A system for three dimensional imaging of motile objects includes an image sensor and a sample holder disposed adjacent to the image sensor. A first illumination source is provided and has a first wavelength and positioned relative to the sample holder at a first location to illuminate the sample. A second illumination source is also provided having a second wavelength, different from the first wavelength, and positioned relative to the sample holder at a second location, different from the first location, to illuminate the sample. The first and second illumination sources are configured to simultaneously, or alternatively, sequentially illuminate the sample contained within the sample holder. Three dimensional positions of the motile objects in each frame are obtained based on digitally reconstructed projection images of the mobile objects obtained from the first and second illumination sources. This positional data is connected for each frame to obtain 3D trajectories of motile objects.