Sub-micrometer Resolution Research Articles

Reverse Offset Printing of Semidried Metal Acetylacetonate Layers and Its Application to a Solution-Processed IGZO TFT Fabrication.

The submicrometer resolution printing of various metal acetylacetonate complex inks including Fe, V, Mn, Co, Ni, Zn, Zr, Mo, and In was enabled by a robust ink formulation scheme which adopted a ternary solvent system where solubility, surface wettability, and drying as well as absorption behavior on a polydimethylsiloxane sheet were optimized. Hydrogen plasma in heated conditions resulted in bombarded, resistive, or conductive state depending on the temperature and the metal species. With a conductivity-bestowed layer of MoO x and a plasma-protecting layer of ZrO x situated on the top of an IGZO layer, a solution-processed TFT exhibiting an average mobility of 0.17 cm2/(V s) is demonstrated.

Tracing and analysis of the whole mouse brain vasculature with systematic cleaning to remove and consolidate erroneous images.

Whole mouse brain microvascular images at submicrometer scale can be obtained by Knife-Edge Scanning Microscopy (KESM). However, due to the large size of the image dataset and the noise from the serial sectioning process of the KESM, whole mouse brain vascular reconstruction and analysis with submicrometer resolution have not been achieved yet, while several previous studies demonstrated manually selected small noise-free portion of the KESM dataset. In addition to the KESM dataset, there have been studies for vessel reconstruction and analysis of the whole mouse brain at lower resolution or of partial brain regions at submicrometer resolution. However, to the best of our knowledge, there has been no study for vessel reconstruction and analysis of the whole mouse brain at submicrometer resolution. In this paper, we propose a framework for the 3D reconstruction and analysis of the whole KESM mouse brain vasculature dataset with rich vasculature information extracted at submicrometer resolution. The framework consists of two methods. The propose methods provide the systematic cleaning to remove and consolidate erroneous images automatically, which enables the full tracing and analysis of the whole KESM mouse brain dataset with richer vasculature information.

High-throughput imaging flow cytometry by optofluidic time-stretch microscopy.

The ability to rapidly assay morphological and intracellular molecular variations within large heterogeneous populations of cells is essential for understanding and exploiting cellular heterogeneity. Optofluidic time-stretch microscopy is a powerful method for meeting this goal, as it enables high-throughput imaging flow cytometry for large-scale single-cell analysis of various cell types ranging from human blood to algae, enabling a unique class of biological, medical, pharmaceutical, and green energy applications. Here, we describe how to perform high-throughput imaging flow cytometry by optofluidic time-stretch microscopy. Specifically, this protocol provides step-by-step instructions on how to build an optical time-stretch microscope and a cell-focusing microfluidic device for optofluidic time-stretch microscopy, use it for high-throughput single-cell image acquisition with sub-micrometer resolution at >10,000 cells per s, conduct image construction and enhancement, perform image analysis for large-scale single-cell analysis, and use computational tools such as compressive sensing and machine learning for handling the cellular 'big data'. Assuming all components are readily available, a research team of three to four members with an intermediate level of experience with optics, electronics, microfluidics, digital signal processing, and sample preparation can complete this protocol in a time frame of 1 month.

NEWSdm: an emulsion-based directional Dark Matter experiment

In the field of direct Dark Matter search, a variety of experiments have been developed over the past decades aiming at detecting Weakly Interactive Massive Particles (WIMPs) via their scattering in a detector medium and, in the last years, several experimental efforts are concentrated on the directionality approach: the observation of the incoming apparent direction of WIMPs would in fact provide a new and unambiguous signature and the proof of the galactic origin of Dark Matter. Furthermore the directionality appears as the only way to overcome the neutrino background that is expected to finally prevent standard techniques to further lower cross-section limits. The NEWSdm experiment has been proposed to measure the direction of WIMP-induced nuclear recoils by using a novel emulsion technology with improved spatial resolution. In this paper we will present the status of the experiment, the performances of the newly developed read-out systems reaching sub-micrometric resolution and we will discuss the expected sensitivity and discovery potential.

