Neurolucida is useful for semi-automated and manual tracing of neurons and is installed on the offline workstations.
For full information on the capabilities of Neurolucida, as well as training videos please visit their website.
In addition to tracing processes and identifying spines on individual neurons, Neurolucida can also be used to automatically contour and count cells on images scanned by the slide scanners. This kind of analysis enables:
- Rapid and automatic contouring of individual sections and subregions
- Automatic detection of labelled cells based on fluorescence intensity or bright field colour maker within selected regions
- 3D reconstruction of contoured sections and detected spots
- Tools for quick and easy navigation of full data set during and after analysis.
- Allows for multiple slide images to be included in a single analysis
Using Neurolucida with Slide Scanner Images:
1. Create a working profile specifically for brain mapping (Dan or I can help you do this). This profile should include 1)macro view, 2) serial section manager, 3) image organiser
2. Load your data into Neurolucida, it is possible to load several image files at once.
3. Contouring – There are two automated options to draw contours around tissue and regions of interest.
- Automatic Object detection
- In the top menu select Image > Automatic Object Detection > Outline Detected Objects
- Adjust object detection parameters as necessary to accurately detect individual tissue sections – request help if you have trouble with this
- Click “outline objects”
- Once you have some reliable parameters you are able to save these for easy reuse
- Automatic Contour Tracing
- Right click on image – choose automatic contouring
- Click to place the first few points of the contour – this gives the software an idea of what you are trying to trace
- Start by clicking play
- Have autoclose contour enabled in the advanced settings (within 20px)
- Tracing a 3D section
- When tracing contours in a section that is more than one plane thick the number of contours may make it difficult to keep tracing.
- To set the depth of what contours are displayed – in the menu Display> Display Settings> Accessories > Orthagonal View then choose Z filter, select Symmetric – use the arrows on the right of the orthagonal view window to select the depth range for contour visibility
- https://support.mbfbioscience.com/index.php?action=kb&article=81
4. Editing Contours
- Click the “Where is mode” button (box with ? mark) – this shows all contours
- click “edit mode” (arrow)
- Draw a box over parts of the contour you don’t want
- Delete those points by pressing delete and the contour will update
- Repeat this for all contours which need adjustment
- https://support.mbfbioscience.com/index.php?action=kb&article=94
5. Detect Cells of Interest in contours
- In the top menu select Image > Automatic Object Detection > Mark Detected Objects
- Select “use existing ROIs” – then click on each tissue section ROI you would like to include in the cell count analysis
- Once the analysis has finished you can use Where is mode to see where the objects were found
- You can use “go to” to instantly move to an area of interest on the image
6. Aligning Contours for 3D analysis
- Associate each section with a Z position
- Run Serial Section Setup from the serial section manager
- Add information on the section cut thickness, evaluation interval and the number of sections
- The serial section manager will the list these sections
- Add contours to the appropriate tissue section by clicking on a contour using the arrow tool (this is more easily done when in where is mode), then right clicking and choosing add contour to section
- Instantly center all contours
- It is possible to instantly centre all contours by pressing Ctrl-F
- This will align the contours for each section over the centre of the very first section
- Manually align contours
- Choose show only selected section in the serial section manager
- This allows you to scroll through sections using the up and down arrow keys
- Keeps the position the same as you move through sections so is a great way to follow a structure of population of cells
- Then choose display flanking section
- Allows you to see image + tracing + tracings of neighbouring contours
- Select move images and tracing
- Align image and contours with previous section – do this for each subsequent section
- Choose show only selected section in the serial section manager
Related Tutorial Videos:
Using contours & markers
Shortcuts:
Alt-N / Alt-E = cycles up and down through markers or contours
It is possible to instantly centre all contours by pressing Ctrl-F