Human perpetual exposure to several pollutants such as smoke, radiation, and toxic chemical substances, causes an excessive production of reactive oxygen species (ROS), that leads to the onset of neurodegeneration diseases. This knowledge has highlighted the importance of frequent monitoring people health with innovative biosensors able to detect pathological conditions at the initial stage. Indeed, it is mainly stated that neurodegenerative diseases can be effectively treated only if diagnosed very early. In this context, the structural aggregation of biomolecules in different districts of the brain, seem to play a key role in the neurodegeneration development mechanism becoming eligible targets for an early diagnosis. Hereby, we propose an innovative technique for detecting such biomolecules, e.g. Tau, by exploiting a pyro-electrohydrodynamic effect that is able to generate and accumulate tiny droplets of analyte on the surface of a reactive glass slide. We call the technique p-jet and we tested it in case of serial dilutions of Tau protein to demonstrate the consistency of the procedure under an immunodetection-based protocol.
Liquid biopsy is an emerging and promising biomedical tool that aims to the early cancer diagnosis and the definition of personalized therapies in non-invasive and cost-effective way, since it is based on the blood sample analysis. Several strategies have been tested to implement an effective liquid biopsy system. Among them, searching of circulating tumor cells (CTCs) released by the tumor into the bloodstream can be a valid solution. Within a blood sample, CTCs can be considered as rare cells due to their extremely low percentage with respect to white blood cells (WBCs). Therefore, a technology able to perform an advanced single-cell analysis is requested for implementing a CTCs-based liquid biopsy. Recently, tomographic phase imaging flow cytometry (TPIFC) has been developed as a technique for the reconstruction of the 3D volumetric distribution of the refractive indices (RIs) of single cells flowing along a microfluidic channel. Hence, TPIFC allows collecting large datasets of single cells thanks to the flow-cytometry high-throughput property in 3D and quantitative manner. Moreover, TPIFC works in label-free modality as no exogenous marker is employed, thus avoiding the limitations of marker-based techniques. For this reason, here we investigate the possibility of exploiting the 3D dataset of single cells recorded by TPIFC to feed a machine learning model, in order to recognize tumor cells with respect to a background of monocytes, which are the most similar cells among the WBCs in terms of morphology. Reported results aim to emulate a real scenario for the label-free liquid biopsy based on TPIFC.
The presence of microgravity and ionizing radiation during spaceflight missions causes excessive Reactive Oxygen Species (ROS) production that contributes to oxidative cellular stress and multifunctional damage in astronauts. This knowledge has underlined the importance of frequent monitoring of astronaut’s health to have early diagnoses. In this scenario, the biosensor diagnostic devices could offer the necessary analytical performance to study pathological astronaut conditions. Herein, we propose an innovative biosensor for detecting highly diluted biomarkers at picogram level by using the pyro-electrohydrodynamic jet (p-jet) system. The detection limit of the system was confirmed using a model protein as the Bovine Serum Albumin (BSA) by optimizing its deposition on different functionalized glass substrates through different chemical reactions starting with a manual procedure. Based on these results, the epoxy glass activated surface was chosen as the best slide for p-jet experiments. The characterization of the processes was performed through different spectroscopic techniques such as infrared-spectroscopy (IR) or confocal fluorescence. In the context of long-term human missions, our revolutionary approach could be extremely useful to monitor the astronaut health.
With the improvement in industrial production technologies, many products related to thin-film materials have been produced, especially in the field of daily chemistry. Because of its special physical and chemical properties, film material has become the best carrier, and a detailed measurement of its characteristics is highly required. The thickness characterizing of the thin film is a long-term challenge, one of the well-known methods is the interferometry. Recently, digital holographic approaches have been considered as one of the best candidates for thin film thickness mapping; it allows real-time, contactless, label-free, and full-field thickness measurement. Thanks to above features, holography-based thin film fabrication paradigm has been established rapidly. In this framework, we present a strategy for forming free-standing thin liquid film under the monitoring of Digital Holography (DH): a customized iris diaphragm has been used to stretch the liquid droplet inside to a thin liquid film. Under the condition of quantitatively adjusting the opening speed and radius of the iris, the precise manufacturing of the desired thin film can be achieved. In this case, DH is implemented to provide the thickness distribution of the droplet during stretching; the real-time thickness mapping of thin film builds up a close loop controlling for fabrication process. Based on this strategy, we performed a series experiments of thin liquid films fabrication and the opening process of thin film have been studied by spatiotemporal modeling. The results show that customized iris diaphragm is a good strategy for quantitative fabrication of thin liquid films.