Characterization of a submicro-X-ray fluorescence setup on the B16 beamline at Diamond Light Source.

An X-ray fluorescence setup has been tested on the B16 beamline at the Diamond Light Source synchrotron with two different excitation energies (12.7 and 17 keV). This setup allows the scanning of thin samples (thicknesses up to several micrometers) with a sub-micrometer resolution (beam size of 500 nm × 600 nm determined with a 50 µm Au wire). Sensitivities and detection limits reaching values of 249 counts s-1 fg-1 and 4 ag in 1000 s, respectively (for As Kα excited with 17 keV), are presented in order to demonstrate the capabilities of this setup. Sample measurements of a human bone and a single cell performed at B16 are presented in order to illustrate the suitability of the setup in biological applications.

Morphological and chemical information in fresh and vitrified ovarian tissues revealed by X-ray Microscopy and Fluorescence: observational study

Many clinical circumstances impose the necessity of collection and prolonged storage of gametes and/or ovarian tissue in order to preserve the reproduction potential of subjects. This is particularly appropriate in the case of young women and pre-pubertal girls undergoing chemotherapeutic treatments. The success of later assisted fertilization will depend on the suitable cooling protocols minimizing cryo-damages and preserving their biological function. The freeze-thaw processes of cryopreservation may induce, in fact, morphological and structural damages of oocytes and tissue mainly due to the formation of intracellular ice and to the toxicity of cryoprotectant. The most used cryo-protocol is the slow freezing procedure, but recently many authors have proposed vitrification as an alternative, because of its simplicity. The damage extent and the quality of follicles after cryopreservation are usually evaluated morphologically by conventional histological procedures, light and electron microscopy. Our laboratory, to further improve the evaluation and to better investigate damages, is adopting a combination of Synchrotron soft X-ray Microscopy (at TwinMic – Elettra) and XRF at different incident energies (at TwinMic – Elettra and ID21 – ESRF). X-ray techniques were performed on histological sections at micro and sub-micron resolution. Phase contrast and absorption images revealed changes in the compactness of the tissues, as well as cellular abnormalities revealed at sub-micrometric resolution. The distributions of the elements detected at 7.3 and 1.5 keV were compared and particularly Cl resulted to be indicative of follicle integrity. The results demonstrate the utility and the potential of X-ray microscopy and fluorescence in this research field.

Robust Cell Detection for Large-Scale 3D Microscopy Using GPU-Accelerated Iterative Voting.

High-throughput imaging techniques, such as Knife-Edge Scanning Microscopy (KESM),are capable of acquiring three-dimensional whole-organ images at sub-micrometer resolution. These images are challenging to segment since they can exceed several terabytes (TB) in size, requiring extremely fast and fully automated algorithms. Staining techniques are limited to contrast agents that can be applied to large samples and imaged in a single pass. This requires maximizing the number of structures labeled in a single channel, resulting in images that are densely packed with spatial features. In this paper, we propose a three-dimensional approach for locating cells based on iterative voting. Due to the computational complexity of this algorithm, a highly efficient GPU implementation is required to make it practical on large data sets. The proposed algorithm has a limited number of input parameters and is highly parallel.

Residual stress and adhesion of thermal spray coatings: Microscopic view by solidification and crystallisation analysis in the epitaxial CoNiCrAlY single splat

A new approach is proposed to achieve an in-depth understanding of crystallisation, residual stress and adhesion in epitaxial splats obtained by Combustion Flame Spray. Modelling of the fundamental process mechanisms is achieved with the help of experimental observations providing details with a sub-micrometre spatial resolution. At this scope, High Angular Resolution Electron Backscatter Diffraction and Transmission Electron Microscopy analysis are employed to provide insights into crystallisation and residual stress levels, while FIB-milled microcantilever beam bending is used for fracture strength measurements in the case of single splats. A comparison to fully-developed coatings is achieved by employing the X-ray Diffraction sin2ψ technique and pull-off methods for residual stress and fracture strength, respectively. The methodology is applied to metallic CoNiCrAlY material sprayed onto a Ni-based superalloy substrate. The establishment of different crystallisation regions: epitaxial and polycrystalline, is the result of variations in the heat flux direction at the solidification front. Significant dislocation density is also reported, indicating the relevance of impact dynamics and plastic deformation mechanisms. The comparison with fully-developed coatings suggests a reduction in inter-splat bonding at splat overlapping.

