Imaging Of Biological Materials Research Articles

Super-resolution model-based iterative reconstruction for lens-coupled micro-CT imaging

Lens-coupled high-resolution micro-computed tomography (micro-CT) uses a visible light magnification system behind the x-ray path to achieve higher resolution imaging than conventional micro-CT. However, the spatial resolution is theoretically limited by optical diffraction and mechanical control precision. As a result, the current system resolution is still insufficient for some applications, such as the imaging of biological materials whose structures are on the nanometer scale. To overcome this limitation, a super-resolution algorithm can be employed to improve the image resolution beyond the theoretical upper bound of the ideal spatial resolution of the system. In this work, a super-resolution model-based iterative reconstruction (SR-MBIR) algorithm is proposed based on a lens-coupled high-resolution micro-CT system and a high-precision nano-stage attached to the rotation stage of the system. The algorithm employs a scanning program that dithers the object via the nano-stage to obtain multiple sets of projection images with sub-pixel information. The blur and noise statistical models are introduced into the physical model for iterative reconstruction, allowing for super-resolution, deblurring, and noise suppression. The results of simulation data and actual data show that the SR-MBIR algorithm has a prominent effect in improving image resolution. The reconstructed images have sharper edges, better details, higher signal-to-noise ratio, and can effectively suppress the systematic blur and noise in the imaging process, thus achieving superior interior reconstruction quality.

Scanning electron microscopy of Escherichia coli encapsulated in a spacerized graphene sandwich.

Electron microscopy of biological materials such as bacteria allows multifaceted analysis to understand their structure and function with high resolution, which is difficult to achieve with optical microscopy. However, the samples are damaged or broken by electron beam irradiation and by the vacuum environment. Here, we observed bacteria in a suspension encapsulated in a graphene sandwich that prevents electron beam damage without the need for fixation. Specifically, we demonstrated in situ scanning electron microscopy observation of Escherichia coli in a graphene sandwich containing a perforated membrane as a spacer, encapsulating non-immobilized E. coli between the graphene layers. However, E. coli activity, such as division, was not observed, although the irradiated cells grew slightly when resuspended under optimal culture conditions. Our findings suggest that the graphene sandwich methodology enables the observation of wet E. coli cells by electron microscopy but requires refinement to allow the live imaging of biological materials.

Coherent X-ray imaging technology

Imaging of biological materials and cells with X-ray microscopy, which has high application potential, can break the diffraction limit barrier of traditional optical microscopy and exceed the transmission depth of electron microscopy. In this study, we introduce the examples of X-ray microscopy technology, such as transmission X-ray microscopy (TXM), small-angle X-ray scattering (SAXS), coherent X-ray diffraction imaging (CDI/CXDI), and X-ray holography. In addition, we demonstrate the combination of different technologies to achieve optimized imaging results such as the combination of STXM with CDI, the combination of scanning SAXS with ptychography, and the combination of X-ray microscopy with fluorescence imaging. Relevant improvements in rapid positioning, algorithms, X-ray sources, and sample preparation are also discussed.

Luminescent AlN:Mn nanoparticles for optical imaging of biological materials

Background: Elaboration of new luminescent nanomaterials for imaging of biological materials including cells of living organisms and their parts is highly actual. These materials must meet a number of requirements such as low toxicity, inherence of intensive luminescence, low costs of raw material and symple synthesis methods. AlN nanopowder is one of such prospective materials fitting the above requirements. Our long time investigations on spectral characteristics for III group element nitrides allows chose of doped AlN nanopowder as prospective candidate for developing of luminescent markers for imaging of biological materials. Objectives: The aim of the present study is spectral characterization of AlN nanopowder doped with Mn and evaluation of its use as luminescent marker for biological materials. Materials and methods: AlN nanopowder with average size of polycrystalline grains of 60 nm and the same doped with Mn were sythesized in Institue of Inorganic Chemistry, Riga Technical University. Photoluminescence and its excitation spectra of the materials were studied at room temperature using a self-made set-up. Results: It was found that in undoped AlN nanopowder at room temperature luminescence of native defects forms a wide and complex band peaking at 415 nm. This blue luminescence can be excited with ultraviolet light from two spectral regions around 315–340 nm and 260 nm. Two luminescence mechanisms are proposed dependent on the spectral region of exciting light. The first of them results in the intra-center luminescence, but the second one is recombination luminescence. Incorporation of Mn atoms in the crystalline lattice of AlN nanopowder forming AlN:Mn NP results in appearance of intensive red luminescence at 600 nm, which can be excited with light from two excitation bands at 260 and 480 nm. Two mechanisms responsible for an appearence of the red luminescence of Mn are proposed. They are the intra-center luminescence and recombination luminescence mechanisms. In this case the red Mn luminiscence prevails and the blue luminescence characterizing the host material has not been observed. Conclusion: AlN nanopowder doped with Mn atoms is a prospective material for use as luminescent marker for imaging of biological materials. Properties of this material are in a good agreement with the main requirements obligated to biological materials: i) AlN NP has low toxicity; ii) AlN:Mn NP possesses intensive red luminescence at 600 nm, which can be excited either with the ultraviolet light around 260 nm or with visible light around 480 nm; iii) it is relatively cheep material and it can be synthesized using simple synthesis methods.

