A customizable digital holographic microscope.

We propose a compact, portable, and low-cost holographic microscope designed for the characterization of micrometric particles suspended in a liquid. This system is built around a commercial optical microscope by substituting its illumination source (a light-emitting diode) with a collimated laser beam. Similarly, a quartz flow cell replaces the microscope glass slide using a 3D-printed custom mount. With the hardware presented in this paper, the holographic imaging of the electromagnetic fields emitted by the particles that intercept the laser beam achieves a resolution close to that of optical microscopes but with a greater depth of field. Several morphological and optical features can be extracted from the holograms, including particle projected section, aspect ratio, and extinction cross-section. Additionally, we introduce a remote system control that enables users to process the acquired holograms on a remote computational device. This work provides a comprehensive description of the methodology of image processing in holographic microscopy and a series of validation measurements conducted using calibrated particles. This technique is suitable for the characterization of airborne particles found in snow, firn, and ice; here we report experimental results obtained from Alpine ice cores.

Standard-Deviation-Based Adaptive Median Filter for Elimination of Batwing Effects in Step Microstructure Measurement Using Digital Holography.

Digital holography has transformative potential for the measurement of stacked-chip microstructures due to its non-invasive, single-shot, full-field characteristics. However, significant light scattering and diffraction at steep edges in step microstructures cause the batwing effect, leading to measurement errors. Herein, we propose a standard-deviation-based adaptive median filter to eliminate batwing effects in step microstructure measurement using digital holography. The standard deviation determines the positions of the steps and the range of the batwing effect. During filtering, the filter window size varies: it adjusts according to the center's position within the batwing effect range and reduces outside this range to prevent distortion in other regions. Filtering weights are set to maintain information integrity while using larger filter windows. Experiments on the Standard Resolution Target USAF 1951 and the standard step height target show that our method successfully eliminates batwings while preserving the integrity of the remaining profile.

Quantitative phase imaging techniques for measuring scattering properties of cells and tissues: a review-part II.

Significance: Quantitative phase imaging (QPI) is a non-invasive, label-free technique that provides intrinsic information about the sample under study. Such information includes the structure, function, and dynamics of the sample. QPI overcomes the limitations of conventional fluorescence microscopy in terms of phototoxicity to the sample and photobleaching of the fluorophore. As such, the application of QPI in estimating the three-dimensional (3D) structure and dynamics is well-suited for a range of samples from intracellular organelles to highly scattering multicellular samples while allowing for longer observation windows. Aim: We aim to provide a comprehensive review of 3D QPI and related phase-based measurement techniques along with a discussion of methods for the estimation of sample dynamics. Approach: We present information collected from 106 publications that cover the theoretical description of 3D light scattering and the implementation of related measurement techniques for the study of the structure and dynamics of the sample. We conclude with a discussion of the applications of the reviewed techniques in the biomedical field. Results: QPI has been successfully applied to 3D sample imaging. The scattering-based contrast provides measurements of intrinsic quantities of the sample that are indicative of disease state, stage of growth, or overall dynamics. Conclusions: We reviewed state-of-the-art QPI techniques for 3D imaging and dynamics estimation of biological samples. Both theoretical and experimental aspects of various techniques were discussed. We also presented the applications of the discussed techniques as applied to biomedicine and biology research.

Correlative Raman Imaging: Development and Cancer Applications.

Despite extensive research efforts, cancer continues to stand as one of the leading causes of death on a global scale. To gain profound insights into the intricate mechanisms underlying cancer onset and progression, it is imperative to possess methodologies that allow the study of cancer cells at the single-cell level, focusing on critical parameters such as cell morphology, metabolism, and molecular characteristics. These insights are essential for effectively discerning between healthy and cancerous cells and comprehending tumoral progression. Recent advancements in microscopy techniques have significantly advanced the study of cancer cells, with Raman microspectroscopy (RM) emerging as a particularly powerful tool. Indeed, RM can provide both biochemical and spatial details at the single-cell level without the need for labels or causing disruptions to cell integrity. Moreover, RM can be correlated with other microscopy techniques, creating a synergy that offers a spectrum of complementary insights into cancer cell morphology and biology. This review aims to explore the correlation between RM and other microscopy techniques such as confocal fluoresce microscopy (CFM), atomic force microscopy (AFM), digital holography microscopy (DHM), and mass spectrometry imaging (MSI). Each of these techniques has their own strengths, providing different perspectives and parameters about cancer cell features. The correlation between information from these various analysis methods is a valuable tool for physicians and researchers, aiding in the comprehension of cancer cell morphology and biology, unraveling mechanisms underlying cancer progression, and facilitating the development of early diagnosis and/or monitoring cancer progression.