Creating a Structurally Realistic Finite Element Geometric Model of a Cardiomyocyte to Study the Role of Cellular Architecture in Cardiomyocyte Systems Biology.

With the advent of three-dimensional (3D) imaging technologies such as electron tomography, serial-block-face scanning electron microscopy and confocal microscopy, the scientific community has unprecedented access to large datasets at sub-micrometer resolution that characterize the architectural remodeling that accompanies changes in cardiomyocyte function in health and disease. However, these datasets have been under-utilized forinvestigating the role of cellular architecture remodeling in cardiomyocyte function. The purpose of this protocol is to outline how to create an accurate finite element model of a cardiomyocyte using high resolution electron microscopy and confocal microscopy images. A detailed and accurate model of cellular architecture has significant potential to provide new insights into cardiomyocyte biology, more than experiments alone can garner. The power of this method lies in its ability to computationally fuse information from two disparate imaging modalities of cardiomyocyte ultrastructure to develop one unified and detailed model of the cardiomyocyte. This protocol outlines steps to integrate electron tomography and confocal microscopy images of adult male Wistar (name for a specific breed of albino rat) rat cardiomyocytes to develop a half-sarcomere finite element model of the cardiomyocyte. The procedure generates a 3D finite element model that contains an accurate, high-resolution depiction (on the order of ~35 nm) of the distribution of mitochondria, myofibrils and ryanodine receptor clusters that release the necessary calcium for cardiomyocyte contraction from the sarcoplasmic reticular network (SR) into the myofibril and cytosolic compartment. The model generated here as an illustration does not incorporate details of the transverse-tubule architecture or the sarcoplasmic reticular network and is therefore a minimal model of the cardiomyocyte. Nevertheless, the model can already be applied in simulation-based investigations into the role of cell structure in calcium signaling and mitochondrial bioenergetics, which is illustrated and discussed using two case studies that are presented following the detailed protocol.

Real time quantitative analysis of lipid storage and lipolysis pathways by confocal spectral imaging of intracellular micropolarity

Organisms store fatty acids in triacylglycerols in the form of lipid droplets, or hydrolyze triacylglycerols in response to energetic demands via activation of lipolytic or storage pathways. These pathways are complex sets of sequential reactions that are finely regulated in different cell types. Here we present a high spatial and temporal resolution-based method for the quantification of the turnover of fatty acids into triglycerides in live cells without introducing sample preparation artifacts.We performed confocal spectral imaging of intracellular micropolarity in cultured insulin secreting beta cells to detect micropolarity variations as they occur in time and at different pixels of microscope images. Acquired data are then analyzed in the framework of the spectral phasors technique.The method furnishes a metabolic parameter, which quantitatively assesses fatty acids - triacylglycerols turnover and the activation of lipolysis and storage pathways. Moreover, it provides a polarity profile, which represents the contribution of hyperpolar, polar and non-polar classes of lipids. These three different classes can be visualized on the image at a submicrometer resolution, revealing the spatial localization of lipids in cells under physiological and pathological settings.This new method allows for a fine-tuned, real-time visualization of the turnover of fatty acids into triglycerides in live cells with submicrometric resolution. It also detects imbalances between lipid storage and usage, which may lead to metabolic disorders within living cells and organisms.

Smart-Dust-Nanorice for Enhancement of Endogenous Raman Signal, Contrast in Photoacoustic Imaging, and T2-Shortening in Magnetic Resonance Imaging.

