Click Surpass Add New Group on the main toolbar or click the Group button in the objects toolbar Group is added to the Surpass Scene 2 Click on another object in the objects area and drag it onto the group object Once released the object is added to the group Like all other objects Groups have a visibility check box This box affects the visibility of all objects in the Group 1 To display the Light Source in the viewing area highlight Light Source from the tree list The Light Source becomes visible if it is checked 2 o move the Light Source in the viewing area choose Select mode in the right frame bar and left click and drag the cone 3 Click on the cylinder above the cone and move it closer or further away from the object to change the light intensity 4 To add a new Light Source select Surpass Add New Light Source ri from the main toolbar or click amp in the Objects toolbar Multiple light sources can be added to an object Imaris 4 1 Operating Instructions BITPLANE AG 5 To change the color of the light highlight the Light Source in the objects tre
182. the maximum distance An important characteristic of both spot tracking algorithms is that by construction they produce tracks without splits and merges Fig 61 Spot tracks In threshold tracking Surface objects are computed from an intensity threshold and linked into track groups To perform threshold tracking first create an IsoSurface object with settings that produce good segmentations of the biological objects Then press the Create Track button in the Settings dialog of the IsoSurface After calculation the tree in the Objects Area will contain a new group called Track Group containing one or several Track objects Each Track in return contains several Surface objects that have been linked into the same track by the tracking algorithm The different tracks by default are assigned different colors in which they visualize their Surface objects This coloring serves to allow for an immediate visual inspection of the result Page 81 BITPLANE AG Manual Tracking Track Editor Page 82 Operating Instructions Besides the different Track objects the Track Group generally contains a group of Surface objects that were not linked to other objects by the tracking algorithm A second way to perform threshold tracking is to create a Track object and then press the Create from Threshold button on the Create tab This will display the IsoSurface Setup dialog where a threshold can be selected see section ls
183. the objects area or click Surpass Add New Volume in the main toolbar The volume is displayed in the viewing area A data set can have only one volume In the Properties area specify the Mode Blend blends all values along the viewing direction and includes their transparency or MIP Maximum Intensity Projection maximum intensity of all layers along the viewing direction Define the range of data to be displayed with the Threshold parameters Select cut low usual procedure or cut high in order to display transparently the voxels with gray values below or above the specified threshold limit Select the threshold by means of the slider or a direct entry of the value in the Threshold field Page 65 BITPLANE AG Operating Instructions After each change the volume is recalculated directly and displayed with the new settings ff retina ims Imaris 3 3 Eile Edit View Image Processing Surpass Help s u e NN Open Save as Snapshot Slice Section Gallery Easy 3D Ful 3D e Coloc Bitplane AG gt mw Surpass Volume Properties Camera Mode Blend C MIP Threshold for first visible channel 255 000 cutlow C cuthigh 17 000 Zoom Pointer Select Navigate View Key Frame Animation Full Screen ii Camera Type C Orthogonal Perspective Objects Delete
184. tic values e g tree order The point is selected from the trunk position with the biggest radius Press Next to execute the Automatic Graph Compilation Press Back to refine the Connective Threshold Press Finish to terminate the segmentation and build the filament graph manually the current step will not be executed Fig 81 Ratio of branch length B to trunk radius R Step 4 Finish To validate the automatically generated graph and the prior segmentation the user can inspect the result together with numerous Surpass modules ortho slicer volume iso surface etc E g he can optionally superimpose the segments with the original slices of the image stack using Imaris OrthoSlicers OrthoSlicers are planes which are perpendicular to any of the three spatial axes and extend throughout the entire image volume 16 11 04 Page 111 BITPLANE AG 6 C2C20Small ims Imaris 4 0 File Edit View Image Processing Surpass Help Operating Instructions 101 gt Open Snapshot Filament 1 Properties Build Filaments for all Time Paints Delete Working Channel Back Nest Create Objects Ev Surpass Scene be Light Source 1 v Frame v Filament 1 N pe cec N NEN he Sy 6 2 4 te OCA Slice Section Gallery Easy3D Full3D Surpass Coloc Bitplane AG Navigate View Key Fr
