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Imaris software user manual

Date of first issue: 21 January 2015

Date of review: 04 March 2016

For assistance or to report an issue Email: Igmm-imaginghelpdesk@igmm.ed.ac.uk Website: www.igmm-imaging.com

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IMARIS (8.0.1)

User Manual

Contents

Imaris Introduction ................................................................................... 3

Opening Images (most formats supported) ....................................................... 3

Viewing and adjusting image display settings .................................................... 4

Adding a Scale bar to an image ..................................................................... 5

Crop a 3D Data set .................................................................................... 6

Creating a View Snapshot ........................................................................... 7

Making movies from 3D data sets.................................................................. 8

Creating image analysis protocols ................................................................. 9

Introduction to the Surpass Scene Toolbar ......................................................................... 9

Manually measure 3D distances ......................................................................................... 9

Count discrete point-like objects (Spots) .......................................................................... 11

Measure objects in 3D automatically (Surfaces) ............................................................... 15

Manually draw & measure objects in 3D (Surfaces) .......................................................... 19

Analyse and present statistical data (Vantage) ................................................ 21

Batch Processing (run an analysis on multiple images) ....................................... 22

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Imaris Introduction

Imaris runs on a server licence sited in the ECRC but the licence can be used on a number of computers within the IGMM imaging facility workstation room (1.26) Holycow (PC), Holybones (PC) and Laxton (Mac). Imaris Structure IMARIS is composed of the core application and additional licenses each of which is designed for a specific purpose. The core features of imaris do not require any of the licenses to be opened, thus allowing multiple users to access the software simultaneously. Currently we have one copy of each license, see below (most frequently used highlighted in bold): FilamentTracer Batch Cell Coloc Track Vantage InPress Vantage XT

Opening Images (most formats supported)

1. At startup do not select any licenses to open at this stage. Imaris will open in the Arena View. This is the interface used to organise, visualise and analyse your images.

2. To get started click on the Assay icon, this is the top hierarchical level within the Arena. Define a new assay whenever you have a new analysis on a different set of images to carry out. Name the assay when prompted.

3. All content in the Arena is organised into a tree structure accessed within the Browser column on

the left hand side of the window.

4. To add images to the Assay click on the Image icon. You can highlight multiple images or a whole folder to add them in batch. You can further organise images into different groups which are essentially folders.

5. Each imported image will display as a thumbnail view within Arena as shown in the image below. In

this case the Assay is called “test” and contains two groups (folders), “cropped nuclei” and “test group. When you select a group you will see a preview of the images contained within. Left click twice on an image to open it.

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Viewing and adjusting image display settings

1. Double click on an image thumbnail to open it. The image will open in Imaris’s image viewer known as Surpass and will default to the 3D rendered view if the image contains XYZ data.

2. You can change to various 2D views by selecting the slice icon. Select a 2D view from the drop down menu: Slice-A single Z plane is displayed, use the vertical scroll bar to change plane. Section-Displays the XY plus orthogonal (XZ, YZ) views. Gallery-A thumbnail of each Z plane is displayed. Easy 3D-Maximum intensity Projection

3. The cursor by default is in Navigate mode which means left clicking and

moving the mouse will rotate the volume. Use the mouse scroll wheel to zoom in or out. You can change the pointer to select mode in the Camera toolbar on the right hand side of the window. When select is active you can select objects in the image or add annotations/drawing/measurement lines.

4. The InMotion feature continuously rocks the image from side to side in the Y

dimension.

5. Camera Type allows you to change the perspective of the rendered image.

6. The Display Adjustment window allows you to switch wavelength channels on/off and alter the images brightness and gamma settings. If it isn’t currently open select More…>Edit>Show Display Adjustment or CTRL-D on the keyboard.

7. Toggle the displayed channels using the tick boxes.

8. Change the colour of a specific channel by clicking on the channel name.

9. To delete a channel select More>Edit>Delete Channels

10. You can change the brightness/contrast settings of each channel individually by moving the black triangles on each side of the coloured bar or tick “select all channels” to apply the same settings to all channels.