Raman microspectroscopy provides chemo-selective image contrast, sub-micrometer resolution, and multiplexing capabilities. However, it suffers from weak signals resulting in image-acquisition times of up to several hours. Surface-enhanced Raman scattering (SERS) can dramatically enhance signals of molecules in close vicinity of metallic surfaces and overcome this limitation. Multimodal, SERS-active nanoparticles are usually labeled with Raman marker molecules, limiting SERS to the coating material. In order to realize multimodal imaging while acquiring the rich endogenous vibronic information of the specimen, a core-shell particle based on "Nanorice", where a spindle-shaped iron oxide core is encapsulated by a closed gold shell, is developed. An ultrathin layer of silica prevents agglomeration and unwanted chemical interaction with the specimen. This approach provides Raman signal enhancement due to plasmon resonance effects of the shell while the optical absorption in the near-infrared spectral region provides contrast in photoacoustic tomography. Finally, T2-relaxation of a magnetic resonance imaging (MRI) experiment is altered by taking advantage of the iron oxide core. The feasibility for Raman imaging is evaluated by nearfield simulations and experimental studies on the primate cell line COS1. MRI and photoacoustics are demonstrated in agarose phantoms illustrating the promising translational nature of this strategy for clinical applications in radiology.

In-Process Measurement of Thickness of Cured Resin in Evanescent-Wave-Based Nano-stereolithography Using Critical Angle Reflection

Stereolithography is one of the most powerful ways to fabricate complex three-dimensional polymeric-based structures layer-by-layer using optical power. Evanescent-wave-based nano-stereolithography using ultra-thin field distribution of evanescent wave to solidify photosensitive resin can provide a sub-micrometer vertical resolution of each layer. To meet strong demands for in-process thickness measurement of cured resin in evanescent-wave-based nano-stereolithography, a measurement method that utilizes variations of resin’s refractive index after polymerization and a high sensitivity of total internal reflection at the critical angle has been proposed. By launching the a measurement light from a substrate to resin at the critical angle and detecting reflections from the resin, slight change in refractive index and thickness of cured resin that have great influences on reflectivity can be in-process measured. This method has been firstly examined by simulation using the rigorous coupled wave analysis method. Here, we show that an increase in cured resin’s thickness induces a decrease in the reflectivity. In experiments, the largest reflectivity contrast between cured and uncured resins has been proved at the critical angle. In addition, the relationship between reflectivity drop and thickness has been calibrated and a linear relationship within a certain thickness range has been experimentally confirmed. Furthermore, the difference of curing process between continuous and discontinuous exposure has been investigated by using the proposed measurement method. At last, image subtraction and a median filter have been applied in imaging processing to remove influences of uneven illumination and background noises.

Glassomer-Processing Fused Silica Glass Like a Polymer.

Fused silica glass is one of the most important high-performance materials for scientific research, industry, and society. However due to its high chemical and thermal resistance as well as high hardness, fused silica glass is notoriously difficult to structure. This work introduces Glassomer, a solid nanocomposite, which can be structured using polymer molding and subtractive technologies at submicrometer resolution. After polymer processing Glassomer is turned into optical grade fused silica glass during a final heat treatment. The resulting glass has the same optical transparency as commercial fused silica and a smooth surface with a roughness of a few nanometers. This work makes high-performance fused silica glass components accessible to high-throughput fabrication technologies and will enable numerous optical, photonic and medical applications in science and industry.

High-resolution Volume Imaging of Neurons by the Use of Fluorescence eXclusion Method and Dedicated Microfluidic Devices.

Volume is an important parameter regarding physiological and pathological characteristics of neurons at different time scales. Neurons are quite unique cells regarding their extended ramified morphologies and consequently raise several methodological challenges for volume measurement. In the particular case of in vitro neuronal growth, the chosen methodology should include sub-micrometric axial resolution combined with full-field observation on time scales from minutes to hours or days. Unlike other methods like cell shape reconstruction using confocal imaging, electrically-based measurements or Atomic Force Microscopy, the recently developed Fluorescence eXclusion method (FXm) has the potential to fulfill these challenges. However, although being simple in its principle, implementation of a high-resolution FXm for neurons requires multiple adjustments and a dedicated methodology. We present here a method based on the combination of fluorescence exclusion, low-roughness multi-compartments microfluidic devices, and finally micropatterning to achieve in vitro measurements of local neuronal volume. The high resolution provided by the device allowed us to measure the local volume of neuronal processes (neurites) and the volume of some specific structures involved in neuronal growth, such as growth cones (GCs).

