Controls and standards:
Often times it is easy to get lost in the process of image quantitation rather than controlling the parameters. For example, it is easy to start writing the specifications of the camera, intensity of light, how many pixels per inch while having no control of what can be done.
Next time doing image analysis think about the parameters that are under your control and those that are not. Measurement of geological data from air obviously has much more out of their control than working an a microscope but in both cases as someone famous has said “There are known unknown’s and then there are unknown unknowns”
Controls and standards help decrease the risk of uncontrollable factors changing the quantitation and interpretation but is not fool proof. It is also amazing to note that with imaging how many people forget the controls instead stating that “If I see a green color under a microscope then I will know that the protein is present” Use beads, standard fluorescent probes or anything that you can use as standards and match it with appropriate controls.