Optical thickness measurement of occluded samples by lens-less Fourier transform digital holography, thermal loading, and machine learning.

Thickness measurements of objects, especially transparent and semi-transparent objects, are essential for their characterization and identification. However, in the case of occluded objects, the optical thickness determination becomes difficult, and an indirect way must be devised. Thermal loading of the objects changes their opto-thermal properties, which will be reflected as a change in their optical thickness. The key to quantifying such occluded objects lies in collecting these opto-thermal signatures. This could be achieved by imaging the changes occurring to a probe wavefront passing through the object while it is being thermally loaded. Digital holographic interferometry is an ideal tool for observing phase changes, as it can be used to compare wavefronts recorded at different instances of time. Lens-less Fourier transform digital holographic imaging provides the phase information from a single Fourier transform of the recorded hologram and can be used to quantify occluded phase objects. Here we describe a technique for the measurement of change in optical thickness of thermally loaded occluded phase samples using lens-less Fourier transform digital holography and machine learning. The advantage of the proposed technique is that it is a single shot, lens-less imaging modality for quasi-real-time quantification of phase samples behind thin occlusions.

LED based large field of view off-axis quantitative phase contrast microscopy by hologram multiplexing.

In this manuscript, we describe the development of a single shot, self-referencing wavefront division, multiplexing digital holographic microscope employing LED sources for large field of view quantitative phase imaging of biological samples. To address the difficulties arising while performing interferometry with low temporally coherent sources, an optical arrangement utilizing multiple Fresnel Biprisms is used for hologram multiplexing, enhancing the field of view and increasing the signal to noise ratio. Biprisms offers the ease of obtaining interference patterns by automatically matching the path length between the two off-axis beams. The use of low temporally coherent sources reduces the speckle noise and the cost, and the form factor of the setup. The developed technique was implemented using both visible and UV LEDs and tested on polystyrene microspheres and human erythrocytes.

Roadmap on digital holography [Invited].

This Roadmap article on digital holography provides an overview of a vast array of research activities in the field of digital holography. The paper consists of a series of 25 sections from the prominent experts in digital holography presenting various aspects of the field on sensing, 3D imaging and displays, virtual and augmented reality, microscopy, cell identification, tomography, label-free live cell imaging, and other applications. Each section represents the vision of its author to describe the significant progress, potential impact, important developments, and challenging issues in the field of digital holography.

Angular spectrum matching for digital holographic microscopy under extremely low light conditions.

Digital holographic microscopy (DHM) is a future three-dimensional (3D) microscopy due to its high-resolution and high-precision 3D images. Thus, it is getting attention in bioinformatics, semiconductor defect detection, etc. However, some limitations still exist. Especially, high-speed holographic imaging requires high-power lasers, which are difficult to image on highly absorbent or light-sensitive samples. To overcome these issues, we propose a new, to the best of our knowledge, digital hologram recovery algorithm called angular spectrum matching (ASM), which achieves hologram imitation to recover holograms in digital holography at low light intensities. The hologram used for the background phase comparison is recorded without objects; thus, no power limitation is required. The ASM utilizes this background hologram to recover dark holograms. We present experimental results showing improved DHM numerical reconstructions and recovered holograms under extremely low light conditions.

Successful surgical management of massive ovarian teratoma in a rhesus macaque (Macaca mulatta).

A 5 years-old Rhesus macaque was presented with abdominal enlargement. The clinical, radiographic and ultrasonographic findings suggested that a massive mass occupying the whole of the abdomen. The mass was surgically removed, and histopathology confirmed ovarian teratoma. The macaque recovered uneventfully; however, the management to avoid self-mutilation of skin sutures was challenging.

Sickle cell disease diagnosis based on spatio-temporal cell dynamics analysis using 3D printed shearing digital holographic microscopy.

We present a spatio-temporal analysis of cell membrane fluctuations to distinguish healthy patients from patients with sickle cell disease. A video hologram containing either healthy red blood cells (h-RBCs) or sickle cell disease red blood cells (SCD-RBCs) was recorded using a low-cost, compact, 3D printed shearing interferometer. Reconstructions were created for each hologram frame (time steps), forming a spatio-temporal data cube. Features were extracted by computing the standard deviations and the mean of the height fluctuations over time and for every location on the cell membrane, resulting in two-dimensional standard deviation and mean maps, followed by taking the standard deviations of these maps. The optical flow algorithm was used to estimate the apparent motion fields between subsequent frames (reconstructions). The standard deviation of the magnitude of the optical flow vectors across all frames was then computed. In addition, seven morphological cell (spatial) features based on optical path length were extracted from the cells to further improve the classification accuracy. A random forest classifier was trained to perform cell identification to distinguish between SCD-RBCs and h-RBCs. To the best of our knowledge, this is the first report of machine learning assisted cell identification and diagnosis of sickle cell disease based on cell membrane fluctuations and morphology using both spatio-temporal and spatial analysis.

