Digital In-line Holographic Microscopy Research Articles

Advancing the science of dynamic airborne nanosized particles using Nano-DIHM

In situ and real-time characterization of aerosols is vital to several fundamental and applied research domains including atmospheric chemistry, air quality monitoring, or climate change studies. To date, digital holographic microscopy is commonly used to characterize dynamic nanosized particles, but optical traps are required. In this study, a novel integrated digital in-line holographic microscope coupled with a flow tube (Nano-DIHM) is demonstrated to characterize particle phase, shape, morphology, 4D dynamic trajectories, and 3D dimensions of airborne particles ranging from the nanoscale to the microscale. We demonstrate the application of Nano-DIHM for nanosized particles (≤200 nm) in dynamic systems without optical traps. The Nano-DIHM allows observation of moving particles in 3D space and simultaneous measurement of each particle’s three dimensions. As a proof of concept, we report the real-time observation of 100 nm and 200 nm particles, i.e. polystyrene latex spheres and the mixture of metal oxide nanoparticles, in air and aqueous/solid/heterogeneous phases in stationary and dynamic modes. Our observations are validated by high-resolution scanning/transmission electron microscopy and aerosol sizers. The complete automation of software (Octopus/Stingray) with Nano-DIHM permits the reconstruction of thousands of holograms within an hour with 62.5 millisecond time resolution for each hologram, allowing to explore the complex physical and chemical processes of aerosols.

Digital In-Line Holography for Large-Volume Analysis of Vertical Motion of Microscale Marine Plankton and Other Particles

Measuring the distribution, characteristics and dynamics of marine microscale plankton and other particulate matter is essential to understand the vertical flux of elements in the marine environment. Digital holographic microscopy is a powerful approach for measuring these and studying their 3-D trajectories in a relatively large observation volume. This article demonstrates a compact, in-line digital holographic microscope that allows large-volume and high-resolution recording of marine particles through combining a continuous wave laser and a short exposure CMOS camera with efficient global shutters. A resolution of better than 10 μm is demonstrated in air and the minimum distinguishable size of targets recorded in water is approximately 20 μm. The maximum volumetric throughput of the setup is 1904 mL/s. The microscope can take motion blur free holograms of particles moving at up to 490 mm/s in theory, and has been tested in the ∼200-mm/s flowing water. The orientation of the measured volume improves the ability of digital holography in profiling sinking rates and active vertical migration. The system was tested onboard a research vessel to record a range of live plankton and other particles. The motion of some samples, including the sinking motion and swimming motion, was analyzed using custom developed image processing software. The experimental results show that the combination of high resolution and a large volume over which motion of sparse-distribution particles can be tracked, can improve the ability to differentiate between different types of marine particle and identify behaviors of live plankton.

Using differential phase for 3D localization of tracer particles in digital inline holographic microscopy PIV/PTV (DIHM-PIV/PTV)

Digital inline holographic microscopy PIV/PTV (DIHM-PIV/PTV) has the ability to provide 4-dimensional (4D), i.e. time-resolved, 3-component 3-dimensional (3C-3D) flow measurement with high spatial and temporal resolution, compact optical setup and minimal calibration Sun et al. (2020) compared to most other volumetric techniques such as tomo-PIV, defocusing PIV, etc. Despite all these advantages DIHMPIV/PTV has not yet developed into a standard laboratory tool due to some major limitations such as the extended depth-of-focus (DOF) problem and the virtual image effect which cause artefacts in the standard reconstruction volume limiting the seeding concentration and thus the achievable velocity spatial resolution. In order to mitigate the above-mentioned limitations we present a novel particle localization and extraction methodology which allows the minimization of these artefacts from the standard reconstruction and perform PIV/PTV analysis on the particle volume fields only. The proposed algorithm is based on the differential phase, which is the axial phase shift of the object wave compared to the reference plane wave propagation.