11. Gamma changes the relationship between low and high intensity pixels. If

gamma is set to one, there is a linear relationship between low and high intensities. Any value other than one means that either low intensity pixels are made brighter while high intensity pixels are left unchanged or vice versa. If you alter the gamma from a value of one and you publish the images you must state the change in gamma.

12. Reset will reset the display back to default values.

13. Auto Blend will automatically determine the Min and Max values that should be

used for the selected channel or all channels if “select all channels” is ticked.

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Adding a Scale bar to an image

1. Confocal image files should already have the correct micron/pixel calibration as it is contained within the files metadata. To check that the value is correct or to add a new value firstly open an image in the surpass view. Now select More>Edit>Image Properties

2. In the Geometry menu you can adjust the voxel (3D pixel) size in XYZ.

3. Now select More>Preferences and highlight the Display menu in the left margin.

4. Under the Coordinate Axis/Scale Bar/TimeColor heading you can select to show the scale bar

and/or length label. Change the scale colour by altering the Measurement Color option.

5. When the scale bar is visible on the image you can adjust its position, length, thickness and text size by clicking on different parts of the scale bar.

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Crop a 3D Data set Cropping allows you to select a region of interest within the image and save it as a new image. This can be useful when you want to reduce the file size of a dataset for processing or for movie making purposes.

1. Open the image to be cropped from the Arena.

2. Select More>Edit>Crop 3D.

3. The area to be cropped is contained within the yellow bounding box. You can adjust the size of the box either by dragging any of its sides entering specific values into the Select Crop Dimensions text boxes. Easily reposition the box by clicking anywhere in the image (ROI will centre around cursors position).

4. To save the cropped data set you can either click Store which will overwrite the original Arena image and only the cropped version will remain or select Store As and give the image a different name. This will create a new image file within the Arena view.

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Creating a View Snapshot

Snapshots are used to create a copy of the image exactly as it appears in the display, similar to a print screen function. The snapshots are saved as an image data type that can be read by any application.

1. Open an image and select the Snapshot icon.

2. Snapshot Preview displays how the snapshot image will appear.

3. Select the size in pixels the snapshot should take from the dropdown list. The “dataset” option will produce a snapshot that is the same size as the original image.

4. Resolution determines the size of the image when physically printed onto

paper. An image of 512x512 set to 100dpi would measure 5.12x5.12 inches on the printed page.

5. When “Snapshot size from window size” is selected, the snapshots

dimensions equal those of the viewing area.

6. 100% Snapshot ensures multiple snapshots have the same scale factor (pixel ratio). Zoom factor set to one pixel per voxel.

7. Image Output contains a tick box to Save to File, select a location for the image. Copy to Clipboard

to allow the image to be accessed from the system clipboard (Edit>Paste menu in any application).

8. Do Snapshot will acquire the image if a save location has been configured.

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Making movies from 3D data sets

1. Open an image and select the Animation icon to open the Key Frame Animation window (opens below image). 3D mode is always selected for animations.

2. A movie is created by assembling a number of Key Frames. A Key Frame is equivalent to a single frame on a roll of film. A movie is produced by capturing multiple frames in which the scene changes in each. There are some default animation options selectable from the dropdown list shown here e.g 360° horizontal. Ensure the image is positioned at the orientation/position you’d like the animation to start before choosing a pre-defined animation.

3. Key Frames appear as the thicker vertical cyan lines as shown here. To speed up the transition

between frames, move the key frames closer together by clicking and dragging. To extend or shorted the entire movie sequence adjust the Number of Frames option.

4. To create a custom animation sequence, start by positioning the image as it should appear at the start of the movie. Then press the Add button to add a frame. Move the image to its next position. An example might be to start with the image zoomed out, add a key frame then zoom in, add another key frame and so on. The time taken to transition between key frames is the same by default but it can be made asymmetric.

