Transparent Specimens Research Articles

Whole Organ Blood and Lymphatic Vessels Imaging (WOBLI)

Thin section histology is limited in providing 3D structural information, particularly of the intricate morphology of the vasculature. Availability of high spatial resolution imaging for thick samples, would overcome the restriction dictated by low light penetration. Our study aimed at optimizing the procedure for efficient and affordable tissue clearing, along with an appropriate immunofluorescence labeling that will be applicable for high resolution imaging of blood and lymphatic vessels. The new procedure, termed whole organ blood and lymphatic vessels imaging (WOBLI), is based on two previously reported methods, CLARITY and ScaleA2. We used this procedure for the analysis of isolated whole ovary, uterus, lung and liver. These organs were subjected to passive clearing, following fixation, immunolabeling and embedding in hydrogel. Cleared specimens were immersed in ScaleA2 solution until transparency was achieved and imaged using light sheet microscopy. We demonstrate that WOBLI allows detailed analysis and generation of structural information of the lymphatic and blood vasculature from thick slices and more importantly, from whole organs. We conclude that WOBLI offers the advantages of morphology and fluorescence preservation with efficient clearing. Furthermore, WOBLI provides a robust, cost-effective method for generation of transparent specimens, allowing high resolution, 3D-imaging of blood and lymphatic vessels networks.

Taxonomy of multi-focal nematode image stacks by a CNN based image fusion approach

Background and objectiveIn the biomedical field, digital multi-focal images are very important for documentation and communication of specimen data, because the morphological information for a transparent specimen can be captured in form of a stack of high-quality images. Given biomedical image stacks containing multi-focal images, how to efficiently extract effective features from all layers to classify the image stacks is still an open question. MethodsWe present to use a deep convolutional neural network (CNN) image fusion based multilinear approach for the taxonomy of multi-focal image stacks. A deep CNN based image fusion technique is used to combine relevant information of multi-focal images within a given image stack into a single image, which is more informative and complete than any single image in the given stack. Besides, multi-focal images within a stack are fused along 3 orthogonal directions, and multiple features extracted from the fused images along different directions are combined by canonical correlation analysis (CCA). Because multi-focal image stacks represent the effect of different factors - texture, shape, different instances within the same class and different classes of objects, we embed the deep CNN based image fusion method within a multilinear framework to propose an image fusion based multilinear classifier. ResultsThe experimental results on nematode multi-focal image stacks demonstrated that the deep CNN image fusion based multilinear classifier can reach a higher classification rate (95.7%) than that by the previous multilinear based approach (88.7%), even we only use the texture feature instead of the combination of texture and shape features as in the previous work. ConclusionsThe proposed deep CNN image fusion based multilinear approach shows great potential in building an automated nematode taxonomy system for nematologists. It is effective to classify multi-focal image stacks.

Quantitative phase microscopy for cellular dynamics based on transport of intensity equation.

We demonstrate a simple method for quantitative phase imaging of tiny transparent objects such as living cells based on the transport of intensity equation. The experiments are performed using an inverted bright field microscope upgraded with a flipping imaging module, which enables to simultaneously create two laterally separated images with unequal defocus distances. This add-on module does not include any lenses or gratings and is cost-effective and easy-to-alignment. The validity of this method is confirmed by the measurement of microlens array and human osteoblastic cells in culture, indicating its potential in the applications of dynamically measuring living cells and other transparent specimens in a quantitative, non-invasive and label-free manner.

Dynamic spatial filtering using a digital micromirror device for high-speed optical diffraction tomography.

Optical diffraction tomography (ODT) is an emerging microscopy technique for three-dimensional (3D) refractive index (RI) mapping of transparent specimens. Recently, the digital micromirror device (DMD) based scheme for angle-controlled plane wave illumination has been proposed to improve the imaging speed and stability of ODT. However, undesired diffraction noise always exists in the reported DMD-based illumination scheme, which leads to a limited contrast ratio of the measurement fringe and hence inaccurate RI mapping. Here we present a novel spatial filtering method, based on a second DMD, to dynamically remove the diffraction noise. The reported results illustrate significantly enhanced image quality of the obtained interferograms and the subsequently derived phase maps. And moreover, with this method, we demonstrate mapping of 3D RI distribution of polystyrene beads as well as biological cells with high accuracy. Importantly, with the proper hardware configuration, our method does not compromise the 3D imaging speed advantage promised by the DMD-based illumination scheme. Specifically, we have been able to successfully obtain interferograms at over 1 kHz speed, which is critical for potential high-throughput label-free 3D image cytometry applications.

