S. Now, to begin tracing, move through the image stack to find the start point of a path then click there with the left mouse button. ### II. Pick A Subsequent Point A small circle should appear, highlighting the start of the path. Move through the stack to find a subsequent point further along the same structure to be traced (neuron, blood vessel, etc.), and click there. If a path between the two points cannot be found immediately, you may see the animated progress of the search. You can scroll through the stack while such a search progresses: If it appears to not be making good progress, it's probably best to press the "Cancel/Esc" button (shortcut:
Esc) and pick a point closer to the start point.
Increase”“Z”” in the ““Cursor Auto-snapping”” panel for automated Z-navigation on signal mouseover.
Y) that this path is following the route through the image that you expect. If it is not, then click "No"
Nand you'll go back to the [previous step](#II._Picking_a_Subsequent_Point "wikilink"). Assuming you confirmed the path, the confirmed path will appear in red. Now you are essentially back at [step II](#II._Picking_a_Subsequent_Point "wikilink"). Normally you will go on to pick further points along the structure. However, if you have finished with that path, click "Finish Path"
Fand you will go back to [step I](#I._Picking_a_Start_Point "wikilink"). If you completed that path it would be shown in magenta.
Gwhile the mouse pointer is near the path. When the path is first selected, it will be shown in the default green color. ### II. Select The Fork Point To force the start of the new path to be a branch of the selected path, hold down the
Alt\|Shiftkeys while you move the mouse to find the branch point under the red cross-hairs, now decorated with a "Fork Point" annotation. With
Alt\|Shiftheld down, click with the left mouse button. Finally, release the keys. Note that it is also possible to zoom into the branch point, right-click on the image and choose *Fork at Nearest Node* from the contextual menu. ### III. Extend The Path From this point on, you can carry on adding nodes to the branched path as [above](#II._Pick_A_Subsequent_Point "wikilink"), i.e., Create a temporary path and confirm it. When you decide to complete the path you should see in the Path Manager that it has been recorded as a child of the existing path.
Supposing you want the end of a path that you're tracing to join onto an existing path, you just use the same modifier key when selecting the end point of the last part of the path. To go into that in more detail, if you're halfway through tracing a path like :
... and you want the final part of that path to join up with the existing path running from the top-left to top-right of the image. First, select that path in the path list (or using the
Now hold down
If you're happy with the result, confirming the temporary path will automatically complete the whole path, since if you're creating an end join there cannot be any more to the path. Note that the path list indicates that this new Path (1) ends on the existing Path (0). The result will look like :
G[shortcut](SNT__Key_Shortcuts) and activate the second merging node by hovering over it 4. Open the contextual menu and select the initial path from the *Connect To (Start Join)* / *Connect To (End Join)* menu. If both nodes are terminal, the paths are merged together. Otherwise, one path will become a child of the other. Note that one of the nodes must be terminal, to ensure no loops are created.
W[shortcut](SNT__Key_Shortcuts#Legacy_3D_Viewer)) in the main ImageJ toolbar and click over the region you want to trace. Tracing works the same way as in the 2.5D view, i.e., click somewhere in the image to create a starting point, then click further along the structure of interest to find a path between the two points, then confirm or deny the temporary segment as described [above](#Tracing "wikilink"). Similarly, branching occurs as [described for 2D canvas(es)](#Branching:_Start_A_Path_On_An_Existing_Path "wikilink"), by holding the
Alt\|Shiftmodifier. - **Rotation** Either use the *Hand tool* (
H[shortcut](SNT__Key_Shortcuts#Legacy_3D_Viewer)) tool and left-click while dragging the mouse or drag mouse while holding the scroll wheel. - **Translation** Hold
Shiftand the scroll wheel while dragging the mouse. - **Adjusting zoom depth** Scroll the mouse wheel. Once you have selected each of these tools (Wand and Hand) once, you should be able to switch between them by pressing the
Esckey. See [Key Shortcuts](SNT__Key_Shortcuts#Legacy_3D_Viewer) for the list of all supported shortcuts. !(/media/Fiji-toolbar-wand-and-hand-2.png "fig:Fiji-toolbar-wand-and-hand-2.png") Filling ======= There is a simple facility in this plugin for "filling out" paths to volumes. This is not particularly sophisticated, but often good enough for a rough visualization of the shape of a neuron beyond what can be seen from the traced path. ### I.Starting the Fill First, select the one or more paths that you want to fill out from in the Path Manager and select "Fill -> Fill Out..." in the Path Manager menu options. The selected paths are shown in green so that you can check that you're starting from the right ones. After a couple of seconds if you scroll through the stack you should be able to see a thick green surround the path: The filler continues to explore the image starting from the path until you click "Pause" or "Stop" in the dialog. However, the fill which is shown only includes those points up to a certain threshold distance from the path. (Note that in this section "distance" doesn't mean a real physical distance, but a measure which takes into account the intensity values of the pixels which must be passed through when moving away from the path.) Information about the current threshold and the progress of the search is shown in the dialog. ![The filling-related part of the dialog.](/media/Snt-filling-statistics-2.png "fig:The filling-related part of the dialog.") The "Cursor position:" state under "Search Status" is updated as you move your mouse over the image. If the point under the mouse has been reached by the search then it will show you that point's distance from the path. Otherwise, it will read "Not reached by search yet". Two lines above, the "Current threshold distance:" shows your current threshold distance: so if this is set to 0.2 then that means that all points less than 0.2 from the path are included in the fill (and shown in green in the image.) The "Max. explored distance:" state under this line shows the maximum distance from the path that has been completely explored. ### II.Adjusting the Fill Threshold You can change the fill threshold in one of three ways: - Clicking on a point in the image that has been reached by the search (This is the most intuitive way of altering the threshold). It may be helpful to disable the "Enable Snapping within: XY-Z" feature for the cursor while doing this. - Manually changing the threshold in the "Specify manually:" input box and clicking the "Apply" button beside it. - Clicking the "Use explored maximum" button below the threshold input box and click "Apply", which sets the threshold to the maximum explored distance (the value shown in "Max. explored distance:" under the "Search Status" dialog). Anyway, assuming that you want to use the first of these approaches, you should use the approach described below. It is difficult to set the threshold accurately from the image unless you zoom in, so first zoom on part of the path that you want to set the threshold for. Since the solid green fill obscures the intensity value of the points in the fill, I suggest that you switch to the semi-transparent view of the fill at this stage by selecting the "Transparent overlay" option in the "Rendering Options" dialog. Note that this may slow down filling. As you can see in the middle image, the threshold is set too far from the path, since there are many completely dark points under the green fill, as well as points on different paths than those of interest. Experiment with clicking on different points closer to the path in order to adjust the threshold until you are happy with it. You might end up with something like the rightmost image:
To get the spatial calibration for your image, go to Image › Properties…
I. Now the pathfinding will occur on the secondary image. To view a MIP of the secondary image "over" the original image during tracing, toggle the "Render in overlay at X% opacity" checkbox.