185. tion In Blend mode with light enabled click the Settings button The Edit Set tings dialog box displays Edit Settings E x Light Directional Intensity I 000 Ambient Intensity 0 000 Channel Opacity Combination C Opaque Translucent C Custom 10 000 Cancel Fig 26 Edit Settings box Directional Intensity controls the brightness of structures that are illumi nated from the light source Ambient Intensity controls the brightness of structures that are in the shadow The default 0 means that structures completely in the shadow are black The higher the value the brighter the picture and the lower the shadowing contrasts Channel Opacity Combination allows the user to control the opacity of the image Select Opaque Transparent or Custom opacity It affects only the behaviour of interleaving structures of two or more channels Time If the slider of the time bar is moved after calculating an image at a certain point in time a new image is automatically calculated When returning to the previous image it is immediately displayed without being recalcu lated Page 41 BITPLANE AG Operating Instructions Full 3D The Full 3Dview creates an entirely user defined 3D view of the data set including orientation material and light settings Animations of the dataset can also be created A preview section allows the user to pre arrange the settings and check their effects on the
186. tistics BITPLANE AG Create a Spots object and highlight the object in the tree list See the Spots section for details Click the Statistics tab in the object area to see the measurements Create a Spots track and highlight the Track group in the tree list See the Track section for details Click the Statistics tab in the object area to see the measurements Fig 70 Spots Statistics box Exporting Statistics The values can be exported in tabulated form 1 Highlight the Group containing the required object s or select the entire Surpass Scene Click Export Statistics in the object properties area The Export Statistics box is displayed Select the directory and enter the name for the file to be saved and click OK The data is written into a text file csv file comma separated values The data contains the Spots Statistics and or Spots Track Statistics Exporting Statistics to MS Excel The values can be directly imported into MS Excel 1 Highlight the Group containing the required object s or select the entire Surpass Scene Click Excel in the object properties area You are asked whether or not you want to use pivot tables with MS Excel some configurations do not support this feature The data is exported to MS Excel and sorted in different sheets The data contains the Spots Statistics and Spots Track Statistics The Contour Surface allows the user to extract a 3D object by manually drawing
187. urface objects will appear in the Track Editor Window and they all become children of the corresponding Track In addition connections are automatically created between all the selected Surface objects in the order in which they were selected e g first to second second to third third to fourth Page 83 BITPLANE AG Page 84 Operating Instructions Add Connect to create connections between Surface objects If sev eral Surface objects of the track that is being edited are selected they then appear highlighted in the Track Editor Window and the Add Connect button is pressed the effect is that connections between the selected Surface objects are created in the order in which they were selected e g first to second second to third third to fourth Merge Connect of a single Surface object If a Surface object to be added to a Track is the child of another Track it may have connections to other Surface objects within that other Track If this Surface object is selected and you press the Merge Connect button the Surface object will be added to the Track and all other Surface objects to which it was previously connected in the other Track will also be added Furthermore the connections between these Surface objects will be restored in the new track exactly as they existed in the track they came from Merge WConnect of multiple Surface objects Several Surface objects can be merged into a Track at once To do so use mult
188. ussian Filter 2 Edge Preserving Filter 3 Median Filter Imaris 4 1 Operating Instructions Gaussian Filter Gaussian Filter Edge Preserving Filter Median Filter 16 11 04 BITPLANE AG The following paragraphs describe the user interface to these filters For details on their function please refer to the Reference section in this manual The Gaussian filter for a data set can have only one value This value can be applied separately to any one channel or to all channels Structures smaller than the filter width will be removed All Channal g Filter width 4 006 um Fig 15 Gaussian Filter box Inthe menu bar select Image Processing Image Smoothing Gaus sian Filler The Gaussian Filter box is displayed Select the channels to be filtered by marking the check box on the left of the channel Enter the requested filter width in the parameter field and click Apply The effect of the change can be seen on the screen f necessary readjust the value When the results are satisfactory click the OK button The data set is modified To restore the initial image click Cancel The Edge Preserving Filter is used in the same way as the Gaussian Fil ter Median Filter E All Channal s Filter size 3x3xl C 3x3x3 Channel 1 name not specified 7 omen Fig 16 Median Filter Dialog Box Page 27 BITPLANE AG Operating Instructions The
