Scaling is a measure of how many microns a pixel represents. Scaling may be different for each axis and is automatically defined for the X and Y axes of each lens when you calibrate the system (Calibration overview). If you're working with an image stack, the Z scaling (also known as the Z step) is equal to the distance between images.
Scaling is embedded for:
Scaling is generally NOT embedded for TIFF files. Make sure that the proper scaling is applied to obtain valid measurements.
You need to know the scaling used at the time of acquisition.
Make adjustments as needed.
Tracing from images without the correct scaling may result in invalid data.