5. Select the Play button to see a preview of the movie. Press the Record button to create a movie file. The standard movie formats are AVI (PC) or Quicktime (Mac). Increasing the compression level will decrease the file size but the quality will also be reduced.

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Creating image analysis protocols

Introduction to the Surpass Scene Toolbar

1. The Scene toolbar contains a wizard for creating an analysis protocol. The default components of a scene are Light source, Frame and Volume. Toggling the frame check box will remove the bounding grid surrounding the image volume. Toggling volume will hide the image itself.

2. The icons along the top of the scene window indicate the different features available. These elements are called objects. The number of objects visible will depend on which Imaris licenses you have opened at startup. Click on an object to add it to the scene list.

Manually measure 3D distances

1. Read the section on the Surpass Scene Toolbar above before

continuing.

2. Ensure you have 3DView mode open.

3. Select the Measurement Points icon to add a Measurement Points object to the scene. Double click the text to change its name.

4. The Measurement points object has 5 sub-menus. Select the Settings sub-menu and choose the point shape to be used.

5. Select whether a pair of points or polygon points are drawn.

6. Point diameter and line width is self-explanatory.

7. Label Properties changes what information is physically added to the

image.

8. Select the Edit sub-menu and choose the Intersect with Specific Channel option.

9. Change the Mouse Pointer option to Select mode.

10. A 3D box will appear in place of the cursor on the image. You can

change the size of the box using the mouse’s scroll wheel. If you were measuring the distance between two discrete spots then aim to make the 3D as large as the spot.

11. If you hold down the Alt key this toggles the mouse back to Navigate curser mode so you can

rotate and zoom the volume to position the point you’d like to measure. Hold down the shift key and left click to place the point, repeat to place the second point. The line between these points will be automatically drawn and measured. Physical coordinate placement of the points is based on the highest intensity voxel (3D pixel) of the selected channel.

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12. If you have segmented (thresholding) the image and created surfaces, you can also measure the

distance between two surfaces, either surface to surface or centre to centre of the objects.

13. To edit the position or delete a point, ensure the Edit submenu is displayed and the Select cursor is active. Click on the point to be moved or deleted then hold the shift key and drag it to its new position or press the Delete Selected Point button in the edit sub-menu.

14. The Statistics sub-menu displays and organises all the length

measurements. The Overall tab displays the total number of points.

15. The Detailed tab displays either specific length values if you select Specific Values from the dropdown list or Average values if selected. You can also select which measurement to view distance, angle, intensity etc from the lower dropdown list.

16. Export the measurement results to Excel using the Export Statistics

button.

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Count discrete point-like objects (Spots)

The spots object is most suited to counting point-like objects such as particles, puncta or FISH probes. It has very limited options for measuring volumes or intensity, use the Surfaces object for this purpose.

1. Read the section on the Surpass Scene Toolbar above before continuing.

2. Add a Spots object to the Surpass Scene by clicking on the spots object. Double click on the default name to change it.

3. The spots object has 3 sub-menus. Begin with the Create menu. Unless you select otherwise a wizard is used to create the protocol. You can edit manually by selecting Skip automatic creation, edit manually.

4. If you have created a spots protocol before you can recall in from disk

in the Favourite Create Parameters dropdown menu.

5. Under Algorithm Settings if you select Segment only a region of interest you will be able to define a ROI in the next step of the wizard. Otherwise the whole image will be analysed. Process entire image finally can be used to apply the wizard settings to the entire image even though it has been configured to identify objects based on a ROI.

6. Different spot sizes (Region Growing) Without this option enabled spots are identified using local

maxima detection only, bright regions found against a relatively dark background. If you would like to estimate the size (Volume) of the spots then use the region growing option. Region growing defines a region directly by placing a seed point at the centre of each object. Pixels next to the seed points are examined and determines whether the neighbouring pixels should be added to the region. This process is iterated until an edge or border is found.

