The signals overview tab shows an aggregate of the positions of nuclear signals. However, this is just based on drawing circles of about the right size at the position of the signal centre of mass:
Irregularly shaped signals may look different. Signal warping can be used to fit the signals from one nucleus onto the shape of another nucleus. The method is described in more detail in our paper here.
When warping signals, there are a number of choices:
- the dataset to take nuclei from - this is the dataset with the signals
- the dataset to warp the signals onto - this is a dataset with a consensus nucleus. It can be the same as the source dataset
- the signal group to be warped - these are the particular FISH signals you want to warp
How to warp signals
Select the datasets to ba available in the populations panel. This means select the datasets you want to take signals from, and any datasets you want to warp signals onto. All selected datasets must have a consensus nucleus. All selected datasets must have the same segmentation pattern.
Switch to the nuclear signals overview tab, and click the 'Warp signals' button. The button will be greyed out if the selected datasets are not suitable for warping.
The signal warping dialog will open. Configure the warping you want to run.
|Source dataset||The dataset that FISH signals are taken from|
|Signal group||The FISH signal group to use|
|Target dataset||The consensus shape to warp signals onto|
|Min threshold||Threshold signal images before warping; removes any signal below the given threshold. This can help to remove background if the images are very noisy. The default value is the threshold initially used for signal detection.|
|Binarise||Should images be converted to binary before warping. This can help avoid skewing due to different signal intensities in the images.|
|Only include cells with signals||Tick to only warp nuclei for which signals were detected, and are drawn in the nuclear signal overview chart. This allows an extra level of background filtering. Untick if you know your signals were difficult to detect.|
- Once you have set up the analysis, click 'Run'. A progress bar will show the warping progress. When the warping is complete, the warped result will be added to the left table.
Visualising warped signals
- Click a row in the table to load the warped image. Images can be adjusted via the 'Display' options.
|Pseudocolour signals||If ticked, the warped signal will be pseudocoloured with the signal group colour (this can be changed in the signal overview tab by double clicking the signal group colour). If unticked, the warped signal will be drawn in greyscale, with black representing the most intense signal. If multiple warped signals are selected, pseudocolours help show the overlap of territories.|
|Enhance signals||This has no effect if a single warped image is selected. If multiple warped images are selected, the pseudocoloured signals are rescaled to make them stand out more against each other.|
|Threshold||Apply a minimum threshold to a warped signal to explore the region of greatest signal intensity. The default is to have no threshold|
Select multiple rows to see a composite image of the warped signals
If at least two warped images onto the same target shape are present, the 'MS-SSIM' can be used to calculate the multi-scale structural similarity index between them.
##Exporting warped signals
Images can be exported in two ways by right-clicking the image:
1) 'Export image' will save a TIFF file of the warped signals without the nuclear outline or chart decorations.
2) 'Save as>PNG' will save an image of the chart including the nuclear outline and chart decorations.
See the screenshots below for examples of the difference.
|The Nuclear signals overview tab. The consensus nucleus with basic signal positions from each nucleus in the dataset|
|The signal warping dialog window.|
|The final result of the signal warping. All signals have been aggregated onto the consensus. The result is a heat-map of where signals were observed in the population of nuclei|
|Resulting images can be exported in two ways. Right-click the image; 'Export image' will save a TIFF file of the warped signals without the nuclear outline or chart decorations. 'Save as>PNG' will save an image of the chart including the nuclear outline and chart decorations.|
|Example of the exported image without chart decoration|
|Example of the exported image with chart decoration|