Quantitative phase imaging techniques for measuring scattering properties of cells and tissues: a review-part I.

Significance: Quantitative phase imaging (QPI) techniques offer intrinsic information about the sample of interest in a label-free, noninvasive manner and have an enormous potential for wide biomedical applications with negligible perturbations to the natural state of the sample in vitro. Aim: We aim to present an in-depth review of the scattering formulation of light-matter interactions as applied to biological samples such as cells and tissues, discuss the relevant quantitative phase measurement techniques, and present a summary of various reported applications. Approach: We start with scattering theory and scattering properties of biological samples followed by an exploration of various microscopy configurations for 2D QPI for measurement of structure and dynamics. Results: We reviewed 157 publications and presented a range of QPI techniques and discussed suitable applications for each. We also presented the theoretical frameworks for phase reconstruction associated with the discussed techniques and highlighted their domains of validity. Conclusions: We provide detailed theoretical as well as system-level information for a wide range of QPI techniques. Our study can serve as a guideline for new researchers looking for an exhaustive literature review of QPI methods and relevant applications.

Coherent light scattering from cellular dynamics in living tissues.

This review examines the biological physics of intracellular transport probed by the coherent optics of dynamic light scattering from optically thick living tissues. Cells and their constituents are in constant motion, composed of a broad range of speeds spanning many orders of magnitude that reflect the wide array of functions and mechanisms that maintain cellular health. From the organelle scale of tens of nanometers and upward in size, the motion inside living tissue is actively driven rather than thermal, propelled by the hydrolysis of bioenergetic molecules and the forces of molecular motors. Active transport can mimic the random walks of thermal Brownian motion, but mean-squared displacements are far from thermal equilibrium and can display anomalous diffusion through Lévy or fractional Brownian walks. Despite the average isotropic three-dimensional environment of cells and tissues, active cellular or intracellular transport of single light-scattering objects is often pseudo-one-dimensional, for instance as organelle displacement persists along cytoskeletal tracks or as membranes displace along the normal to cell surfaces, albeit isotropically oriented in three dimensions. Coherent light scattering is a natural tool to characterize such tissue dynamics because persistent directed transport induces Doppler shifts in the scattered light. The many frequency-shifted partial waves from the complex and dynamic media interfere to produce dynamic speckle that reveals tissue-scale processes through speckle contrast imaging and fluctuation spectroscopy. Low-coherence interferometry, dynamic optical coherence tomography, diffusing-wave spectroscopy, diffuse-correlation spectroscopy, differential dynamic microscopy and digital holography offer coherent detection methods that shed light on intracellular processes. In health-care applications, altered states of cellular health and disease display altered cellular motions that imprint on the statistical fluctuations of the scattered light. For instance, the efficacy of medical therapeutics can be monitored by measuring the changes they induce in the Doppler spectra of livingex vivocancer biopsies.

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.

Dual Field-of-View Off-Axis Spatially Multiplexed Digital Holography Using Fresnel's Bi-Mirror.