7. Select the Next button to move on to the next stage of the wizard.

8. Select which colour channel is to be used for spot identification from the Source Channel dropdown menu.

9. The Spot Detection option will automatically estimate the average XY diameter but you can change the value if necessary. It is important to note that if you don’t select the region growing option above all spots placed on the image will have the diameter specified in this step. To estimate the XY diameter of the spots select the Slice view from the top menu. Alter the Z plane viewed with the vertical scroll bar if necessary.

10. The Measure toolbar appears automatically in the right margin of the Slice view, the Line option is selected by default. Click on two points on an object to measure its length. The length is displayed at the bottom of the Measure toolbar.

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11. Return to the 3D view. The Detect Ellipsoids option can be used if your spots are elongated in the Z

axis which is usually the case due to the inherent lower axial resolution. Again Z diameter is calculated from the raw data but can be edited however you can’t draw a line through Z to accurately estimate the length.

12. Background Subtraction essentially smooths the image and can make it easier to identify

foreground objects of interest over the background fluorescence which is always present in an image (uses Gaussian filtered versions of the original image for subtraction). The result of the subtraction is not displayed on screen but is applied while segmentation is carried out.

13. Select Next and you will see that a point surface is added to each object the algorithm has

identified.

14. Use the Settings menu to change the point style, rendering quality or size of the point.

15. Use the Color menu to change the default colour of the rendered points.

16. In this step of the Create menu you can add filters to the analysis

to detect more or less spots if they have not all been identified automatically. Quality is the default filter type added initially and represents the intensity at the centre of the spots.

17. An automatic threshold is applied by default. If this hasn’t

detected all of the spots you can manually adjust the threshold by hovering the cursor over the boundary between the grey and yellow regions on the histogram and then dragging the yellow region to the left or right. Moving the yellow inclusive region to the left will allow spots with lower intensity values to be identified.

18. To toggle between automatic and manual thresholding, click on the green A or M symbol.

19. Add more filters by selecting from the Filter Type dropdown list then click on the +Add button

above. Most of the additional filters are self-explanatory.

20. When all the relevant spots have been identified click on the Finish or Next button.

Which button is visible depends on whether the Different Spot Sizes (Region Growing) option was selected in one of the previous steps. If it wasn’t selected the wizard will finish otherwise read points 20-22 below.

21. The Spot Region Type menu has two options for how the region growing

step is carried out. Spot regions from Absolute Intensity will use the raw pixel values contained within the image without any morphological filters being applied. The Local Contrast setting should only be selected if Absolute Intensity doesn’t properly segment all spots. This option applies a Gaussian filter and background subtraction in order to estimate the intensity value of each voxel. Essentially a Gaussian filter is a smoothing

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function and in this case each new voxel value is a weighted sum of a particular voxel and its neighbouring voxels. Local contrast requires more memory but may held when noise is limiting spot identification.

22. Click on the Next button.

23. The Spot Regions threshold window will appear and an automatic threshold is applied in white. Adjust the area of the histogram that is covered by the yellow section to manually set a threshold.

24. When the Finish button is pressed the regions will grow out of each

spot seed point until they fill the white regions and reach a border. Read point 24 before doing this.

25. The Diameter from option determines how the spot radius is calculated

either from the Region Volume (spot volume itself) or the Region Border (smallest distance from each spot centre to the region border). The Create Color Coded Region Channel will add an additional colour channel to the Display adjustment window. When this channel is active it displays the result of the region growing step and indicates the contour used to measure the volume of each spot which you can use as an accuracy test.

26. Once the wizard has finished you will see 6 icons displayed in the surpass view. The Spots sub-menu was described earlier. The Wand sub-menu allows you to go back and edit the creation parameters, click on Rebuild to do this but note that it takes you back to the start of the wizard but maintains your previous settings. The Store Parameters for Batch permits saving the protocol either to the Arena view or as a Favourite Creation Parameter. Save to Arena if you intend to batch process images but it might be useful to save using both options as they’re both useful in different situations.