Scanning frequency comb microscopy—A new method in scanning probe microscopy

A method for superimposing a microwave frequency comb (MFC) on the DC tunneling current in a scanning tunneling microscope (STM) is described in which a mode-locked laser is focused on the tunneling junction. The MFC is caused by optical rectification of the regular sequence of laser pulses due to the nonlinear current-voltage characteristics of the tunneling junction. Hundreds of harmonics, at integer multiples of the laser pulse-repetition frequency, are generated with a metal tip and sample. However, the harmonics have less power with a resistive sample due to the loss in its spreading resistance. The microwave power is greatest at a tip-sample distance that is unique for each sample resistivity. This distance may be set by using different pairs of the applied DC bias and the set-point for the DC tunneling current. However, the laser, and not the applied DC bias or the DC tunneling current, is the source of energy for the MFC so they are not required. Feedback control of the tip-sample distance may be based on maximizing the attowatt-level microwave power of the harmonics, which have a signal-to-noise ratio of 20 dB. This method shows promise for nondestructive carrier profiling of semiconductors with true sub-nanometer resolution which is essential in the continued progress below the 40-nm technology node. It may enable carrier profiling with 2-D materials such as graphene, and it also shows promise for finer resolution in images of biological materials or other resistive samples.

Imaging of Biological Materials and Cells by X-ray Scattering and Diffraction.

Cells and biological materials are large objects in comparison to the size of internal components such as organelles and proteins. An understanding of the functions of these nanoscale elements is key to elucidating cellular function. In this review, we describe the advances in X-ray scattering and diffraction techniques for imaging biological systems at the nanoscale. We present a number of principal technological advances in X-ray optics and development of sample environments. We identify radiation damage as one of the most severe challenges in the field, thus rendering the dose an important parameter when putting different X-ray methods in perspective. Furthermore, we describe different successful approaches, including scanning and full-field techniques, along with prominent examples. Finally, we present a few recent studies that combined several techniques in one experiment in order to collect highly complementary data for a multidimensional sample characterization.

Biomedical surface analysis: Evolution and future directions (Review).

This review describes some of the major advances made in biomedical surface analysis over the past 30–40 years. Starting from a single technique analysis of homogeneous surfaces, it has been developed into a complementary, multitechnique approach for obtaining detailed, comprehensive information about a wide range of surfaces and interfaces of interest to the biomedical community. Significant advances have been made in each surface analysis technique, as well as how the techniques are combined to provide detailed information about biological surfaces and interfaces. The driving force for these advances has been that the surface of a biomaterial is the interface between the biological environment and the biomaterial, and so, the state-of-the-art in instrumentation, experimental protocols, and data analysis methods need to be developed so that the detailed surface structure and composition of biomedical devices can be determined and related to their biological performance. Examples of these advances, as well as areas for future developments, are described for immobilized proteins, complex biomedical surfaces, nanoparticles, and 2D/3D imaging of biological materials.

Liquid Cell Transmission Electron Microscopy.

Liquid cell transmission electron microscopy (TEM) has attracted significant interest in recent years. With nanofabricated liquid cells, it has been possible to image through liquids using TEM with subnanometer resolution, and many previously unseen materials dynamics have been revealed. Liquid cell TEM has been applied to many areas of research, ranging from chemistry to physics, materials science, and biology. So far, topics of study include nanoparticle growth and assembly, electrochemical deposition and lithiation for batteries, tracking and manipulation of nanoparticles, catalysis, and imaging of biological materials. In this article, we first review the development of liquid cell TEM and then highlight progress in various areas of research. In the study of nanoparticle growth, the electron beam can serve both as the illumination source for imaging and as the input energy for reactions. However, many other research topics require the control of electron beam effects to minimize electron beam damage. We discuss efforts to understand electron beam-liquid matter interactions. Finally, we provide a perspective on future challenges and opportunities in liquid cell TEM.

Spatially resolved NEXAFS spectroscopy of siderophores in biological matrices

Iron is an essential nutrient for almost all forms of life. In the presence of oxygen iron is present in its ferric form which precipitates under formation of rather insoluble oxide-hydroxide polymers. Hence the bioavailability of iron is extremely low (< 10−17 M at pH 7 for Fe3+). Under such conditions almost all microorganisms synthesize siderophores as iron chelating agents, thus solubilizing ferric iron from rather insoluble iron sources. Siderophores form soluble complexes with Fe3+. The present study aims at developing a methodology that would allow for the specific detection and localization of such iron chelators in their natural environment. The applicability of spatially resolved NEXAFS spectroscopy in the soft X-ray energy (E < 1 keV) range was evaluated for localization of typical fungal hydroxamate siderophores like ferrichrome or coprogen, which can be present in various biological materials. Results obtained with the scanning transmission X-ray microscopes at beamlines 11.0.2 and 5.3.2 of the ALS have shown characteristic signatures for siderophores. Thus NEXAFS spectroscopy at the carbon K-edge, nitrogen K-edge and iron L-edge with high spatial resolution has proven to be extremely useful for their identification in their natural environment. Spectra of different siderophores as well as spectra and images of biological material containing siderophores are presented.

