The femtosecond Coherent X-ray Diffraction Image (CXDI) technique can employ a ‘diffraction before destruction’ principle. An ultrafast and extremely intense X-ray beam of a free electron laser probes a biological sample. Biological samples (e.g. viruses, organelles, entire cells or single macromolecules) are injected into a high vacuum chamber and shortly thereafter exposed to a bright coherent X-ray pulse. The diffraction images can be acquired before radiation damage occurs. We aim to characterize and control bio-samples during the sample injection process to confirm their structural integrity during the diffraction measurement. The optical properties of fluorescent proteins are a sensitive indicator of their conformational state. We therefore measure fluorescence in vacuum from proteins injected into a high vacuum environment. The results show that the proteins are fluorescent in flight at the interaction region where they would be imaged in a CXDI experiment. In the present experiments the fluorescence was measured from multiple proteins in each aerosol droplet. In future experiments we will pursue the single molecule limit.