189. utton The color selection box is displayed Select a color To define a custom color click the Define Custom Color button Adjust the parameters using the cross and or the slider control or type in the values directly Click OK when finished Repeat the process for other channels For information about color maps please refer to the Reference section Preparations for visualization Preparation for visualization includes the following steps gt Cropping the data set gt Filtering the image Adjusting the contrast gt nverting or flipping the image if necessary gt Saving the data set in the Imaris file format Page 24 Imaris 4 1 Operating Instructions BITPLANE AG The Crop dialog Cropping the data set allows you to crop the images down to the region of interest Cropping reduces the size of the data set and makes it easier and faster to handle the viewing and storing of the images Cancel 2 Fig 13 Crop selection box Inthe menu bar select Edit Crop The Crop selection box is displayed showing a sectional view of the actual image Current time point A rectangle representing the region of interest ROI is overlaid on all three views Modify the size and the position of the region of interest by entering the direct values in the corresponding x y and z fields or as follows Move the ROI Click inside the rectangle with the left mouse button hold it down an
190. ve Measurement Points are connected by a line AB CD etc e Polygon All Points in the Group are connected consecutively P1 P2 P3 P4 e Origin All points in Group are connected with the first Point in a Group P1 P2 P1 P3 P1 P4 Page 100 Imaris 4 1 Operating Instructions Attached Measure ment Points 16 11 04 BITPLANE AG Lines between points display in the viewing area Lines can be displayed between e Visible points e All Points No Points The measured distance between two points is displayed next to the line connecting the two points Check the Hide Distance Labels box if you do not want to see the distances in the viewing area Click Hide Labels if you do not want to see the point numbers in the view ing area Distances If there are more than two measurement points the object properties area displays the sum of all distances Click Export to save the coordinates of each Measurement Point as well as a list of all distances in the Group Data is written into a text file that can be imported by MS Excel See the statistics section above Converting csv format to MS Excel Open the csv file in MS Excel Highlight the entire first column Click Data Test to Columns on the toolbar Select Delimited and click Next Select Semi Colon and click Finish Save in MS Excel format to retain the formatting changes QD Measurement points can be attached to two dimensional objects such as an OrthoS
191. ved on the disk and an entry is added to the database Check Copy to Clipboard Open another application and select the paste function The image is pasted into the new application Imaris 4 1 Operating Instructions BITPLANE AG 30 set Revised 3D data sets can be saved as a new file Save in Images amp amp wobbel ims pollen inns retina ims File name retina red Save as type Bitplane Imaris 3 uncompressed imi Cancel Data Output Fig 87 Save As box e Inthe menu bar select File Save As or click the Save button in the toolbar The Save As box is displayed Select the directory and enter the name for the file to be saved Select the file type to save as and click Save The data set is saved The following types can be saved e maris uncompressed ims Hecommended e maris Classic ims e series tif tiff Saves gray scale images for each channel Tiff series Channels merged to one RGBA color image ICS file ics ids e Olympus Cell R Open Microscopy Environment Xml OME e BMP series bmp Saves series as 2 D bitmap files e Movie file slice animation avi In Z direction from bottom up 16 11 04 Page 121 BITPLANE AG Operating Instructions Saving as an avi file creates an animation of the slices of the image file This is the view seen when clicking the arrow gt at the
192. ves the quality of the image by in terpolating harsh transitions between poorly sampled voxels where intensities vary greatly between voxels Interpolation puts in more voxels with mid intensities to smooth the transition The rendering process will be much slower with this option enabled The option is es pecially useful if the zooming factor is larger than 2 Ray Cutoff The Ray Cutoff value determines when the calcula tion along one ray pixel should be terminated A value of 95 indicates that the process is stopped when at least 95 of the correct value has been reached Ray Increment The Ray Increment determines the sampling rate along the ray A smaller value indicates higher sam pling and improved quality A sampling rate of 0 5 is optimal Page 45 BITPLANE AG Operating Instructions Color Calculation Calculation can be performed in 24 bit or 96 bit mode 96 bit mode is faster and results in a slightly better image However it uses 4x more memory Click the OK button when finished Edit Settings Light Enable Direction 0 38680 0 2168 0 89630 Intensity 1 000000 Shadow Rendering quality Low ie Medium High C User defined Advanced Quality Settings nterpoelation Ray cutoff 0 950000 increment 000000 Color Calculation E 2A bi Se bit Fig 30 Full 3D Edit Settings box lf Blend mode was selected Imaris offe
