Since microvascular flow varies between capillaries and changes over a time course of seconds, a method for the continuous quantification of flow across entire fields is needed. Staff enables a nearly continuous measurement of flow velocities across entire microscope fields, within hours, that would otherwise take months to obtain manually. Although these procedures are straightforward, visual demonstration will significantly help new users navigate staff for the first time.
To use TrakEM2 to define the vascular network, first select File, New, and TrakEM2 Blank to setup a new blank TrakEM2 project, and select the new folder containing the single image from the movie as the project folder. The TrakEM2 windows will open. Right-click in the main work area and select Import and Import Image.
Navigate to the single saved image and select the image. Right-click in the main work area again and select Display and Auto Resize Canvas/LayerSet. Then, left-click in the main work window.
The image will fill the work area. To select the areas in the image that contain the vascular network, in the smaller TrakEM2 window, right-click on template Anything, and select Add New Child and Area List. In the smaller TrakEM2 window, drag template Anything onto Project Objects, untitled project.
Then drag template Anything, area list, onto Project Objects, untitled projects, Anything. Under Project Objects, untitled project, the Anything area list will now be present. In the main TrakEM2 window under the Z Space tab, a bar labeled Area List will have been created.
Click to select the list. In the main TrakEM2 window, select the paintbrush tool and click Shift while rolling the mouse wheel to select a paintbrush size that is smaller than the diameter of the vasculature of interest. Press Control S to save the project.
Using the paintbrush tool, paint the vascular network, excluding any out of focus regions. When the labeling is complete, right-click in the main TrakEM2 window and select Export and Area Lists as Labels. In the pop-up window, select Scale, 100%and export all Area List.
Close the TrakEM2 windows and select Yes to save the project. The image of Area List will open and may appear as a blank, black image. In the the main Fiji menu, select Image, Adjust, and Brightness/Contrast.
In the Brightness and Contrast window, click Auto and the Area List will become visible. In the main Fiji menu, select Image, Lookup Tables, and Invert Lookup Table. Save this image of black labels on white background as a t-i-f file and close the image.
Open the Labels file in Fiji and select Plugins, Skeleton, and Skeleton Eyes. Then, save the Skeleton Eyes image as a t-i-f file. To create a new staff project, select Plugins, Macros, and Open/Create Project and follow the prompts to create or update a configuration file.
From the Open/Create project menu, navigate to and select the project directory Input File folder and the Input Movie and Skeleton files. Input the values for the Maximum Measured Speed, the Maximum Speed Mapped, the Minimum Segment Length, the Pixel Size, and the Frame Rate. Check the Flicker correction box if the images have periodic background intensity flicker and click OK.Adjust the parameters for the best visualization of the data.
Set the Maximum Speed Mapped to include about 95 percent of the data, so that the data is mapped across the full range of the color scale. To analyze the skeleton, select Plugins, Macros, and Analyze Skeleton. The skeleton file will open and the Region of Interest manager will open and run.
In the Region of Interest manager, click Show All and Label and click OK.To select the time intervals, select Plugins, Macros, and Edit Time Intervals and wait for the macro to generate a kymograph from a single segment over the total time for the movie. Use the Control plus and minus keys to increase the size of the kymograph as needed and use the Rectangle selection tool to draw a rectangle around each time interval. Press the T key or click the Add button to record a Time Interval selection and repeat the selection for as many time intervals as desired.
To evaluate the parameter choices, select three to four time intervals and complete the analysis for just those intervals. Click OK when the time interval selection has been completed. A pop-up window will appear when the selected intervals have been saved in the project folder.
Click OK.To analyze the flow, select Plugins, Macros, and Analyze Flow. The Analyze Flow Parameters dialogue box will open and display the values entered in the Open/Create Project step. Edit these values if necessary and click OK.The Output File Names dialogue box will open and display the names entered in the Open/Create Project step.
Edit these names as necessary and click OK to begin the Flow Analysis. When a dialogue box appears indicating that the flow has been analyzed, click OK to store the analysis results in CSV Spreadsheet files. To produce spatial maps, select Plugins, Macros, and Produce Spatial Map.
The Spatial Map Parameters window will open displaying the values entered in the Open/Create Project step. Edit these values as necessary and click OK.A temporal sequence of Spatial Maps of Flow Velocities will be generated with each color indicating a flow speed. Scroll through the generated stack of one image per interval to visualize the spatial and temporal variations in flow and save the stack as an a-v-i file to share the file as a movie.
Staff Analysis generates the complete census of the microvascular velocities across entire microscope fields, over periods of time extending from seconds to minutes. For example, using a single image in this time series of the microvascular network in the liver of a mouse, the generated skeletonized image was used to define the axis of the microvascular flow, allowing the Staff generated map of individual vascular segments to be identified for subsequent flow quantification. From the individual vascular segments generated from the skeletonized image, kymographs can be generated representing the intensity along each line segment over time.
The Skeleton and User Supply time intervals can be used to break the kymograph of each segment into individual segment time intervals. Staff then identifies the predominant angle in the kymograph from each segment time interval and provides the calculated velocity measurements as CSV data files. Staff CSV output can be used to analyze the overall velocity distribution to enable plotting of the velocity in the individual vascular segments over time.
And can be visualized as stacks of color-coded velocity map images. Note that the results that came from Staff depend upon the quality of the image series, with respect to the sample stability, frame rate, resolution, and signal-to-noise ratio. The Staff Velocity Map Outputs allow an intuitive exploration of spatial flow patterning while Staff CSV outputs enable the statistical analysis of flow velocities, both within and between treatment groups.
