2.1	Draw a vertical line, a few pixels wide crossing the reflection line in the image, in its middle. Get a line profile.
2.2	Record the peak values for each time point and calculate the mean value and the short term variation.
2.3	Compare the mean value with those obtained previously, with exactly the same instrument settings (Figure 4).
2.4	Repeat measurements close to the two ends of the reflection line. Differences from the central value reflect the flatness of field. This is particularly important if intensity of small objects dispersed in the image is measured: the ones on the periphery may appear less intense than the ones in the middle (Figure 6).
2.5	Follow the variation with time of the position of the maximum on the graph’s x-axis, corresponding to the z position of the coverslip’s surface. This may be caused by mechanical instability in the stage, the galvo control of the stage or variations in temperature affecting the refractive index of the immersion medium. Record the magnitude of any observed fluctuations or drift (constant movement in one direction). This is particularly important to know when attempting time lapse live cell experiments (Figure 4).
2.6	Repeat measurements for all channels/laser lines.
2.7	Measure the distance on the x-axis between the maxima of each channel to the next. Any distance between peaks of different channels indicates lack of registration between channels on the z-axis. These values are particularly important to know when attempting to measure colocalization in a confocal stack. Axial registration (along the z-axis) is more difficult to achieve than lateral (in x and y) (Figure 5).