Digital holography (DH) is an important method for three-dimensional (3D) imaging since it allows for the recording and reconstruction of an object's amplitude and phase information. However, the field of view (FOV) of a DH system is typically restricted by the finite size of the pixel pitch of the digital image sensor. We proposed a new configuration of the DH system based on Fresnel's bi-mirror to achieve doubling the camera FOV of the existing off-axis DH system which leveraged single-shot acquisition and a common-path optical framework. The dual FOV was obtained by spatial frequency multiplexing corresponding to two different information-carrying beams from an object. Experimental evidence of the proposed dual FOV-DH system's viability was provided by imaging two different areas of the test object and an application to surface profilometry by measuring the step height of the resolution chart which showed excellent agreement with an optical profiler. Due to the simple configuration, the proposed system could find a wide range of applications, including in microscopy and optical metrology.

Moving grating-based laser heterodyne digital holographic microscopy system for measuring dynamic phase of living cell attachment.

We propose a laser heterodyne digital holography microscopy system based on a moving grating, which uses the Doppler principle between a moving grating and beam to achieve a low-frequency bias between the diffracted beams, abandoning traditional heterodyne digital holography that requires multiple acousto-optic modulators. The dynamic phase distribution obtained using the laser heterodyne digital holography phase-reconstruction algorithm was more realistic and analyzable than the results of the angular spectrum algorithm. The structure and algorithm were used to capture the shape characteristics of mouse fibroblasts after ~2 h of incubation (37°C, 5% CO2), and the dynamic phase distribution of the cells was monitored in real-time during the attachment process. The system proposed in this study, with its high spatial resolution and high-precision phase measurement capability, is suitable for both static and live cells.

Dielectric Metasurface Enabled Compact, Single-Shot Digital Holography for Quantitative Phase Imaging.

Quantitative phase imaging (QPI) enables nondestructive, real-time, label-free imaging of transparent specimens and can reveal information about their fundamental properties such as cell size and morphology, mass density, particle dynamics, and cellular fluctuations. Development of high-performance and low-cost quantitative phase imaging systems is thus required in many fields, including on-site biomedical imaging and industrial inspection. Here, we propose an ultracompact, highly stable interferometer based on a single-layer dielectric metasurface for common path off-axis digital holography and experimentally demonstrate quantitative phase imaging. The interferometric imaging system leveraging an ultrathin multifunctional metasurface captures image plane holograms in a single shot and provides quantitative phase information on the test samples for extraction of its physical properties. With the benefits of planar engineering and high integrability, the proposed metasurface-based method establishes a stable miniaturized QPI system for reliable and cost-effective point-of-care devices, live cell imaging, 3D topography, and edge detection for optical computing.

Synchronous Phase-Shifting Interference for High Precision Phase Imaging of Objects Using Common Optics.

Quantitative phase imaging and measurement of surface topography and fluid dynamics for objects, especially for moving objects, is critical in various fields. Although effective, existing synchronous phase-shifting methods may introduce additional phase changes in the light field due to differences in optical paths or need specific optics to implement synchronous phase-shifting, such as the beamsplitter with additional anti-reflective coating and a micro-polarizer array. Therefore, we propose a synchronous phase-shifting method based on the Mach-Zehnder interferometer to tackle these issues in existing methods. The proposed method uses common optics to simultaneously acquire four phase-shifted digital holograms with equal optical paths for object and reference waves. Therefore, it can be used to reconstruct the phase distribution of static and dynamic objects with high precision and high resolution. In the experiment, the theoretical resolution of the proposed system was 1.064 µm while the actual resolution could achieve 1.381 µm, which was confirmed by measuring a phase-only resolution chart. Besides, the dynamic phase imaging of a moving standard object was completed to verify the proposed system's effectiveness. The experimental results show that our proposed method is suitable and promising in dynamic phase imaging and measurement of moving objects using phase-shifting digital holography.

Surface topographic characterization of optical storage devices by Digital Holographic Microscopy.

In this study, we have used Digital Holographic Microscopy (DHM) for surface topographic characterization of the optical storage devices. Optical storage devices like Compact Discs (CDs) and Digital Versatile discs (DVDs) are used worldwide for memory storage, grating, sensing and spectroscopy applications. These devices can store the information in binary form on a spiral track in the data recording layer. We demonstrate that these data tracks can be characterized through DHM and one can decode the binary data in transmission mode. The experimental results are shown for blank (without data) and burned (with data) areas of CDs and DVDs. The average width of the CD and DVD data track is experimentally found to be 600 ± 30 nm & 230 ± 20 nm, respectively and the thickness of the data recording track is obtained as 80 ± 10 nm. The obtained results are verified by Field Emission Scanning Electron Microscopy (FESEM) measurements and found a very close agreement between the two results. In addition, the proposed method can also be used for manufacturing defect identification and measurement.

