Ratio Output Process Description

Ratio Output Process Description:

This process allows you to export the ratio images you have created in step 10 (or step 11, if photobleach correction has been applied) as .tif images. This requires multiplying the images by a large number called a "scale factor". This is necessary because the ratio images themselves are small, non-integer values (usually between 0.5 and 5), while .tif images can only store positive integer values. If these ratios are converted to integers without multiplying by a scale factor, there would be severe rounding error in the images. Alternatively, you can use the ratio images themselves, which are stored as floating-point MATLAB .mat files in the movie's output directory.

Parameter Descriptions:

Ratio Channel:

This box allows you to select the channel which is the NUMERATOR of the ratio images you want to export (usually the FRET channel).

TIF export options:

Customize color limits

If checked, it enables the "color limits options" panel, and allows you to manually specify the color scale of the ratio output tiff images.

Color limits options:

Manually set color limits by values

If checked, it allows you to manually specify the color scale limits by values. If unchecked, it enables the "manually set color limits by percentages as decimals" panel, allowing you to manually specify the lower and upper color scale limits based on the percentage cut-offs.

Low-end color limit value:

This allows you to specify the lower bound of the color scale. The minimum allowed value is the lowest color scale value across all ratio output images.

High-end color limit value:

This allows you to specify the upper bound of the color scale. The maximum allowed value is the highest color scale value across all ratio output images.

Low-end color limit cut-off:

This allows you to specify the lower cut-off for the color scale as a decimal percentage. For example, setting this to 0.05 will replace the bottom 5% of data in the color scale range with the new minimum.

High-end color limit cut-off

This allows you to specify the upper cut-off for the color scale as a decimal percentage. For example, setting this to 0.1 will replace the top 10% of data in the color scale range with the new maximum.

Select Path:

This allows you to specify the directory to save the .tif ratio images to. They will be saved to a subdirectory of this folder called "ratio_tiffs", with one .tif file per ratio image.

Make ratio movie:

This allows you to export the ratio output as a movie (in .avi and/or .mov format).

Movie options:

Use a constant scale throughout the movie:

If checked, the color scale used for the first image will be used throughout the movie. Otherwise, the range will be selected separately for each frame.

Display color bar:

If checked, a bar showing the color scale, i.e., the ratio value associated with each color in the movie, will be displayed.

Create .avi/.mov movie:

If checked, the movie will be saved as a .avi/.mov.