Three-Dimensional Quantitative Intracellular Visualization of Graphene Oxide Nanoparticles by Tomographic Flow Cytometry.

Interaction of nanoparticles (NPs) with cells is of fundamental importance in biology and biomedical sciences. NPs can be taken up by cells, thus interacting with their intracellular elements, modifying the life cycle pathways, and possibly inducing death. Therefore, there is a great interest in understanding and visualizing the process of cellular uptake itself or even secondary effects, for example, toxicity. Nowadays, no method is reported yet in which 3D imaging of NPs distribution can be achieved for suspended cells in flow-cytometry. Here we show that, by means of label-free tomographic flow-cytometry, it is possible to obtain full 3D quantitative spatial distribution of nanographene oxide (nGO) inside each single flowing cell. This can allow the setting of a class of biomarkers that characterize the 3D spatial intracellular deployment of nGO or other NPs clusters, thus opening the route for quantitative descriptions to discover new insights in the realm of NP-cell interactions.

Rolling angle recovery of flowing cells in holographic tomography exploiting the phase similarity.

Holographic tomography allows the 3D mapping of the refractive index of biological samples thanks to reconstruction methods based on the knowledge of illumination directions or rotation angles of the imaged sample. Recently, phase contrast tomographic flow cytometry by digital holography has been demonstrated to reconstruct the three-dimensional refractive index distribution of single cells while they are flowing along microfluidic channels. In this system, the illumination direction is fixed while the sample's rotation is not deterministically known a priori but induced by hydrodynamic forces. We propose here a technique to retrieve the rolling angles, based on a new phase images similarity metric that is capable of identifying a cell's orientations from its 3D positioning while it is flowing along the microfluidic channel. The method is experimentally tested and also validated through appropriate numerical simulations. We provide demonstration of concept by achieving reconstruction of breast cancer cells tomography.

Learning Diatoms Classification from a Dry Test Slide by Holographic Microscopy.

Diatoms are among the dominant phytoplankters in marine and freshwater habitats, and important biomarkers of water quality, making their identification and classification one of the current challenges for environmental monitoring. To date, taxonomy of the species populating a water column is still conducted by marine biologists on the basis of their own experience. On the other hand, deep learning is recognized as the elective technique for solving image classification problems. However, a large amount of training data is usually needed, thus requiring the synthetic enlargement of the dataset through data augmentation. In the case of microalgae, the large variety of species that populate the marine environments makes it arduous to perform an exhaustive training that considers all the possible classes. However, commercial test slides containing one diatom element per class fixed in between two glasses are available on the market. These are usually prepared by expert diatomists for taxonomy purposes, thus constituting libraries of the populations that can be found in oceans. Here we show that such test slides are very useful for training accurate deep Convolutional Neural Networks (CNNs). We demonstrate the successful classification of diatoms based on a proper CNNs ensemble and a fully augmented dataset, i.e., creation starting from one single image per class available from a commercial glass slide containing 50 fixed species in a dry setting. This approach avoids the time-consuming steps of water sampling and labeling by skilled marine biologists. To accomplish this goal, we exploit the holographic imaging modality, which permits the accessing of a quantitative phase-contrast maps and a posteriori flexible refocusing due to its intrinsic 3D imaging capability. The network model is then validated by using holographic recordings of live diatoms imaged in water samples i.e., in their natural wet environmental condition.

Label-Free Optical Marker for Red-Blood-Cell Phenotyping of Inherited Anemias.

The gold-standard methods for anemia diagnosis are complete blood counts and peripheral-smear observations. However, these do not allow for a complete differential diagnosis as that requires biochemical assays, which are label-dependent techniques. On the other hand, recent studies focus on label-free quantitative phase imaging (QPI) of blood samples to investigate blood diseases by using video-based morphological methods. However, when sick cells are very similar to healthy ones in terms of morphometric features, identification of a blood disease becomes challenging even with QPI. Here, we introduce a label-free optical marker (LOM) to detect red-blood-cell (RBC) phenotypes, demonstrating that a single set of all-optical parameters can clearly identify a signature directly related to an erythrocyte disease through modeling each RBC as a biological lens. We tested this novel biophotonic analysis by proving that several inherited anemias, such as iron-deficiency anemia, thalassemia, hereditary spherocytosis, and congenital dyserythropoietic anemia, can be identified and sorted, thus opening a novel route for blood diagnosis on a completely different concept based on LOMs.

Biolens behavior of RBCs under optically-induced mechanical stress.

