Yasuharu Takaku

A thin polymer membrane, nano-suit, enhancing survival across the continuum between air and high vacuum

Most multicellular organisms can only survive under atmospheric pressure. The reduced pressure of a high vacuum usually leads to rapid dehydration and death. Here we show that a simple surface modification can render multicellular organisms strongly tolerant to high vacuum. Animals that collapsed under high vacuum continued to move following exposure of their natural extracellular surface layer (or that of an artificial coat-like polysorbitan monolaurate) to an electron beam or plasma ionization (i.e., conditions known to enhance polymer formation). Transmission electron microscopic observations revealed the existence of a thin polymerized extra layer on the surface of the animal. The layer acts as a flexible “nano-suit” barrier to the passage of gases and liquids and thus protects the organism. Furthermore, the biocompatible molecule, the component of the nano-suit, was fabricated into a “biomimetic” free-standing membrane. This concept will allow biology-related fields especially to use these membranes for several applications.

Function and Evolutionary Origin of Unicellular Camera-Type Eye Structure

The ocelloid is an extraordinary eyespot organelle found only in the dinoflagellate family Warnowiaceae. It contains retina- and lens-like structures called the retinal body and the hyalosome. The ocelloid has been an evolutionary enigma because of its remarkable resemblance to the multicellular camera-type eye. To determine if the ocelloid is functionally photoreceptive, we investigated the warnowiid dinoflagellate Erythropsidinium. Here, we show that the morphology of the retinal body changed depending on different illumination conditions and the hyalosome manifests the refractile nature. Identifying a rhodopsin gene fragment in Erythropsidinium ESTs that is expressed in the retinal body by in situ hybridization, we also show that ocelloids are actually light sensitive photoreceptors. The rhodopsin gene identified is most closely related to bacterial rhodopsins. Taken together, we suggest that the ocelloid is an intracellular camera-type eye, which might be originated from endosymbiotic origin.

Dressing living organisms in a thin polymer membrane, the NanoSuit, for high-vacuum FE-SEM observation

Scanning electron microscopy (SEM) has made remarkable progress and has become an essential tool for observing biological materials at microscopic level. However, various complex procedures have precluded observation of living organisms to date. Here, a new method is presented by which living organisms can be observed by field emission (FE)-SEM. Using this method, active movements of living animals were observed in vacuo (10(-5)-10(-7) Pa) by protecting them with a coating of thin polymer membrane, a NanoSuit, and it was found that the surface fine structure of living organisms is very different from that of traditionally fixed samples. After observation of mosquito larvae in the high vacuum of the FE-SEM, it was possible to rear them subsequently in normal culture conditions. This method will be useful for numerous applications, particularly for electron microscopic observations in the life sciences.

In Situ Preparation of Biomimetic Thin Films and Their Surface-Shielding Effect for Organisms in High Vacuum

Self-standing biocompatible films have yet to be prepared by physical or chemical vapor deposition assisted by plasma polymerization because gaseous monomers have thus far been used to create only polymer membranes. Using a nongaseous monomer, we previously found a simple fabrication method for a free-standing thin film prepared from solution by plasma polymerization, and a nano-suit made by polyoxyethylene (20) sorbitan monolaurate can render multicellular organisms highly tolerant to high vacuum. Here we report thin films prepared by plasma polymerization from various monomer solutions. The films had a flat surface at the irradiated site and were similar to films produced by vapor deposition of gaseous monomers. However, they also exhibited unique characteristics, such as a pinhole-free surface, transparency, solvent stability, flexibility, and a unique out-of-plane molecular density gradient from the irradiated to the unirradiated surface of the film. Additionally, covering mosquito larvae with the films protected the shape of the organism and kept them alive under the high vacuum conditions in a field emission-scanning electron microscope. Our method will be useful for numerous applications, particularly in the biological sciences.

A ‘NanoSuit’ surface shield successfully protects organisms in high vacuum: observations on living organisms in an FE-SEM

Although extremely useful for a wide range of investigations, the field emission scanning electron microscope (FE-SEM) has not allowed researchers to observe living organisms. However, we have recently reported that a simple surface modification consisting of a thin extra layer, termed ‘NanoSuit’, can keep organisms alive in the high vacuum (10−5 to 10−7 Pa) of the SEM. This paper further explores the protective properties of the NanoSuit surface-shield. We found that a NanoSuit formed with the optimum concentration of Tween 20 faithfully preserves the integrity of an organisms surface without interfering with SEM imaging. We also found that electrostatic charging was absent as long as the organisms were alive, even if they had not been coated with electrically conducting materials. This result suggests that living organisms possess their own electrical conductors and/or rely on certain properties of the surface to inhibit charging. The NanoSuit seems to prolong the charge-free condition and increase survival time under vacuum. These findings should encourage the development of more sophisticated observation methods for studying living organisms in an FE-SEM.

A modified ‘NanoSuit®’ preserves wet samples in high vacuum: direct observations on cells and tissues in field-emission scanning electron microscopy

Although field-emission scanning electron microscopy (FE-SEM) has proven very useful in biomedical research, the high vacuum required (10−3 to 10−7u2009Pa) precludes direct observations of living cells and tissues at high resolution and often produces unwanted structural changes. We have previously described a method that allows the investigator to keep a variety of insect larvae alive in the high vacuum environment of the electron microscope by encasing the organisms in a thin, vacuum-proof suit, the ‘NanoSuit®. However, it was impossible to protect wet tissues freshly excised from intact organisms or cultured cells. Here we describe an improved ‘NanoSuit technique to overcome this limitation. We protected the specimens with a surface shield enhancer (SSE) solution that consists of glycerine and electrolytes and found that the fine structure of the SSE-treated specimens is superior to that of conventionally prepared specimens. The SSE-based NanoSuit affords a much stronger barrier to gas and/or liquid loss than the previous NanoSuit did and, since it allows more detailed images, it could significantly help to elucidate the ‘real organization of cells and their functions.