Visualization of Three-Dimensional Acoustic Streaming Flow Patterns around an Inclined Triangular Obstruction using Digital In-Line Holographic Micro-Particle Tracking Velocimetry

Acoustic Streaming is a flow phenomenon with many applications in the field of microfluidics, such as micro mixing[1, 2] and particle manipulation[3]. With the manufacturing techniques evolves, more complicated geometries can be designed for microfluidic device and 3-D acoustic streaming patterns may occurs. In this study, 3-D trajectories of particle induced by acoustic streaming around an inclined triangular obstruction in a microchannel were visualized by a volumetric tracking method using Digital Inline Holographic Microscopy (DIHM)[4-6]. The triangular obstruction has a tip angle of 20° and an inclined angle of 30°. The acoustic streaming is created under 12 kHz oscillation of a piezo plate driven by 20V voltage. Illuminated by a 450nm continuous laser, the magnified hologram of the motion of 1.79μm tracer particles was recorded by a low-cost 10X industrial microscope with a machine vision camera of 10 fps (frames per second). Using RayleighSommerfeld back-propagation method[7], particle locations was reconstructed frame by frame and 3-D tracking of individual particles was performed afterwards. The trajectories of each particle were reconstructed to reveal the vortical structure of the acoustic streaming flow. For the current system setup, the measurable range was estimated to be 550×685×840 μm3. The 3-D location reconstruction accuracy was verified with a calibration target and the location sensitivity was found to be linear throughout the measurable range. Reconstruction at different depth locations show that the dick-shaped calibration dots and the spherical polystyrene particles have different intensity profiles. The calibration dots show local minimum of intensity at the correct depth location, while polystyrene particles show local maximum of intensity instead. Resolved particle trajectories show that the acoustic streaming flows cause particles to move with 3-D spiral shaped motions near the side of the triangular obstruction, while particles away from the obstruction shows planar motions.

Plankton classification with high-throughput submersible holographic microscopy and transfer learning

BackgroundPlankton are foundational to marine food webs and an important feature for characterizing ocean health. Recent developments in quantitative imaging devices provide in-flow high-throughput sampling from bulk volumes—opening new ecological challenges exploring microbial eukaryotic variation and diversity, alongside technical hurdles to automate classification from large datasets. However, a limited number of deployable imaging instruments have been coupled with the most prominent classification algorithms—effectively limiting the extraction of curated observations from field deployments. Holography offers relatively simple coherent microscopy designs with non-intrusive 3-D image information, and rapid frame rates that support data-driven plankton imaging tasks. Classification benchmarks across different domains have been set with transfer learning approaches, focused on repurposing pre-trained, state-of-the-art deep learning models as classifiers to learn new image features without protracted model training times. Combining the data production of holography, digital image processing, and computer vision could improve in-situ monitoring of plankton communities and contribute to sampling the diversity of microbial eukaryotes.ResultsHere we use a light and portable digital in-line holographic microscope (The HoloSea) with maximum optical resolution of 1.5 μm, intensity-based object detection through a volume, and four different pre-trained convolutional neural networks to classify > 3800 micro-mesoplankton (> 20 μm) images across 19 classes. The maximum classifier performance was quickly achieved for each convolutional neural network during training and reached F1-scores > 89%. Taking classification further, we show that off-the-shelf classifiers perform strongly across every decision threshold for ranking a majority of the plankton classes.ConclusionThese results show compelling baselines for classifying holographic plankton images, both rare and plentiful, including several dinoflagellate and diatom groups. These results also support a broader potential for deployable holographic microscopes to sample diverse microbial eukaryotic communities, and its use for high-throughput plankton monitoring.

HologLev: A Hybrid Magnetic Levitation Platform Integrated with Lensless Holographic Microscopy for Density-Based Cell Analysis.

