In recent years, in-line holography as originally proposed by Gabor, supplemented with numerical reconstruction, has been perfected to the point at which wavelength resolution both laterally and in depth is routinely achieved with light by using digital in-line holographic microscopy (DIHM). The advantages of DIHM are: (1) simplicity of the hardware (laser- pinhole-CCD camera), (2) magnification is obtained in the numerical reconstruction, (3) maximum information of the 3-D structure with a depth of field of millimeters, (4) changes in the specimen and the simultaneous motion of many species, can be followed in 4-D at the camera frame rate. We present results obtained with DIHM in biological and microfluidic applications. By taking advantage of the large depth of field and the plane-to-plane reconstruction capability of DIHM, we can produce 3D representations of the paths followed by micron-sized objects such as suspensions of microspheres and biological samples (cells, algae, protozoa, bacteria). Examples from biology include a study of the motion of bacteria in a diatom and the track of algae and paramecium. In microfluidic applications we observe micro-channel flow, motion of bubbles in water and evolution in electrolysis. The paper finishes with new results from an underwater version of DIHM.