Oblique illumination in microscopy: A quantitative evaluation

Many biological objects are barely distinguished with the brightfield microscope because they appear transparent, translucent and colourless. One simple way to make such specimens visible without compromising contrast and resolution is by controlling the amount and the directionality of the illumination light. Oblique illumination is an old technique described by many scientists and microscopists that however has been largely neglected in favour of other alternative methods. Oblique lighting (OL) is created by illuminating the sample by only a portion of the light coming from the condenser. If properly used it can improve the resolution and contrast of transparent specimens such as diatoms. In this paper a quantitative evaluation of OL in brigthfield microscopy is presented. Several feature descriptors were selected for characterising contrast and sharpness showing that in general OL provides better performance for distinguishing minute details compared to other lighting modalities. Oblique lighting is capable to produce directionally shadowed differential contrast images allowing to observe phase details in a similar way to differential contrast images (DIC) but at lower cost. The main advantage of OL is that the resolution of the light microscope can be increased by effectively doubling the angular aperture. OL appears as a cost-effective technique both for the amateur and professional scientist that can be used as a replacement of DIC or phase contrast when resources are scarce.

Multi-focal nematode image stack classification using a projection-based multi-linear method

In digital multi-focal images (DMIs), morphological and topological information for a transparent specimen can be captured in the form of a stack of high-quality images. We propose to use projection methods such as coefficient of variation projection to exploit the entire information of a given DMI stack using its projection images from different directions. Besides, multiple features extracted from the projection images along different directions are combined by using canonical correlation analysis. Because DMI stacks represent the effect of different factors—texture, the directions of projection, different instances within the same class and different classes of objects, we embed the projection method within a multi-linear analysis framework to propose a multiple direction projection-based multi-linear classification approach. The experimental results on the nematode data show that our proposed classifier can achieve very reliable recognition rate (98.5%) on a real-life database, even we only use the texture feature instead of the combination of texture and shape features as in a previous work.

Three-dimensional hard and soft tissue imaging of the human cochlea by scanning laser optical tomography (SLOT)

The present study focuses on the application of scanning laser optical tomography (SLOT) for visualization of anatomical structures inside the human cochlea ex vivo. SLOT is a laser-based highly efficient microscopy technique which allows for tomographic imaging of the internal structure of transparent specimens. Thus, in the field of otology this technique is best convenient for an ex vivo study of the inner ear anatomy. For this purpose, the preparation before imaging comprises decalcification, dehydration as well as optical clearing of the cochlea samples in toto. Here, we demonstrate results of SLOT imaging visualizing hard and soft tissue structures with an optical resolution of down to 15 μm using extinction and autofluorescence as contrast mechanisms. Furthermore, the internal structure can be analyzed nondestructively and quantitatively in detail by sectioning of the three-dimensional datasets. The method of X-ray Micro Computed Tomography (μCT) has been previously applied to explanted cochlea and is solely based on absorption contrast. An advantage of SLOT is that it uses visible light for image formation and thus provides a variety of contrast mechanisms known from other light microscopy techniques, such as fluorescence or scattering. We show that SLOT data is consistent with μCT anatomical data and provides additional information by using fluorescence. We demonstrate that SLOT is applicable for cochlea with metallic cochlear implants (CI) that would lead to significant artifacts in μCT imaging. In conclusion, the present study demonstrates the capability of SLOT for resolution visualization of cleared human cochleae ex vivo using multiple contrast mechanisms and lays the foundation for a broad variety of additional studies.

Human pancreatic neuro-insular network in health and fatty infiltration.

