Once a population of nuclei has been imported (either by a new analysis or loading an existing dataset), an attempt can be made to find nuclear signals.
Right click the population of nuclei in the populations panel and select 'Add nuclear signal'.
You will be asked to choose a folder containing the images with signals. A detection window will then be displayed, similar to the new analysis setup. The outlines of nuclei detected previously will be shown in blue, and the detected signals will be outlined in yellow (if they meet filtering criteria) or red (if they fail to meet filtering criteria).
Set information about the images to be analysed.
|H&E||Is the image H&E stained, or fluorescence?|
|Channel||The RGB colour channel containing the signal (set to Greyscale if the image is black and white)|
|Scale||The number of pixels per micron. Allows the real size of images to be entered, so statistics can be presented in microns as well as in pixels. This can be updated after an analysis is complete. By default, this is copied from the nuclei.|
|Threshold||The pixels must be equal to or brighter than this to be counted|
|Method||One of the available methods for finding a signal, described below and in the dialog window.|
|Forward||Takes all objects with pixels over the threshold, meeting the size requirements. If there is a lot of bright background, it can mistake this for signal.|
|Reverse||Starts with the brightest pixels (intensity 255), and tries to detect objects meeting size and shape criteria. If it fails, it looks at pixels with intensity 254 or above. This recurses until either a signal is found, or the signal threshold is reached.|
|Adaptive||The intensity histogram within the nuclear bounding box is trimmed to the minimum signal threshold defined in the options, then scanned for the position with maximum dropoff (formally, in the delta profile, the local minimum (a) below zero (b) with an absolute value greater than 10% of the total intensity range of the trimmed profile (c) with the highest index). Since this position lies in the middle of the dropoff, a (currently) fixed offset is added to the index to remove remaining background. This index is used as the new threshold for the detector. If a suitable position is not found, we fall back to the minimum signal threshold defined in the options.|
|Min area||Signals must have an area in pixels greater than this|
|Max fraction||The signal cannot be larger than this fraction of the nuclear area|
|Min and max circ||Circularity measures (see nucleus detection). Default is to accept anything.|
Start the analysis
When ready, click the 'Proceed with detection' button in the lower right to be begin finding signals.
The images must have the same names as the DAPI images from which the nuclei were originally found. IE, if a nucleus came from an image named
P22.tif, the signal must also be in an image named
After the signal detection has run, the resulting populations are displayed in the populations panel. New child populations are created for the nuclei with signals in the channel, and for nuclei without signals in the channel.