Optofluidic microscopy has been an open challenge during past decade; it is also a well-established paradigm where precise control of microfluidic streams is smartly exploited. Digital Holography (DH) has been proved as one of the optimal tools for flow-cytometry, cell sorting and classification, cell counting and study of cell mechanics. In this framework, Space-Time Digital Holography (STDH) is a convenient complement to conventional holographic cell imaging. Thanks to a spatiotemporal reassembling strategy, one single space-time hologram can efficiently store information of a series of time-lapse holograms using a small subset of detecting elements, e.g. a linear sensor array. In this case, the modulated pattern of interference fringes is projected onto a new hybrid space-time domain and reassembled by time series. Here we propose a phase-retrieval process in STDH for optofluidics, which allows the quantitative phase information reconstruction for flowing cells in different focus planes simultaneously with extended field of view. For a space-time hologram storing information from flowing cells, a unique flow velocity meeting the matching condition of STDH would enable accurate space-time phase shifting. In the case of mismatches between cells speed and recording frame rate, an ad-hoc reconstruction algorithm is developed that compensates for the mismatch and retrieves the correct phase-contrast map of the sample by smartly adapting the method to the microfluidic speed. Based on the proposed strategy, we show the 4D mapping of flowing cells in space-time domain; in other words, the ASTDH is able to encode efficiently a 4D information in a 2D map, self-adapting to unexpected variations of the flow profiles.
In Digital Holography (DH) modality for lab-on-chip applications, the cells passing through the Field of View (FOV) of a microscope can be detected and analyzed even if they are flowing at different depths in a microfluidic channel. If the cells rotate while flowing along the channel, they can be probed by light beams from many different directions while they cross the holographic FOV, thus, it is possible to retrieve the 3D refractive index map of each flowing cell, i.e., a 3D phase-contrast tomogram. Since in biological samples many cells flow close to each other along the FOV, so giving the possibility of increasing the throughput of the system, it is important to establish how close the cells can be to avoid mutual disturbing effects on their rotation due to hydrodynamic interactions. Here, we investigate by means of direct numerical simulations the effects of the hydrodynamic interactions among several cells on their rotational behavior and mechanical deformation during the flow along a microfluidic channel, which are two essential aspects connected to the possibility of recovering the tomograms.
Human health and disease prevention are among the priorities to safeguard astronauts and, in the next future, space tourists. There is a great demand of new reliable biotechnologies that would be eventually implemented on spacecrafts to observe the space-induced effect on humans. One of the main risks is related to the radiation exposure, that is significantly higher than on Earth. For this reason, space agencies are pushing to develop strategies to quantify, oversee and limiting such risks. Here we present an approach based on the combination of microfluidics and stain-free imaging also aided by artificial intelligence to monitor the effect on ionizing radiation on blood cells. The system is based on the Holographic Image Flow Cytometry system where Quantitative Phase Contrast images are retrieved for cell flowing and rotating into a microfluidics circuits. Proof of concept is demonstrated where morphological parameters are identified able to distinguish cell population irradiated at different radiation doses and at different time from the radiation exposure. Blood cell will be analyzed. The presented approach has main advantages respect to standard and already existent technologies for single cell analysis. The first one is the no-need of fluorescence staining thus opening to faster and easier operation steps. The second one is related to cell rotation into the field of view, allowing to acquire images at different rotation angle and thus collecting a broader dataset useful for the application of artificial intelligence network. Furthermore, the system can be miniaturized to a scale portable out of the laboratory environment.
We present the multimodal characterization of thin polymeric membrane by digital holography-based methods. Herein, two microscope techniques had been chosen to reveal the morphology of membranes, which are conventional off-axis Digital Holography (DH) and Space-Time Digital Holography (STDH). The complementary features of the different methods allow for a bottom-up analysis of the related membranes. Meanwhile, the dynamic forming process of polymeric membrane at the air-water interface is revealed in real-time by CDH. By comparing the imaging results of different methods, the application range of different imaging methods is analyzed in detail.