Identification of a pancreatic neuro-insular network in mice suggests that a similar integration of islets and nerves may be present in the human pancreas. To characterise the neuro-insular network and the intra-pancreatic ganglia in a clinically related setting, we examined human pancreases in health and with fatty infiltration via 3-dimensional (3D) histology and compared the human pancreatic microenvironment with its counterpart in mice. Human pancreatic specimens from individuals with normal BMI, high BMI (≥25) and type 2 diabetes were used to investigate the neuro-insular network. Transparent specimens were prepared by tissue clearing for transmitted light and deep-tissue fluorescence imaging to simultaneously visualise infiltrated adipocytes, islets and neurovascular networks. High-definition images of human islets reveal that both the sympathetic and parasympathetic nerves enter the islet core and reside in the immediate microenvironment of islet cells. Around the islets, the neuro-insular network is visualised with 3D histology to identify the intra-pancreatic ganglia (peri-lobular and intra-parenchymal ganglia) and the islet-ganglionic association. In humans, but not in mice, pancreatic fatty infiltration (BMI dependent) features adipocytes infiltrating into the parenchyma and accumulating in the peri-lobular space, in which the peri-lobular ganglia also reside. We identified the formation of adipose-ganglionic complexes in the peri-lobular space and enlargement of ganglia around adipocytes. In the specimen from the individual with type 2 diabetes, an increase in the number of nerve projections from the intra-parenchymal ganglia is associated with severe fatty infiltration. We present new perspectives of human pancreas and islet innervation via 3D histology. Our results strongly suggest that fatty infiltration in the human pancreas creates a neurotrophic microenvironment and promotes remodelling of pancreatic innervation.

Smartphone-based multi-contrast microscope using color-multiplexed illumination

We present a portable multi-contrast microscope capable of producing bright-field, dark-field, and differential phase contrast images of thin biological specimens on a smartphone platform. The microscopy method is based on an imaging scheme termed “color-coded light-emitting-diode (LED) microscopy (cLEDscope),” in which a specimen is illuminated with a color-coded LED array and light transmitted through the specimen is recorded by a color image sensor. Decomposition of the image into red, green, and blue colors and subsequent computation enable multi-contrast imaging in a single shot. In order to transform a smartphone into a multi-contrast imaging device, we developed an add-on module composed of a patterned color micro-LED array, specimen stage, and miniature objective. Simple installation of this module onto a smartphone enables multi-contrast imaging of transparent specimens. In addition, an Android-based app was implemented to acquire an image, perform the associated computation, and display the multi-contrast images in real time. Herein, the details of our smartphone module and experimental demonstrations with various biological specimens are presented.

Thermal joining of foamed thermoplastic injection-moulded parts using laser radiation

The foam injection moulding is an established process to produce plastic components with reduced density. Due to the fragile foam structure, contactless welding methods such as laser transmission welding are suitable for foamed components. In a research project, the correlation between the injection moulding parameters and the resulting foam structure is determined in preliminary tests. Specimens with four different foam structures are produced, which differ in terms of their surface layer thickness and cell density. Measurements of the transmittance, as well as the intensity profile after irradiation of the transparent specimen, show that the transmission of the foamed test specimens is lower than compared to compact specimens. In particular, foam structures with low cell density and low surface layer thickness show a low transmittance. The weld strength is influenced only by the foam structure of the transparent joining partner. Additionally, a possible post-expansion of the enclosed gas during the welding process has to be taken into account. The temperature within the laser-absorbing joining partner is locally increased above the melting point of the plastic. Once the surrounding material is melted, the gas can expand due to the temperature increase and defects during the solidification of the melt are formed.

Crista Supraventricularis Purkinje Network and Its Relation to Intraseptal Purkinje Network.

Using transparent specimens with a dual color injection, microscopy, and computer tomography, this report shows that the right and left ventricular subendocardial Purkinje networks are connected by an extensive septal network in the bovine heart. The septal network is present along the entire septum except at a free zone below ventricular valves. Being the only communication of the basal right septum with the right free wall, the supraventricular crest is an enigmatic but not, by any means, hidden muscular structure. It is one of the last structures to be activated in human heart. It is shown here that the supraventricular crest Purkinje network connects the anterosuperior right ventricular basal free wall Purkinje network to anterior right ventricular basal septal Purkinje network. It is suggested that the stimulus initiated at middle left ventricular endocardium will activate the supraventricular crest. The intraseptal connection found between the basal left ventricular subendocardial septal Purkinje network and the right ventricular basal septal Purkinje network is, probably, the pathway for the stimulus. An anatomic basis is provided to explain why the inflow tract contracts earlier than the outflow tract in the right ventricle systole. Anat Rec, 2017. © 2017 Wiley Periodicals, Inc. Anat Rec, 300:1793-1801, 2017. © 2017 Wiley Periodicals, Inc.

Hyperspectral imaging acousto-optic system with spatial filtering for optical phase visualization.

