We first briefly review the state of the art of digital in-line holographic microscopy (DIHM) with numerical reconstruction and then discuss some technical issues, such as lateral and depth resolution, depth of field, twin image, four-dimensional tracking, and reconstruction algorithm. We then present a host of examples from microfluidics and biology of tracking the motion of spheres, algae, and bacteria. Finally, we introduce an underwater version of DIHM that is suitable for in situ studies in an ocean environment that show the motion of various plankton species.

Few instruments exist that can image microscopic marine organisms in their natural environment so that their locomotion mechanisms, feeding habits, and interactions with surfaces, such as biofouling, can be investigated in situ. We describe here the design and performance of a simple submersible digital in-line holographic microscope that can image organisms and their motion with micron resolution and that can be deployed from small vessels. Holograms and reconstructed images of several microscopic aquatic organisms were successfully obtained down to a depth of 20m. Important microscope characteristics such as the effect of camera pixel size on lateral and depth resolutions as well as the maximum sample volume that can be imaged with a given resolution are discussed in detail.

The utilization of microscope objectives (MOs) in digital holographic microscopy (DHM) has associated effects that are not present in conventional optical microscopy. The remaining phase curvature, which can ruin the quantitative phase imaging, is the most evident and analyzed. As phase imaging is considered, this interest has made possible the development of different methods of overcoming its undesired consequences. Additionally to the effects in phase imaging, there exist a set of less obvious conditions that have to be accounted for as MOs are utilized in DHM to achieve diffraction-limit operation. These conditions have to be considered even in the case in which only amplitude or intensity imaging is of interest. In this paper, a thorough analysis of the physical parameters that control the appropriate utilization of MOs in DHM is presented. A regular DHM system is theoretically modeled on the basis of the imaging theory. The Fourier spectrum of the recorded hologram is analyzed to evaluate the performance of the DHM. A set of the criteria that consider the microscope features and the recording parameters to achieve DHM operation at the diffraction limit is derived. Numerical modeling and experimental results are shown to validate our findings.

Digital in-line holographic microscopy is a promising new tool for high resolution imaging. We demonstrate, by using latex beads, that a considerable increase in numerical aperture, and, therefore, resolution can be achieved if the space between a source and a CCD camera chip is filled with a high refractive index medium. The high refractive index medium implies a shorter effective wavelength so that submicrometer resolution can be obtained with laser light in the visible range.

In this work, Fourier integral microscope (FIMic), an ultimate design of 3D-integral microscopy, is presented. By placing a multiplexing microlens array at the aperture stop of the microscope objective of the host microscope, FIMic shows extended depth of field and enhanced lateral resolution in comparison with regular integral microscopy. As FIMic directly produces a set of orthographic views of the 3D-micrometer-sized sample, it is suitable for real-time imaging. Following regular integral-imaging reconstruction algorithms, a 2.75-fold enhanced depth of field and [Formula: see text]-time better spatial resolution in comparison with conventional integral microscopy is reported. Our claims are supported by theoretical analysis and experimental images of a resolution test target, cotton fibers, and in-vivo 3D-imaging of biological specimens.