27. You can use the Edit menu to highlight a particular spot and see its coordinate position in the volume or its XY or Z diameter which you can edit here directly if necessary. To select a spot on the image ensure that the cursor is set to Select mode, this will turn the cursor into a cube and left click on a spot in the image and it will turn yellow to indicate it has been selected.

28. The Filter sub-menu is used to isolate a number of spots based on filters you apply. For example if you wanted to only generate statistical data concerning a subset of spots. Use the +Add button to insert a filter. Once you have filtered the spots of interest you can create a new Spots item to add to the Surpass tree by clicking Duplicate Selection to New Spots.

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29. The Statistics sub-menu contains any measurements made as part of the analysis protocol. The Overall tab displays only the number of spots identified. The Detailed tab can display specific values (e.g. spot volume) for each spot or average values. The Selection tab will only show data for the spot that has been selected in the image.

30. To export statistics data use the Disk icons at the bottom of the Statistics window. The two options are export statistics on tab display (only the stats currently visible) or export all statistics (All stats generated by the protocol). Saving as .Xls and .csv are supported.

31. The Spots object performs segmentation and

measurement on the specific colour channel selected during creation of the protocol. If you would like to apply the same protocol on a different channel you can either export the statistics from one channel and then rebuild the protocol changing just the Source Channel but you will lose the original stats unless exported. The other option is to create a second Spots object in the Surpass view. Click on the new spots object and in the Create sub-menu choose the specific Favourite Creation Parameter to apply, you will just need to edit the selected Source channel.

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Measure objects in 3D automatically (Surfaces)

The Surfaces object is suitable for identifying most cellular structures. Use Surfaces for most objects unless you’re measuring point like spots in which case refer to the section on measuring spots above.

1. Read the section on the Surpass Scene Toolbar above before continuing.

2. Start by opening an image and add a Surfaces object to the Surpass Tree.

3. Double click on the default “Surfaces1” name to rename.

4. The spots object has 3 sub-menus. Begin with the Create menu. Unless you select otherwise a wizard is used to create the protocol. You can edit manually by selecting Skip automatic creation, edit manually.

5. If you have created a spots protocol before you can recall in from disk in the Favourite Create Parameters dropdown menu.

6. Under Algorithm Settings if you select Segment only a region of interest you will be able to define a ROI in the next step of the wizard. Otherwise the whole image will be analysed. Process entire image finally can be used to apply the wizard settings to the entire image even though it has been configured to identify objects based on a ROI.

7. Select the Next button to move on to the next stage of the wizard.

8. Select which colour channel is to be used for surface identification from the Source Channel dropdown menu.

9. Check the Smooth option to apply a Gaussian filter to the data set. Smoothing may make object identification easier for noisy images. If smooth is selected the Surface Area Detail Level option is available. A value for this parameter is determined automatically from the data set but this can be altered.

10. Thresholding is either based on Absolute Intensity (pixel values left unaltered) or Background Subtraction (Local Contrast). The Background Subtraction option is often required when objects are touching. If Background Subtraction is selected a Gaussian filter is applied to the data set which estimates the background intensity of each voxel. This variable background is then subtracted from each voxel in the image. The width of the Gaussian filter is estimated by subtracting the diameter of the largest sphere that fits into the object. This is estimated automatically from the raw data or you can switch to Slice view and draw a line across the diameter of the object and base the sphere diameter on this measurement.

11. Select the Next button to move on to the next stage of the wizard.

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12. The Create sub-menu is where you can adjust the threshold setting. By default an Automatatic threshold is applied. Increase/decrease the area of the histogram covered by the yellow region if the automatic threshold setting is not satisfactory. You can switch back to the automatic threshold by clicking the green A-M button.

