Publications (76) View all
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Article: Direct observation of biological molecules in liquid by environmental phase-plate transmission electron microscopy.
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ABSTRACT: We have been developing a combination method for environmental TEM (E-TEM) and phase-plate TEM (P-TEM) that enables direct observations of the structure of biological molecules in aqueous solution. It is clearly demonstrated that the biological molecules in a water layer can be imaged by the combined method without any stain. The spatial resolution obtained in the present study was about 10nm. This should be improved by using energy filtering. The image contrast of the specimen in water was reduced in comparison with that in vacuum. A model calculation that includes the effects of beam broadening, intensity decrease, and background increase caused by scattering from the water layer around the specimen shows that an increase in the thickness of the water layer reduces the contrast, intensity, and resolution of the image.Micron 02/2012; 43(11):1091-8. · 1.53 Impact Factor -
Article: Zernike phase contrast cryo-electron tomography of whole mounted frozen cells.
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ABSTRACT: Cryo-electron tomography of frozen hydrated cells has provided cell biologists with an indispensable tool for delineating three-dimensional arrangements of cellular ultrastructure. To avoid the damage induced by electron irradiation, images of frozen hydrated biological specimens are generally acquired under low-dose conditions, resulting in weakly contrasted images that are difficult to interpret, and in which ultrastructural details remain ambiguous. Zernike phase contrast transmission electron microscopy can improve contrast, and can also fix a fatal problem related to the inherent low contrast of conventional electron microscopy, namely, image modulation due to the unavoidable setting of deep defocus. In this study, we applied cryo-electron tomography enhanced with a Zernike phase plate, which avoids image modulation by allowing in-focus setting. The Zernike phase contrast cryo-electron tomography has a potential to suppress grainy background generation. Due to the smoother background in comparison with defocus phase contrast cryo-electron tomography, Zernike phase contrast cryo-electron tomography could yield higher visibility for particulate or filamentous ultrastructure inside the cells, and allowed us to clearly recognize membrane protein structures.Journal of Structural Biology 11/2011; 177(2):484-9. · 3.41 Impact Factor -
Article: Optimizing the phase shift and the cut-on periodicity of phase plates for TEM.
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ABSTRACT: Images acquired with a phase plate often exhibit fringing and/or contrast reversal artifacts. The two basic parameters controlling the performance of the phase plate are phase shift and cut-on periodicity. We investigate theoretically and numerically the effect of these parameters on the image quality. The analysis covers not just the typical negative phase shift phase plates but also positive phase shift ones. The theoretical study derives formulas for calculating the optimal phase plate phase shift and for the maximum achievable contrast with a given specimen. Two figures of merit - fidelity and contrast - were defined and used to quantify the numerical results. Larger cut-on periodicities provide better performance with higher contrast and less artifacts in the images. Both, the theoretical results and the simulations indicate that positive phase shift phase plates generate higher contrast with better linearity and are free from contrast reversal artifacts. However, with such phase plates the amplitude and the phase contrast components are opposed to each other and the simulations show stronger fringing outside of objects. Based on these results it is difficult to predict if and to what extent the positive phase shift phase plates will be advantageous in practice. Two methods for reduction of fringing artifacts were compared-tapered phase plate and low-frequency amplification software filter. Overall the software solution produced better results and is much easier to implement than modifying the hardware of the phase plate to realize the taper.Ultramicroscopy 07/2011; 111(8):1305-15. · 2.47 Impact Factor -
Article: Systemic delivery of siRNA to tumors using a lipid nanoparticle containing a tumor-specific cleavable PEG-lipid.
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ABSTRACT: Previously, we developed a multifunctional envelope-type nano device (MEND) for efficient delivery of nucleic acids. For tumor delivery of a MEND, PEGylation is a useful method, which confers a longer systemic circulation and tumor accumulation via the enhanced permeability and retention (EPR) effect. However, PEGylation inhibits cellular uptake and subsequent endosomal escape. To overcome this, we developed a PEG-peptide-DOPE (PPD) that is cleaved in a matrix metalloproteinase (MMP)-rich environment. In this study, we report on the systemic delivery of siRNA to tumors by employing a MEND that is modified with PPD (PPD-MEND). An in vitro study revealed that PPD modification accelerated both cellular uptake and endosomal escape, compared to a conventional PEG modified MEND. To balance both systemic stability and efficient activity, PPD-MEND was further co-modified with PEG-DSPE. As a result, the systemic administration of the optimized PPD-MEND resulted in an approximately 70% silencing activity in tumors, compared to non-treatment. Finally, a safety evaluation showed that the PPD-MEND showed no hepatotoxicity and innate immune stimulation. Furthermore, in a DNA microarray analysis in liver and spleen tissue, less gene alternation was found for the PPD-MEND compared to that for the PEG-unmodified MEND due to less accumulation in liver and spleen.Biomaterials 03/2011; 32(18):4306-16. · 7.40 Impact Factor -
Article: Another 60 years in electron microscopy: development of phase-plate electron microscopy and biological applications.
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ABSTRACT: It has been six decades since the concept of phase-plate electron microscopy was first reported by Boersch, but an experimental report on a phase plate with a theoretically rational performance has only recently been released by a group including the present author. Currently, many laboratories around the world are attempting to develop a wide range of phase plates to enhance the capabilities of transmission electron microscopy. They are reporting not only advantages of their own developments but also a fundamental problem inherent to electron beam devices, namely charging, i.e. the accumulation of electrostatic charge. In this report, we review the 60-year history of phase-plate development, with a particular focus on the fundamental issue of phase-plate charging. Next, we review biological applications of qualified phase plates, which have been successful in avoiding charging to some extent. Finally, we compare and discuss electron microscopic images, taken with or without phase plates, of biological targets such as proteins (GroEL and TRPV4), protein complexes (flagellar motor), viruses (T4 phage, ε-15 phage and herpes simplex virus), bacterial (cyanobacteria) and mammalian (PtK2) cells.Journal of electron microscopy 01/2011; 60 Suppl 1:S43-62. · 1.31 Impact Factor
