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.
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.
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.
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.
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.
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.
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.
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].
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.
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