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.

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.

Aberrations and the distortions due to the imaging optics can be compensated in quantitative phase microscopy of thin phase objects by digital holography using a single hologram. The reconstructed quantitative phase microscopy phase distribution map can be directly corrected in the reconstructed image plane by a numerical method. To remove this unwanted aberration, in the special case of thin objects, the authors perform a two-dimensional fit with the Zernike polynomials of the reconstructed unwrapped phase. Subtraction of the fitted polynomial from the original phase map gives quantitative phase microscopy phase map free of aberrations.

The perspective of using live cells as lenses could open new revolutionary and intriguing scenarios in the future of biophotonics and biomedical sciences for endoscopic vision, local laser treatments via optical fibres and diagnostics. Here we show that a suspended red blood cell (RBC) behaves as an adaptive liquid-lens at microscale, thus demonstrating its imaging capability and tunable focal length. In fact, thanks to the intrinsic elastic properties, the RBC can swell up from disk volume of 90 fl up to a sphere reaching 150 fl, varying focal length from negative to positive values. These live optofluidic lenses can be fully controlled by triggering the liquid buffer’s chemistry. Real-time accurate measurement of tunable focus capability of RBCs is reported through dynamic wavefront characterization, showing agreement with numerical modelling. Moreover, in analogy to adaptive optics testing, blood diagnosis is demonstrated by screening abnormal cells through focal-spot analysis applied to an RBC ensemble as a microlens array.

Sperm morphology is regarded as a significant prognostic factor for fertilization, as abnormal sperm structure is one of the most common factors in male infertility. Furthermore, obtaining accurate morphological information is an important issue with strong implications in zoo-technical industries, for example to perform sorting of species X from species Y. A challenging step forward would be the availability of a fast, high-throughput and label-free system for the measurement of physical parameters and visualization of the 3D shape of such biological specimens. Here we show a quantitative imaging approach to estimate simply and quickly the biovolume of sperm cells, combining the optical tweezers technique with digital holography, in a single and integrated set-up for a biotechnology assay process on the lab-on-a-chip scale. This approach can open the way for fast and high-throughput analysis in label-free microfluidic based “cytofluorimeters” and prognostic examination based on sperm morphology, thus allowing advancements in reproductive science.