A ‘NanoSuit’ successfully protects petals of cherry blossoms in high vacuum: examination of living plants in an FE-SEM

Land plants have evolved on dry land and developed surface barriers to protect themselves from environmental stresses. We have previously reported that polymerization of a natural extracellular substance (ECS) on the outer surface of animals by electron beam or plasma irradiation, can give rise to a nano-scale layer, termed the “NanoSuit”, which can keep small animals alive under the high vacuum of a scanning electron microscope (SEM). In the present research, we have focused on plants, using petals of cherry blossoms, as experimental specimens and examined their behavior under high vacuum conditions. Experiments on healthy living petals have demonstrated that without any pre-treatment, the overall morphology of specimens is well preserved and intact after imaging in an SEM, suggesting that natural substances on the petal surface behave like animal ECS and form a NanoSuit following irradiation with an electron beam. Furthermore, we have shown that the surface material can be extracted with chloroform and polymerized into a free-standing membrane by plasma irradiation. From our results, we conclude that surface materials, which have the ability to prevent water loss under natural conditions, increase the barrier ability and can protect plants under high vacuum conditions.

A thin polymer membrane 'NanoSuit' allows living organisms to survive in the harsh conditions of electron microscopy

We present a new method to observe living organisms by a common scanning electron microscope (SEM) including a field emission SEM. A simple surface modification to extracellular substances by electron beams or plasmas can equip some multicellular organisms with a thin extra layer, coined the “ NanoSuit(R)”, and hence can keep them alive under the high vacuum (10-3-10-7 Pa) conditions. The “NanoSuit(R)” acts as a flexible ‘Nano-spacesuit’ barrier to the passage of gases and liquids and thus protects the organism. Using this method, it was found that the surface fine structure of living organisms is very different from that of traditionally treated samples, and the active movements of living animals are also observed in an SEM. We next invented the coating method by the “biomimetic NanoSuit” based on artificial substance for the organism which lack the natural extracellular substances. After observation of living organisms by an SEM, despite the high vacuum it is possible to rear many of them subsequently in normal culture conditions. In addition to this method, it is now available to observe human tissues using new surface shield enhancer NanoSuit(R). Those new “ NanoSuit(R)” methods will be useful for numerous applications, particularly in the life sciences.

Abstract 1862: Direct observation of colorectal cancers using field-emission scanning electron microscopy with a thin polymer membrane, the NanoSuit

Background: Field-emission scanning electron microscopy (FE-SEM) enables us to observe nano-sized objects with great depth of focus and high resolution. However, the observation of biological samples including colorectal tissues using an FE-SEM has been difficult because it requires to evacuate its inside to prevent electron scattering, therefore all organisms containing ca. 70% water are rapidly evaporated and consequently caused structural disruption and collapse. To overcome the limitations of the conventional SEM, equipment such as low-vacuum scanning electron microscopy and environmental scanning electron microscope was developed. However, they are not reliable enough to investigate living organisms or wet tissues with high resolution level. We have recently reported that a simple surface modification consisting of a thin extra layer, coined the term ‘NanoSuit ® ’, can keep organisms alive in the high vacuum (10 −3 to 10 −7 Pa) of the SEM. We now modified the technique and developed a new solution, which enables FE-SEM observations of wet tissues. In this study, we utilized this technique to observe real images of colorectal cancers and their adjacent normal mucosae at high resolution. Materials and methods: Colorectal cancer tissues and their adjacent normal mucosa were cut with a scalpel from specimens surgically resected. All patients enrolled in this study provided written informed consent. Observations were carried out with an FE-SEM (S-4800, Hitachi or JEM-7100F, JEOL, Japan) at acceleration voltage of 1.0 kV. The vacuum level of the observation chamber was 10 -3 - 10 -7 Pa. The newly developed surface shield enhancer (SSE) solution was used to make NanoSuit ® for wet tissue observation. To form the NanoSuit ® , the specimens were dipped for 1 min into the SSE solution and blotted briefly thereafter put on dry filter paper to remove excess solution. Specimens were then introduced directly into an FE-SEM to form a NanoSuit ® following irradiation of the electron beam. Results: Fine structures of intestinal crypt and villi were observed in normal colon mucosa using FE-SEM with a NanoSuit ® , whereas fixed specimens prepared with conventional method showed obvious structural damage. Comparing with the region of normal colon mucosa, colorectal cancer lesion had relatively amorphous surface, therefore the border between non-cancerous mucosa and cancer lesions in colon tissues are able to be distinguished under high magnification. In addition, fiber-like structure was observed at the border between noncancerous mucosa and cancer lesions, suggesting an invasive front of colorectal cancer. Conclusions: We successfully observed the real mucosal surfaces and cancer lesions of colorectum with high resolution by an FE-SEM using a newly developed vacuum-proof suit, the “NanoSuit ® ”. This novel technique will enable us to investigate further physiopathology of GI tract including cancers. Citation Format: Hirotoshi Kikuchi, Tomohiro Matsumoto, Takanori Hiraide, Yusuke Ozaki, Amane Hirotsu, Tomohiro Murakami, Toshiki Kawabata, Yoshihiro Hiramatsu, Kinji Kamiya, Takanori Sakaguchi, Yasuharu Takaku, Isao Ohta, Takahiro Hariyama, Hiroyuki Konno. Direct observation of colorectal cancers using field-emission scanning electron microscopy with a thin polymer membrane, the NanoSuit [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1862. doi:10.1158/1538-7445.AM2017-1862