In clinical practice, a variety of diagnostic applications require the identification of target cells. Density has been used as a physical marker to distinguish cell populations since metabolic activities could alter the cell densities. Magnetic levitation offers great promise for separating cells at the single cell level within heterogeneous populations with respect to cell densities. Traditional magnetic levitation platforms need bulky and precise optical microscopes to visualize levitated cells. Moreover, the evaluation process of cell densities is cumbersome, which also requires trained personnel for operation. In this work, we introduce a device (HologLev) as a fusion of the magnetic levitation principle and lensless digital inline holographic microscopy (LDIHM). LDIHM provides ease of use by getting rid of bulky and expensive optics. By placing an imaging sensor just beneath the microcapillary channel without any lenses, recorded holograms are processed for determining cell densities through a fully automated digital image processing scheme. The device costs less than $100 and has a compact design that can fit into a pocket. We perform viability tests on the device by levitating three different cell lines (MDA-MB-231, U937, D1 ORL UVA) and comparing them against their dead correspondents. We also tested the differentiation of mouse osteoblastic (7F2) cells by monitoring characteristic variations in their density. Last, the response of MDA-MB-231 cancer cells to a chemotherapy drug was demonstrated in our platform. HologLev provides cost-effective, label-free, fully automated cell analysis in a compact design that could be highly desirable for laboratory and point-of-care testing applications.

Design and Experiments with a Robot-Driven Underwater Holographic Microscope for Low-Cost In Situ Particle Measurements

Microscopic analysis of microparticles in situ in diverse water environments is necessary for monitoring water quality and localizing contamination sources. Conventional sensors such as optical microscopes and fluorometers often require complex sample preparation, are restricted to small sample volumes, and are unable to simultaneously capture all pertinent details of a sample such as particle size, shape, concentration, and three-dimensional motion. This paper proposes a novel and cost-effective robotic system for mobile microscopic analysis of particles in situ at various depths which are fully controlled by the robot system itself. A miniature underwater digital in-line holographic microscope (DIHM) performs high-resolution imaging of microparticles (e.g., algae cells, plastic debris, sediments) while movement allows measurement of particle distributions covering a large area of water.

Digital holographic microscopy for domain structure investigation of triglycine sulfate monocrystals doped with Nd

The domain structure is an important characteristic of ferroelectrics. One of the most intensively studied ferroelectrics is triglycine sulfate (TGS). Although many methods have been developed for the observation of the domain structure in TGS, it still cannot be visualized routinely. A new method for observation of the domains in TGS, in particular digital in-line holographic microscopy, was applied for investigation of the domain structure of TGS monocrystals doped with Nd. Monocrystals of TGS with different concentration of Nd in the solution of growth were investigated. The size of the domains was calculated using appropriate software. The results show that the average size of domains increases with the increase of the Nd concentration in the doped TGS monocrystals. Correlation analysis was applied to study the effect of Nd concentration on the domains area. A very strong, positive effect of Nd concentration on the area was established (R = 0.958 *). The established influence of the dopant concentration on the domains area is presented in analytical form through a mathematical model.

Separating twin images in digital holographic microscopy using weak scatterers.

When using inline digital holographic microscopy (DHM) and placing the hologram plane within a particle suspension, both real and virtual images come into focus during reconstruction, limiting our ability to resolve three-dimensional (3D) particle distribution. Here, we propose a new method to distinguish between real and virtual images in the 3D reconstruction field. This new method is based on the use of weak scatterers, and the fact that the real and virtual images of weak scatterers display distinct intensity distributions along the optical axis. We experimentally demonstrate this method by localizing and tracking 1 µm particles in a 3D volume with a particle concentration ranging from 200 to 6000particles/mm3. Unlike previous approaches to address the virtual image problem, this method does not require the recording of multiple holograms or the insertion of additional optical components. The proposed method allows the hologram plane to be placed within the sample volume, and extends the capability of DHM to measure the 3D movements of particles in deep samples far away from the optical window.

Height distribution and orientation of colloidal dumbbells near a wall.

Geometric confinement strongly influences the behavior of microparticles in liquid environments. However, to date, nonspherical particle behaviors close to confining boundaries, even as simple as planar walls, remain largely unexplored. Here, we measure the height distribution and orientation of colloidal dumbbells above walls by means of digital in-line holographic microscopy. We find that while larger dumbbells are oriented almost parallel to the wall, smaller dumbbells of the same material are surprisingly oriented at preferred angles. We determine the total height-dependent force acting on the dumbbells by considering gravitational effects and electrostatic particle-wall interactions. Our modeling reveals that at specific heights both net forces and torques on the dumbbells are simultaneously below the thermal force and energy, respectively, which makes the observed orientations possible. Our results highlight the rich near-wall dynamics of nonspherical particles and can further contribute to the development of quantitative frameworks for arbitrarily shaped microparticle dynamics in confinement.

