Quantification of diabetic macular ischemia using novel three‐dimensional optical coherence tomography angiography metrics

We applied three‐dimensional (3D) analysis to optical coherence tomography angiography (OCTA) to measure macular ischemia in eyes affected by non‐proliferative diabetic retinopathy (DR). A previously validated algorithm was applied to OCTA data in order to obtain 3D visualization of the retinal vasculature. Successively, a global thresholding algorithm was applied and two novel quantitative metrics were introduced: 3D vascular volume and 3D perfusion density. Two‐dimensional (2D) OCTA metrics were also obtained with different binarization thresholds for comparison. Of the 30 patients included, 15 were diagnosed with DR and 15 were controls. The 3D vascular volume and 3D perfusion density were reduced in DR eyes (P < .0001). The 2D variables also significantly differ between groups. The 3D perfusion density had the highest area under the receiver operating characteristic curve (0.964) among tested variables. Assessing quantitative perfusion using 3D analysis is reliable and promising, and with an elevated diagnostic efficacy in identifying DR eyes.     

Efficient super‐resolution volumetric imaging by radial fluctuation Bayesian analysis light‐sheet microscopy

Various computational super‐resolution methods are available based on the analysis of fluorescence fluctuation behind acquired frames. However, dilemmas often exist in the balance of fluorophore characteristics, computation cost, and achievable resolution. Here we present an approach that uses a super‐resolution radial fluctuations (SRRF) image to guide the Bayesian analysis of fluorophore blinking and bleaching (3B) events, allowing greatly accelerated localization of overlapping fluorophores with high accuracy. This radial fluctuation Bayesian analysis (RFBA) approach is also extended to three dimensions for the first time and combined with light‐sheet fluorescence microscopy, to achieve super‐resolution volumetric imaging of thick samples densely labeled with common fluorophores. For example, a 700‐nm thin Bessel plane illumination is developed to optically section the Drosophila brain, providing a high‐contrast 3D image of rhythmic neurons. RFBA analyzes 30 serial volumes to reconstruct a super‐resolved 3D image at 4‐times higher resolutions (~70 and 170 nm), and precisely resolve the axon terminals. The computation is over 2‐orders faster than conventional 3B analysis microscopy. The capability of RFBA is also verified through dual‐color imaging of cell nucleus in live Drosophila brain. The spatial co‐localization patterns of the nuclear envelope and DNA in a neuron deep inside the brain can be precisely extracted by our approach.     

Two‐dimensional correlation analysis of Raman microspectroscopy of subcellular interactions of drugs in vitro

Two‐dimensional (2D) correlation analysis is explored to data mine the time evolution of the characteristic Raman microspectroscopic signatures of the subcellular responses of the nucleoli of human lung cancer cells to the uptake of doxorubicin. A simulated dataset of experimental control spectra, perturbed with systematically time‐dependent spectral changes, constituted by a short‐term response which represents the initial binding of the drug in the nucleolus, followed by a longer term response of the organelle metabolism, is used to validate the analysis protocol. Applying 2D correlation analysis, the in phase, synchronous correlation coefficients are seen to contain contributions of both response profiles, whereas they can be independently extracted from the out of phase, asynchronous correlation coefficients. The methodology is applied to experimental data of the uptake of doxorubicin in human lung cell lines to differentiate the signatures of chemical binding and subsequent cellular response.      

Computed tomography for in vivo deep over‐1000 nm near‐infrared fluorescence imaging

This study aims to develop a novel cross‐sectional imaging of fluorescence in over‐1000 nm near‐infrared (OTN‐NIR), which allows in vivo deep imaging, using computed tomography (CT) system. Cylindrical specimens of composite of OTN‐NIR fluorophore, NaGdF₄ co‐doped with Yb³⁺ and Ho³⁺ (ex: 980 nm, em: 1150 nm), were embedded in cubic agar (10.5–12 mm) or in the peritoneal cavity of mice and placed on a rotatable stage. When the fluorescence from inside of the samples was serially captured from multiple angles, the images were disrupted by the reflection and refraction of emitted light on the sample‐air interface. Immersing the sample into water filled in a rectangular bath suppressed the disruption at the interface and successfully reconstructed the position and concentration of OTN‐NIR fluorophores on the cross‐sectional images using a CT technique. This is promising as a novel three‐dimensional imaging technique for OTN‐NIR fluorescent image projections of small animals captured from multiple angles.     

