TITLE: Vesselucida 360 Protocol for Segmenting and Analyzing Human Islet Microvasculature
AUTHORS: Martha Campbell Thompson, Malavika Nair, Aidan Sullivan

[STEPS]
?. [Set-up]
Download and install Vesselucida 360 application (RRID:SCR_017320).
?. [Set-up]
Launch the application and set-up SciCrunch connection.
?. [Set-up]
Go to https://scicrunch.org/ and click REGISTER in the top menu. Fill out the registration form to create an account. From "My account", click API KEYS. Click GENERATE AN API KEY. Copy the key and return to Vesselucida 360.
?. [Set-up]
Select the "SPARC Vocabulary Services" icon from the Trace ribbon.
?. [Set-up]
Right-click to paste the API key in the dialog, then click SET API KEY.
?. [Get Started]
Open microscopy image data (2D or 3D) in the application.
?. [Get Started]
When the SPARC Vocabulary Services dialog appears, select the appropriate items from the drop-down lists and enter the appropriate information regarding the subject to be segmented. Then select BEGIN.
?. [Get Started]
Subject information.Indicate the species, subject ID, sex, and age of the subject. All entered metadata will be stored o the XML data file. Independent from the cirteria for anatmoic terms selected. Note the "blinded to condition" options for sex and age.
?. [Get Started]
Criteria for anatomic terms.ORGAN: Select the organ term list you are interested in annotating with.SPECIES: The species you are interested in annotating with.PARCELLATION: The atlas parcellation scheme you are interested in annotating with. If no parcellation exists for the organ and species criteria, select Species Independent from the drop-down.
?. [Vascular Reconstruction: 3D Semi-Automatic Tracing Methods]
In the 3D Environment window, click the VESSEL icon to display the Trace Vessels panel.
?. [Vascular Reconstruction: 3D Semi-Automatic Tracing Methods]
Under TRACING MODE, select USER-GUIDED.
?. [Vascular Reconstruction: 3D Semi-Automatic Tracing Methods]
Select a TRACING METHOD: Directional Kernels, Rayburst Crawl, or Voxel Scooping.Note: The three tracing methods are based on algorithms that work by tracing along the vessel one point at a time, and by producing three measurments at each point: (X,Y,Z) coordinate, thickness, estimated position of the next point.
?. [Vascular Reconstruction: 3D Semi-Automatic Tracing Methods]
Hover over the area of the vessel where you want to place the first point. Click to place the first point. A sphrere represents the first point. Continue to hover over the process. A series of circles is displayed, representing the path detected by the software. When you reach the end of the vessel, click to place the last point. Continue. Right-click once to end the branch.The traced branch is displayed in 3D.
?. [Vascular Reconstruction: 3D Semi-Automatic Tracing Methods]
To create a bifurcating vessel, hover over an existing traced branch (branch A) at the location where the process bifurcates in the image data. A partial sphere appears on branch A (indicating a potential connecting point). Click to place the bifurcation point. Hover over the area of the bifurcating vessel and trace as you did in step 8. Note: all vessel branches can be traced in either direction, in any order.
?. [Vascular Reconstruction: 3D Automatic Tracing Methods]
In the 3D Environment window, click the VESSEL icon to display the Trace Vessels panel.
?. [Vascular Reconstruction: 3D Automatic Tracing Methods]
Under TRACING MODE, select AUTOMATIC.
?. [Vascular Reconstruction: 3D Automatic Tracing Methods]
Select a TRACING METHOD: Directional Kernels, Rayburst Crawl, or Voxel Scooping.Note: The three tracing methods are based on algorithms that work by tracing along the vessel one point at a time, and by producing three measurments at each point: (X,Y,Z) coordinate, thickness, estimated position of the next point.
?. [Vascular Reconstruction: 3D Automatic Tracing Methods]
Click the SHOW SETTINGS button.
?. [Vascular Reconstruction: 3D Automatic Tracing Methods]
In the DISPLAY SEEDS tab, click the DISPLAY SEEDS button.Optional: To change the seed color, use the color picker next to the HIDE SEEDS button.To adjust the number of seeds, use the slider and the density buttons.
?. [Vascular Reconstruction: 3D Automatic Tracing Methods]
In the REFINE SEEDS tab, click the VALIDATE SEEDS button.Optional: to decrease the number of unwanted seeds, increase the REFINE FILTER value. Add or remove seeds manually.
?. [Vascular Reconstruction: 3D Automatic Tracing Methods]
In the TRACE tab, adjust the SENSITIVITY, GAP TOLERANCE, REMOVE TRACES SHORTER THAN, AND CONNECT BRANCH SEGMENTS settings. Then click the TRACE button to start the automatic trace.
?. [Editing Vessels]
Selecting Vessels.To select a single vessel, click the vessel. To select all the vessels, click the SELECT ALL button.To select several adjacent vessels, press the CTRL key and draw a marquee around the vessels of interest.To deselect, click away from the selected object.
?. [Editing Vessels]
Selecting Points.To select a signle point on a vessel, click the POINTS button then click the point of interest.To select multiple, adjacent points, draw a marquee around the points of interest.To deselect, click away from the selected options.