Lensless In-Line Holographic Microscopy With Light Source of Low Spatio-Temporal Coherence

Lensless microscopy with coherent or partially coherent light sources is a well known imaging technique, commonly referred as digital in-line holographic microscopy. In the established methods, both the spatial and temporal coherence of light play a crucial role in determining the resolution of reconstructed object. We report lensless microscopy with a spatially extended white LED, a light source of low spatial and very low temporal coherence. The wave-field propagation between two parallel planes can be obtained using a convolution operation, where the convolution kernel depends on the object-sensor distance and the characteristics of the light source. For a light source of unknown characteristics, this kernel is an unknown function. In the proposed reconstruction method, we decompose an unknown convolution kernel of very large size (128 X 128) into a small unknown light-source-specific kernel (size 9 X 9) and a known light-source-independent kernel (size 128 X 128). This drastically reduces the number of parameters to be estimated at the system identification step, which has been performed here by one time imaging of the known microscopic objects. Final unknown object estimation has been performed using the upper-bound constrained deconvolution. A lateral resolution of ~1-2 micrometer has been demonstrated.

Three-dimensional volumetric monitoring of settling particulate matters on a leaf using digital in-line holographic microscopy

Plants are considered as a possible modality to reduce particulate matter (PM) particles from ambient air in an ecofriendly manner. A new precise monitoring technique that can explore interactions between individual PM particles and a leaf surface is necessary to understand the underlying mechanisms of PM removal of plant leaves. In this study, a digital in-line holographic microscopy (DIHM) was employed to experimentally investigate the settling motions of PM particles over the leaf surface. The in-plane positions and sizes of opaque PMs with irregular shapes were obtained from the projection images of numerically reconstructed holographic images. The depth positions of PMs were determined by using proper selection of an autofocusing criterion with automatic segmentation method. The edge of a hairy Perilla frutescens leaf was detected by adopting several digital imaging processing techniques. The DIHM technique was applied in this study to accurately detect 3D settling trajectories of PMs with velocity information of PMs in the midair and near leaf surface, simultaneously.

Optically Based Hand-Held Sensor for Visualization and Quantification of Cryptosporidium Parvum

The ingestion of the protozoan parasite Cryptosporidium, resulting in the associated disease cryptosporidiosis, can cause major problems if introduced into a water supply for both animals and humans. This research focuses on the creation of a sensor to track and quantify the parasite with a digital camera system using Digital Inline Holographic Microscopy (DIHM) and software reconstruction. Since Cryptosporidium oocysts are not single celled and can be around 4–8 µm in diameter, they are easily detected with the DIHM at various depths within a 3.5 mL cuvette. As the sensor is improved by modifications, it could pick up behavior of movement and sizes of particles measured to positively identify the particle(s) being tracked as Cryptosporidium as others have achieved on Escherichia coli and other microorganisms. A significant advantage of this technique is the resulting sensor costs significantly less than traditional microscopes used in laboratory tests for detecting Cryptosporidium, or other methods like USEPA Method 1623 and can provide results within minutes. The sensor is a stellar alternative to current methods in detecting and quantifying Cryptosporidium.

Lensless inline digital holography versus Fourier ptychography: phase estimation of a large transparent bead

Lensless inline digital holographic microscopy (LI-DHM) and Fourier ptychographic microscopy (FPM) are two widespread quantitative phase imaging (QPI) techniques. They have been employed in various fields, especially for biological slice imaging because of their simplicity in use, stability in structure, and also large field of view. Spherical phase response (for example from HeLa cells) is commonly observed in biological imagery. As a consequence, for calibration and validation purposes, small (several to tenth of microns in diameter) transparent microbeads have been used as standards. Phase imaging of their large counterparts (hundreds of microns in diameter) using either LI-DHM or FPM has not been reported so far. We are aiming to analyze the phase response of a 146-μm soda-lime microsphere. It has been immersed in Canada balsam to reduce phase difference and to avoid overexposed diffraction rings. The phase estimation issue has been tackled using approaches that involve either Gerchberg–Saxton type algorithms or an inverse problem-based procedure. Confronting the results confirms the QPI capability for both imaging techniques to assess phase responses from such a large transparent object.