Digital Holography (DH) is a label-free optical microscopy technique which allows reconstructing the Quantitative Phase Maps (QPMs) of transparent biological specimens. In a QPM, the phase-contrast is endogenous and is due to the Refractive Index (RI) and thickness differences. Although phase-contrast allows a quantitative characterization of the whole biological sample, it is often not enough to ensure an adequate intracellular segmentation, also because of the lack of exogenous markers, e.g., fluorescent dyes. Here we investigate a biological strategy for increasing the intracellular contrast inside epidermal onion cells to recognize their nuclei within the QPMs. Plant cells continuously undergo dehydration-hydration loops during their lifetime since dehydration is reversible when plasmolysis is not reached. Therefore, by setting specific environmental temperature and humidity, we can induce dehydration, thus provoking the water evaporation from the vacuole and therefore increasing the nucleus-cytoplasm contrast. Moreover, the reduction of the turgor pressure causes a rearrangement of the cytoskeleton, thus allowing nuclear roto-translations. We exploit an ad-hoc algorithm to estimate the nucleus rolling angles around the image plane. Then, we perform phase-contrast tomography to reconstruct the three-dimensional (3D) RI distribution of the plant cells’ nuclei by operating in complete reversible conditions, i.e., before plasmolysis when no cell damage has occurred. Finally, we segment the nuclear tomograms to isolate the 3D nucleoli, thus providing quantitative measurements about their volumes, dry masses, and RI statistics. In this way, DH can be further exploited for the label-free and non-invasive analysis of several plant cell lines at the nuclear and sub-nuclear level.
Polymeric thin films represent an emerging industrial area driven by their enormous technological and commercial potential in interdisciplinary sectors such as chemistry, material science, engineering, and physics. The large selection in terms of materials/composites and the wide range of technological solutions that could be used for their fabrication could create confusion for the final user requiring a quantitative characterization of their properties. This analysis could be even more complex in the case of functionalized polymeric films such as the samples reported in this work. Here we present how thin polymer films can be wholly characterized by applying a multiplicity of optical methods. Films were realized by a special liquid one-step process. Moreover, such polymer films were functionalized here for the first time by mesoporous silica nanoparticles. The nanoparticles were added to a polymeric matrix. We show that a full characterization was achieved by employing three different microscope techniques, i.e., scanning electron microscope, digital holography (DH), and space-time DH. Exploiting such a multimodal methodology can be of great benefit for characterizing the functionalized polymeric thin films. In fact, multiple characterization in different conditions was possible. The results reported in terms of morphological information, thickness distribution, three-dimensional (3D) mapping, large field of view, high magnification, and super resolution of the zoomed area offer a good solution for testing materials and obtaining a quantitative characterization and whole inspection in the case of complex polymeric samples.
The digital holography method has been implemented to several industrial systems with the aim of improving new products by quantitative measurement. Nowadays, digital holography (DH) has been considered an important measurement tool, owing to the abilities of non-contact, label-free, quantitative, high-resolution and real-time. The inherent characteristics of DH makes it a powerful tool for visualization and testing of soft matter, as well as in-situ and real-time characterization of bottom-up fabrication processes. Herein, we report the most useful applications of soft matter, where the capabilities offered by DH, such as three-dimensional (3D) imaging, extended focus, 3D tracking, full-field analysis, high sensitivity, and wide measurement’s range, permit completely non-invasive characterizations on a full-scale. Taking the advantages of DH measurement, the possibility of numerically managing the complex wavefront scattered or transmitted by the sample under investigation allows the extraction of all information through a full-digital modality. Meanwhile, the numerical diffraction propagation process allows object imaging well-in-focus during dynamic process. This also creates the possibility to retrieve phase-contrast maps that enable quantitative measurements of the sample in full-field mode and 3D. Moreover, DH measurement has good ability to manage and remove aberrations in the optical system using simple and flexible methods, thus simplifying the optical apparatus and measurement operations. Owing to these unique features of DH, we have possibility to better study the world of soft-matter.
The detection of CTCs in a blood sample is a challenging task due to their rarity and variety. We develop a new label-free and all-optical approach at the lab-on-chip scale for the detection of CTCs based on morphological biomarkers. In particular, we design a microfluidic device to be combined with a phase-contrast tomography system to carry out quantitative measurements of the three-dimensional structure of each single cell in a blood sample. In such device, two aspects are conjugated: on the one hand, the cells need to perform at least one complete rotation within the field of view of the imaging apparatus; on the other hand, the highest possible throughput has to be achieved, yet without deforming the cells significantly, which would impede their tomographic reconstruction. In this contribution, the finite-element-simulation-based preliminary design of a microfluidic device that would allow the achievement of the aforementioned objectives for cells with different shape and deformability is presented.
