Mitsuru Koizumi

Reprint of: Atmospheric scanning electron microscope observes cells and tissues in open medium through silicon nitride film.

Direct observation of subcellular structures and their characterization is essential for understanding their physiological functions. To observe them in open environment, we have developed an inverted scanning electron microscope with a detachable, open-culture dish, capable of 8 nm resolution, and combined with a fluorescence microscope quasi-simultaneously observing the same area from the top. For scanning electron microscopy from the bottom, a silicon nitride film window in the base of the dish maintains a vacuum between electron gun and open sample dish while allowing electrons to pass through. Electrons are backscattered from the sample and captured by a detector under the dish. Cells cultured on the open dish can be externally manipulated under optical microscopy, fixed, and observed using scanning electron microscopy. Once fine structures have been revealed by scanning electron microscopy, their component proteins may be identified by comparison with separately prepared fluorescence-labeled optical microscopic images of the candidate proteins, with their heavy-metal-labeled or stained ASEM images. Furthermore, cell nuclei in a tissue block stained with platinum-blue were successfully observed without thin-sectioning, which suggests the applicability of this inverted scanning electron microscope to cancer diagnosis. This microscope visualizes mesoscopic-scale structures, and is also applicable to non-bioscience fields including polymer chemistry.

Atmospheric scanning electron microscope system with an open sample chamber: configuration and applications.

An atmospheric scanning electron microscope (ASEM) with an open sample chamber and optical microscope (OM) is described and recent developments are reported. In this ClairScope system, the base of the open sample dish is sealed to the top of the inverted SEM column, allowing the liquid-immersed sample to be observed by OM from above and by SEM from below. The optical axes of the two microscopes are aligned, ensuring that the same sample areas are imaged to realize quasi-simultaneous correlative microscopy in solution. For example, the cathodoluminescence of ZnO particles was directly demonstrated. The improved system has (i) a fully motorized sample stage, (ii) a column protection system in the case of accidental window breakage, and (iii) an OM/SEM operation system controlled by a graphical user interface. The open sample chamber allows the external administration of reagents during sample observation. We monitored the influence of added NaCl on the random motion of silica particles in liquid. Further, using fluorescence as a transfection marker, the effect of small interfering RNA-mediated knockdown of endogenous Varp on Tyrp1 trafficking in melanocytes was examined. A temperature-regulated titanium ASEM dish allowed the dynamic observation of colloidal silver nanoparticles as they were heated to 240°C and sintered.

Progress in Electron Beam Mastering of 100 Gbit/inch2 Density Disc

We developed an electron beam recorder (EBR) capable of recording master discs under atmospheric conditions using a novel differential pumping head. Using the EBR and optimized fabrication process for Si-etched discs with reactive ion etching (RIE), a bottom signal jitter of 9.6% was obtained from a 36 Gbit/inch2 density disc, readout using a near-field optical pickup with an effective numerical aperture (NA) of 1.85 and a wavelength of 405 nm. We also obtained the eye patterns from a 70 Gbit/inch2 density disc readout using an optical pickup with a 2.05 NA and the same wavelength, and showed almost the same modulation ratio as the simulation value. Moreover, the capability of producing pit patterns corresponding to a 104 Gbit/inch2 density is demonstrated.

Electron Beam Recording with a Novel Differential Pumping Head Realizing More than 50 GB/Layer Capacity Disc

We developed an electron beam recorder with a novel differential pumping head, which produces a local high-vacuum area for an electron beam by balancing nitrogen gas flow into and out of the head. Regarding recording speed, it was confirmed to be as high as 4.92 m/s while maintaining a 10-3 Pa vacuum condition. In order to achieve a high speed recording velocity, we introduced an electron gun with a low acceleration voltage (15 kV) and a chemically amplified resist to the mastering to make resist sensitivity very high. In addition, it takes only 2 min to exchange the Si substrate. As for the performance of the electron beam mastering, a bottom jitter value appropriate for mass production was obtained from a 25 GB/layer capacity disc. We also fabricated a 50 GB/layer capacity disc and confirmed the HF signal using a near-field optical pick up for the first time. Furthermore, we were able to fabricate a 100 GB/layer capacity disc using our electron beam recorder.

New Scanning Electron Microscope Capable of Observing Cells in Solution

Optical microscope in combination with fluorescent stain is a powerful devise for real time observation of cell macro-organelles. However, its resolution is limited upto 100 nm level, which is insufficient for observing smaller subcellular structures. Scanning Electron Microscope (SEM) is a powerful tool to obtain high-resolution image, however, the sample should be in vacuum. Recently, the capsule for liquid SEM imaging was developed. The film of polyimide [1] or silicon nitride (SiN) [2] was set as a window of an air-filled capsule facing the cells inside, and located in the sample chamber of SEM. The capsule is closed system, however, is limited in capacity to 15 μl, and does not allow for prolonged cultivation or external drug administration.

Recent progress in electron beam mastering for 100Gb/in2 and beyond

We improved the electron beam recorder with a differential pumping head for higher density discs and mass production. The beam diameters were improved by exchanging the aperture size of the objective lens and beam stability were also improved by adding a sound proof case. As for the performance of the improved electron beam recorder, we showed that a 104Gb/in2 (150GB capacity/layer) density disc with EFM plus modulation codes can be fabricated. We also improved the pit shape uniformity and a margin of the process by introducing the appropriate write strategy that is simulated by the Monte Carlo simulation to the recording pulses.

Electron beam mastering process realized over a 100-GB'layer capacity disk

We demonstrate the capability of 100-GB density recording by electron beam mastering and readout by a near-field optical pickup with an effective NA of 2.05 and a blue LD of 405-nm wavelength. A silicon (Si) disk of 100-GB density is fabricated by an optimized Si etching process condition to form suitable pit pattern shapes for the near-field readout.

Electron beam mastering process realizing over 100-GB/layer capacity disc

We have demonstrated the capability of 100GB density recording by the electron beam mastering and readout by a near-field optical pick-up with an effective NA of 2.05 and a blue LD of 405 nm wavelength. The Si disc of 100GB density was fabricated by the optimized Si etching process condition to form suitable pit pattern shape for the near-field readout.