AI-aided holographic flow cytometry for label-free identification of ovarian cancer cells in the presence of unbalanced datasets.

Liquid biopsy is a valuable emerging alternative to tissue biopsy with great potential in the noninvasive early diagnostics of cancer. Liquid biopsy based on single cell analysis can be a powerful approach to identify circulating tumor cells (CTCs) in the bloodstream and could provide new opportunities to be implemented in routine screening programs. Since CTCs are very rare, the accurate classification based on high-throughput and highly informative microscopy methods should minimize the false negative rates. Here, we show that holographic flow cytometry is a valuable instrument to obtain quantitative phase-contrast maps as input data for artificial intelligence (AI)-based classifiers. We tackle the problem of discriminating between A2780 ovarian cancer cells and THP1 monocyte cells based on the phase-contrast images obtained in flow cytometry mode. We compare conventional machine learning analysis and deep learning architectures in the non-ideal case of having a dataset with unbalanced populations for the AI training step. The results show the capacity of AI-aided holographic flow cytometry to discriminate between the two cell lines and highlight the important role played by the phase-contrast signature of the cells to guarantee accurate classification.

Phase contrast tomography at lab on chip scale by digital holography.

High-throughput single-cell analysis is a challenging target for implementing advanced biomedical applications. An excellent candidate for this aim is label-free tomographic phase microscopy (TPM). In this paper, some of the methods used to obtain TPM are reviewed, analyzing advantages and disadvantages of each of them. Moreover, an alternative tomographic technique is described for live cells analysis, and future trends of the method are foreseen. In particular, by exploiting random rolling of cells while they are flowing along a microfluidic channel, it is possible to obtain phase-contrast tomography thus obtaining complete retrieval of both 3D-position and orientation of rotating cells. Thus, a priori knowledge of such information is no longer needed. This approach extremely simplifies the optical system avoiding any mechanical/optical scanning of light source. The proof is given for different classes of biosamples, red-blood-cells (RBCs) and diatom algae. Accurate characterization of each type of cells is reported and compared to that obtained by other tomographic techniques.

Red blood cell as an adaptive optofluidic microlens.

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.

Active accumulation of very diluted biomolecules by nano-dispensing for easy detection below the femtomolar range.

Highly sensitive detection of biomolecules is of paramount interest in many fields including biomedicine, safety and eco-pollution. Conventional analyses use well-established techniques with detection limits ~1 pM. Here we propose a pyro-concentrator able to accumulate biomolecules directly onto a conventional binding surface. The operation principle is relatively simple but very effective. Tiny droplets are drawn pyro-electro-dynamically and released onto a specific site, thus increasing the sensitivity. The reliability of the technique is demonstrated in case of labelled oligonucleotides diluted serially. The results show the possibility to detect very diluted oligonucleotides, down to a few hundreds of attomoles. Excellent results are shown also in case of a sample of clinical interest, the gliadin, where a 60-fold improved detection limit is reached, compared with standard ELISA. This method could open the way to a mass-based technology for sensing molecules at very low concentrations, in environmental as well as in diagnostics applications.

Particle tracking by full-field complex wavefront subtraction in digital holography microscopy.

The 3D tracking of micro-objects, based on digital holography, is proposed through the analysis of the complex wavefront of the light scattered by the micro-samples. Exploiting the advantages of the off-axis full-field holographic interferometry, the tracking of multiple objects is achieved by a direct wavefront analysis at the focal plane overcoming the limitation of the conventional back focal plane interferometry in which only one object at a time can be tracked. Furthermore, the method proposed and demonstrated here is a step forward with respect to other holographic tracking tools. The approach is tested in two experiments, the first investigates the Brownian motion of particles trapped by holographic optical tweezers, while the second relates to the cell motility in a 3D collagen matrix, thus showing its usefulness for lab-on-chip systems in typical bioassay testing.

Graded-size microlens array by the pyro-electrohydrodynamic continuous printing method.

In the present work, the pyro-electrohydrodynamic technique was used for the realization of tunable-size microlens arrays. Poly(methyl methacrylate) dissolved in different solvent mixtures was used as the polymeric material for the realization of the microstructures. By controlling the experimental parameters and in particular, the volume of the drop reservoir, graded-size square arrays of tens of microlenses with focal length in the range 1.5-3 mm were produced. Moreover, the optical quality and geometrical features were investigated by profilometric and interferometric analysis.

Digital holography as a method for 3D imaging and estimating the biovolume of motile cells.

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.

Imaging live humans through smoke and flames using far-infrared digital holography.

The ability to see behind flames is a key challenge for the industrial field and particularly for the safety field. Development of new technologies to detect live people through smoke and flames in fire scenes is an extremely desirable goal since it can save human lives. The latest technologies, including equipment adopted by fire departments, use infrared bolometers for infrared digital cameras that allow users to see through smoke. However, such detectors are blinded by flame-emitted radiation. Here we show a completely different approach that makes use of lensless digital holography technology in the infrared range for successful imaging through smoke and flames. Notably, we demonstrate that digital holography with a cw laser allows the recording of dynamic human-size targets. In this work, easy detection of live, moving people is achieved through both smoke and flames, thus demonstrating the capability of digital holography at 10.6 µm.

Printing of polymer microlenses by a pyroelectrohydrodynamic dispensing approach.

The investigation of a method for fabricating microlenses by a nozzle-free inkjet printing approach is reported. The new method, based on a pyroelectrohydrodynamic mechanism, is also able to dispense viscous liquids and to draw liquid phase drops directly from the reservoir. Specifically, by dispensing optical grade polymer dissolved in different solvent mixtures, microlenses were printed with a pattern defined directly through this deposition method. The reliability of the microlenses and the tunability of their focal properties were demonstrated through profilometric and inteferometric analyses.