High-resolution Volume Imaging of Neurons by the Use of Fluorescence eXclusion Method and Dedicated Microfluidic Devices

Volume is an important parameter regarding physiological and pathological characteristics of neurons at different time scales. Neurons are quite unique cells regarding their extended ramified morphologies and consequently raise several methodological challenges for volume measurement. In the particular case of in vitro neuronal growth, the chosen methodology should include sub-micrometric axial resolution combined with full-field observation on time scales from minutes to hours or days. Unlike other methods like cell shape reconstruction using confocal imaging, electrically-based measurements or Atomic Force Microscopy, the recently developed Fluorescence eXclusion method (FXm) has the potential to fulfill these challenges. However, although being simple in its principle, implementation of a high-resolution FXm for neurons requires multiple adjustments and a dedicated methodology. We present here a method based on the combination of fluorescence exclusion, low-roughness multi-compartments microfluidic devices, and finally micropatterning to achieve in vitro measurements of local neuronal volume. The high resolution provided by the device allowed us to measure the local volume of neuronal processes (neurites) and the volume of some specific structures involved in neuronal growth, such as growth cones (GCs).

Resonant nonlinear microscopy reveals changes in molecular level chirality in native biological tissues

Chirality is a fundamental property of biochemical molecules and often dictates their functionality. Conventionally, molecular chirality is studied by linear optical activity effects. However, poor contrast and artifacts due to anisotropy limit such studies to purified molecules not in their original microenvironments, potentially modifying their conformations. Here, we demonstrate that resonant second-harmonic-generation circular dichroism (SHG-CD) microscopy provides not only tissue imaging with improved chiral contrast, but also molecular chirality information of collagen, the most abundant protein in mammals, at its native state. Gradual protein denaturation shows that the resonant SHG-CD is dominated by the microscopic chirality related to collagen structures smaller than the spatial resolution of the microscope, i.e. to the protein conformation and microfibril organization, while the effects due to fiber orientation/anisotropy are mostly responsible of the non-resonant part. This result agrees well with a simple and intuitive model we propose to explain the resonant behavior and the consequent numerical SHG-CD simulations. Our results demonstrate the possibility to study molecular chirality in intact bio-tissues with nearly-unity contrast and sub-micrometer resolution, which will be useful in a broad range of biological and biochemical applications.

3D-glass molds for facile production of complex droplet microfluidic chips.

In order to leverage the immense potential of droplet microfluidics, it is necessary to simplify the process of chip design and fabrication. While polydimethylsiloxane (PDMS) replica molding has greatly revolutionized the chip-production process, its dependence on 2D-limited photolithography has restricted the design possibilities, as well as further dissemination of microfluidics to non-specialized labs. To break free from these restrictions while keeping fabrication straighforward, we introduce an approach to produce complex multi-height (3D) droplet microfluidic glass molds and subsequent chip production by PDMS replica molding. The glass molds are fabricated with sub-micrometric resolution using femtosecond laser machining technology, which allows directly realizing designs with multiple levels or even continuously changing heights. The presented technique significantly expands the experimental capabilities of the droplet microfluidic chip. It allows direct fabrication of multilevel structures such as droplet traps for prolonged observation and optical fiber integration for fluorescence detection. Furthermore, the fabrication of novel structures based on sloped channels (ramps) enables improved droplet reinjection and picoinjection or even a multi-parallelized drop generator based on gradients of confinement. The fabrication of these and other 3D-features is currently only available at such resolution by the presented strategy. Together with the simplicity of PDMS replica molding, this provides an accessible solution for both specialized and non-specialized labs to customize microfluidic experimentation and expand their possibilities.

Fabrication of 3D Capillary Vessel Models with Circulatory Connection Ports.