In this work, the optical behavior of Red Blood Cells (RBCs) under an optically-induced mechanical stress was studied. Exploiting the new findings concerning the optical lens-like behavior of RBCs, the variations of the wavefront refracted by optically-deformed RBCs were further investigated. Experimental analysis have been performed through the combination of digital holography and numerical analysis based on Zernike polynomials, while the biological lens is deformed under the action of multiple dynamic optical tweezers. Detailed wavefront analysis provides comprehensive information about the aberrations induced by the applied mechanical stress. By this approach it was shown that the optical properties of RBCs in their discocyte form can be affected in a different way depending on the geometry of the deformation. In analogy to classical optical testing procedures, optical parameters can be correlated to a particular mechanical deformation. This could open new routes for analyzing cell elasticity by examining optical parameters instead of direct but with low resolution strain analysis, thanks to the high sensitivity of the interferometric tool. Future application of this approach could lead to early detection and diagnosis of blood diseases through a single-step wavefront analysis for evaluating different cells elasticity. © 2017 International Society for Advancement of Cytometry.

3D morphometry of red blood cells by digital holography.

Three dimensional (3D) morphometric analysis of flowing and not-adherent cells is an important aspect for diagnostic purposes. However, diagnostics tools need to be quantitative, label-free and, as much as possible, accurate. Recently, a simple holographic approach, based on shape from silhouette algorithm, has been demonstrated for accurate calculation of cells biovolume and displaying their 3D shapes. Such approach has been adopted in combination with holographic optical tweezers and successfully applied to cells with convex shape. Nevertheless, unfortunately, the method fails in case of specimen with concave surfaces. Here, we propose an effective approach to achieve correct 3D shape measurement that can be extended in case of cells having concave surfaces, thus overcoming the limit of the previous technique. We prove the new procedure for healthy red blood cells (RBCs) (i.e., discocytes) having a concave surface in their central region. Comparative analysis of experimental results with a theoretical 3D geometrical model of RBC is discussed in order to evaluate accuracy of the proposed approach. Finally, we show that the method can be also useful to classify, in terms of morphology, different varieties of RBCs.

3D lithography by rapid curing of the liquid instabilities at nanoscale.

In liquids realm, surface tension and capillarity are the key forces driving the formation of the shapes pervading the nature. The steady dew drops appearing on plant leaves and spider webs result from the minimization of the overall surface energy [Zheng Y, et al. (2010) Nature 463:640-643]. Thanks to the surface tension, the interfaces of such spontaneous structures exhibit extremely good spherical shape and consequently worthy optical quality. Also nanofluidic instabilities generate a variety of fascinating liquid silhouettes, but they are however intrinsically short-lived. Here we show that such unsteady liquid structures, shaped in polymeric liquids by an electrohydrodynamic pressure, can be rapidly cured by appropriate thermal treatments. The fabrication of many solid microstructures exploitable in photonics is demonstrated, thus leading to a new concept in 3D lithography. The applicability of specific structures as optical tweezers and as novel remotely excitable quantum dots-embedded microresonators is presented.

Differential diagnosis of hereditary anemias from a fraction of blood drop by digital holography and hierarchical machine learning.

Anemia affects about the 25% of the global population and can provoke severe diseases, ranging from weakness and dizziness to pregnancy problems, arrhythmias and hearth failures. About 10% of the patients are affected by rare anemias of which 80% are hereditary. Early differential diagnosis of anemia enables prescribing patients a proper treatment and diet, which is effective to mitigate the associated symptoms. Nevertheless, the differential diagnosis of these conditions is often difficult due to shared and overlapping phenotypes. Indeed, the complete blood count and unaided peripheral blood smear observation cannot always provide a reliable differential diagnosis, so that biomedical assays and genetic tests are needed. These procedures are not error-free, require skilled personnel, and severely impact the financial resources of national health systems. Here we show a differential screening system for hereditary anemias that relies on holographic imaging and artificial intelligence. Label-free holographic imaging is aided by a hierarchical machine learning decider that works even in the presence of a very limited dataset but is enough accurate for discerning between different anemia classes with minimal morphological dissimilarities. It is worth to notice that only a few tens of cells from each patient are sufficient to obtain a correct diagnosis, with the advantage of significantly limiting the volume of blood drawn. This work paves the way to a wider use of home screening systems for point of care blood testing and telemedicine with lab-on-chip platforms.

Stain-free identification of cell nuclei using tomographic phase microscopy in flow cytometry.

