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.

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.

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.

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.

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.

Digital Holography and 3D Imaging 2020: introduction to the feature issue.

This feature issue of JOSA A and Applied Optics is dedicated to the fourteenth OSA Topical Meeting "Digital Holography and 3D Imaging" held 22-26 June 2020 in a virtual meeting. The conference, taking place every year, is a focal point for global technical interchange in the field of digital holography and 3D imaging, providing premier opportunities for people working in the field to present their new advances in research and development. Papers presented at the meeting highlight current research in digital holography and three-dimensional imaging, including interferometry, phase microscopy, phase retrieval, novel holographic processes, 3D and novel holographic displays, integral imaging, computer-generated holograms, compressive holography, 3D holographic display, AR display, full-field tomography, specific image and signal processing, and holography with various light sources, including coherent to incoherent and x-ray to terahertz waves. Techniques of digital holography and of 3D imaging have numerous applications, such as the state-of-the-art technological developments that are currently underway and stimulate further novel applications of digital holography and 3D imaging in biomedicine, deep learning, and scientific and industrial metrologies.

Digital Holography and 3D Imaging 2020: introduction to the feature issue.

This feature issue of JOSA A and Applied Optics is dedicated to the fourteenth OSA Topical Meeting "Digital Holography and 3D Imaging" held 22-26 June 2020 in a virtual meeting. The conference, taking place every year, is a focal point for global technical interchange in the field of digital holography and 3D imaging, providing premier opportunities for people working in the field to present their new advances in research and development. Papers presented at the meeting highlight current research in digital holography and three-dimensional imaging, including interferometry, phase microscopy, phase retrieval, novel holographic processes, 3D and novel holographic displays, integral imaging, computer-generated holograms, compressive holography, 3D holographic display, AR display, full-field tomography, specific image and signal processing, and holography with various light sources, including coherent to incoherent and x-ray to terahertz waves. Techniques of digital holography and of 3D imaging have numerous applications, such as the state-of-the-art technological developments that are currently underway and have also stimulated further novel applications of digital holography and 3D imaging in biomedicine, deep learning, and scientific and industrial metrologies.

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.

Comparative study of multi-look processing for phase map de-noising in digital Fresnel holographic interferometry.

This paper presents a comparative study of multi-look approaches for de-noising phase maps from digital holographic interferometry. A database of 160 simulated phase fringe patterns with eight different phase fringe patterns with fringe diversity was computed. For each fringe pattern, 20 realistic noise realizations are generated in order to simulate a multi-look process with 20 inputs. A set of 22 de-noising algorithms was selected and processed for each simulation. Three approaches for multi-look processing are evaluated. Quantitative appraisal is obtained using two metrics. The results show good agreement for algorithm rankings obtained with both metrics. One singular and highly practical result of the study is that a multi-look approach with average looks before noise processing performs better than averaging computed with all de-noised looks. The results also demonstrate that the two-dimensional windowed Fourier transform filtering exhibits the best performance in all cases and that the block-matching 3D (BM3D) algorithm is second in the ranking.

Adaptive and automatic diffraction order filtering by singular value decomposition in off-axis digital holographic microscopy.

Digital holography is widely used in many fields for imaging, display, and metrology by exploiting its capability to furnish quantitative phase contrast maps. The entire processing pipeline that permits achievement of phase contrast images can be obtained by a cascade of numerical processing, such as zero-order and twin-image suppression, automatic refocusing, phase extraction by aberration compensation, and, if necessary, phase unwrapping. In this paper, we propose a new method, to the best of our knowledge, based on singular value decomposition filtering, to suppress zero-order and twin images in off-axis configuration, thus, automatically selecting the desired real diffraction order. We demonstrate the proposed approach in the case of lack of knowledge about the reference beam's frequency and curvature, which typically occurs in portable off-axis holographic microscope systems for lab-on-a-chip applications. We validate the proposed strategy by a comparison with common Fourier spatial filtering in the case of different experimental conditions and for several biological samples.

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.

On-speckle suppression in IR digital holography.

