The Pixel Colocalization recipe takes in three input channels and can generate up to three output channels. An output channel can be turned off by de-selecting the checkbox next to its name in the Input and Output section.
There are three inputs to the recipe, and their descriptions are summarized in the table below.
Input Channel | Description |
---|---|
Input Mask Channel | This input defines the image region that the colocalization analysis will be applied to. The Mask channel serves as an initial region-based filter for colocalization (for example: to demarcate cells from extracellular regions when analyzing spatial overlap between intracellular proteins). Colocalized signal that falls outside of the Mask channel region will not be included in the recipe output. Typically, this input is the same as one of the two input colocalization channels. |
Input Coloc Channel 1 | This input specifies the first of the two input image channels used for analyzing pixel colocalization. |
Input Coloc Channel 2 | This input specifies the second of the two input image channels used for analyzing pixel colocalization. |
An example of how each input channel is used is shown on the image on the right.
There are three possible outputs from the recipe which can be specified in the Input and Output section of the recipe. Descriptions of the outputs are in the table below.
Output Channel | Description |
---|---|
Colocalized Pixels Ch1 | This output contains all image regions from Input Coloc Channel 1 that are above the user-defined threshold; pixels that are below the threshold or outside of the defined Mask Channel area are suppressed. This output is turned on by default. |
Colocalized Pixels Ch2 | This output contains all image regions from Input Coloc Channel 2 that are above the user-defined threshold; pixels that are below the threshold or outside of the defined Mask Channel area are suppressed. This output is turned on by default. |
Combined Coloc Pixels | This output contains all image regions whose pixel intensities are above the user-defined threshold for both Input Coloc Channels 1 and 2 (or colocalized). The pixel intensity of this output is the average intensity of both Input Coloc Channels. |
Recipe parameters for Pixel Colocalization and their descriptions are summarized in the table below.
Preset Group | Parameter Name | Min Value | Max Value | Description |
---|---|---|---|---|
Mask Detection | Min Intensity Threshold | 0 | 255 (8-bit) 65,535 (16-bit) | Specifies the minimum intensity for image region detection; the image region defines the area that the colocalization analysis will take place; a lower value will incorporate a larger area of the image for colocalization analysis |
Fill Holes Size | 0 | 50,000 | Adjusts the maximum size threshold for filling in gaps inside the detected image region; a lower value will preserve more holes in the detection | |
Smoothing Factor | 1 | 100 | Adjusts the amount of smoothing applied to the surface reconstructions of the detected image region; a lower value will preserve more of the region's morphological features | |
Channel 1 Detection | Min Intensity Threshold | 0 | 255 (8-bit) 65,535 (16-bit) | Specifies the minimum signal intensity for the first of the two input image channels used for colocalization analysis; a lower value will detect more, dimmer signals |
Min Colocalized Pixels | 0 | 50,000 | Specifies the minimum area of contiguous pixels to be included in the analysis output | |
Channel 2 Detection | Min Intensity Threshold | 0 | 255 (8-bit) 65,535 (16-bit) | Specifies the minimum signal intensity for the second of the two input image channels used for colocalization analysis; a lower value will detect more, dimmer signals |
Min Colocalized Pixels | 0 | 50,000 | Specifies the minimum area of contiguous pixels to be included in the analysis output |
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