Compact and field-portable 3D printed shearing digital holographic microscope for automated cell identification.

We propose a low-cost, compact, and field-portable 3D printed holographic microscope for automated cell identification based on a common path shearing interferometer setup. Once a hologram is captured from the portable setup, a 3D reconstructed height profile of the cell is created. We extract several morphological cell features from the reconstructed 3D height profiles, including mean physical cell thickness, coefficient of variation, optical volume (OV) of the cell, projected area of the cell (PA), ratio of PA to OV, cell thickness kurtosis, cell thickness skewness, and the dry mass of the cell for identification using the random forest (RF) classifier. The 3D printed prototype can serve as a low-cost alternative for the developing world, where access to laboratory facilities for disease diagnosis are limited. Additionally, a cell phone sensor is used to capture the digital holograms. This enables the user to send the acquired holograms over the internet to a computational device located remotely for cellular identification and classification (analysis). The 3D printed system presented in this paper can be used as a low-cost, stable, and field-portable digital holographic microscope as well as an automated cell identification system. To the best of our knowledge, this is the first research paper presenting automatic cell identification using a low-cost 3D printed digital holographic microscopy setup based on common path shearing interferometry.

Microsphere-assisted super-resolved Mirau digital holographic microscopy for cell identification.

In this paper, we use a glass microsphere incorporated into a digital holographic microscope to increase the effective resolution of the system, aiming at precise cell identification. A Mirau interferometric objective is employed in the experiments, which can be used for a common-path digital holographic microscopy (DHMicroscopy) arrangement. High-magnification Mirau objectives are expensive and suffer from low working distances, yet the commonly used low-magnification Mirau objectives do not have high lateral resolutions. We show that by placing a glass microsphere within the working distance of a low-magnification Mirau objective, its effective numerical aperture can be increased, leading to super-resolved three-dimensional images. The improvement in the lateral resolution depends on the size and vertical position of microsphere, and by varying these parameters, the lateral resolution and magnification may be adjusted. We used the information from the super-resolution DHMicroscopy to identify thalassemia minor red blood cells (tRBCs). Identification is done by comparing the volumetric measurements with those of healthy RBCs. Our results show that microsphere-assisted super-resolved Mirau DHMicroscopy, being common path and off-axis in nature, has the potential to serve as a benchtop device for cell identification and biomedical measurements.

Highly stable digital holographic microscope using Sagnac interferometer.

Interferometric microscopy has grown into a very potent tool for quantitative phase imaging of biological samples. Among the interfermetric methods, microscopy by digital holography is one of the most effective techniques, especially for studying dynamics of cells. Imaging of cell fluctuations requires digital holographic setups with high temporal stability. Common path setups in which the object and the reference beams encounter the same set of optical elements provide better temporal stability compared to two-beam setups. Here, we present a compact, easy-to-implement, common path digital holographic microscope based on Sagnac interferometer geometry. The microscope is implemented using a diode laser module employing a CCD array or a webcam sensor to record holograms. The system was tested for three-dimensional imaging capability, numerical focusing ability, and temporal stability. Sub-nanometer temporal stability without external vibration isolation components was obtained in both cases. The higher temporal stability makes the microscope compatible to image cell fluctuations, which is demonstrated by imaging the oscillation of the cell membrane of human red blood cells.

Digital holographic microscopy with coupled optical fiber trap for cell measurement and manipulation.

We present an integrated optical system for three-dimensional (3D) imaging of micrometer-sized samples, while immobilizing and manipulating the samples by means of an optical fiber trap. Optical traps allow us to apply and measure pico-Newton-sized forces, and perform detailed measurements of micrometer-sized dielectric systems in the field of biology. The integrated 3D system can be used as a major tool in the field of biophysics. The trap is built using a tapered optical fiber to enhance the effective numerical aperture of the fiber. The trapping system is mounted on a conventional microscope, in which the two eyepieces' output ports are used as the paths of an off-axis self-referencing digital holographic microscopy (DHM) setup. The trap is calibrated using a high-speed camera, and trap stiffness is determined through the power spectrum method. The compact setup provides an elegant apparatus for temporally stable DHM for 3D imaging of optically controlled samples. Three-dimensional information and quantitative phase contrast images of the trapped samples are obtained by postprocessing the recorded digital holograms. Experiments were performed on lipids and red blood cells. Quantitative phase contrast images and temporal evolution of optical thickness of trapped samples are presented.

