Region of Interest (ROI) Tools (Galvo)

SciScan provides several tools to aid in monitoring and conducting experiments on specific regions of interest.

Select the ROI Tab in the channels bar. The channel to work on can be selected using the menu on the right hand side of the image window.

Three main tools are available:

  • Move to ROI
  • Zoom to ROI
  • ROI Monitoring

Move to ROI

The move to ROI function allows users to select a point on the reference image and move that point to the centre of the field of view, which can be very useful in centring a structure to zoom in on.

Move to ROI
Point to move to centre of field marked with green crosshair.

Use

Select the single point ROI tool from the ROI tool menu and click on the point in the image that you would like to centre. Click “Move to Point”. SciScan will move the XY motors to re-position the selected structure in the middle of the field.

 

Same point now centred
Same point now centred

Please note: If system moves opposite to expected, reverse the incorrect axis using the reverse XYZ buttons on in the motor control tabs.

Move to ROI function relies on a correct calibration of the field of view size.  A warning will prompt users to ensure calibration is correct before continuing.

ROI Callibration Warning
ROI Callibration Warning

Zoom to ROI

The Zoom to ROI function was developed with laser ablation experiments in mind but can also be used to image specific structures within the field of view. When used, SciScan will automatically update the frame geometry to zoom in and scan a selected region.

Use

Select the rectangle ROI tool from the right hand menu and draw a box around the desired region. Click the “Zoom to ROI” button.

Marked ROI
Marked ROI

 

Zoomed in ROI Region
Zoomed in ROI Region

SciScan will adjust the number of lines (Y Resolution) to match the aspect ratio of the selected region while maintaining the current X resolution and pixel size. This may result in a change in frame rate depending on the aspect ratio of the ROI. Scanning parameters can be adjusted once the ROI has been zoomed into using the arbitrary frame scan settings in the Analogue Input Tab but may also affect the size and shape of the ROI.

Adjusted frame scanning parameters to fit with selected ROI.
Adjusted frame scanning parameters to fit with selected ROI.

ROI Monitoring

SciScan’s ROI monitoring feature allows you to draw a region of interest and plots a graph of the mean grey value of the pixels within that region over time. Multiple ROIs can be selected and monitored simultaneously. This can give a good indication of how fluorescence changes over time e.g. In response to electrical or sensory stimulation; particularly useful for calcium imaging.

ROI Monitor Tab (Resonant)
ROI Monitor Tab (Galvo)

Please note: This feature is intended only for online guidance during the experiment and should not replace a detailed offline analysis of the recorded images.

Please note: At very high frame rates SciScan prioritises saving and displaying data and the module may not be capable of calculating the mean pixel intensity fast enough. Data points on the graph will be dropped from the graph.

Use

Select the ROI monitoring feature by click on the ROI tab in the channels bar

  • Click on channel (right hand side of display window) to select which PMT you would like to monitor then click Focus to bring up a live image
  • Select any of the available tools (freehand, polygon, ellipse ect…) to add a ROI to the image. All ROIs must be closed loops, SciScan will not measure pixel intensities over time for a line
  • Hold the CTRL key to add additional ROIs
  • Clicking anywhere on the image without CTRL pressed will remove all active ROIs
  • The graph at the bottom of the pane will update automatically with the mean grey values for the pixels within each ROI, assigning a different colour to each for easy recognition.
  • Right click on the graph to export as a simple image or .csv file

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