Drop-on-Demand Pyro-Electrohydrodynamic Printing of Nematic Liquid Crystal Microlenses.

Inkjet printing of liquid crystal (LC) microlens arrays is particularly appealing for the development of switchable 2D/3D organic light-emitting diode (OLED) displays, as the printing process ensures that the lenses can be deposited directly and on-demand onto the pixelated OLED layer without the need for additional steps, thus simplifying fabrication complexity. Even if different fabrication technologies have been employed and good results in LC direct printing have already been achieved, all the systems used require costly equipment and heated nozzles to reduce the LC solution's viscosity. Here, we present the direct printing of a nematic LC (NLC) lens by a Drop-on-Demand (DoD) inkjet printing by a pyro-electrohydrodynamic effect for the first time. The method works at ambient temperature and avoids dispensing nozzles, thus offering a noncontact manipulation approach of liquid with high resolution and good repeatability on different kinds of substrates. NLC microlenses are printed on different substrates and fully characterized. Polarization properties are evaluated for various samples, i.e., NLC lenses on unaligned and indium-tin oxide (ITO) aligned. Moreover, an in-depth characterization of the NLC lenses is reported by polarized optical microscopy and by analyzing the birefringence in digital holographic microscopy.

Classifying breast cancer and fibroadenoma tissue biopsies from paraffined stain-free slides by fractal biomarkers in Fourier Ptychographic Microscopy.

Breast cancer is one of the most spread and monitored pathologies in high-income countries. After breast biopsy, histological tissue is stored in paraffin, sectioned and mounted. Conventional inspection of tissue slides under benchtop light microscopes involves paraffin removal and staining, typically with H&E. Then, expert pathologists are called to judge the stained slides. However, paraffin removal and staining are operator-dependent, time and resources consuming processes that can generate ambiguities due to non-uniform staining. Here we propose a novel method that can work directly on paraffined stain-free slides. We use Fourier Ptychography as a quantitative phase-contrast microscopy method, which allows accessing a very wide field of view (i.e., mm2) in one single image while guaranteeing high lateral resolution (i.e., 0.5 µm). This imaging method is multi-scale, since it enables looking at the big picture, i.e. the complex tissue structure and connections, with the possibility to zoom-in up to the single-cell level. To handle this informative image content, we introduce elements of fractal geometry as multi-scale analysis method. We show the effectiveness of fractal features in describing and classifying fibroadenoma and breast cancer tissue slides from ten patients with very high accuracy. We reach 94.0 ± 4.2% test accuracy in classifying single images. Above all, we show that combining the decisions of the single images, each patient's slide can be classified with no error. Besides, fractal geometry returns a guide map to help pathologist to judge the different tissue portions based on the likelihood these can be associated to a breast cancer or fibroadenoma biomarker. The proposed automatic method could significantly simplify the steps of tissue analysis and make it independent from the sample preparation, the skills of the lab operator and the pathologist.

All-optical dual module platform for motility-based functional scrutiny of microencapsulated probiotic bacteria.

Probiotic bacteria are widely used in pharmaceutics to offer health benefits. Microencapsulation is used to deliver probiotics into the human body. Capsules in the stomach have to keep bacteria constrained until release occurs in the intestine. Once outside, bacteria must maintain enough motility to reach the intestine walls. Here, we develop a platform based on two label-free optical modules for rapidly screening and ranking probiotic candidates in the laboratory. Bio-speckle dynamics assay tests the microencapsulation effectiveness by simulating the gastrointestinal transit. Then, a digital holographic microscope 3D-tracks their motility profiles at a single element level to rank the strains.

Multi-scale fractal Fourier Ptychographic microscopy to assess the dose-dependent impact of copper pollution on living diatoms.

