Abstract Cold atom experiments often use images of the atom clouds as their exclusive source of experimental information. The most commonly used technique is absorption imaging, which provides accurate information about the shapes of the atom clouds, but requires care when seeking the absolute atom number for small atom samples. In this paper, we present an independent, absolute calibration of the atom numbers. We directly compare the atom number detected using dark-ground imaging to the one observed by fluorescence imaging of the same atoms in a magneto-optical trap. We normalise the signal using single-atom resolved fluorescence imaging. In order to be able to image the absorption of the very low atom numbers involved, we use diffractive dark-ground imaging as a novel, ultra-sensitive method of in situ imaging for untrapped atom clouds down to only 100 atoms. We demonstrate that the Doppler shift due to the acceleration of the atoms by the probe beam has to be taken into account when measuring the atom-number.