Assessment of holographic microscopy for quantifying marine particle size and concentration

Holographic microscopy has emerged as a tool for in situ imaging of microscopic organisms and other particles in the marine environment: appealing because of the relatively larger sampling volume and simpler optical configuration compared to other imaging systems. However, its quantitative capabilities have so far remained uncertain, in part because hologram reconstruction and image recognition have required manual operation. Here, we assess the quantitative skill of our automated hologram processing pipeline (CCV Pipeline), to evaluate the size and concentration measurements of environmental and cultured assemblages of marine plankton particles, and microspheres. Over 1 million particles, ranging from 10 to 200 μm in equivalent spherical diameter, imaged by the 4‐Deep HoloSea digital inline holographic microscope (DIHM) are analyzed. These measurements were collected in parallel with a FlowCam (FC), Imaging FlowCytobot (IFCB), and manual microscope identification. Once corrections for particle location and nonuniform illumination were developed and applied, the DIHM showed an underestimate in ESD of about 3% to 10%, but successfully reproduced the size spectral slope from environmental samples, and the size distribution of cultures (Dunaliella tertiolecta, Heterosigma akashiwo, and Prorocentrum micans) and microspheres. DIHM concentrations (order 1 to 1000 particles ml−1) showed a linear agreement (r 2 = 0.73) with the other instruments, but individual comparisons at times had large uncertainty. Overall, we found the DIHM and the CCV Pipeline required extensive manual correction, but once corrected, provided concentration and size estimates comparable to the other imaging systems assessed in this study. Holographic cameras are mechanically simple, autonomous, can operate at very high pressures, and provide a larger sampling volume than comparable lens‐based tools. Thus, we anticipate that these characterization efforts will be rewarded with novel discovery in new oceanic environments.

Optically Based Bacteria Hand-Held Sensor: From Fundamentals to Proof of Concept

A prototype of a hand-held particle sensor based on digital inline holographic microscopy was developed to work as an early warning device to detect bacteria Escherichia coli (E. coli) in water within minutes. The aim of the device is to record holographic images of water samples and perform rapid analyses (less than 20 min) of these samples to give timely information about the levels of particulates, with the goal of detecting and quantifying pathogens such as E. coli in water samples. Using the hand-held sensor, laboratory trials were performed to compare the effectiveness of the sensor with traditional methods of detecting E. coli in water. To address the linearity of the sensor signal, a dilution series covering nine decades of bacterial suspensions were analyzed and a linear regression coefficient of determination (R2) of 86.4% was revealed. Preliminary studies have shown that the sensor can detect average values of E. coli levels from 7 colony forming units (CFU)/mL to 106 CFU/mL in deionized water spiked with the bacteria. The device can be essential for accurate and precise bacteria counts in drinking water systems inline or at home. The sensor’s major advantages are its portability, low cost, ease of operation, low power consumption rate, and no consumption of chemical reagents is needed to visualize and count E. coli.

Axial displacement measurement with high resolution of particle movement based on compound digital holographic microscopy

In-line digital holographic particle imaging is gifted with high axial resolution but axial displacement of nearly-in-focus or in-focus particle is unable to be measured because of twin-image problem. Both in-focus and out-of-focus particles can be measured by off-axis digital holographic particle imaging but with poor axial resolution. These two make particle tracking in three-dimensional more complicated. This paper proposes a compound method of in-line and off-axis digital holographic microscopy to measure the axial displacement of both the in-focus and out-of-focus particle with improved resolution up to nanometer scale collectively. In the proposed compound digital holographic microscopy (CDHM), the critical reconstruction distance (CRD) is calculated. The twin image effect can be resolved by ensuring the reconstruction distance is larger than the CRD, then in-line digital holographic particle imaging can be effectively applied. If the reconstruction distance is smaller than the CRD, the ring pixels of optical path length (RPOPL) method is applied to improve resolution. The axial displacement of silica particles 5 μm and 10 μm in diameter fixed on the piezoelectric stage were successfully measured using CDHM method at resolution up to 4 nm (in-focus particle) and 2 nm (out-of-focus particle).

