If you set up your online ratio calculation and select the Single wavelength (F/F0) method for the calculation of the online ratio, you need to define a reference image.

1. You are setting up an online ratio calculation (see also Setting up the Online Ratio Calculation).
2. You have selected the Single wavelength (F/F0) as the calculation method.

1. In the Online Ratio tab, in the input field of Reference Image Set-up, enter the number of images from which the reference image should be averaged.
2. Click on Define.

1. The images are acquired, and a reference image is calculated from them. You can now continue to set up the online ratio calculation.