Accumulation of bioavailable heavy metals in aquatic environment poses a serious threat to marine communities and human health due to possible trophic transfers through the food chain of toxic, non-degradable, exogenous pollutants. Copper (Cu) is one of the most spread heavy metals in water, and can severely affect primary producers at high doses. Here we show a novel imaging test to assay the dose-dependent effects of Cu on live microalgae identifying stress conditions when they are still capable of sustaining a positive growth. The method relies on Fourier Ptychographic Microscopy (FPM), capable to image large field of view in label-free phase-contrast mode attaining submicron lateral resolution. We uniquely combine FPM with a new multi-scale analysis method based on fractal geometry. The system is able to provide ensemble measurements of thousands of diatoms in the liquid sample simultaneously, while ensuring at same time single-cell imaging and analysis for each diatom. Through new image descriptors, we demonstrate that fractal analysis is suitable for handling the complexity and informative power of such multiscale FPM modality. We successfully tested this new approach by measuring how different concentrations of Cu impact on Skeletonema pseudocostatum diatom populations isolated from the Sarno River mouth.

Investigation on lysosomal accumulation by a quantitative analysis of 2D phase-maps in digital holography microscopy.

Lysosomes are the terminal end of catabolic pathways in the cell, as well as signaling centers performing important functions such as the recycling of macromolecules, organelles, and nutrient adaptation. The importance of lysosomes in human health is supported by the fact that the deficiency of most lysosomal genes causes monogenic diseases called as a group Lysosomal Storage Diseases (LSDs). A common phenotypic hallmark of LSDs is the expansion of the lysosomal compartment that can be detected by using conventional imaging methods based on immunofluorescence protocols or overexpression of tagged lysosomal proteins. These methods require the alteration of the cellular architecture (i.e., due to fixation methods), can alter the behavior of cells (i.e., by the overexpression of proteins), and require sample preparation and the accurate selection of compatible fluorescent markers in relation to the type of analysis, therefore limiting the possibility of characterizing cellular status with simplicity. Therefore, a quantitative and label-free methodology, such as Quantitative Phase Imaging through Digital Holographic (QPI-DH), for the microscopic imaging of lysosomes in health and disease conditions may represent an important advance to study and effectively diagnose the presence of lysosomal storage in human disease. Here we proof the effectiveness of the QPI-DH method in accomplishing the detection of the lysosomal compartment using mouse embryonic fibroblasts (MEFs) derived from a Mucopolysaccharidosis type III-A (MSP-IIIA) mouse model, and comparing them with wild-type (WT) MEFs. We found that it is possible to identify label-free biomarkers able to supply a first pre-screening of the two populations, thus showing that QPI-DH can be a suitable candidate to surpass fluorescent drawbacks in the detection of lysosomes dysfunction. An appropriate numerical procedure was developed for detecting and evaluate such cellular substructures from in vitro cells cultures. Results reported in this study are encouraging about the further development of the proposed QPI-DH approach for such type of investigations about LSDs.

Label-free cell classification in holographic flow cytometry through an unbiased learning strategy.

Nowadays, label-free imaging flow cytometry at the single-cell level is considered the stepforward lab-on-a-chip technology to address challenges in clinical diagnostics, biology, life sciences and healthcare. In this framework, digital holography in microscopy promises to be a powerful imaging modality thanks to its multi-refocusing and label-free quantitative phase imaging capabilities, along with the encoding of the highest information content within the imaged samples. Moreover, the recent achievements of new data analysis tools for cell classification based on deep/machine learning, combined with holographic imaging, are urging these systems toward the effective implementation of point of care devices. However, the generalization capabilities of learning-based models may be limited from biases caused by data obtained from other holographic imaging settings and/or different processing approaches. In this paper, we propose a combination of a Mask R-CNN to detect the cells, a convolutional auto-encoder, used to the image feature extraction and operating on unlabelled data, thus overcoming the bias due to data coming from different experimental settings, and a feedforward neural network for single cell classification, that operates on the above extracted features. We demonstrate the proposed approach in the challenging classification task related to the identification of drug-resistant endometrial cancer cells.

Beyond fluorescence: advances in computational label-free full specificity in 3D quantitative phase microscopy.

Despite remarkable progresses in quantitative phase imaging (QPI) microscopes, their wide acceptance is limited due to the lack of specificity compared with the well-established fluorescence microscopy. In fact, the absence of fluorescent tag prevents to identify subcellular structures in single cells, making challenging the interpretation of label-free 2D and 3D phase-contrast data. Great effort has been made by many groups worldwide to address and overcome such limitation. Different computational methods have been proposed and many more are currently under investigation to achieve label-free microscopic imaging at single-cell level to recognize and quantify different subcellular compartments. This route promises to bridge the gap between QPI and FM for real-world applications.