We report a method for phase visualization in the images of transparent specimens using analog image processing in incoherent light. The experimental technique is based on adaptive bandpass spatial filtering with an amplitude mask matched with an acousto-optic tunable filter in a telecentric optical system. We demonstrate the processing of microscopic images of unstained and stained histological sections of human thyroid tumor with improved contrast.

The pathologic mechanisms underlying lumbar distraction spinal cord injury in rabbits

Background ContextA reliable experimental rabbit model of distraction spinal cord injury (SCI) was established to successfully simulate gradable and replicable distraction SCI. However, further research is needed to elucidate the pathologic mechanisms underlying distraction SCI. PurposeThe aim of this study was to investigate the pathologic mechanisms underlying lumbar distraction SCI in rabbits. Study DesignThis is an animal laboratory study. MethodsUsing a self-designed spine distractor, the experimental animals were divided into a control group and 10%, 20%, and 30% distraction groups. Pathologic changes to the spinal cord microvessels in the early stage of distraction SCI were identified by perfusion of the spinal cord vasculature with ink, production of transparent specimens, observation by light microscopy, and observation of corrosion casts of the spinal cord microvascular architecture by scanning electron microscopy. Malondialdehyde (MDA) and superoxide dismutase (SOD) concentrations in the injured spinal cord tissue were measured after 8 hours. ResultsWith an increasing degree and duration of distraction, the spinal cord microvessels were only partially filled and had the appearance of spasm until rupture and hemorrhage were observed. The MDA concentration increased and the SOD concentration decreased in the spinal cord tissue. ConclusionsChanges to the internal and external spinal cord vessels led to spinal cord ischemia, which is a primary pathologic mechanism of distraction SCI. Lipid peroxidation mediated by free radicals took part in secondary pathologic damage of distraction SCI.

The specialisation of the third metacarpal and hand in arboreal frogs: Adaptation for arboreal habitat?

AbstractSkeletal morphology is directly associated with habitat characteristics. To investigate the arboreal adaptation, we studied the osteological morphology of the forelimbs and the third metacarpals of arboreal frogs (Rhacophoridae and Hylidae) and non‐arboreal frogs (Bombinatoridae, Bufonidae, Megophryidae, Ranidae, and Microhylidae) using transparent specimens or X‐rays of skeletons. Our results revealed that the bony knob on the third metacarpal, which formed by a dilated and elongated lateral articular cartilage (AL) through endochondral ossification, occurred only in species of Rhacophorinae. The results of the phylogenetic comparative methods and correlation analysis strongly supported the conclusion that the bony knob is a phylogenetic independent evolution trait and had a significant correlation with the arboreal habitat. Furthermore, anatomical observation showed that a muscle adhered to the bony knob. Therefore, we speculated that the bony knob might act as an enlarged attachment point for larger or more musculatures to help with grasping. In addition, the relative length of the hand showed a significant difference between arboreal and non‐arboreal species (p = .007), suggesting that longer hands might be an arboreal adaptive trait. Overall, this study leads to a deeper understanding of the arboreal adaptation.

Single-exposure quantitative phase imaging in color-coded LED microscopy.

We demonstrate single-shot quantitative phase imaging (QPI) in a platform of color-coded LED microscopy (cLEDscope). The light source in a conventional microscope is replaced by a circular LED pattern that is trisected into subregions with equal area, assigned to red, green, and blue colors. Image acquisition with a color image sensor and subsequent computation based on weak object transfer functions allow for the QPI of a transparent specimen. We also provide a correction method for color-leakage, which may be encountered in implementing our method with consumer-grade LEDs and image sensors. Most commercially available LEDs and image sensors do not provide spectrally isolated emissions and pixel responses, generating significant error in phase estimation in our method. We describe the correction scheme for this color-leakage issue, and demonstrate improved phase measurement accuracy. The computational model and single-exposure QPI capability of our method are presented by showing images of calibrated phase samples and cellular specimens.

Phase-shifting infrared digital holographic microscopy based on an all-fiber variable phase shifter.

Phase-shifting infrared digital holographic microscopy based on a homemade all-fiber variable phase shifter is presented to quantitatively obtain the phase distribution of an object wave carrying the information of a transparent specimen in the infrared band. The all-fiber variable phase shifter, which consists of a tubular piezoelectric transducer (PZT) and a single-mode fiber, can accurately produce any phase shift between 0 and 2π by modulating the driving voltage of the tubular PZT. Taking measurements of different staircase structures on a silicon wafer as samples, two configurations are presented based on different phase-shifting implementations: one is a slight off-axis two-step phase shift and the other is an in-line four-step phase shift. The reconstructed results prove the validity of this method.