The goal of the SensApp FET-Open project is to develop an innovative super-sensor that will be able to detect Alzheimer’s disease (AD) biomarkers (β-amyloid, Tau and pTAU) in peripheral blood. Considering that nowadays an accurate diagnosis of AD requires the highly invasive withdrawal and analysis of cerebrospinal fluid, SensApp will represent a breakthrough in the field of AD diagnosis thanks to the ability to detect the early stage of the disease by a simple blood collection. We call Droplet-Split-and-Stack (DSS) the new technology that will emerge from SensApp. The achievement of SensApp goal is enabled by the interdisciplinary cooperation between different research institutions and one company involved in the key fields of the project, Vrije Universiteit Brussels, VTT Technical Research Centre of Finland, University of Linz, Ginolis Ltd, IRCCS Centre “Bonino Pulejo”, under the coordination of CNR-Institute of Applied Sciences and Intelligent Systems. This communication will illustrate the progress of the activities.
Quantitative measurement for thin-film thickness has been a widely studied issue, and, visualizing and characterizing for full-field thin-film evolution can provide effective data support for membrane science, polymer chemistry, biomaterials, applied Chemistry, etc. Interferometry was considered the most useful tool to measure film thickness in past decades. However, there were deficiencies in imaging speed and field of view for conventical interferometric measurement. In this paper, we proposed a fusion method to achieve full-field and quantitative analysis of the thin liquid film at nanoscale by using Digital Holography (DH) and White Light Interferometry (WLI) simultaneously. Compared with our previous holographic studies on thin film measurement, this new method can overcome the shortcomings of the holographic method related to registration and reconstruction. In addition, these parallel experiments can also provide a reference when we have to make a choice between DH and WLI. For the thickness reconstruction process, the entire life cycle registration is no longer required to obtain the thickness information. Moreover, the region where the thin film thickness is less than half the wavelength is now measurable. Thanks to the fusion processing, the film thickness calibration can be obtained without any background subtraction, in the meantime, the background hologram becomes not essential in the holographic recording process.
Visualizing the intracellular dynamics of plant cells has been an open challenge for modern botany, agronomy and pharmacy. In this paper, we proposed an approach to improve the phase contrast during plant cell holographic imaging by cells’ dehydration, and used this method to realize the observation of cytoplasmic circulation inside the living onion epithelial cell. The dehydration process can be seen as a sort of label-free contrast agent for better imaging biological processes. We have investigated live onion epidermal cells, observing their inner dynamics during long time recordings using a digital holographic microscopy system. For the experiments, an off-axis digital holography setup in transmission configuration with double spherical wave interference was used to record the digital holograms of onion cells. Then, we performed long-term time lapse holographic recordings of onion epidermal cells, and the results show that the intracellular tissue structure and the dynamic behavior of the cytoskeleton features and nuclei can be better exhibited via high-contrast phase imaging under cell dehydration conditions. In this case, the movements of intracellular filaments and the nucleus are observed via dynamical high-contrast phase imaging during the dehydration process. The experimental results clearly show the positive effect of dehydration process on intracellular imaging quality, and create the possibility to track the movement of plant organelles. In sum, thanks to the dehydration process of plant cells, holographic phase contrast enhancement imaging is realized.
KEYWORDS: Microfluidics, Tomography, Holography, 3D modeling, 3D image processing, Reconstruction algorithms, Microscopy, Digital holography, Detection and tracking algorithms, Intelligence systems
The complete cells characterization in microfluidic flow can be achieved by using the quantitative phase imaging by
digital holography as imaging tool. In fact, by assuring the complete 3D rotation of flowing cells, it is possible to recover their 3D refractive index mapping by using the tomographic phase-contrast reconstruction. In this paper, we investigate all steps need to obtain the tomographic reconstruction of flowing cells. In particular, we employ a holographic 3D tracking algorithm to follow each cells that moves in the field of view, along with a suitable tracking angle method for the cell’s tumbling. Moreover, a fluid modeling is used to characterize the cell rotation effect. We test the proposed processing pipeline for circulating tumor cells.
Suspensions carrying deformable inclusions are ubiquitous in nature and applications. Hence, high-throughput characterization of the mechanical properties of soft particles is of great interest. Recently, a non-invasive optofluidic technique has been developed for the measurement of the interfacial tension between two immiscible liquids.1, 2 We adapt such technique to the case of soft solid beads, thus designing a non-invasive optofluidic device for the measurement of the mechanical properties of deformable particles from real-time optical imaging of their deformation. The device consists of a cylindrical microfluidic channel with a cross-section reduction in which we make initially spherical soft beads flow suspended in a Newtonian carrier. By imaging the deformation of a particle in real time while it goes through the constriction, it is possible to get a measure of its elastic modulus through a theoretically derived-correlation. We provide both experimental and numerical validation of our device.
