Use case: tracing electrode tracks

You can trace electrode tracks in Lasagna with Plugins > Annotation plugins > Add line plugin. On an Apple laptop you will need an external mouse.

Steps

Load image stacks

• Load the image stacks with File > Load ingredient > New image stack. The whole stack is loaded into RAM. During loading the GUI is unresponsive. Just wait and it will load.
• You may load multiple channels and they will be automatically overlaid in different colours.
• If you see nothing when the images are loaded, just change the look-up table by dragging the slider box in the histogram.
• If you wish to change the displayed orientation of the sample go to the "axis" tab and use the "flip stacks" button. e.g. if the sample is flipped in the coronal plane, click the third button.
• Lasagna assumes samples are isotropic. Say your data were acquired at 5x5x20 microns. To get square images you should go to the Axis tab and set "View 2" to 4 and "View 3" to 0.25

Starting the plugin

• Start the plugin: Plugins > annotation_plugins > add_line_plugin
• Click on the "Points" tab.
• Select "Piecewise" from the drop-down selector box under Add/Remove.

Exploring the stack

Scan through the stack to get a feel for where the track is. Following the instructions in Navigating data to pan, zoom, and slice the data. Note that with the plugin started, a left-click will add a point but you can still click and drag without adding a point. The autofluoresence may well look grainy, but the tracks should be bright.

Tracing a track

• If you have multiple tracks, name your first track in the GUI.
• Use left-click to place the first point at the brain surface where the electrode entered. You may click on any of the axes, but the coronal plane may be easiest.
• Continue scrolling through the brain and adding points. New points are appended to the end of the line.
• You can extend the range of depths over which the line and points are visible by altering the "z-spread" values. Separate values for each axis. Likely you will want quite large Z-spread numbers for the non-coronal views as the program currently uses distance to the nearest clicked point to decide what to show.
• You change the colour of the points or lines by clicking on the "ingredient" in the list (the points are called "addLine_currentLine") then changing the color, line width, etc, with the options below. The line is "addLine_fit_currentLine. We are aware this is naming system is not intuitive.

Modifying the track

• If you make a mistake, you can delete a point by selecting the ""Remove" radio button. The highlighted point nearest the mouse will be removed when you right-click. Return to "Add" mode when done.
• You may go back an insert points between others by right-clicking on a point. It now becomes highlighted. You can highlight a different point, again with a right-click, if needed. You may cancel insert mode by right-clicking again near the highlighted point. The next point you add by left-clicking will be added between the highlighted point and the one before it.

Committing the track

Once you are happy with the track hit "Add it!" in the add_line_plugin GUI. The points and fit will change colour and be named according to the name you previously entered in the plugin window. Save the track by right-clicking on the ingredient containing the string _pts. The plugin is new and might crash: save tracks once created. To a new line, enter a new name in the plugin GUI and start clicking on a new track.