2 Calculation and visualization
Although there are many parameters exposed in the API, most of them should stay as default.
A few of them are useful, but require no adjustment:
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LRstack: input your wide-field reference; -
pixelSize: input your pixel size;
There are only two parameters should be adjusted according to your data and specific usage.
Scale bar: 5μm.
This parameter should be selected to remove background area calculation but avoiding to overfilter the image content.
Although the rFRC allows evaluation at the pixel level, the most delicate scale of detectable errors can only reach the highest resolution allowed by the system, which satisfies the Nyquist-Shannon sampling theorem. Thus, the smallest error should be larger than 3 × 3 pixels. Therefore, we can skip 2~4 pixels for each rolling operation to accelerate the mapping calculation 4~16 times. The rFRC map will be resized to the original image size by a interpolation for better visualization.
Choosing a proper color map to visualize error maps is another tricky question. The existing popular color maps, such as Jet, use blue to red to index the different error magnitudes. However, people usually tend to define black (dark color) as small magnitude and white (light color) as large magnitude, which is identical to the logic of the gray color map. In this sense, the Jet color map may be incompatible with human intuition. On the other hand, human vision is insensitive to light or dark gray levels and sensitive to different colors. As a result, we intend to create a color map using color to index the magnitudes and with black/white zone to visualize the smallest/largest values.
First, because human eyes are more sensitive to green color, we use green to highlight errors of large magnitude. Second, human instinct usually regards bright color (white) as an effect of large magnitude and dark color (black) for small magnitudes. Therefore, we involve a black zone (0, 0, 0) and a white zone (1, 1, 1) in the color map to visualize the smallest and largest values. Taken together, we shift the Jet colormap (left panel) to create the shifted Jet (sJet) color map (right panel). Along with the extension of the blue color component in this sJet color map, we obtain a white zone to represent the most significant error (even larger than those highlighted in green). Because the background in the rFRC map means no error, we use the black zone for the display. As shown in the following figure, our sJet color map is more intuitive for visualizing errors than the original Jet color map.
In addition to the sJet colormap, we also provided another alternative colormap, i.e., Jet with the black zone (bJet, middle panel) while using red color to represent large magnitude. The readers are encouraged to try these colormaps and select their favorite ones.
The user can find here, the sJet.lut and bJet.lut at here. Please right click the file name and then "save target as" to download the file to your local drive.