Label-Free Intracellular Multi-Specificity in Yeast Cells by Phase-Contrast Tomographic Flow Cytometry.

In-flow phase-contrast tomography provides a 3D refractive index of label-free cells in cytometry systems. Its major limitation, as with any quantitative phase imaging approach, is the lack of specificity compared to fluorescence microscopy, thus restraining its huge potentialities in single-cell analysis and diagnostics. Remarkable results in introducing specificity are obtained through artificial intelligence (AI), but only for adherent cells. However, accessing the 3D fluorescence ground truth and obtaining accurate voxel-level co-registration of image pairs for AI training is not viable for high-throughput cytometry. The recent statistical inference approach is a significant step forward for label-free specificity but remains limited to cells' nuclei. Here, a generalized computational strategy based on a self-consistent statistical inference to achieve intracellular multi-specificity is shown. Various subcellular compartments (i.e., nuclei, cytoplasmic vacuoles, the peri-vacuolar membrane area, cytoplasm, vacuole-nucleus contact site) can be identified and characterized quantitatively at different phases of the cells life cycle by using yeast cells as a biological model. Moreover, for the first time, virtual reality is introduced for handling the information content of multi-specificity in single cells. Full fruition is proofed for exploring and interacting with 3D quantitative biophysical parameters of the identified compartments on demand, thus opening the route to a metaverse for 3D microscopy.

Phenotyping neuroblastoma cells through intelligent scrutiny of stain-free biomarkers in holographic flow cytometry.

To efficiently tackle certain tumor types, finding new biomarkers for rapid and complete phenotyping of cancer cells is highly demanded. This is especially the case for the most common pediatric solid tumor of the sympathetic nervous system, namely, neuroblastoma (NB). Liquid biopsy is in principle a very promising tool for this purpose, but usually enrichment and isolation of circulating tumor cells in such patients remain difficult due to the unavailability of universal NB cell-specific surface markers. Here, we show that rapid screening and phenotyping of NB cells through stain-free biomarkers supported by artificial intelligence is a viable route for liquid biopsy. We demonstrate the concept through a flow cytometry based on label-free holographic quantitative phase-contrast microscopy empowered by machine learning. In detail, we exploit a hierarchical decision scheme where at first level NB cells are classified from monocytes with 97.9% accuracy. Then we demonstrate that different phenotypes are discriminated within NB class. Indeed, for each cell classified as NB its belonging to one of four NB sub-populations (i.e., CHP212, SKNBE2, SHSY5Y, and SKNSH) is evaluated thus achieving accuracy in the range 73.6%-89.1%. The achieved results solve the realistic problem related to the identification circulating tumor cell, i.e., the possibility to recognize and detect tumor cells morphologically similar to blood cells, which is the core issue in liquid biopsy based on stain-free microscopy. The presented approach operates at lab-on-chip scale and emulates real-world scenarios, thus representing a future route for liquid biopsy by exploiting intelligent biomedical imaging.

Digital holographic approaches to the detection and characterization of microplastics in water environments.

Microplastic (MP) pollution is seriously threatening the environmental health of the world, which has accelerated the development of new identification and characterization methods. Digital holography (DH) is one of the emerging tools to detect MPs in a high-throughput flow. Here, we review advances in MP screening by DH. We examine the problem from both the hardware and software viewpoints. Automatic analysis based on smart DH processing is reported by highlighting the role played by artificial intelligence for classification and regression tasks. In this framework, the continuous development and availability in recent years of field-portable holographic flow cytometers for water monitoring also is discussed.

Label-free liquid biopsy through the identification of tumor cells by machine learning-powered tomographic phase imaging flow cytometry.