ALIGNMENT AND IMAGING OF AN EYE WITH AN ULTRASONIC SCANNER

A method and apparatus are disclosed for generating accurate and precise ultrasonic images of biological materials or animate objects, such as the cornea and lens of the eye, and, in particular, to an ultrasonic scanning apparatus that can position its virtual center of curvature such that its ultrasonic transducer will emit pulses that reflect substantially perpendicularly from a curved specular surface of interest within the eye. This invention can allow real time imaging of a lens as it accommodates and can better enable researchers and ophthalmic surgeons to develop, fit, implant and diagnose performance of accommodative lenses.

Intravital imaging of gastrointestinal diseases in preclinical models using two-photon laser scanning microscopy

Two-photon laser scanning microscopy (TPLSM), relying on two-photon excitation restricted to the focal plane, has become the gold standard in biomedical research because of its ability to produce high-resolution, higher penetrating imaging of biological materials over an extended duration without an significant photobleaching. Intravital (in vivo) imaging using TPLSM for intra-abdominal organs has long been a technical challenge because of the difficulty of achieving high-quality and higher magnification imaging with acceptable cardiac and respiratory motion artifacts. A method of intravital TPLSM was developed using an organ-stabilizing system for imaging the intra-abdominal organs of green fluorescent protein transgenic mice. This method was further refined for the time-series imaging of intra-abdominal organs in the same mouse model using intravital TPLSM. These procedures allow the observation of not only a single cell or tissue microenvironment at a higher penetrating depth for a longer period of time but also to observe the same organ of the same mouse at multiple time points in preclinical models. This report presents the general principles and properties of TPLSM for biomedical research. In addition, the methods and the usefulness of time-series intravital TPLSM imaging for preclinical gastrointestinal diseases is discussed.

Quantitative phase microscopy with asynchronous digital holography

We demonstrate a new method of measuring quantitative phase in imaging of biological materials. This method, asynchronous digital holography, employs knowledge of a moving fringe created by acousto-optic modulators to execute phase-shifting interferometry using two near-simultaneous interferograms. The method can be used to obtain quantitative phase images of dynamic biological samples on millisecond time scales. We present results on a standard sample, and on live cell samples.

3F1030 クライオ原子間力顕微鏡による生体試料のイメージング : フリーズフラクチャー・エッチング技術の適用

3F1030 クライオ原子間力顕微鏡による生体試料のイメージング : フリーズフラクチャー・エッチング技術の適用

Eels Imaging of Biological Materials

ABSTRACTParallel-detection electron energy loss spectroscopy (EELS) in the scanning transmission electron microscope provides a very sensitive means of detecting specific elements in biological systems. By analyzing EELS spectrum-image data recorded from rapidly-frozen and cryosectioned tissue it is possible to map quantitatively the distribution of the biologically important element, calcium, which is typically present at concentrations of only a few parts per million in cellular structures some tens of nanometers in diameter. A significant improvement (factor of four) in calcium detectability has been demonstrated for EELS compared with energy-dispersive x-ray spectroscopy. The spectrum-imaging technique has also been applied to map water distributions in hydrated biological specimens by utilizing the valence electron excitations.

Effect of hydration on the morphology of enzyme powder

We report the first direct images of the hydration of protein powders. Using an environmental scanning electron microscope (ESEM) we have taken a series of micrographs of a region of the enzyme (subtilisin) power whilst hydrating the sample. In addition, the sample has been viewed during exposure to toluene vapors. The ESEM is a remarkable new instrument that will have wide applicability in imaging of biological materials in their native environments.

X-Ray Microscopy Using Collimated and Focussed Synchrotron Radiation*

X-ray microscopy is a field that has developed rapidly in recent years. Two different approaches have been used. Zone plates have been employed to produce focussed beams with sizes as low as 0.07 pm for x-ray energies below 1 keV. Images of biological materials and elemental maps for major and minor low Z have been produced using above and below absorption edge differences. At higher energies collimators and focussing mirrors have been used to make small diameter beams for excitation of characteristic K— or L-x rays of all elements in the periodic table.

Nanosecond Exposure Time Soft X-Ray Contact Imaging

Soft x-ray contact microscopy has been pursued for many years because of the potential advantages of 1 to 10 nm electro-magetic radiation for imaging biological materials. These are the lower damage and greater penetration than electrons and considerably higher resolution than visible light microscopy, both possible without exotic optics.The technique is similar to contact printing. The object to be viewed is placed as close as possible to the recording medium which is then exposed to a suitable x-ray source.. The sample is removed from the recording medium which is then chemically developed. The result is map of the x-ray opacity of the original specimen.The high resolution has only become reality in recent years with the use of x-ray photo-resists as the recording material. These were origionally developed for use in the electronics industry but have been used for contact images of biological material.