193. ware that you may not see all files in the directory Apply Press this button to use the regular expression to the left and select all files in the current directory that match the criterion i e tif selects all files with the ending tif selects all files that start with and are followed by any letter or digit and by the extension tif Page 20 Imaris 4 1 Operating Instructions BITPLANE AG myfile tif selects all files that start with myfile and are followed by two letters or digits and by the extension tif e Note Depending on the filter not all files in the directory may be visible Selecting the required file may take some time because every file is opened but only files with identical xy dimension are chosen Dimensions Define the dimensions of the image starting with Slices Z Channels C and Time Points T em Note The total number of files in the series as defined by your selection criteria is displayed below the Time Points input box Selecting the required file may take some time because every file is opened but only files with identical xy dimension are chosen Dimension Sequence Defines how the individual images which are sorted alphabetically are to be assigned to Slices Z Channels C and Time Points T Click OK to return to the File Open dialog box Click Resampling Open to open the Resampling dialog box or click Open to open the im
194. wo different functions The so called AutoPath function automatically computes all the paths from a user defined starting point to the end of the structure The second function which is called AutoDepth adds the automatic computation of the depth while doing manual segmentation e g if the manual segmentation is done while looking at xy plane z coordinate will be automatically computed Page 107 BITPLANE AG Operating Instructions User Interface There is a menu item and a button to create a new Filament object The user can add as many Filament objects to the Surpass scene as he likes 8 C2CZ0Small ims Imaris 4 0 File Edit view Image Processing Surpass Help y man jM ES he Open Saveas Snapshot Slice Section Gallery Easy 3D Full 3D Surpass Filament 1 Properties Select Source Channel Channel 1 name not specified gt Feature Preprocessing Fit v Enable Feature Preprocessing m Pointer Appr Filament Diameter 0 441 um C Navigate Preserve Edges View Key Frame Animation Full Screen Camera Type Orthogonal Perspective El VI Surpass Scene 9 Light Source 1 Draw Style ME Frame Fui Featurt v Filament 1 box move smooth lines Set Center Stereo INUM 2 Zoom 1 8 Fig
195. zation detection Imaris 4 1 Operating Instructions Selecting the channels Setting the thresholds 16 11 04 BITPLANE AG Look in Sy wobbel ims amp P wobbel TOO ims wobbel TOO inns wobbel TO12 ins wobbel TC wobbel TOOL ins ES wobbel_TOO ims wobbel TO13 inns wobbel TC wobbel TOO ins ES wobbel_TOOS ims wabbel TO14 ims wobbel TC wobbel TOO3 ims ES wobbel TODO ims wobbel TO15 ims ms wobbel TC wobbel TOO4 ims 22 wobbel TO 10 ims wobbel TO16 ims URS wobbel TC wobbel TOOS ins 22 wobbel_TO1 1 ims wobbel TO17 ims wobbel TC Cancel pi File name wobbel T OOO ins Files of type Bitplanie maris 3 Header Configuration Read only one Time Point settings Resampling Open 2 Fig 103 File open dialog 1 In the main toolbar select File Open The Open box is displayed 2 Select file type from the Files of Type pull down menu Select a file name from the list and click Open or double click on the requested file entry The file is loaded The two channels for colocalization detection must be selected first 6 Select channel A and channel B from the pull down lists The 1D and 2D intensity histogram displays are updated 7 Modify the Histogram options if necessary in order to display an expressive voxel intensity representation Specify Selection mode In order to be able to set the thresholds the user must check the Selection mode Thr
196. zontal or vertical drawing offsets the camera is set to a perpendicular position with an orthogonal projection In this case the camera can only zoom or pan but not rotate Fig 84 Drawing manually filaments The grey tube was previously drawn or generated by the creation wizard The thin white line is being currently drawn the yellow dot is the drawing pencil Page 114 Drawing in Manual mode Select Manual in the Draw box press shift and hold down the left mouse button to begin drawing The circle on the drawing plane indicates the filament diameter The diameter can also be changed using the mouse wheel To make a connection to an existing filament start painting on this filament If the filamentous structure runs perpendicular to the drawing plane use the right mouse button to alter the plane orientation Drawing in AutoPath mode Select AutoPath in the Draw box and shift right click on the desired starting point of your structure e g in a neuron picture it would typically be Imaris 4 1 Operating Instructions Mouse Selects Edge Segment C Branch 16 11 04 Global Selection rel Branch abs Branch Loops Invert Path Clear Process Selection for this mode Delete _5 e Filament Unconnected parts of the filament can be E split into separate filament objects The new objects Export will appear as new items in the surpass tree This al Settings Dr

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