Long-IR wavelength is the best option for capturing digital holograms of large-size, real-world objects. However, the coherent noise level in a long-IR hologram is by far larger than that of a visible wavelength recording, thus resulting in a poor quality of both numerical and optical reconstructions. In this Letter, we show how such coherent noise can be efficiently suppressed by employing an optical scanning multi-look approach, in combination with 3D block matching numerical filtering. Results demonstrate the possibility to obtain near noise-free numerical reconstructions of IR digital holograms of large-size objects, while preserving resolution. We applied this method to the holograms of a rotating statuette. It will be shown that a remarkable contrast enhancement is achievable along with the recovery of object details that otherwise would be lost because of large speckle grains intrinsically due to the source coherence.

Improving holographic reconstruction by automatic Butterworth filtering for microelectromechanical systems characterization.

Digital holographic microscopy is an important interferometric tool in optical metrology allowing the investigation of engineered surfaces with microscale lateral resolution and nanoscale axial precision. In particular, microelectromechanical systems (MEMS) surface analysis, conducted by holographic characterization, requires high accuracy for functional testing. The main issues related to MEMS inspection are the superficial roughness and the complex geometry resulting from the several fabrication steps. Here, an automatic procedure, particularly suited in the case of high-roughness surfaces, is presented to selectively filter the spectrum, providing very low-noise reconstructed images. The numerical procedure is based on Butterworth filtering, and the obtained results demonstrate a significant increase in the images' quality and in the accuracy of the measurements, making our technique highly applicable for quantitative phase imaging in MEMS analysis. Furthermore, our method is fully tunable to the spectrum under investigation and automatic. This makes it highly suitable for real-time applications. Several experimental tests show the suitability of the proposed approach.

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.

Encoding multiple holograms for speckle-noise reduction in optical display.

In digital holography (DH) a mixture of speckle and incoherent additive noise, which appears in numerical as well as in optical reconstruction, typically degrades the information of the object wavefront. Several methods have been proposed in order to suppress the noise contributions during recording or even during the reconstruction steps. Many of them are based on the incoherent combination of multiple holographic reconstructions achieving remarkable improvement, but only in the numerical reconstruction i.e. visualization on a pc monitor. So far, it has not been shown the direct synthesis of a digital hologram which provides the denoised optical reconstruction. Here, we propose a new effective method for encoding in a single complex wavefront the contribution of multiple incoherent reconstructions, thus allowing to obtain a single synthetic digital hologram that show significant speckle-reduction when optically projected by a Spatial Light Modulator (SLM).

Refocusing criterion via sparsity measurements in digital holography.

Several automatic approaches have been proposed in the past to compute the refocus distance in digital holography (DH). However most of them are based on a maximization or minimization of a suitable amplitude image contrast measure, regarded as a function of the reconstruction distance parameter. Here we show that, by using the sparsity measure coefficient regarded as a refocusing criterion in the holographic reconstruction, it is possible to recover the focus plane and, at the same time, establish the degree of sparsity of digital holograms, when samples of the diffraction Fresnel propagation integral are used as a sparse signal representation. We employ a sparsity measurement coefficient known as Gini's index thus showing for the first time, to the best of our knowledge, its application in DH, as an effective refocusing criterion. Demonstration is provided for different holographic configurations (i.e., lens and lensless apparatus) and for completely different objects (i.e., a thin pure phase microscopic object as an in vitro cell, and macroscopic puppets) preparation.

Holographic tracking of living cells by three-dimensional reconstructed complex wavefronts alignment.

We propose here a new three-dimensional (3D) holographic tracking method capable to track, simultaneously and in a single step, all the spatial coordinates of micro-objects. The approach is based on the enhanced correlation coefficient (ECC) maximization method but applied, for the first time to the best of our knowledge, directly on the holographic reconstructed complex wave fields. The key novelty of the proposed strategy is its ability to calculate simultaneously the 3D coordinates of cells, without decoupling the contribution of amplitude and phase. The proposed strategy is tested on living cells (i.e., NIH 3T3 mouse fibroblast) flowing into a microfluidic channel and compared with classical holographic tracking approach. Theoretical description and experimental validation of the proposed strategy are reported.

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.