32. Split touching objects (Region Growing) The Enable check box will allow objects which are touching to be separated and viewed as different objects. This method stems from region growing segmentation where by seed points are placed at local maxima. This seed then grows outwards in all directions until a border is detected. If this option is enabled you will need to define the seed point diameter. This should be estimated as the diameter of the smallest sphere that will fit within the object. To estimate the XY diameter of the spots select the Slice view from the top menu. Alter the Z plane viewed with the vertical scroll bar if necessary. The Measure toolbar appears automatically in the right margin of the Slice view, the Line option is selected by default. Click on two points on an object to measure its length. The length is displayed at the bottom of the Measure toolbar.

13. The Settings sub-menu allows you to change the colour and method for visualising the surface applied to every identified object in the Surfaces Preview section. Isosurface is the most common option but volume rendering would also be suitable. Seed Points View can be used to change how the seed points (local maxima) are displayed. Note that you will not be able to see the seed points on the isosurface setting unless you make it transparent.

14. Select the Next button to move on to the next stage of the wizard.

15. In this step of the Create menu is Classify Seed Points. Here you can add filters to the analysis to filter out objects that could interfere with interpretation of results. Quality is the default filter type added initially and represents the intensity at the centre of the spots.

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16. Below is an example of when to use this feature. The image to the left shows the objects found both as rendered objects (red) and their corresponding seed points (yellow). If we only wanted to render and measure objects over a certain quality threshold we could alter the Quality filter for seed points by moving the yellow region further to the right on the histogram. The result of this is shown on the lower centre image. Now only 3 seed points are visible. When you proceed to the next step in the wizard the rendered objects with a quality lower than the threshold limit set are removed as shown in the image on the right.

17. Select the Next button to move on to the next stage of the wizard.

18. Classify Surfaces is used to further filter the segmented objects but the filtering options are more exhaustive compared to classify seed points. The filters fit within these overall categories size, shape, intensity and position.

19. Select the Finish button to complete the wizard.

20. Once the wizard has finished you will see 7 icons displayed in the surpass view. The Surfaces sub-menu was described earlier. The Wand sub-menu allows you to go back and edit the creation parameters, click on Rebuild to do this but note that it takes you back to the start of the wizard but maintains your previous settings. The Store Parameters for Batch permits saving the protocol either to the Arena view or as a Favourite Creation Parameter. Save to Arena if you intend to batch process images but it might be useful to save using both options as they’re both useful in different situations.

21. The Draw sub-menu is used to semi-automatically create further surface objects. Select the Add/Delete tab. Cursor intersect describes which channel or object is used to determine the position of the cursor within the volume. Picking the right position/depth to click at within the volume would be difficult without this. Select the Color Channel that contains the objects you want to identify.

22. Ensure the cursor is in select mode, the cursor will become a cube whose size can be adjusted with the mouse scroll wheel.

23. Two techniques are available for adding new surfaces. Marching Cubes is optimised for objects with a rough complex surface. A marching cube type surface generation is applied within the volume of the cursor box on the image. Move the cursor to the centre of the object to be segmented, adjust the size of the box to envelope the object using the scroll wheel. Holding Shift plus left click will begin surface generation within that area. The Magic Wand technique is optimised for single complex objects which are located over a large region of the image. The threshold is taken from the cursor position when shift-click used.

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24. The Contour tab is used to manually draw around 3D objects to segment them. You would use this if automated segmentation methods have failed. This feature is explained elsewhere in the manual.

25. You can use the Edit menu to highlight a particular segmented object

in the image and delete it if necessary. To select a surface on the image ensure that the cursor is set to Select mode, this will turn the cursor into a cube and left click on a spot in the image and it will turn yellow to indicate it has been selected.

26. The Filter sub-menu is used to isolate a number of segmented objects based on filters you apply. For example if you wanted to only generate statistical data concerning a subset of surfaces. Use the +Add button to insert a filter. Once you have filtered the surfaces of interest you can create a new surfaces item to add to the Surpass tree by clicking Duplicate selection to new Surfaces.

