High-throughput single-cell analysis is a challenging task. Label-free tomographic phase microscopy is an excellent candidate to perform this task. However, in-line tomography is very difficult to implement in practice because it requires a complex set-up for rotating the sample and examining the cell along several directions. We demonstrate that by exploiting the random rolling of cells while they are flowing along a microfluidic channel, it is possible to obtain in-line phase-contrast tomography, if smart strategies for wavefront analysis are adopted. In fact, surprisingly, a priori knowledge of the three-dimensional position and orientation of rotating cells is no longer needed because this information can be completely retrieved through digital holography wavefront numerical analysis. This approach makes continuous-flow cytotomography suitable for practical operation in real-world, single-cell analysis and with a substantial simplification of the optical system; that is, no mechanical scanning or multi-direction probing is required. A demonstration is given for two completely different classes of biosamples: red blood cells and diatom algae. An accurate characterization of both types of cells is reported, despite their very different nature and material content, thus showing that the proposed method can be extended by adopting two alternate strategies of wavefront analysis to many classes of cells.

An approach is proposed for removing the wave front curvature introduced by the microscope imaging objective in digital holography, which otherwise hinders the phase contrast imaging at reconstruction planes. The unwanted curvature is compensated by evaluating a correcting wave front at the hologram plane with no need for knowledge of the optical parameters, focal length of the imaging lens, or distances in the setup. Most importantly it is shown that a correction effect can be obtained at all reconstruction planes. Three different methods have been applied to evaluate the correction wave front and the methods are discussed in detail. The proposed approach is demonstrated by applying digital holography as a method of coherent microscopy for imaging amplitude and phase contrast of microstructures.

This Roadmap article on digital holography provides an overview of a vast array of research activities in the field of digital holography. The paper consists of a series of 25 sections from the prominent experts in digital holography presenting various aspects of the field on sensing, 3D imaging and displays, virtual and augmented reality, microscopy, cell identification, tomography, label-free live cell imaging, and other applications. Each section represents the vision of its author to describe the significant progress, potential impact, important developments, and challenging issues in the field of digital holography.

Particle tracking is a fundamental technique for investigating a variety of biophysical processes, from intracellular dynamics to the characterization of cell motility and migration. However, observing three-dimensional (3D) trajectories of particles is in general a challenging task in classical microscopy owing to the limited imaging depth of field of commercial optical microscopes, which represents a serious drawback for the analysis of time-lapse microscopy image data. Therefore, numerous automated particle-tracking approaches have been developed by many research groups around the world. Recently, digital holography (DH) in microscopy has rapidly gained credit as one of the elective techniques for these applications, mainly due to the uniqueness of the DH to provide a posteriori quantitative multiple refocusing capability and phase-contrast imaging. Starting from this paradigm, a huge amount of 3D holographic tracking approaches have been conceived and investigated for applications in various branches of science, including optofluids, microfluidics, biomedical microscopy, cell mechano-trasduction, and cell migration. Since a wider community of readers could be interested in such a review, i.e., not only scientists working in the fields of optics and photonics but also users of particle-tracking tools, it should be very beneficial to provide a complete review of state-of-the-art holographic 3D particle-tracking methods and their applications in bio-microfluidics.

Digital holography (DH) has emerged as one of the most effective coherent imaging technologies. The technological developments of digital sensors and optical elements have made DH the primary approach in several research fields, from quantitative phase imaging to optical metrology and 3D display technologies, to name a few. Like many other digital imaging techniques, DH must cope with the issue of speckle artifacts, due to the coherent nature of the required light sources. Despite the complexity of the recently proposed de-speckling methods, many have not yet attained the required level of effectiveness. That is, a universal denoising strategy for completely suppressing holographic noise has not yet been established. Thus the removal of speckle noise from holographic images represents a bottleneck for the entire optics and photonics scientific community. This review article provides a broad discussion about the noise issue in DH, with the aim of covering the best-performing noise reduction approaches that have been proposed so far. Quantitative comparisons among these approaches will be presented.