A Method for Detecting the Vacuum Degree of Vacuum Glass Based on Digital Holography.

The vacuum degree is the key parameter reflecting the quality and performance of vacuum glass. This investigation proposed a novel method, based on digital holography, to detect the vacuum degree of vacuum glass. The detection system was composed of an optical pressure sensor, a Mach-Zehnder interferometer and software. The results showed that the deformation of monocrystalline silicon film in an optical pressure sensor could respond to the attenuation of the vacuum degree of vacuum glass. Using 239 groups of experimental data, pressure differences were shown to have a good linear relationship with the optical pressure sensor's deformations; pressure differences were linearly fitted to obtain the numerical relationship between pressure difference and deformation and to calculate the vacuum degree of the vacuum glass. Measuring the vacuum degree of vacuum glass under three different conditions proved that the digital holographic detection system could measure the vacuum degree of vacuum glass quickly and accurately. The optical pressure sensor's deformation measuring range was less than 4.5 µm, the measuring range of the corresponding pressure difference was less than 2600 pa, and the measuring accuracy's order of magnitude was 10 pa. This method has potential market applications.

Digital holographic technique based breast cancer detection using transfer learning method.

The digital holographic technique is an interferometric method that provides comprehensive information on morphological traits such as cell layer thickness and shape as well as access to biophysical attributes of cells like refractive index, dry mass, and volume. This method helps characterize sample structures in three dimensions both statically and dynamically, even for transparent objects like living biological cells. This research work captures the digital holograms of breast tissues and analyzes the malignancy of the tissue using a deep learning technique. It enables dynamic measurement of the sample under investigation. Different transfer learning models such as Inception, DenseNet, SqueezeNet, VGG, and ResNet are incorporated in this work. The parameters accuracy, precision, sensitivity, and F1 score of different models are compared and found that the ResNet model outperforms better compared to other models.

Algal cell viability assessment: The role of environmental factors in phytoplankton population dynamics.

The viability of algal cells is one of the most fundamental issues in marine ecological research. In this work, a method was designed to identify algal cell viability based on digital holography and deep learning, which divided algal cells into three categories: active, weak, and dead cells. This method was applied to measure algal cells in surface waters of the East China Sea in spring, revealing about 4.34 %-23.29 % weak cells and 3.98 %-19.47 % dead cells. Levels of nitrate and chlorophyll a were the main factors affecting the viability of algal cells. Furthermore, algal viability changes during the heating and cooling were observed in laboratory experiments: high temperatures led to an increase in weak algal cells. This may provide an explanation for why most harmful algal blooms occur in warming months. This study provided a novel insight into how to identify the viability of algal cells and understand their significance in the ocean.

Label-Free Long-Term Methods for Live Cell Imaging of Neurons: New Opportunities.

Time-lapse light microscopy combined with in vitro neuronal cultures has provided a significant contribution to the field of Developmental Neuroscience. The establishment of the neuronal polarity, i.e., formation of axons and dendrites, key structures responsible for inter-neuronal signaling, was described in 1988 by Dotti, Sullivan and Banker in a milestone paper that continues to be cited 30 years later. In the following decades, numerous fluorescently labeled tags and dyes were developed for live cell imaging, providing tremendous advancements in terms of resolution, acquisition speed and the ability to track specific cell structures. However, long-term recordings with fluorescence-based approaches remain challenging because of light-induced phototoxicity and/or interference of tags with cell physiology (e.g., perturbed cytoskeletal dynamics) resulting in compromised cell viability leading to cell death. Therefore, a label-free approach remains the most desirable method in long-term imaging of living neurons. In this paper we will focus on label-free high-resolution methods that can be successfully used over a prolonged period. We propose novel tools such as scanning ion conductance microscopy (SICM) or digital holography microscopy (DHM) that could provide new insights into live cell dynamics during neuronal development and regeneration after injury.