?. [Editing Vessels]
Connecting and Detaching Vessels.Connecting VesselsZoom in and rotate as needed.Click the CONNECT button. Vessels are now represented by series of points along colored lines.Click and drag an end point toward the area you want to connect to. The end point briefly turns green and a green line appears.Release the mouse button to connect the segments.Detaching VesselsClick a vessel to select it.Click the DETACH button.Click on the segment to detach.Detaching VesselsClick a vessel to select it.Click the DETACH button.Click on the segment to detach.If you click a single segment, the program splits the segment into two segments.If you click a bifurcation, the program splits the segment into three segments.
?. [Editing Vessels]
Deleting VesselsClick a vessel to select it or click the SELECT ALL button.Press the DELETE key or click the REMOVE button. You can’t undo this action!
?. [Analysis of Reconstruction]
Segment Details and Network AnalysesIn the TRACED STRUCTURES window, select all Directed Networks (vessels). Select ANALYZE> NETWORKS. From the Network Analysis window check ALL NETWORK SUMMARY from the Network tab and SEGMENT DETAILS from the Segment Details tab. Click OK to run the analysis. Right-click and select SAVE TO EXEL for both result tables.
?. [Analysis of Reconstruction]
Segment in Closed SurfaceIn the TRACED STRUCTURES window, select one islet set. Hold control and select all Directed Networks (vessels). Select ANALYZE> CLOSED SURFACE ANALYSIS Right-click the result table and select SAVE TO EXEL
?. [Analysis of Reconstruction]
3D Contour Summary and Contour DetailsIn the TRACED STRUCTURES window, select one islet set.  Select ANALYZE> MARKERS AND REGIONFrom the Markers and Regions Analysis window check CONTOUR DETAILS and 3D CONTOUR SUMMARY from the Segment Details tab. Click OK to run the analysis. Right-click the result table and select SAVE TO EXELRepeat this process for each islet set.
?. [Contouring Islets]
Generating Serial Z SectionsIn the 2D window, Click TRACE > SECTIONS> SERIAL SECTION MANAGER (SSM).Open the image organizer, IMAGE> ORGANIZER. Right-click on the image and select CREATE SECTIONS FROM THIS STACK. This will generate a section in the SSM at each Z plane of the image. Contouring IsletsStarting at the first Z section that the islet comes into view...In the SSM, select SHOW CURRENT SECTION. Select TRACE> CONTOUR> CONTOUR SELECTION.From the CONTOUR SELECTION menu, choose the contour name "Set of pancreatic islets". Select a tracing method by right-clicking in the tracing window. The method chosen is strictly a matter of preference. The tracing method can be changed at any time during the tracing by right-clicking in the tracing window.Contour the islet region: Click to place points along the boundaries of a region of interest; when you're done, right-click and select CLOSE CONTOUR.To adjust the thickness of the contour, scroll the mouse wheel.If the contour is larger than the field of view, it is easiest to trace if MOVE> TOOLS> AUTOMOVE is turned on.If you make a mistake, press Ctrl-Z to erase the last drawn points one by one back to the beginning of the contour.This was carried out for every 4th Z section that the islet(s) were present in. Place contours into setsSelect TRACE> SELECT OBJECTS. Hold control and click to select each contour making up one islet. Right-click and select PLACE CONTOURS INTO SET.Name the set with the islet ID and select OKContouring IsletsStarting at the first Z section that the islet comes into view...In the SSM, select SHOW CURRENT SECTION. Select TRACE> CONTOUR> CONTOUR SELECTION.From the CONTOUR SELECTION menu, choose the contour name "Set of pancreatic islets". Select a tracing method by right-clicking in the tracing window. The method chosen is strictly a matter of preference. The tracing method can be changed at any time during the tracing by right-clicking in the tracing window.Contour the islet region: Click to place points along the boundaries of a region of interest; when you're done, right-click and select CLOSE CONTOUR.To adjust the thickness of the contour, scroll the mouse wheel.If the contour is larger than the field of view, it is easiest to trace if MOVE> TOOLS> AUTOMOVE is turned on.If you make a mistake, press Ctrl-Z to erase the last drawn points one by one back to the beginning of the contour.This was carried out for every 4th Z section that the islet(s) were present in. Place contours into setsSelect TRACE> SELECT OBJECTS. Hold control and click to select each contour making up one islet. Right-click and select PLACE CONTOURS INTO SET.Name the set with the islet ID and select OKTo adjust the thickness of the contour, scroll the mouse wheel.If the contour is larger than the field of view, it is easiest to trace if MOVE> TOOLS> AUTOMOVE is turned on.If you make a mistake, press Ctrl-Z to erase the last drawn points one by one back to the beginning of the contour.This was carried out for every 4th Z section that the islet(s) were present in. Place contours into setsSelect TRACE> SELECT OBJECTS. Hold control and click to select each contour making up one islet. Right-click and select PLACE CONTOURS INTO SET.Name the set with the islet ID and select OK
?. [Analysis of Reconstruction]
Download and install Vesselucida Explorer application (RRID:SCR_017674).Launch the application and open the XML reconstruction data file.
?. [Save Data File]
Select FILE>SAVE AS> DATA FILE to save the file with a new name, the same name but with different options, or with a different file type.Name the datafile with the same name as the associated image. Choose XML from the Save as type dropdown.Use FILE> SAVE> DATA FILE without asking for a filename, if the file already has a name.