Bionic microscopic vessel models can contribute to the development of vascular treatment skills and techniques for clinical training. Most microscopic vessel models are limited to two dimensions, but three-dimensional (3D) models are important for surgery, such as on retina microscopic vessels, for the observation of colon microvessels, for measuring the deformability of red blood cell (RBC), and so on. Therefore, bionic 3D blood vessel models are increasingly in demand. For this reason, it is necessary to establish 3D fabrication techniques for microchannels. In this study, we established two fabrication methods for 3D microfluidic devices for the development of microscopic vessel models. First, we employed an exposure method using photolithographic technology. Second, we employed a 3D method using femtosecond laser and mask hybrid exposure (FMEx). Both methods made it possible to fabricate a millimeter-scale 3D structure with a submicrometer resolution and achieve an easy injection of solution. This is because it was possible to fabricate typical microfluidic channels used for model inlet and outlet ports. Furthermore, in the FMEx method, we employed an acid-diffusion effect using a chemically amplified resist to form a circular channel cross-section. The acid-diffusion effect made it realizable to fabricate a smooth surface independent of the laser scanning line width. Thus, we succeeded in establishing two methods for the fabrication of bionic 3D microfluidic devices with microfluidic channels having diameters of 15–16 µm for mimicking capillary vessels.

Three-dimensional micro-scale strain mapping in living biological soft tissues

Non-invasive characterization of the mechanical micro-environment surrounding cells in biological tissues at multiple length scales is important for the understanding of the role of mechanics in regulating the biosynthesis and phenotype of cells. However, there is a lack of imaging methods that allow for characterization of the cell micro-environment in three-dimensional (3D) space. The aims of this study were (i) to develop a multi-photon laser microscopy protocol capable of imprinting 3D grid lines onto living tissue at a high spatial resolution, and (ii) to develop image processing software capable of analyzing the resulting microscopic images and performing high resolution 3D strain analyses. Using articular cartilage as the biological tissue of interest, we present a novel two-photon excitation imaging technique for measuring the internal 3D kinematics in intact cartilage at sub-micrometer resolution, spanning length scales from the tissue to the cell level. Using custom image processing software, we provide accurate and robust 3D micro-strain analysis that allows for detailed qualitative and quantitative assessment of the 3D tissue kinematics. This novel technique preserves tissue structural integrity post-scanning, therefore allowing for multiple strain measurements at different time points in the same specimen. The proposed technique is versatile and opens doors for experimental and theoretical investigations on the relationship between tissue deformation and cell biosynthesis. Studies of this nature may enhance our understanding of the mechanisms underlying cell mechano-transduction, and thus, adaptation and degeneration of soft connective tissues. Statement of SignificanceWe presented a novel two-photon excitation imaging technique for measuring the internal 3D kinematics in intact cartilage at sub-micrometer resolution, spanning from tissue length scale to cellular length scale. Using a custom image processing software (lsmgridtrack), we provide accurate and robust micro-strain analysis that allowed for detailed qualitative and quantitative assessment of the 3D tissue kinematics. The approach presented here can also be applied to other biological tissues such as meniscus and annulus fibrosus, as well as tissue-engineered tissues for the characterization of their mechanical properties. This imaging technique opens doors for experimental and theoretical investigation on the relationship between tissue deformation and cell biosynthesis. Studies of this nature may enhance our understanding of the mechanisms underlying cell mechano-transduction, and thus, adaptation and degeneration of soft connective tissues.

A sensitive EUV Schwarzschild microscope for plasma studies with sub-micrometer resolution

We present an extreme ultraviolet (EUV) microscope using a Schwarzschild objective which is optimized for single-shot sub-micrometer imaging of laser-plasma targets. The microscope has been designed and constructed for imaging the scattering from an EUV-heated solid-density hydrogen jet. Imaging of a cryogenic hydrogen target was demonstrated using single pulses of the free-electron laser in Hamburg (FLASH) free-electron laser at a wavelength of 13.5 nm. In a single exposure, we observe a hydrogen jet with ice fragments with a spatial resolution in the sub-micrometer range. In situ EUV imaging is expected to enable novel experimental capabilities for warm dense matter studies of micrometer-sized samples in laser-plasma experiments.