Image-based identification of circulating tumor cells in microfluidic cytometry condition is one of the most challenging perspectives in the Liquid Biopsy scenario. Here we show a machine learning-powered tomographic phase imaging flow cytometry system capable to provide high-throughput 3D phase-contrast tomograms of each single cell. In fact, we show that discrimination of tumor cells against white blood cells is potentially achievable with the aid of artificial intelligence in a label-free flow-cyto-tomography method. We propose a hierarchical machine learning decision-maker, working on a set of features calculated from the 3D tomograms of the cells' refractive index. We prove that 3D morphological features are adequately distinctive to identify tumor cells versus the white blood cell background in the first stage and, moreover, in recognizing the tumor type at the second decision step. Proof-of-concept experiments are shown, in which two different tumor cell lines, namely neuroblastoma cancer cells and ovarian cancer cells, are used against monocytes. The reported results allow claiming the identification of tumor cells with a success rate higher than 97% and with an accuracy over 97% in discriminating between the two cancer cell types, thus opening in a near future the route to a new Liquid Biopsy tool for detecting and classifying circulating tumor cells in blood by stain-free method.

Beyond conventional microscopy: Observing kidney tissues by means of fourier ptychography.

Kidney microscopy is a mainstay in studying the morphological structure, physiology and pathology of kidney tissues, as histology provides important results for a reliable diagnosis. A microscopy modality providing at same time high-resolution images and a wide field of view could be very useful for analyzing the whole architecture and the functioning of the renal tissue. Recently, Fourier Ptychography (FP) has been proofed to yield images of biology samples such as tissues and in vitro cells while providing high resolution and large field of view, thus making it a unique and attractive opportunity for histopathology. Moreover, FP offers tissue imaging with high contrast assuring visualization of small desirable features, although with a stain-free mode that avoids any chemical process in histopathology. Here we report an experimental measuring campaign for creating the first comprehensive and extensive collection of images of kidney tissues captured by this FP microscope. We show that FP microscopy unlocks a new opportunity for the physicians to observe and judge renal tissue slides through the novel FP quantitative phase-contrast microscopy. Phase-contrast images of kidney tissue are analyzed by comparing them with the corresponding renal images taken under a conventional bright-field microscope both for stained and unstained tissue samples of different thicknesses. In depth discussion on the advantages and limitations of this new stain-free microscopy modality is reported, showing its usefulness over the classical light microscopy and opening a potential route for using FP in clinical practice for histopathology of kidney.

On monocytes and lymphocytes biolens clustering by in flow holographic microscopy.

Live cells act as biological lenses and can be employed as real-world optical components in bio-hybrid systems. Imaging at nanoscale, optical tweezers, lithography and also photonic waveguiding are some of the already proven functionalities, boosted by the advantage that cells are fully biocompatible for intra-body applications. So far, various cell types have been studied for this purpose, such as red blood cells, bacterial cells, stem cells and yeast cells. White Blood Cells (WBCs) play a very important role in the regulation of the human body activities and are usually monitored for assessing its health. WBCs can be considered bio-lenses but, to the best of our knowledge, characterization of their optical properties have not been investigated yet. Here, we report for the first time an accurate study of two model classes of WBCs (i.e., monocytes and lymphocytes) by means of a digital holographic microscope coupled with a microfluidic system, assuming WBCs bio-lens characteristics. Thus, quantitative phase maps for many WBCs have been retrieved in flow-cytometry (FC) by achieving a significant statistical analysis to prove the enhancement in differentiation among sphere-like bio-lenses according to their sizes (i.e., diameter d) exploiting intensity parameters of the modulated light in proximity of the cell optical axis. We show that the measure of the low intensity area (S: I z < I th z ) in a fixed plane, is a feasible parameter for cell clustering, while achieving robustness against experimental misalignments and allowing to adjust the measurement sensitivity in post-processing. 2D scatterplots of the identified parameters (d-S) show better differentiation respect to the 1D case. The results show that the optical focusing properties of WBCs allow the clustering of the two populations by means of a mere morphological analysis, thus leading to the new concept of cell-optical-fingerprint avoiding fluorescent dyes. This perspective can open new routes in biomedical sciences, such as the chance to find optical-biomarkers at single cell level for label-free diagnosis.

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.

Instant in situ formation of a polymer film at the water-oil interface.