33. The Statistics sub-menu contains any measurements made as part of the analysis protocol. The Overall tab displays only the number of surfaces identified. The Detailed tab can display specific values (e.g. surface volume) for each surface or average values. The Selection tab will only show data for the surface that has been selected in the image.

34. To export statistics data use the Disk icons at the bottom of the Statistics window. The two options are export statistics on tab display (only the stats currently visible) or export all statistics (All stats generated by the protocol). Saving as .Xls and .csv are supported.

27. The surfaces object performs segmentation and measurement on the specific colour channel selected during creation of the protocol. If you would like to apply the same protocol on a different channel you can either export the statistics from one channel and then rebuild the protocol changing just the Source Channel but you will lose the original stats unless exported. The other option is to create a second surfaces object in the surpass view. Click on the new surfaces object and in the create sub-menu choose the specific Favourite Creation Parameter to apply, you will just need to edit the selected Source channel.

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Manually draw & measure objects in 3D (Surfaces)

Only use this functionality if the surfaces creation wizard has failed to identify your objects of interest. It works by generating contours at different planes within the volume based on the drawings you make on the image.

1. Open a data set in the surpass view and select the 3D view mode.

2. Create a new surfaces object if you don’t already have one associated with this image.

3. Select the Draw sub-menu then the Contour tab. To see an

unobstructed slice view disable the volume rendering option in the surpass tree.

4. Select the Board tab within the Contour menu. Here you can alter the slice orientation between XZ, XZ and XY. XY is selected by default.

5. Use the Slice Position scrollbar to change the slice being viewed.

6. The Visibility option defines the visibility of previously drawn contour lines, None, Next or All.

7. Resolution controls the smoothness and complexity of the surface created by the contour lines which can be set to Auto or Manually determined. You only see the result when you click Create Surface.

8. The Lock check box is used to stop the orthogonal slicer yellow box from changing plane while you’re drawing contours.

9. In order to draw anything you have to be in the pointer select mode and in the Active draw mode. Deselecting the draw button switches the cursor to inactive mode so that the cursor can be used to select existing contour lines and delete them.

10. Select the Mode tab and choose a Drawing Mode Click-Clicking within the image inserts a point (vertex) if you click another vertex elsewhere the two vertices are connected with a straight line. Time-Places a vertex wherever the cursor is after the set Insert vertex after delay period. I.e. place a vertex every 300ms. Distance-Places a vertex at set distance intervals as specified in the vertex spacing box. Circle-Draws a circular contour line of a set radius as defined in the radius box. The number of vertices making up the circle can be defined in Number of vertices. Increasing this number will result in a smoother surface render. Isoline-Contour lines created by tracing points of uniform intensity under the mouse cursor. This option autonomously tries to create the contour line that outlines the object in that particular plane. The Reduce density to percentage determines number of vertices used to create the isoline contour shape, e.g 100% = vertex at each pixel along the line, 10% = vertex at every 10 pixels along the line.

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Magic Wand-Creates a contour line by selecting the largest connected area that contains the maximum intensity voxels and all voxels within a user defined tolerance range.

11. Once the appropriate drawing mode has been chosen, select the colour channel that should be used to compute the contour lines.

12. Select a slice position where you would like to create a drawing around a slice of an object.

13. Set Draw to Active mode.

14. Left click on the image to trace your contour lines. Double click to finish.

15. Move to the next z plane using the slice position scrollbar and repeat this process to define the object.

16. Click the Create Surface button to calculate the surface from the contour lines.

17. You can now view statistics about this surface and process further.

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Analyse and present statistical data (Vantage) Imaris has an in-built statistical data visualisation module called Vantage, providing tools to create multi-dimensional plots for presentation. Vantage can be used with measurements created from both the spots, surfaces, cells and filaments objects.