Quantitative Phase Imaging (QPI) has gained popularity in bioimaging because it can avoid the need for cell staining, which in some cases is difficult or impossible. However, as a result, QPI does not provide labelling of various specific intracellular structures. Here we show a novel computational segmentation method based on statistical inference that makes it possible for QPI techniques to identify the cell nucleus. We demonstrate the approach with refractive index tomograms of stain-free cells reconstructed through the tomographic phase microscopy in flow cytometry mode. In particular, by means of numerical simulations and two cancer cell lines, we demonstrate that the nucleus can be accurately distinguished within the stain-free tomograms. We show that our experimental results are consistent with confocal fluorescence microscopy (FM) data and microfluidic cytofluorimeter outputs. This is a significant step towards extracting specific three-dimensional intracellular structures directly from the phase-contrast data in a typical flow cytometry configuration.

Multi-imaging capabilities of a 2D diffraction grating in combination with digital holography.

In this Letter we report on an alternative approach to get multiple images in microscopy, exploiting the capabilities of both a lithium niobate diffraction grating and digital holographic technique. We demonstrate that multi-imaging can be achieved in a lensless configuration by using a hexagonal diffraction grating but overcoming, thanks to digital holography (DH), the many constrains imposed by the grating parameters in multi-imaging with Talbot effect or Talbot array illuminators. In fact, DH permits the numerical reconstruction of the optical field diffracted by the grating, thus obtaining in-focus multiple images in a plane different from the fractional or entire Talbot ones.

Gold Nanorods and Nanoprisms Mediate Different Photothermal Cell Death Mechanisms In Vitro and In Vivo.

Photothermal therapy (PTT) is an efficient method of inducing localized hyperthermia and can be achieved using gold nanoparticles as photothermal agents. However, there are many hurdles to get over before this therapy can safely reach the clinics, including nanoparticles' optimal shape and the accurate prediction of cellular responses. Here, we describe the synthesis of gold nanorods and nanoprisms with similar surface plasmon resonances in the near-infrared (NIR) and comparable photothermal conversion efficiencies and characterize the response to NIR irradiation in two biological systems, melanoma cells and the small invertebrate Hydra vulgaris. By integrating animal, cellular, and molecular biology approaches, we show a diverse outcome of nanorods and nanoprisms on the two systems, sustained by the elicitation of different pathways, from necrosis to programmed cell death mechanisms (apoptosis and necroptosis). The comparative multilevel analysis shows great accuracy of in vivo invertebrate models to predict overall responses to photothermal challenging and superior photothermal performance of nanoprisms. Understanding the molecular pathways of these responses may help develop optimized nanoheaters that, safe by design, may improve PTT efficacy for clinical purposes.

Biological Lenses as a Photomask for Writing Laser Spots into Ferroelectric Crystals.

Red blood cells on the surface of a lithium niobate crystal can be used as optical lenses for direct writing of laser-induced refractive index changes. The writing process by such a photomask made of biological lenses is due to the photorefractive effect. Wavefront analysis by a digital holographic microscope is performed for deep and accurate evaluation of local refractive index changes. Different focusing properties can be imprinted on the crystal depending on which type of RBC is employed, discocytes or spherical-like RBCs. The possibility to fix into a solid material the optical fingerprint of the RBCs will have an impact on both diagnostics and cell\material interfacing.

In vitro cytotoxicity evaluation of cadmium by label-free holographic microscopy.

Among all environmental pollutants, the toxic heavy metal cadmium is considered as a human carcinogen. Cadmium may induce cell death by apoptosis in various cell types, although the underlying mechanisms are still unclear. In this paper we show how a label-free digital holography (DH)-based technique is able to quantify the evolution of key biophysical parameters of cells during the exposure to cadmium for the first time. Murine embryonic fibroblasts NIH 3T3 are chosen here as cellular model for studying the cadmium effects. The results demonstrate that DH is able to retrieve the temporal evolution of different key parameters such as cell volume, projected area, cell thickness and dry mass, thus providing a full quantitative characterization of the cell physical behaviour during cadmium exposure. Our results show that the label-free character of the technique would allow biologists to perform systematic and reliable studies on cell death process induced by cadmium and we believe that more in general this can be easily extended to others heavy metals, thus avoiding the time-consuming, expensive and invasive label-based procedures used nowadays in the field. In fact, pollution by heavy metals is severe issue that needs rapid and reliable methods to be settled.

Full-angle tomographic phase microscopy of flowing quasi-spherical cells.