Acoustofluidics exploits ultrasounds and microfluidic platforms to achieve label-free and contactless manipulation of micro sized objects. Here, we demonstrate the use of off-axis digital holography to investigate the behavior of erythrocytes dispersed in water and exposed to ultrasound standing waves. We consider two different regimes of manipulation. In the first case, the sample is stilled inside the microfluidic channel. Under the influence of acoustic forces, the cells move to the first nodal plane, where they start an aggregation process. We follow the formation of clusters in different regions of the channel, highlighting the different structures that emerge. As a second regime, we monitor the axial position of cells flowing during the application of ultrasuonds. By using a resonance frequency that originates multiple nodal positions, we show how holographic imaging can be used to image the cells distributed in the different nodes.
In global healthcare and point-of-care diagnostics there is an increasing request of medical equipment with devices able to provide fast and reliable testing for clinical diagnosis. In developing countries that lack of adequate facilities, this need is even more urgent. Lab-on-a-Chip devices have undergone a great growth during the last decade, supported by optical imaging techniques more and more refined. Here we present recent progresses in developing imaging tools based on holographic microscopy that can be very useful when applied into bio-microfluidics. Digital Holography (DH) is label-free, non-invasive, potentially high-throughput and, above all, quantitative. We show the recent advancements of DH in transmission microscopy mode, when this is applied to microfluidics to yield 3D imaging capabilities. Holographic flow cytometry through quantitative phase imaging and in-flow tomography for the analysis and manipulation of micro-particles and cells will be shown [1-3]. Medical diagnostic applications based on DH will be also shown. Moreover, we present a portable common-path holographic microscope embedded onboard a microfluidic device that paves the way to the application of DH on the field [4].
Acoustophoresis devices are popular tools for manipulation and diagnostic in microfluidic environments. They offer the opportunity for contactless manipulation of cells. We demonstrate that the combination of acoustic manipulation and holographic imaging provides a suitable system for the simultaneous handling and of biological matter. We employ an acoustofluidic device with a transparent piezo element, to enable optical investigation through the channel. The holographic imaging is thus employed to observe and analyze the behavior of Red Blood Cells during the application of ultrasound radiation. The flexible refocusing, and quantitative phase imaging of single cells and RBCs clusters is reported.
Tomography is one of the most powerful imaging tools for analyzing biological samples, able to furnish complete mapping of the object in 3D. In particular, tomographic phase microscopy (TPM) exploits quantitative phase imaging (QPI) techniques to map the 3D refractive index (RI) of cells, by adopting laser beam deflection, direct mechanical rotation or holographic optical tweezers (HOTs) to probe the sample along a number of controlled directions. To date, all tomographic methods require a high-precision, opto-mechanical and/or opto-electronic device to acquire a set of many images by probing the sample along a large number of controlled directions. Here we report on a smart solution to obtain TPM of samples at lab-on-chip scale, by exploiting their tumbling inside microfluidic channels. This method, recently developed, presents the following advantages: (i) Permits to observe full 360° of rotating cells, this avoiding the limitation in the accuracy of tomograms; (ii) no mechanical contact neither holographic optical tweezers are needed to rotate the sample; (iii) it is suitable for application in flowing conditions with high-throughput performances. This would allow real microfluidic biomedical applications on a large scale. The results shown in a previous work for RBCs and diatoms are here extended to quasi-spherical cells, by exploiting a new algorithm for rolling angle recovery in TPM. In particular, we performed the 3D imaging of 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.
We demonstrate the non-invasive investigation of circulating human breast adenocarcinoma cells in microfluidic environment by implementing the full-angle tomographic phase microscopy (TPM). The proposed approach lies in a completely passive optical system, i.e. avoiding mechanical scanning or multi-direction probing of the sample and exploiting the engineered rolling of cells while they are flowing along a microfluidic channel.
In this paper, we propose digital holography in transmission configuration as an effective method to measure the time-dependent thickness of polymeric films during bubble blowing. We designed a complete set of experiments to measure bubble thickness, including the evaluation of the refractive index of the polymer solution. We report the measurement of thickness distribution along the film during the bubble formation process until the bubble‘s rupture. Based on those data, the variation range and variation trend of bubble film thickness are clearly measured during the process of expansion to fracture is indicated.
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