Two‐dimensional crystals of carboxysome shell proteins recapitulate the hexagonal packing of three‐dimensional crystals

Bacterial microcompartments (BMCs) are large intracellular bodies that serve as simple organelles in many bacteria. They are proteinaceous structures composed of key enzymes encapsulated by a polyhedral protein shell. In previous studies, the organization of these large shells has been inferred from the conserved packing of the component shell proteins in two‐dimensional (2D) layers within the context of three‐dimensional (3D) crystals. Here, we show that well‐ordered, 2D crystals of carboxysome shell proteins assemble spontaneously when His‐tagged proteins bind to a monolayer of nickelated lipid molecules at an air–water interface. The molecular packing within the 2D crystals recapitulates the layered hexagonal sheets observed in 3D crystals. The results reinforce current models for the molecular design of BMC shells.     

Tissue clearing‐based method for unobstructed three‐dimensional imaging of mouse penis with subcellular resolution

Although mice are widely used to elucidate factors contributing to penile disorders and develop treatment options, quantification of tissue changes upon intervention is either limited to minuscule tissue volume (histology) or acquired with limited spatial resolution (MRI/CT). Thus, imaging method suitable for expeditious acquisition of the entire mouse penis with subcellular resolution is described that relies on both aqueous‐ (clear, unobstructed brain imaging cocktails and computational analysis) and solvent‐based (fluorescence‐preserving capability imaging of solvent‐cleared organs) tissue optical clearing (TOC). The combined TOC approach allows to image mouse penis innervation and vasculature with unprecedented detail and, for the first time, reveals the three‐dimensional structure of murine penis fibrocartilage.     

High‐speed X‐ray‐induced luminescence computed tomography

X‐ray‐induced luminescence computed tomography (XLCT) is an emerging molecular imaging. Challenges in improving spatial resolution and reducing the scan time in a whole‐body field of view (FOV) still remain for practical in vivo applications. In this study, we present a novel XLCT technique capable of obtaining three‐dimensional (3D) images from a single snapshot. Specifically, a customed two‐planar‐mirror component is integrated into a cone beam XLCT imaging system to obtain multiple optical views of an object simultaneously. Furthermore, a compressive sensing based algorithm is adopted to improve the efficiency of 3D XLCT image reconstruction. Numerical simulations and experiments were conducted to validate the single snapshot X‐ray‐induced luminescence computed tomography (SS‐XLCT). The results show that the 3D distribution of the nanophosphor targets can be visualized much faster than conventional cone beam XLCT imaging method that was used in our comparisons while maintaining comparable spatial resolution as in conventional XLCT imaging. SS‐XLCT has the potential to harness the power of XLCT for rapid whole‐body in vivo molecular imaging of small animals.     

Bioassembly of three‐dimensional embryonic stem cell‐scaffold complexes using compressed gases

Tissues are composed of multiple cell types in a well‐organized three‐dimensional (3D) microenvironment. To faithfully mimic the tissue in vivo, tissue‐engineered constructs should have well‐defined 3D chemical and spatial control over cell behavior to recapitulate developmental processes in tissue‐ and organ‐specific differentiation and morphogenesis. It is a challenge to build a 3D complex from two‐dimensional (2D) patterned structures with the presence of cells. In this study, embryonic stem (ES) cells grown on polymeric scaffolds with well‐defined microstructure were constructed into a multilayer cell‐scaffold complex using low pressure carbon dioxide (CO₂) and nitrogen (N₂). The mouse ES cells in the assembled constructs were viable, retained the ES cell‐specific gene expression of Oct‐4, and maintained the formation of embryoid bodies (EBs). In particular, cell viability was increased from 80% to 90% when CO₂ was replaced with N₂. The compressed gas‐assisted bioassembly of stem cell‐polymer constructs opens up a new avenue for tissue engineering and cell therapy. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

Scaffold‐free inkjet printing of three‐dimensional zigzag cellular tubes

The capability to print three‐dimensional (3D) cellular tubes is not only a logical first step towards successful organ printing but also a critical indicator of the feasibility of the envisioned organ printing technology. A platform‐assisted 3D inkjet bioprinting system has been proposed to fabricate 3D complex constructs such as zigzag tubes. Fibroblast (3T3 cell)‐based tubes with an overhang structure have been successfully fabricated using the proposed bioprinting system. The post‐printing 3T3 cell viability of printed cellular tubes has been found above 82% (or 93% with the control effect considered) even after a 72‐h incubation period using the identified printing conditions for good droplet formation, indicating the promising application of the proposed bioprinting system. Particularly, it is proved that the tubular overhang structure can be scaffold‐free fabricated using inkjetting, and the maximum achievable height depends on the inclination angle of the overhang structure. As a proof‐of‐concept study, the resulting fabrication knowledge helps print tissue‐engineered blood vessels with complex geometry. Biotechnol. Bioeng. 2012; 109: 3152–3160. © 2012 Wiley Periodicals, Inc.     