Entropy-based clustering of embryonic stem cells using digital holographic microscopy.

Embryonic stem (ES) cells are an important factor in the development of cell-based therapeutic strategies. In this work, the use of digital holographic interferometric microscopy and statistical identification for automatic discrimination of ES cells and fibroblast (FB) cells is discussed in detail. The proposed algorithm first reduces the complex data structure to lower dimensions. Then, based on asymptotic normality, model-based clustering and linear discriminant analysis are applied to the transformed data to obtain the classification between ES and FB cells. The proposed algorithm is robust because it does not depend on parametric assumptions and can be extended to the classification of other cell image data. Experimental results are presented to demonstrate the performance of the system.

Optical encryption using multiple intensity samplings in the axial domain.

Image encryption with optical means has attracted attention due to its inherent multidimensionality and degrees of freedom, including phase, amplitude, polarization, and wavelength. In this paper, we propose an optical encoding system based on multiple intensity samplings of the complex-amplitude wavefront with axial translation of the image sensor. The optical encoding system is developed based on a single optical path, where multiple diffraction patterns, i.e., ciphertexts, are sequentially recorded through the axial translation of a CCD camera. During image decryption, an iterative phase retrieval algorithm is proposed for extracting the plaintext from ciphertexts. The results demonstrate that the proposed phase retrieval algorithm possesses a rapid convergence rate during image decryption, and high security can be achieved in the proposed optical cryptosystem. In addition, other advantages of the proposed method, such as high robustness against ciphertext contaminations, are also analyzed.

Correlation of various dental parameters of mandibular first molar with age, body weight and breed in healthy dogs.

The aim of this study was to investigate the relationship of different radiographic parameters of mandibular first molar with respect to age, body weight and breed in healthy dogs. Overall, 50 dogs with the age from 5 to 156 months and body weight from 6.00 to 45.00 kg of various breeds were made the subject of study. Animals were categorized into different groups based on age, body weight and breed. A new dental parameters measurement technique was standardised which was in line with modified Lind's measurement technique of human dentistry. A significant decrease in least square count means of dental parameters was observed in age group 1 (0 - 12 months) in comparison with group 2 (13 - 60 months) and group 3 (> 60 months). A significant decrease in least square count means of dental parameters was observed in group A (0.00 - 10.00 kg) in comparison with group B (11.00 - 25.00 kg) and group C (> 25.00 kg). The root canal width showed a significant decrease with an increase in age; therefore, it was difficult to approach pulp cavity in older dogs. Small breeds showed decline in root length/crown height (R/C) ratio and mandible height/first mandibular molar height (MH/M1H) ratio in comparison with medium and large breeds because of which there were more chances of tooth loosening in smaller breeds. Regression equations formulated with respect to body weight and age can act as a ready reference to calculate values of different dental parameters at places where dental radiography is not available.

Thoracic and abdominal aortic alterations in dogs affected with systemic hypertension.

Aortic remodeling is the consequence of untreated systemic hypertension along with aortic dilatation as a marker for target organ damage in human literature. Therefore, the present study was planned to detect the changes in aorta at the level of aortic root via echocardiography, thoracic descending aorta via radiography and abdominal aorta via ultrasonography in healthy (n = 46), diseased normotensive (n = 20) and systemically hypertensive dogs (n = 60). The aortic root dimensions were measured at the level of aortic annulus, sinus of valsalva, sino-tubular junction and proximal ascending aorta via left ventricular outflow tract view of echocardiography. The thoracic descending aorta was subjectively assessed for any disparity in size and shape of aorta via lateral and dorso-ventral view of chest radiography. The abdominal aorta was assessed via left and right paralumbar window for calculating the aortic elasticity along with aortic and caudal venacaval dimensions to calculate the aortic-caval ratio. The aortic root measurements were dilated (p < 0.001) in systemically hypertensive dogs with a positive correlation (p < 0.001) with systolic blood pressure (BP). Thoracic descending aorta was also (p < 0.05) altered in the size and shape (undulation) of systemically hypertensive dogs. Abdominal aorta was markedly stiffened with reduced elasticity (p < 0.05) along with dilatation (p < 0.01) in hypertensive dogs. Also, there was a positive correlation (p < 0.001) of aortic diameters and aortic-caval ratio and negative correlation (p < 0.001) of aortic elasticity with systolic BP. Therefore, it was concluded that aorta could be considered as an important target organ damage of systemic hypertension in dogs.