DarkFocus: numerical autofocusing in digital in-line holographic microscopy using variance of computational dark-field gradient

We report on a novel computational technique for automatic numerical refocusing in digital in-line holographic microscopy. It is based on the adaptive filtering of the recorded on-axis hologram to eliminate its background term and extract interference intensity-component connected with light scattered on the sample (interference fringes). Numerical propagation of such filtered hologram yields the computationally generated dark-field imaging coming from the amplitude part of the complex field. We propose a simple measure in the form of the variance of the dark-field gradient, which attains its maximum value in the focal planes for all types of objects (phase, amplitude and mixed phase-amplitude), as a robust indicator of overall sharpness and in-focus quality. Demonstrated novel autofocusing technique is positively validated using experimental data exhibiting significant variation of the magnification factor for both single and double focal plane scenarios. It compares favorably with other well-established automatic numerical refocusing techniques based on the high-pass filtered complex amplitude and edge sparsity, mainly in terms of higher axial resolution and better robustness to hologram low signal-to-noise ratio.

Digital holographic microscopy for red blood cell imaging

Digital holographic microscopy (DHM) can obtain biological parameters and morphological information of cells by reconstructing holograms, which is different from traditional optical microscopy. The DHM is a three-dimensional imaging technology which is effective, non-contact and non-destructive. With the developments of the image sensor and the computing technology, it has made significant progress in the field of living cells detection, especially for red blood cell. Compared with the technologies which are widely used in the field of cell imaging such as con-focal laser scanning microscopy, scanning near-field optical microscopy and optical coherence tomography, the DHM has the advantages including wide FOV and high-resolution to achieve higher imaging and quality. This paper introduces the principle of recording and reconstruction of digital holography, and then analyzes the performance of three reconstruction algorithms using the Fresnel method, the convolution method and the angular spectrum method. The Fresnel method is suitable for the sample size larger than the image sensor. Both the convolution method and the angular spectrum method have an optimal reconstruction distance. When the reconstruction distance is different from the optimal distance, the resolution of the reconstructed image will decrease, and the angular spectrum method is better than the convolution method in overall performance. The DHM system for RBC measurements mainly adopts the convolution algorithm or the angular spectrum algorithm to implement numerical reconstruction. The systems of the in-line DHM, the off-axis DHM and the optical tweezers combining with off-axis DHM are introduced. These techniques use algorithms including Rayleigh-Sommerfeld back-propagation, the sharpness quantification, the watershed segmentation, the numerical refocusing and the thermal fluctuation to determine the focal plane position and obtain the best reconstruction distance of the RBCs, and further detect the shape change of the RBCs and extract the information of high-resolution blood vessel shape and blood flow velocity. These techniques can even achieve the dynamic tracking and measure three-dimensional volume of RBCs in real-time which is helpful for pathological studies such as diabetes, cardiovascular disease and Parkinson's disease. With its unique non-contact and non-destructive characteristics, the DHM realizes real-time and quantitative detection that is difficult to achieve with traditional three-dimensional microscopic imaging technologies.

Variable zoom digital in-line holographic microscopy

We report on a novel layout providing variable zoom in digital in-line holographic microscopy (VZ-DIHM). The implementation is in virtue of an electrically tunable lens (ETL) which enables to slightly shift the illumination source axial position without mechanical movement of any system component. Magnifications ranging from ~15X to ~35X are easily achievable using the same layout and resulting in a substantial variation of the total field of view (FOV). The performance of the proposed setup is, first, validated using a resolution test target where the main parameters are analyzed (theoretically and experimentally) and, second, corroborated analyzing biological sample (prostate cancer cells) showing its application to biomedical imaging.