QSG‐7701 human hepatocytes form polarized acini in three‐dimensional culture

Hepatocytes are polarized and fulfill a variety of liver‐specific functions in vivo; but the polarized tissue structure and many of these functions are lost when the cells are cultured on plastic. To recapitulate the polarized structure and tissue‐specific function of liver cells in culture, we established a three‐dimensional (3D) culture assay with the human hepatocyte line QSG‐7701. In 3D Matrigel culture, QSG‐7701 cells formed polarized spheroids with a center lumen, which is reminiscent of bile canaliculi in the liver. Immunofluoresence analysis showed that F‐actin bundles and radixin were mainly located at the apical membrane and that α6 and β1 integrins were localized basally in 3D culture. Lumen formation was associated with the selective apoptosis of centrally located cells and was accompanied by proliferative suppression during acinar development. Compared to QSG‐7701 cells in 2D or agarose gel cultures, the cells in 3D Matrigel culture maintained a given direction of biliary excretion and acquired higher levels of cytochrome P450 and albumin expression. Our study shows that the immortal human hepatocytes, QSG‐7701, in 3D Matrigel culture reacquire cardinal features of glandular epithelium in vivo, providing an ex vivo model to study liver‐specific function and tumorigenesis. J. Cell. Biochem. 110: 1175–1186, 2010. Published 2010 Wiley‐Liss, Inc.     

Two‐Photon polymerization for microfabrication of three‐dimensional scaffolds for tissue engineering application

In natural tissues cells are embedded in a three‐dimensional fibrous network of biopolymers like collagen, hyaluronic acid etc. This extracellular matrix (ECM) influences the cell fate, the differentiation status, metabolic processes and provides structural integrity. For a three‐dimensional or physiological cell cultivation that are required in biomedical applications (e.g. tissue engineering, BioMEMS) scaffolds are needed. These scaffolds mimic the ECM according to their biocompatibility which comprises aspects of surface compatibility and importantly for tissue engineering applications aspects of structural compatibility. We have evaluated scaffold design parameters for the three‐dimensional cultivation of chondrocytes for the tissue engineering of artificial cartilage. Two‐photon polymerization is a powerful technique for fabrication of polymeric three‐dimensional micro‐ and submicro‐structures. The photoinitiation system for two‐photon polymerization is excited by simultaneous absorption of two photons leading to chemical polymerization reactions. Due to a tight confinement of the excitation volume around the focal point, this method can produce micrometer sized objects maintaining a high spatial resolution down to 100 nm. Two‐photon processes require very high photon densities which are provided by pulsed femtosecond lasers. The potential of this approach for microfabrication of scaffolds for tissue engineering is demonstrated by investigation of the cell response to microstructures with complex three‐dimensional geometry and feature sizes in the range of few micrometers.     

Three‐dimensional ring‐oven washing technique for a paper‐based immunodevice

Washing is a standard step for enzyme‐linked immunosorbent assays (ELISA) performed on a paper‐based chip, in which nonspecific‐binding antibodies and antigens should be removed completely from the paper surface. In this study, a novel three‐dimensional (3D) washing strategy using a heating ring‐oven was carried out on a paper‐based chip. Compared with a plane washing mode by a ring‐oven, this 3D washing strategy obtained a lower background, as gravity played an important role in the washing step. The paper‐based chip was placed on a 3D plastic holder and the waste area was connected to a heating ring. Use of a heating waste area meant that the nonspecific‐binding protein was continuously carried to the waste area through gravity and capillary action. The angle between the plastic holder and the ring plane was carefully selected. The effect of washing on different parts of the detection area was investigated by upconversion fluorescence and chemiluminescence (CL). This novel 3D washing strategy was performed for carcinoembryonic antigen detection through CL and a lower detection limit of 2 pg ml^?1 was obtained. This approach provides an effective washing strategy to remove nonspecific‐binding antibody from a paper‐based immunodevice.     