The water-oil interface is an environment that is often found in many contexts of the natural sciences and technological arenas. This interface has always been considered a special environment as it is rich in different phenomena, thus stimulating numerous studies aimed at understanding the abundance of physico-chemical problems that occur there. The intense research activity and the intriguing results that emerged from these investigations have inspired scientists to consider the water-oil interface even as a suitable setting for bottom-up nanofabrication processes, such as molecular self-assembly, or fabrication of nanofilms or nano-devices. On the other hand, biphasic liquid separation is a key enabling technology in many applications, including water treatment for environmental problems. Here we show for the first time an instant nanofabrication strategy of a thin film of biopolymer at the water-oil interface. The polymer film is fabricated in situ, simply by injecting a drop of polymer solution at the interface. Furthermore, we demonstrate that with an appropriate multiple drop delivery it is also possible to quickly produce a large area film (up to 150 cm2). The film inherently separates the two liquids, thus forming a separation layer between them and remains stable at the interface for a long time. Furthermore, we demonstrate the fabrication with different oils, thus suggesting potential exploitation in different fields (e.g. food, pollution, biotechnology). We believe that the new strategy fabrication could inspire different uses and promote applications among the many scenarios already explored or to be studied in the future at this special interface environment.

Biological Cells as Natural Biophotonic Devices: Fundamental and Applications-introduction to the feature issue.

This feature issue of Biomedical Optics Express presents a cross-section of interesting and emerging work of relevance to the use of biological cells or microorganisms in optics and photonics. The technologies demonstrated here aim to address challenges to meeting the optical imaging, sensing, manipulating and therapy needs in a natural or even endogenous manner. This collection of 15 papers includes the novel results on designs of optical systems or photonic devices, image-assisted diagnosis and treatment, and manipulation or sensing methods, with applications for both ex vivo and in vivo use. These works portray the opportunities for exploring the field crossing biology and photonics in which a natural element can be functionalized for biomedical applications.

Developing a Reliable Holographic Flow Cyto-Tomography Apparatus by Optimizing the Experimental Layout and Computational Processing.

Digital Holographic Tomography (DHT) has recently been established as a means of retrieving the 3D refractive index mapping of single cells. To make DHT a viable system, it is necessary to develop a reliable and robust holographic apparatus in order that such technology can be utilized outside of specialized optics laboratories and operated in the in-flow modality. In this paper, we propose a quasi-common-path lateral-shearing holographic optical set-up to be used, for the first time, for DHT in a flow-cytometer modality. The proposed solution is able to withstand environmental vibrations that can severely affect the interference process. Furthermore, we have scaled down the system while ensuring that a full 360° rotation of the cells occurs in the field-of-view, in order to retrieve 3D phase-contrast tomograms of single cells flowing along a microfluidic channel. This was achieved by setting the camera sensor at 45° with respect to the microfluidic direction. Additional optimizations were made to the computational elements to ensure the reliable retrieval of 3D refractive index distributions by demonstrating an effective method of tomographic reconstruction, based on high-order total variation. The results were first demonstrated using realistic 3D numerical phantom cells to assess the performance of the proposed high-order total variation method in comparison with the gold-standard algorithm for tomographic reconstructions: namely, filtered back projection. Then, the proposed DHT system and the processing pipeline were experimentally validated for monocytes and mouse embryonic fibroblast NIH-3T3 cells lines. Moreover, the repeatability of these tomographic measurements was also investigated by recording the same cell multiple times and quantifying the ability to provide reliable and comparable tomographic reconstructions, as confirmed by a correlation coefficient greater than 95%. The reported results represent various steps forward in several key aspects of in-flow DHT, thus paving the way for its use in real-world applications.

Kinematic analysis and visualization of Tetraselmis microalgae 3D motility by digital holography.