We report a reliable full-angle tomographic phase microscopy (FA-TPM) method for flowing quasi-spherical cells along microfluidic channels. This method lies in a completely passive optical system, i.e. mechanical scanning or multi-direction probing of the sample is avoided. It exploits the engineered rolling of cells while they are flowing along a microfluidic channel. Here we demonstrate significant progress with respect to the state of the art of in-flow TPM by showing a general extension to cells having almost spherical shapes while they are flowing in suspension. In fact, the adopted strategy allows the accurate retrieval of rotation angles through a theoretical model of the cells' rotation in a dynamic microfluidic flow by matching it with phase-contrast images resulting from holographic reconstructions. So far, the proposed method is the first and the only one that permits to get in-flow TPM by probing the cells with full-angle, achieving accurate 3D refractive index mapping and the simplest optical setup, simultaneously. Proof of concept experiments were performed successfully on human breast adenocarcinoma MCF-7 cells, opening the way for the full characterization of circulating tumor cells (CTCs) in the new paradigm of liquid biopsy.

Tomographic flow cytometry by digital holography.

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.

Investigation on dynamics of red blood cells through their behavior as biophotonic lenses.

The possibility to adopt biological matter as photonic optical elements can open scenarios in biophotonics research. Recently, it has been demonstrated that a red blood cell (RBC) can be seen as an optofluidic microlens by showing its imaging capability as well as its focal tunability. Moreover, correlation between an RBC's morphology and its behavior as a refractive optical element has been established and its exploitation for biomedical diagnostic purposes has been foreseen. In fact, any deviation from the healthy RBC morphology can be seen as additional aberration in the optical wavefront passing through the cell. By this concept, accurate localization of focal spots of RBCs can become very useful in the blood disorders identification. We investigate the three-dimensional positioning of such focal spots over time for samples with two different osmolarity conditions, i.e., when they assume discocyte and spherical shapes, respectively. We also demonstrate that a temporal variation of an RBC's focal points along the optical axis is correlated to the temporal fluctuations in the RBC's thickness maps. Furthermore, we show a sort of synchronization of the whole erythrocytes ensemble.

Holographic imaging of unlabelled sperm cells for semen analysis: a review.

Male reproductive health in both humans and animals is an important research field in biological study. In order to characterize the morphology, the motility and the concentration of the sperm cells, which are the most important parameters to feature them, digital holography demonstrated to be an attractive technique. Indeed, it is a label-free, non-invasive and high-resolution method that enables the characterization of live specimen. The review is intended both for summarizing the state-of-art on the semen analysis and recent achievement obtained by means of digital holography and for exploring new possible applications of digital holography in this field. Quantitative phase maps of living swimming spermatozoa.

Imaging adherent cells in the microfluidic channel hidden by flowing RBCs as occluding objects by a holographic method.

Imaging through turbid media is a challenging topic. A liquid is considered turbid when dispersed particles provoke strong light scattering, thus destroying the image formation by any standard optical system. Generally, colloidal solutions belong to the class of turbid media since dispersed particles have dimensions ranging between 0.2 µm and 2 µm. However, in microfluidics, another relevant issue has to be considered in the case of flowing liquid made of a multitude of occluding objects, e.g. red blood cells (RBCs) flowing in veins. In such a case instead of severe scattering processes unpredictable phase delays occur resulting in a wavefront distortion, thus disturbing or even hindering the image formation of objects behind such obstructing layer. In fact RBCs can be considered to be thin transparent phase objects. Here we show that sharp amplitude imaging and phase-contrast mapping of cells hidden behind biological occluding objects, namely RBCs, is possible in harsh noise conditions and with a large field-of view by Multi-Look Digital Holography microscopy (ML-DH). Noteworthy, we demonstrate that ML-DH benefits from the presence of the RBCs, providing enhancement in terms of numerical resolution and noise suppression thus obtaining images whose quality is higher than the quality achievable in the case of a liquid without occlusive objects.

Bartonella henselae in Ixodes ricinus ticks (Acari: Ixodida) removed from humans, Belluno province, Italy.

The potential role of ticks as vectors of Bartonella species has recently been suggested. In this study, we investigated the presence of Bartonella species in 271 ticks removed from humans in Belluno Province, Italy. By using primers derived from the 60-kDa heat shock protein gene sequences, Bartonella DNA was amplified and sequenced from four Ixodes ricinus ticks (1.48%). To confirm this finding, we performed amplification and partial sequencing of the pap31 protein and the cell division protein ftsZ encoding genes. This process allowed us to definitively identify B. henselae (genotype Houston-1) DNA in the four ticks. Detection of B. henselae in these ticks might represent a highly sensitive form of xenodiagnosis. B. henselae is the first human-infecting Bartonella identified from Ixodes ricinus, a common European tick and the vector of various tickborne pathogens. The role of ticks in the transmission of bartonellosis should be further investigated.