Lateral shearing digital holographic imaging of small biological specimens.

A lateral shearing interferometer is used for direct holographic imaging of microorganisms. This is achieved by increasing the shear to be larger than the object size and results in a very simple and inexpensive common-path imaging device that can be easily coupled to the output of an inverted microscope. The shear is created by reflections from the front and back surface of a glass plate. Stability measurements show a standard deviation of the phase measurements of less than 1nm over 8 min. without any vibration compensation. The setup is applied to imaging both microorganisms in a microfluidic channel and red blood cells and reconstructions are presented.

Quantitative phase-contrast imaging with compact digital holographic microscope employing Lloyd's mirror.

Digital holographic microscopy (DHM) is one of the most effective techniques used for quantitative phase imaging of cells. Here we present a compact, easy to implement, portable, and very stable DHM setup employing a self-referencing Lloyd's mirror configuration. The microscope is constructed using a diode laser source and a CMOS sensor, making it cost effective. The reconstruction of recorded holograms yields the amplitude and phase information of the object. The temporal stability of the presented technique was found to be around 0.9 nm without any vibration compensation, which makes it ideal for studying cell profile changes. This aspect of the technique is demonstrated by studying membrane fluctuations of red blood cells.

Surgical retrieval of a metallic foreign body from the spleen of a dog.

This is the first report of a metallic foreign body in the spleen of a dog. The animal had abdominal discomfort with a tucked-up abdomen and occasional vomition. Radiography and ultrasonography were used to identify a splenic foreign body which was surgically removed through a ventral midline celiotomy. Migration of the foreign body through the stomach wall was suspected as the most likely cause.

Pharmacokinetic-Pharmacodynamic Study of Ampicillin-Cloxacillin Combination in Indian Thoroughbred Horses (Equus caballus) and Safety Evaluation of the Computed Dosage Regimen.

The pharmacokinetics of ampicillin-cloxacillin, given as single intravenously dose of 10 mg.kg-1 (5 mg.kg-1 of ampicillin plus 5 mg.kg-1 of cloxacillin) was examined in clinically presented Indian thoroughbred horses (n = 6) in order to design appropriate dosing strategies. Drug concentrations in plasma were determined by high performance liquid chromatography (HPLC) and pharmacokinetic parameters were derived by non-compartmental analysis using WinNonlin software. The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of ampicillin-cloxacillin against quality control strains of Escherichia coli and Staphylococcus aureus, grown in Muller Hinton Broth, were determined by broth microdilution method. For ampicillin, area under plasma drug concentration time curve (AUC) was 15.2 ± 0.54 µg.h.ml-1, mean residence time (MRT) was 1.33 ± 0.06 h and clearance (Cl) was 0.33 ± 0.01 L.h-1.kg-1. For cloxacillin, AUC was 18.0 ± 0.9 µg.h.ml-1, MRT was 1.28 ± 0.02 h and Cl was 0.28 ± 0.01 L.h-1.kg-1. MIC of ampicillin-cloxacillin combination against E. coli and S. aureus was determined to be 0.4 µg.ml-1. PK-PD integration indicated that to maintain %T > MIC value 50% for bacteria with MIC ≤ 0.4 µg.ml-1, an appropriate intravenous dosage regimen of ampicillin-cloxacillin combination in horses would be 15 mg.kg-1 (i.e. 7.5 mg.kg-1 of ampicillin plus 7.5 mg.kg-1 of cloxacillin), to be repeated at 12 h intervals. Safety profile of the recommended regimen did not significantly alter any of the 16 biochemical or haematological parameters studied.

High-resolution quantitative phase microscopic imaging in deep UV with phase retrieval.

High-resolution three-dimensional (3D) microscopic imaging requires the use of short wavelengths. Quantitative 3D imaging techniques, such as digital holographic microscopy, require interference between the object beam and a known reference background for the extraction of phase information. At shorter wavelengths, due to short coherence lengths, it may be difficult to implement a two-beam off-axis setup. Thus, a single-beam technique, which provides complete phase information, may be better suited for short wavelengths. This Letter describes the development of a quantitative microscopy technique at 193 nm using multiple intensity samplings and phase retrieval.