A study on locomotion in a 3D environment of Tetraselmis microalgae by digital holographic microscopy is reported. In particular, a fast and semiautomatic criterion is revealed for tracking and analyzing the swimming path of a microalga (i.e., Tetraselmis species) in a 3D volume. Digital holography (DH) in a microscope off-axis configuration is exploited as a useful method to enable fast autofocusing and recognition of objects in the field of view, thus coupling DH with appropriate numerical algorithms. Through the proposed method we measure, simultaneously, the tri-dimensional paths followed by the flagellate microorganism and the full set of the kinematic parameters that describe the swimming behavior of the analyzed microorganisms by means of a polynomial fitting and segmentation. Furthermore, the method is capable to furnish the accurate morphology of the microorganisms at any instant of time along its 3D trajectory. This work launches a promising trend having as the main objective the combined use of DH and motility microorganism analysis as a label-free and non-invasive environmental monitoring tool, employable also for in situ measurements. Finally, we show that the locomotion can be visualized intriguingly by different modalities to furnish marine biologists with a clear 3D representation of all the parameters of the kinematic set in order to better understand the behavior of the microorganism under investigation.

Speeding up reconstruction of 3D tomograms in holographic flow cytometry via deep learning.

Tomographic flow cytometry by digital holography is an emerging imaging modality capable of collecting multiple views of moving and rotating cells with the aim of recovering their refractive index distribution in 3D. Although this modality allows us to access high-resolution imaging with high-throughput, the huge amount of time-lapse holographic images to be processed (hundreds of digital holograms per cell) constitutes the actual bottleneck. This prevents the system from being suitable for lab-on-a-chip platforms in real-world applications, where fast analysis of measured data is mandatory. Here we demonstrate a significant speeding-up reconstruction of phase-contrast tomograms by introducing in the processing pipeline a multi-scale fully-convolutional context aggregation network. Although it was originally developed in the context of semantic image analysis, we demonstrate for the first time that it can be successfully adapted to a holographic lab-on-chip platform for achieving 3D tomograms through a faster computational process. We trained the network with input-output image pairs to reproduce the end-to-end holographic reconstruction process, i.e. recovering quantitative phase maps (QPMs) of single cells from their digital holograms. Then, the sequence of QPMs of the same rotating cell is used to perform the tomographic reconstruction. The proposed approach significantly reduces the computational time for retrieving tomograms, thus making them available in a few seconds instead of tens of minutes, while essentially preserving the high-content information of tomographic data. Moreover, we have accomplished a compact deep convolutional neural network parameterization that can fit into on-chip SRAM and a small memory footprint, thus demonstrating its possible exploitation to provide onboard computations for lab-on-chip devices with low processing hardware resources.

Intelligent polarization-sensitive holographic flow-cytometer: Towards specificity in classifying natural and microplastic fibers.

Micron size fiber fragments (MFFs), both natural and synthetic, are ubiquitous in our life, especially in textile clothes, being necessary in modern society. In the Earth's aquatic ecosystem, microplastic fibers account for ~91% of microplastic pollution, thus deserving notable attention as one of the most alarming ecological problems. Accurate automatic identification of MFFs discharges in specific upstream locations is highly demanded. Computational microscopy based on Digital Holography (DH) and machine learning has been demonstrated to identify microplastics in respect to microalgae. However, DH is a non-specific optical tool, meaning it cannot distinguish different types of plastic materials. On the other hand, materials-specific assessments are pivotal to establish the environmental impact of different textile products and production processes. Spectroscopic assays can be employed to identify microplastics for their intrinsic specificity, although they are generally low-throughput and require large concentrations to enable effective measurements. Conversely, MFFs are usually finely dispersed within a water sample. Here we rely on a polarization-resolved holographic flow cytometer in a Lab-on-Chip (LoC) platform for analysing MFFs. We demonstrate that two important objectives can be achieved, i.e. adding material specificity through polarization analysis while operating in a microfluidic stream modality. Through a machine learning numerical pipeline, natural fibers (i.e. cotton and wool) can be clearly separated from synthetic microfilaments, namely PA6, PA6.6, PET, PP. Moreover, the proposed system can accurately distinguish between different polymers under investigation, thus fulfilling the specificity goal. We extract and select different features from amplitude, phase and birefringence maps retrieved from the digital holograms. These are shown to typify MFFs without the need for sample pre-treatment or large concentrations. The simplicity of the DH method for identifying MFFs in LoC-based flow cytometers could promote the use of polarization resolved field-portable analysis systems suitable for studying pollution caused by washing processes of synthetic textiles.