Sulforaphane enhances temozolomide‐induced apoptosis because of down‐regulation of miR‐21 via Wnt/β‐catenin signaling in glioblastoma

Temozolomide (TMZ) has been widely used in the treatment of glioblastoma (GBM), although inherent or acquired resistance restricts the application. This study was aimed to evaluate the efficacy of sulforaphane (SFN) to TMZ‐induced apoptosis in GBM cells and the potential mechanism. Biochemical assays and subcutaneous tumor establishment were used to characterize the function of SFN in TMZ‐induced apoptosis. Our results revealed that β‐catenin and miR‐21 were concordantly expressed in GBM cell lines, and SFN significantly reduced miR‐21 expression through inhibiting the Wnt/β‐catenin/TCF4 pathway. Furthermore, down‐regulation of miR‐21 enhanced the pro‐apoptotic efficacy of TMZ in GBM cells. Finally, we observed that SFN strengthened TMZ‐mediated apoptosis in a miR‐21‐dependent manner. In conclusion, SFN effectively enhances TMZ‐induced apoptosis by inhibiting miR‐21 via Wnt/β‐catenin signaling in GBM cells. These findings support the use of SFN for potential therapeutic approach to overcome TMZ resistance in GBM treatment.

     

3D morphological and biophysical changes in a single tachyzoite and its infected cells using three‐dimensional quantitative phase imaging

Toxoplasma gondii is an apicomplexan parasite that causes toxoplasmosis in the human body and commonly infects warm‐blooded organisms. Pathophysiology of its diseases is still an interesting issue to be studied since T gondii can infect nearly all nucleated cells. Imaging techniques are crucial for studying its pathophysiology. In T gondii‐infected cells structural and biochemical alterations occurred. To study that modification, we use digital holotomography to investigate the structure and biochemical alteration of single tachyzoite and its infected cells in a label‐free and quantitative manner. Quantification analysis was done by measuring the refractive index distribution, which provides information about the concentration and dry mass of individual cells. This study showed that holotomography could be effectively used to identify the structural and biochemical alteration in tremendously different cells in supporting pathophysiological research in particular for T gondii‐caused diseases.     

Three‐dimensional static optical coherence elastography based on inverse compositional Gauss‐Newton digital volume correlation

The three‐dimensional (3D) mechanical properties characterization of tissue is essential for physiological and pathological studies, as biological tissue is mostly heterogeneous and anisotropic. A digital volume correlation (DVC)‐based 3D optical coherence elastography (OCE) method is developed to measure the 3D displacement and strain tensors. The DVC algorithm includes a zero‐mean normalized cross‐correlation criterion‐based coarse search regime, an inverse compositional Gauss‐Newton fine search algorithm and a local ternary quadratic polynomial fitting strain calculation method. A 3D optical coherence tomography (OCT) scanning protocol is proposed through theoretical analysis and experimental verification. Measurement errors of the DVC‐based 3D OCE method are evaluated to be less than 2.0 μm for displacements and 0.30% for strains by rigid body motion experiments. The 3D displacements and strains of a phantom and a specimen of chicken breast tissue under compression are measured. Results of the phantom show a good agreement with theoretical analysis and tensile testing. The strains of the chicken breast tissue indicate anisotropic biomechanical properties. This study provides an effective method for 3D biomechanical property studies of soft tissue and improves the development of 3D OCE techniques.     

Inside Cover: Efficient and cost‐effective 3D cellular imaging by sub‐voxel‐resolving light‐sheet add‐on microscopy

A type of compact and cost‐effective light‐sheet imaging device, termed sub‐voxel‐resolving light‐sheet add‐on module (SLAM), is developed to cooperate with conventional 2D epifluorescence microscope, allowing high‐contrast, resolution‐improved 3D imaging of various biological samples at high throughput. Further details can be found in the article by Fang Zhao, Yicong Yang, Yi Li, et al. ( e201960243 ).

     

Hyperspectral characterization of re‐epithelialization in an in vitro wound model

In vitro wound models are useful for research on wound re‐epithelialization. Hyperspectral imaging represents a non‐destructive alternative to histology analysis for detection of re‐epithelialization. This study aims to characterize the main optical behavior of a wound model in order to enable development of detection algorithms. K‐Means clustering and agglomerative analysis were used to group spatial regions based on the spectral behavior, and an inverse photon transport model was used to explain differences in optical properties. Six samples of the wound model were prepared from human tissue and followed over 22 days. Re‐epithelialization occurred at a mean rate of 0.24 mm²/day after day 8 to 10. Suppression of wound spectral features was the main feature characterizing re‐epithelialized and intact tissue. Modeling the photon transport through a diffuse layer placed on top of wound tissue properties reproduced the spectral behavior. The missing top layer represented by wounds is thus optically detectable using hyperspectral imaging.