1. When you open Imaris, ensure you select the Vantage license.

2. Open a data set that contains either a spots or surfaces object.

3. Select the Vantage icon.

4. To create a new plot select Add new vantage plot.

5. This will create a new plot item in the plot input data area. In this case called Vantage3.

6. In the source data section you can choose what data is analysed i.e. a spots or surface object. If there are multiple surface objects associated with the image they will appear in the list with a check box next to each. Tick only the surface/spots objects that you’d like to create plots from.

7. Click Next to move on to the next section of the plot creation wizard.

8. Choose a plot type from the 4 options which one you choose depends on the kind of data you’d like to visualise. In this example an XYZ plot is created. Note that if you want to set your own axes categories then you should use the Scatter plot option. Gallery plot is not available if you have selected to plot multiple surface/spots objects. You can alter the sub-type of plot by selecting the down arrow. Options include 2D or 3D plot and to include extra parameters such as scale and colour.

9. The plot values represent what measurement type is presented on each axis. Depending on the plot sub-type different plot values are available. In this example the XYZ 3D scale and colour plot is used. Choose the measurement category to be used for scale and colour. In this case, scale of the plotted object is set to sphericity and colour is set to volume. Therefore the size of the plotted objects are scaled based on how spherical the objects are and the objects are colour coded according to their volume.

10. Show Box Plots will display box plots on the graph. They are used to

summarise selected statistical variables so that only 5 numbers are displayed. These values are minimum, lower quartile, median, upper quartile and maximum. Shown in this image as the yellow boxed areas.

11. Note that the actual data values used to create the plot are displayed in the Plot Numbers Area. This data can be exported to a CSV or Xls file by clicking on the Disk icon next to the data table.

12. In the Settings menu you can change how the objects are displayed on the plot, either Surface or Centre point. If you enable the Sub Volume option, the raw data is superposed onto the plot view either as maximum intensity projection or blended view. Note that this will change the background colour of the plot to black. To complete the plot creation wizard click the Finish button.

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Batch Processing (run an analysis on multiple images) Imaris contains a batch processing feature which allows a Surfaces, Spots or Cells protocol to be run on a series of images. Due to the variable nature of biological images it is often required that the analysis protocol paramaters are adjusted per image, however using Imaris Batch could save you some time.

1. Ensure that you select the Batch license when you open Imaris.

2. Generate an analysis protocol using one of the available objects-Spots, Surfaces etc as explained elsewhere in this document. If you would like to batch process multiple colour channels then you will need to generate a protocol for each channel.

3. Select the object (protocol) to be used in batch processing in the Surpass tree and select the Creation sub-menu.

4. Select the Store Parameters for Batch button, name the protocol and select Arena as the save location.

5. Remember to repeat steps 2-4 for each colour channel to be analysed.

6. Open the Arena view. The group that contains the image used to

generate the analysis protocol now contains a new Creation Parameters item “green spots”.

7. Right click on the creation parameter item and select Run Batch.

8. While the analysis runs an orange star icon will appear on each image within the group. When the

analysis of that image has finished the star becomes white and contains a tick if the analysis was

successful or is replaced by a sad face if unsuccessful.

9. Another new icon will appear known as a Batch Collection item. This is a file which stores

the images data, batch segmented objects and statistical results. Left click twice to open

(Vantage license must be selected at startup). Right click the item to export the results data to csv

or xls file.

10. To validate the batch segmentation results, right click on an image and select Open with surpass

objects and batch result.

11. The surpass tree will display an additional object which is given the name

of the group that was batch processed. Click on the object to view the

batch results. The no entry sign displayed on the object signifies that this object is for viewing only.

12. If you’re not happy with the segmentation results and you change the analysis protocol, the

resulting segmented objects and results will not appear in the previously generated batch

collection item. The easiest way to modify the protocol is to select the

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Creation menu and Duplicate and Rebuild. This will duplicate the object with a new name and

appears in the surpass tree. Now you can edit the protocol and run the analysis again on this image

alone.

13. Use the Store or Store As buttons to save the image and the newly generated

objects.