Visual Examination of Flexural Cracking Behaviors in a Helicoidally Laminated Composite

Fiber reinforced material is a composite material which is matrix, such as resin, reinforced with fibers. It has light weight, high specific strength and stiffness. Although it is strong, some of these materials show a brittle fracture, e.g. FRP. The purpose of this study is to observe the damage processes by making a transparent specimens that has a helicoidally laminated structure and to consider whether the mitigation of brittle fracture is possible or not. As a result, a helicoidally laminated specimen exhibited a load-displacement relation with mitigated brittleness, and it showed a rather curved crack surface between wires, while a cross-ply specimen showed a brittle relation with a crack surface straight through wires.

Anisotropic contrast optical microscope.

An optical microscope is described that reveals contrast in the Mueller matrix images of a thin, transparent, or semi-transparent specimen located within an anisotropic object plane (anisotropic filter). The specimen changes the anisotropy of the filter and thereby produces contrast within the Mueller matrix images. Here we use an anisotropic filter composed of a semi-transparent, nanostructured thin film with sub-wavelength thickness placed within the object plane. The sample is illuminated as in common optical microscopy but the light is modulated in its polarization using combinations of linear polarizers and phase plate (compensator) to control and analyze the state of polarization. Direct generalized ellipsometry data analysis approaches permit extraction of fundamental Mueller matrix object plane images dispensing with the need of Fourier expansion methods. Generalized ellipsometry model approaches are used for quantitative image analyses. These images are obtained from sets of multiple images obtained under various polarizer, analyzer, and compensator settings. Up to 16 independent Mueller matrix images can be obtained, while our current setup is limited to 11 images normalized by the unpolarized intensity. We demonstrate the anisotropic contrast optical microscope by measuring lithographically defined micro-patterned anisotropic filters, and we quantify the adsorption of an organic self-assembled monolayer film onto the anisotropic filter. Comparison with an isotropic glass slide demonstrates the image enhancement obtained by our method over microscopy without the use of an anisotropic filter. In our current instrument, we estimate the limit of detection for organic volumetric mass within the object plane of ≈49 fg within ≈7 × 7 μm2 object surface area. Compared to a quartz crystal microbalance with dissipation instrumentation, where contemporary limits require a total load of ≈500 pg for detection, the instrumentation demonstrated here improves sensitivity to a total mass required for detection by 4 orders of magnitude. We detail the design and operation principles of the anisotropic contrast optical microscope, and we present further applications to the detection of nanoparticles, to novel approaches for imaging chromatography and to new contrast modalities for observations on living cells.

Mixed mode dynamic crack-fiber bundle interaction using caustics

Caustic method was extended to study the interaction between the mixed mode dynamic matrix crack and the fiber bundle in fiber reinforced composites. First, the dynamic caustic experiments were conducted using transparent specimens with different thicknesses of fiber bundles under dynamic three point bending load. Then, the evolutions of dynamic shadow spots at the crack tip shielded by different thicknesses of fiber bundles were obtained. Moreover, the dynamic stress intensity factors were extracted from the shadow spots. Finally, the crack propagation and bifurcation behaviors were analyzed. It was shown that the stress intensity factors and crack propagation velocities were shielded by the fiber bundles, and the shielding effect increased with the increase of fiber bundle thickness.

Line-Field Swept-Source Interferometer for Simultaneous Measurement of Thickness and Refractive Index Distribution

We present a novel method for simultaneous measurement of the thickness and refractive index distribution of a transparent specimen by a line-field swept-source interferometer. In this study, by illuminating with the line-field beam instead of the point-focused beam, multiple interferences along the line-field distribution are measured simultaneously by using a combination of a novel swept source of 800-nm central region and a Si-based 1024-pixel charge-coupled device (CCD) detector. When the transparent specimen has an uneven thickness or nonuniform refractive index, the whole distribution of these two parameters can be also simultaneously measured over the illuminated region with a one-step comparison process. Using four types of transparent specimens, the thickness and refractive index distribution of each sample and a group sample paired with two specimens are accurately measured during the single wavelength scanning time of 27 ms by using the swept source with a 36-Hz sweeping rate and the CCD detector with a 36-kHz line rate.