Driving and analysis of micro-objects by digital holographic microscope in microfluidics.

We propose an optical configuration in which floating particles in a microfluidic chamber can be characterized by an interference microscopy configuration to obtain quantitative phase-contrast maps. The configuration is simply made by two laser beams from the same laser source. One beam provides the optical forces for driving the particle along appropriate paths, but at same time works as the object illumination beam in the holographic microscope. The second beam plays the role of the reference beam, allowing recording of an interference fringe pattern (i.e., the digital hologram) in an out-of-focus image plane. The system and method are illustrated and experimental results are offered for polymeric particles as well as for in vitro cells with the aim to demonstrate the approach.

Digital self-referencing quantitative phase microscopy by wavefront folding in holographic image reconstruction.

A completely numerical method, named digital self-referencing holography, is described to easily accomplish a quantitative phase microscopy for microfluidic devices by a digital holographic microscope. The approach works through an appropriate numerical manipulation of the retrieved complex wavefront. The self-referencing is obtained by folding the retrieved wavefront in the image plane. The folding operation allows us to obtain the correct phase map by subtracting from the complex region of interest a flat area outside the microfluidic channel. To demonstrate the effectiveness of the method, quantitative phase maps of bovine spermatozoa and in vitro cells are retrieved.

Tunable liquid microlens arrays in electrode-less configuration and their accurate characterization by interference microscopy.

A special class of tunable liquid microlenses is presented here. The microlenses are generated by an electrowetting effect under an electrode-less configuration and they exhibit two different regimes that are named here as separated lens regime (SLR) and wave-like lens regime (WLR). The lens effect is induced by the pyroelectricity of polar dielectric crystals, as was proved in principle in a previous work by the same authors (S. Grilli et al., Opt. Express 16, 8084, 2008). Compared to that work, the improvements to the experimental set-up and procedure allow to reveal the two lens regimes which exhibit different optical properties. A digital holography technique is used to reconstruct the transmitted wavefront during focusing and a focal length variation in the millimetre range is observed. The tunability of such microlenses could be of great interest to the field of micro-optics thanks to the possibility to achieve focus tuning without moving parts and thus favouring the miniaturization of the optical systems.

Numerical multiplexing and demultiplexing of digital holographic information for remote reconstruction in amplitude and phase.

We investigate the possibility to multiplexing (Mux) and demultiplexing (de-Mux) numerically digital holograms (DHs) with the aim of optimizing their storage and/or transmission process. The DHs are multiplexed and demultiplexed thanks to the unique property of the digital holography to numerically manage the complex wavefields. We show that it is possible to retrieve correctly quantitative information about the amplitude and phase of one hundred DHs. This result can be useful to transmit efficiently, in terms of reduced amount of data, the DHs from the recording head to a remote display unit.

Liquid micro-lens array activated by selective electrowetting on lithium niobate substrates.

Lens effect was obtained in an open microfluidic system by using a thin layer of liquid on a polar electric crystal like LiNbO3. An array of liquid micro-lenses was generated by electrowetting effect in pyroelectric periodically poled crystals. Compared to conventional electrowetting devices, the pyroelectric effect allowed to have an electrode-less and circuit-less configuration. An interferometric technique was used to characterize the curvature of the micro-lenses and the corresponding results are presented and discussed. The preliminary results concerning the imaging capability of the micro-lens array are also reported.

Phase map retrieval in digital holography: avoiding the undersampling effect by a lateral shear approach.

In digital holography (DH) the numerical reconstruction of the whole wavefront allows one to extract the wrapped phase map mod, 2 pi. It can occur that the reconstructed wrapped phase map in the image plane is undersampled because of the limited pixel size in that plane. In such a case the phase distribution cannot be retrieved correctly by the usual unwrapping procedures. We show that the use of the digital lateral-shearing interferometry approach in DH provides the correct reconstruction of the phase map in the image plane, even in extreme cases where the phase profile changes very rapidly. We demonstrate the effectiveness of the method in a particular case where the profile of a highly curved silicon microelectromechanical system membrane has to be reconstructed.

Amplitude and phase reconstruction of photorefractive spatial bright-soliton in LiNbO3 during its dynamic formation by digital holography.

The time behaviour of bright spatial solitons in congruent undoped lithium niobate crystal is experimentally investigated. Full field characterization of the optical wavefront emerging from the crystal during the soliton formation process is performed by digital holographic method. Experimental results of the amplitude and phase maps of the field distribution at the exit face of the crystal allow the real-time monitoring of the evolution of the soliton beam from the application of the external field to the end of the process when the generation of the channel waveguide appears to be stable. The features of the dynamics of the soliton formation are visualized, analyzed and compared to a time-dependent numerical model.

Quantitative Phase Microscopy of microstructures with extended measurement range and correction of chromatic aberrations by multiwavelength digital holography.

Quantitative Phase Microscopy (QPM) by interferometric techniques can require a multiwavelength configuration to remove 2pi ambiguity and improve accuracy. However, severe chromatic aberration can affect the resulting phase-contrast map. By means of classical interference microscope configuration it is quite unpractical to correct such aberration. We propose and demonstrate that by Digital Holography (DH) in a microscope configuration it is possible to clear out the QPM map from the chromatic aberration in a simpler and more effective way with respect to other approaches. The proposed method takes benefit of the unique feature of DH to record in a plane out-of-focus and subsequently reconstruct numerically at the right focal image plane. In fact, the main effect of the chromatic aberration is to shift differently the correct focal image plane at each wavelength and this can be readily compensated by adjusting the corresponding reconstruction distance for each wavelength. A procedure is described in order to determine easily the relative focal shift among different imaging wavelengths by performing a scanning of the numerical reconstruction along the optical axis, to find out the focus and to remove at the same time the chromatic aberration.