Investigation of the effects of the magnetic field on the anodic dissolution of alloy 690 in SO4 2- + SCN- solution using digital holography.

Digital holography has been employed for in situ observation of dynamic processes occurring at the electrode|electrolyte interface during the anodic dissolution of Alloy 690 in solutions containing SO4 2- + SCN- with or without magnetic field (MF). It was found that MF increased the anodic current of Alloy 690 in 0.5 M Na2SO4 + 5 mM KSCN solution but showed a decreased value when evaluated in 0.5 M H2SO4 + 5 mM KSCN solution. For each solution, as a result of the stirring effect due to Lorentz force, MF showed a decreased localized damage further preventing pitting corrosion. The content of nickel and iron at grain boundaries is higher than that on the grain body, in accordance with the Cr-depletion theory. MF increased the anodic dissolution of nickel and iron, which in turn increased the anodic dissolution at grain boundaries. In situ inline digital holography revealed that IGC begins at one grain boundary and progresses to adjacent grain boundaries with or without MF.

Phase-sensitive detection of anomalous diffusion dynamics in the neuronal membrane induced by ion channel gating.

Non-ergodicity of neuronal dynamics from rapid ion channel gating through the membrane induces membrane displacement statistics that deviate from Brownian motion. The membrane dynamics from ion channel gating were imaged by phase-sensitive optical coherence microscopy. The distribution of optical displacements of the neuronal membrane showed a Lévy-like distribution and the memory effect of the membrane dynamics by the ionic gating was estimated. The alternation of the correlation time was observed when neurons were exposed to channel-blocking molecules. Non-invasive optophysiology by detecting the anomalous diffusion characteristics of dynamic images is demonstrated.

Multi-Class Classification and Multi-Output Regression of Three-Dimensional Objects Using Artificial Intelligence Applied to Digital Holographic Information.

Digital holographically sensed 3D data processing, which is useful for AI-based vision, is demonstrated. Three prominent methods of learning from datasets such as sensed holograms, computationally retrieved intensity and phase from holograms forming concatenated intensity-phase (whole information) images, and phase-only images (depth information) were utilized for the proposed multi-class classification and multi-output regression tasks of the chosen 3D objects in supervised learning. Each dataset comprised 2268 images obtained from the chosen eighteen 3D objects. The efficacy of our approaches was validated on experimentally generated digital holographic data then further quantified and compared using specific evaluation matrices. The machine learning classifiers had better AUC values for different classes on the holograms and whole information datasets compared to the CNN, whereas the CNN had a better performance on the phase-only image dataset compared to these classifiers. The MLP regressor was found to have a stable prediction in the test and validation sets with a fixed EV regression score of 0.00 compared to the CNN, the other regressors for holograms, and the phase-only image datasets, whereas the RF regressor showed a better performance in the validation set for the whole information dataset with a fixed EV regression score of 0.01 compared to the CNN and other regressors.

Multi-Illumination Single-Holographic-Exposure Lensless Fresnel (MISHELF) Microscopy: Principles and Biomedical Applications.

Lensless holographic microscopy (LHM) comes out as a promising label-free technique since it supplies high-quality imaging and adaptive magnification in a lens-free, compact and cost-effective way. Compact sizes and reduced prices of LHMs make them a perfect instrument for point-of-care diagnosis and increase their usability in limited-resource laboratories, remote areas, and poor countries. LHM can provide excellent intensity and phase imaging when the twin image is removed. In that sense, multi-illumination single-holographic-exposure lensless Fresnel (MISHELF) microscopy appears as a single-shot and phase-retrieved imaging technique employing multiple illumination/detection channels and a fast-iterative phase-retrieval algorithm. In this contribution, we review MISHELF microscopy through the description of the principles, the analysis of the performance, the presentation of the microscope prototypes and the inclusion of the main biomedical applications reported so far.