Only 2D or 3D grayscale images are supported. Processing log is shown in Console.", visibility="MESSAGE") msg # @File(label="Directory containing your images", style="directory") input_dir # @String(label="Consider only filenames containing",description="Clear field for no filtering",value="") name_filter # @boolean(label="Include subdirectories") recursive # @float(label="Size of structures to be filtered (spatially calibrated units)",description="The filter kernel will be determined from the average voxel size") scale # @LogService log # @StatusService status # @UIService uiservice """ file: Filter_Multiple_Images.py author: Tiago Ferreira, Cameron Arshadi info: Bulk filtering of image files using Frangi Vesselness """ from net.imagej.axis import Axes import os def get_image_files(directory, filtering_string, extension): """Returns a list containing the paths of files in the specified directory. The list will only include files with the supplied extension whose filename contains the specified string.""" files =  for (dirpath, dirnames, filenames) in os.walk(directory): for f in filenames: if os.path.basename(f).startswith('.'): continue if filtering_string in f and f.lower().endswith(extension): files.append(os.path.join(dirpath, f)) if not recursive: break # do not process subdirectories return files def run(): # First check that scale parameter is > 0, exiting if not if scale <= 0: log.error('Please select a value > 0 for the scale parameter. Exiting...') return # Get all files with specified name filter and extension in the input directory d = str(input_dir) extension = ".tif" files = get_image_files(d, name_filter, extension) if not files or len(files) == 0: uiservice.showDialog("No files matched the specified criteria", "Error") return processed = 0 skipped = 0 for f in files: basename = os.path.basename(f) msg = 'Processing file %s: %s...' % (processed + skipped + 1, basename) status.showStatus(msg) log.info(msg) # Load the input image input_image = ij.io().open(f) # Verify that the image is 2D/3D and grayscale, skipping it if not num_dimensions = input_image.numDimensions() if num_dimensions > 3 or int(input_image.getChannels()) > 1: log.error('Could not process %s...Only 2D/3D grayscale images are supported' % basename) skipped += 1 continue # Convert input image to float, since we are dealing with derivatives float_input = ij.op().run("convert.float32", input_image) # Obtain spatial calibration of the image x_spacing = float_input.averageScale(0) y_spacing = float_input.averageScale(1) spacing = [x_spacing, y_spacing] if num_dimensions == 3 and float_input.axis(2).type() == Axes.Z: z_spacing = float_input.averageScale(2) spacing.append(z_spacing) # Create placeholder image for the output then run the Frangi Vesselness op output = ij.op().run("create.img", float_input) pixel_scale = scale / (sum(spacing) / len(spacing)) # average voxel size: convert scale to pixel units ij.op().run("frangiVesselness", output, float_input, spacing, pixel_scale) # Save the result using the same basename as the image, adding "[Frangi].tif" # For example, the output for "OP_1.tif" would be named "OP_1[Frangi].tif" l = len(f) el = len(extension) output_filepath = f[0:l - el] + "[Frangi].tif" ij.io().save(output, output_filepath) processed += 1 log.info('Done. %s file(s) processed. %s file(s) skipped...' % (processed, skipped)) run() Accurate Point Placement ======================== This section describes methods to increase the accuracy of node placement.
Accurate node placement requires XY, ZY and XZ views to be visible. You can do so at startup, by making sure that Default: XY, ZY and XZ views is selected, or by clicking in Display ZY/XZ Views in the Options tab if you have already started SNT.
Find the approximate location of your start point by moving your mouse in the XY window, holding down
When you press
Locate the center of the structure to be clicked on. If cursor auto-snapping is enabled, simply mouse over the structure, otherwise, try moving the mouse and scroll wheel in each of the panes (holding down
When you're happy with the point under the crosshairs, left-click to start the path:
Zoom out again with the
The path along the neuron may not follow the center line perfectly:
... but you can later improve that with the Refine/Fit › Fit Path... option in the Path Manager, which tries to align the path to the midline of the structure to sub-voxel accuracy: