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Journal Articles

Correlative imaging of live biological cells with a soft X-ray microscope and a fluorescence microscope

Kado, Masataka; Kishimoto, Maki; Tamotsu, Satoshi*; Yasuda, Keiko*; Aoyama, Masato*; Tone, Shigenobu*; Shinohara, Kunio*

AIP Conference Proceedings 1696, p.020019_1 - 020019_4, 2016/01

 Times Cited Count:2 Percentile:80.58

Soft X-ray microscope is a very powerful tool to observe cellular organelles of living biological cells and many works have demonstrated imaging of inner structures of the cells. However the inner structures are very complicated and it is difficult to identify the organelles obtained with the soft X-ray microscopes. We have proposed a hybrid imaging method with a soft X-ray microscope and a fluorescence microscope that is to observe the same biological cells with the both microscopes at the same time. Using the information of the cellular organelles obtained with the fluorescence microscope, inner structures obtained with the soft X-ray microscope are accurately identified. We have observed living biological cells by the hybrid imaging method. Since the soft X-ray microscope has higher spatial resolution than that of the fluorescence microscope, fine structures of the cellular organelles in the living biological cells were discussed.

Journal Articles

In situ observation of cellular organelles with a contact X-ray microscope

Kado, Masataka; Kishimoto, Maki; Tamotsu, Satoshi*; Yasuda, Keiko*; Shinohara, Kunio*

Journal of Physics; Conference Series, 463, p.012056_1 - 012056_4, 2013/10

 Times Cited Count:13 Percentile:97.61

A contact X-ray microscope coupled with a high intense laser plasma soft X-ray source has been developed and in situ observations of cellular organelles have been conducted. The soft X-ray source were generated by a high power laser pulse onto a thin foiled gold target at the photon numbers of 1.3$$times$$10$$^{15}$$ photons/sr to be able to capture an image of live wet biological cells. The cells were cultured on PMMA photoresists that were formed on transparent glass plates to make optical microscope observation possible. The cells were observed by both of optical microscope and soft X-ray microscope. The obtained soft X-ray images were directly compared with corresponding fluorescent optical images. Cellular organelles such as mitochondria and cytoskeleton in the soft X-ray images were identified referencing the information obtained from the fluorescent images.

Journal Articles

Imaging of fine structures of cellular organelles in hydrated biological cells by a soft X-ray microscope combined with a fluorescence microscope

Kado, Masataka; Kishimoto, Maki; Tamotsu, Satoshi*; Yasuda, Keiko*; Aoyama, Masato*; Shinohara, Kunio*

Proceedings of SPIE, Vol.8849, p.88490C_1 - 88490C_7, 2013/09

 Times Cited Count:1 Percentile:59.67

We have proposed to use a fluorescence microscope to identify the cellular organelles in the images obtained with the soft X-ray microscope observing the same cells with both microscopes. The cells were stained with several fluorescent dyes such as Mito-tracker, Phalloidin, and DAPI and after taking many fluorescence images of cellular organelles the cells were exposed to the flash soft X-rays. The obtained soft X-ray images and fluorescence images of the cells were directly compared and each of the cellular organelles such as mitochondria, actin filaments, and chromosomes in the soft X-ray images was clearly identified. Since the soft X-ray microscope has higher spatial resolution than that of the fluorescence microscope, fine structures of the cellular organelles in the hydrated biological cells were observed for the first time.

Journal Articles

Development of single shot soft X-ray contact microscopy system for nano-scale dynamics measurement of living biological specimen

Kishimoto, Maki; Kado, Masataka; Ishino, Masahiko; Tamotsu, Satoshi*; Yasuda, Keiko*; Shinohara, Kunio*

AIP Conference Proceedings 1465, p.43 - 47, 2012/07

 Times Cited Count:6 Percentile:91.07

We have been developing a picosecond single shot soft X-ray contact microscopy system for observing the nanometer-scale inner structure of the living biological specimen in a hydrated condition. The microscopy system consists of an intense IR pump laser system for generating soft X-rays and X-ray microscope chamber. The pump laser system employs OPCPA technique to generate water-window X-rays effectively. The X-ray microscope chamber is composed of a vacuum chamber, a focusing lens, a metal film target, an in-vacuum type sample holder. The soft X-rays from the laser-induced plasma generated by pump laser pulse illuminates bio-specimens on the PMMA photo resist set in the in-vacuum sample holder. The photo resist is developed and the X-ray transmission imageis read out by AFM. We took X-ray images of hydrated Leydig cells from mouse testicle and demonstrated that the developed X-ray microscopy system has a spatial resolution of about 100 nm.

Journal Articles

Observation of organelle by a laser plasma X-ray microscope

Kado, Masataka; Kishimoto, Maki; Ishino, Masahiko; Tamotsu, Satoshi*; Yasuda, Keiko*; Shinohara, Kunio*

AIP Conference Proceedings 1465, p.246 - 250, 2012/07

 Times Cited Count:5 Percentile:87.84

Contact X-ray microscopy has a potential to image wet biological specimens in natural condition. It is very important to identify obtained features in the X-ray images, since X-ray microscopes have potential to image features that have not been visualized yet. We have proposed to compare the X-ray images of the biological specimens with the fluorescence images and to identify the features found in the X-ray images based on the features found in the fluorescence images. Comparing the X-ray images to the fluorescence images of the set biological cells, fine structures of the mitochondria in the X-ray images have been able to be identified.

Journal Articles

Observation of organelles in Leydig cells by contact soft X-ray microscopy with a laser plasma X-ray source

Kado, Masataka; Ishino, Masahiko; Tamotsu, Satoshi*; Yasuda, Keiko*; Kishimoto, Maki; Nishikino, Masaharu; Kinjo, Yasuhito*; Shinohara, Kunio*

AIP Conference Proceedings 1365, p.391 - 394, 2011/09

 Times Cited Count:6 Percentile:89.64

Contact X-ray microscopy has achieved a single-shot imaging of wet biological specimens in natural condition and succeeded in imaging live mouse macrophages with hair-like structures, which was not observed before. It is very important to identify obtained features in the X-ray images, since X-ray microscopes have potential to image features that have not been visualized yet. Here, we demonstrate to image the same biological specimens both by confocal laser microscopy and soft X-ray microscopy. Staining biological specimens with well-established techniques makes easy to identify features in the fluorescence images obtained with confocal laser microscope. Comparing the X-ray images of the specimens with the fluorescence images, features found in the fluorescence images could also be identified in the X-ray images. Comparing the X-ray images to the fluorescence images, fine structures of the actin filaments in the X-ray images were identified.

Journal Articles

Development of a specimen holder combined with ultra thin film laser plasma X-ray source for compact contact-type soft X-ray microscope to observe hydrated living biologocal cells

Ishino, Masahiko; Kado, Masataka; Shinohara, Kunio*; Yamamoto, Yoshimasa*; Hirai, Itaru*; Kishimoto, Maki; Nishikino, Masaharu; Hasegawa, Noboru; Tamotsu, Satoshi*; Yasuda, Keiko*; et al.

Proceedings of SPIE Europe Optics + Optoelectronics 2011, Vol.8139, p.81390R_1 - 81390R_8, 2011/09

 Times Cited Count:0 Percentile:0.01

Ultra thin gold films are favorable laser plasma targets for a soft X-ray microscopy, because the thin films emit intense soft X-rays at the wavelength of water window region. Using rear side emissions, the distance between the X-ray source and the specimens can be reduced so that the X-ray flux on specimens increases. The microscope system can be designed to be compact when the specimen holder and X-ray source are combined in one piece. The biological specimen holder combined with an ultra thin film target has been developed. This X-ray microscope system needs not any X-ray optics which causes a decrease in X-ray photons for imaging. X-ray images of hydrated living cells have been obtained successfully by use of the newly developed specimen holder. Specimen holder combined with plasma X-ray source will be a key component of a compact soft X-ray microscope using in a laboratory.

Journal Articles

Flash imaging of fine structures of cellular organelles by contact X-ray microscopy with a high intensity laser plasma X-ray source

Kado, Masataka; Ishino, Masahiko; Kishimoto, Maki; Tamotsu, Satoshi*; Yasuda, Keiko*; Kinjo, Yasuhito*; Shinohara, Kunio*

Proceedings of SPIE Europe Optics + Optoelectronics 2011, Vol.8139, p.81390O_1 - 81390O_7, 2011/09

Laser plasma X-ray sources have high intensity and short pulse duration, and are suitable for X-ray microscopy in biology. They make wet live biological specimens possible to be imaged with a single shot X-ray exposure and several works have been done to image them. However there were no reports on the imaging of fine structures of cellular organelles in a live biological cell since higher X-ray intensity is needed for it. We have developed a high intensity laser plasma X-ray source, cooperating it with contact X-ray microscopy, and observed fine structures of cellular organelles in a wet biological cells. Comparing the X-ray images and the fluorescence images of cellular organelles such as actin filaments and mitochondria we have been clearly able to identify organelles in the X-ray images and observed fine structures.

Journal Articles

Observation of actin filaments in Leydig cells with a contact-type soft X-ray microscope with laser plasma X-ray source

Kado, Masataka; Ishino, Masahiko; Tamotsu, Satoshi*; Yasuda, Keiko*; Kishimoto, Maki; Nishikino, Masaharu; Kinjo, Yasuhito*; Shinohara, Kunio*

Denki Gakkai Rombunshi, C, 130(10), p.1774 - 1778, 2010/10

Actin filaments in Leydig cells from mouse testes have been observed with a contact-type soft X-ray microscope with laser plasma X-ray source. The Leydig cells were fixed with paraformaldehyde, stained with Phalloidin, and observed with a confocal laser microscope prior to the observation with X-ray microscope. Obtained images by both of the confocal laser microscopy and the X-ray microscopy were directly compared and revealed that not only position of actin filaments but also the shapes can be identified each other. The actin filaments in the X-ray images were clearly recognized and their structures were obtained in more detail compared to those in the confocal laser microscope images.

Journal Articles

Observations of the intense soft X-ray emissions from ultra thin Au films irradiated with high contrast laser pulses

Ishino, Masahiko; Kado, Masataka; Nishikino, Masaharu; Shinohara, Kunio*; Tamotsu, Satoshi*; Yasuda, Keiko*; Hasegawa, Noboru; Kishimoto, Maki; Oba, Toshiyuki; Kawachi, Tetsuya

Proceedings of SPIE, Vol.7589, p.75891B_1 - 75891B_8, 2010/02

 Times Cited Count:2 Percentile:75.92

Soft X-ray microscopes operating in the water window are capable of imaging living hydrated biological specimens. Laser produced plasmas are attractive soft X-ray sources, because of their short duration time. Based on the minimum dose calculation, soft X-ray photons more than 10$$^{5}$$ photons/$$mu$$m$$^{2}$$ at the sample surface are needed to acquire an image of the biological specimens with spatial resolution up to 100 nm. The observations of soft X-ray emissions from laser produced plasmas using ultra thin film targets have been carried out. Au thin films were irradiated by a high contrast Nd:glass laser pulses. The spectral properties of emitted soft X-rays were monitored by an X-ray spectrograph from the rear side with respect to the surface of laser irradiation. The observed emission intensities had an obvious dependence on the film thickness, and the most intense emissions were obtained at the thickness of 28 nm. The experimental results have suggested that the most of the laser energy irradiated is absorbed by the film target, and it is resulted an efficient energy deposition from laser to X-rays.

Oral presentation

Rapid analysis of radionuclides using porous polymer sheet functionalized by radiation-induced graft polymerization

Asai, Shiho; Magara, Masaaki; Suzuki, Daisuke; Shinohara, Nobuo; Saito, Kyoichi*; Sugo, Takanobu*; Takami, Michiko*; Shiraishi, Kunio*

no journal, , 

no abstracts in English

Oral presentation

Preparation of access-restricted separation material for analysis of bio-samples

Shibahara, Ryuji*; Asai, Shiho; Hagiwara, Kyohei*; Takami, Michiko*; Shiraishi, Kunio*; Umeno, Daisuke*; Shinohara, Nobuo; Sugo, Takanobu*; Saito, Kyoichi*

no journal, , 

no abstracts in English

Oral presentation

Observation of biological cells with a soft X-ray microscope using laser plasma X-ray sources

Kado, Masataka; Ishino, Masahiko; Nishikino, Masaharu; Hasegawa, Noboru; Kawachi, Tetsuya; Mizutani, Haruo*; Shinohara, Kunio*

no journal, , 

no abstracts in English

Oral presentation

Observations of living cells by use of a soft X-ray microscope and direct comparison of cell images obtained with a fluorescence microscope and a soft X-ray microscope

Ishino, Masahiko; Kado, Masataka; Kishimoto, Maki; Nishikino, Masaharu; Hasegawa, Noboru; Oba, Toshiyuki; Kawachi, Tetsuya; Tamotsu, Satoshi*; Yasuda, Keiko*; Yamamoto, Yoshimasa*; et al.

no journal, , 

no abstracts in English

Oral presentation

Biological imaging by soft X ray, 2; Imaging of organelles

Kado, Masataka; Ishino, Masahiko; Kishimoto, Maki; Shinohara, Kunio*; Tamotsu, Satoshi*; Yasuda, Keiko*; Yamamoto, Yoshimasa*; Kinjo, Yasuhito*

no journal, , 

Contact soft X-ray microscopes with laser plasma X-ray sources are anticipated technology to be able to observe live hydrated biological cells with high spatial resolution. However there were no works reported which identified specific organelles clearly, because the obtained X-ray images are too complicated due to overlapping of cell structures. We have observed the same biological cells with both of a contact soft X-ray microscope and a confocal laser microscope and compared images obtained with both microscopes in order to identify organelles obtained with the soft X-ray microscope. As the results we succeeded to identify actin filaments and mitochondria clearly and found that the organelles obtained with the soft X-ray microscope were more detailed than those with the confocal laser microscope.

Oral presentation

Direct comparison of images of organelles obtained with a fluorescence microscope and a soft X-ray microscope

Ishino, Masahiko; Kado, Masataka; Tamotsu, Satoshi*; Yasuda, Keiko*; Shinohara, Kunio*; Mikata, Yuji*; Kishimoto, Maki; Nishikino, Masaharu; Oba, Toshiyuki; Kaihori, Takeshi; et al.

no journal, , 

no abstracts in English

Oral presentation

Observation of cellular organelles by a contact-type soft X-ray microscope with laser plasma X-ray sources

Kado, Masataka; Kishimoto, Maki; Ishino, Masahiko; Tamotsu, Satoshi*; Yasuda, Keiko*; Shinohara, Kunio*

no journal, , 

Soft X-ray microscope have been expected as dream technique which can observe cellular organelles in vivo. However in order to realize it very high intensity soft X-ray sources have been required. Most studies had to use freezing technique to avoid degradation of spatial resolution and/or radiation damage since long exposure time such as several seconds to several minutes were required. We have succeeded to generate high intensity soft X-rays irradiating double layered target consisted of a 100nm thick silicon nitride film and a 20nm thick gold film with high contrast and high power laser pulses. Irradiating the high intensity soft X-rays to biological cells cultivated directory onto PMMA photo resists we have succeeded to observe mitochondria and cytoskeleton inside of live cells in vivo.

Oral presentation

Observation of apoptotic nuclei by a laser-plasma soft X-ray microscope

Kado, Masataka; Kishimoto, Maki; Tone, Shigenobu*; Tamotsu, Satoshi*; Yasuda, Keiko*; Aoyama, Masato*; Shinohara, Kunio*

no journal, , 

Laser plasma soft X-ray microscope using a laser plasma X-ray source has high spatial resolution of 100 nm and high temporal resolution of 1 nm and has advantage to be able to observe live hydrated biological cells. We have succeeded to observe cellular organelles such as mitochondria, cytoskeleton, and chromatin structures. We also invented to use a fluorescent microscope along with the soft X-ray microscope in order to identify cellular organelles obtained in the soft X-ray images. As the next application for the soft X-ray microscope Apoptotic nuclei have been observed. Since the laser plasma soft X-ray microscope has higher spatial resolution than the fluorescent microscope and can observe the structural change directory, it is expected to accomplish important role to understand apoptotic mechanism.

Oral presentation

Observation of apoptotic nuclei with a laser plasma soft X-ray microscope, 2

Kado, Masataka; Kishimoto, Maki; Tone, Shigenobu*; Tamotsu, Satoshi*; Yasuda, Keiko*; Aoyama, Masato*; Shinohara, Kunio*

no journal, , 

Laser plasma soft X-ray microscope, which is a contact type soft X-ray microscope coupled with a highly intense and short pulsed laser plasma soft X-ray source, has higher spatial resolution better than 100 nm and high temporal resolution shorter than 1 nm and it is possible to observe inner structures of live biological cells. We have developed the laser plasma soft X-ray microscope and have applied for various life science phenomenons especially apoptotic cell nuclei. It has been observed with transmission electron microscope and fluorescence microscope that chromatin which was usually folded in nucleus was condensed along nucleus membrane and changed its shape to ring, necklace, and finally collapses when the apoptosis occurred. However there are many things to be studied in order to understand detailed mechanism caused such apoptotic deformation. We have studied detailed structural deformation from ring to necklace which is important to understand apoptosis.

Oral presentation

Structural study of cellular organelles with a laser-plasma soft X-ray microscope

Kado, Masataka; Kishimoto, Maki; Tamotsu, Satoshi*; Yasuda, Keiko*; Aoyama, Masato*; Shinohara, Kunio*

no journal, , 

Laser plasma soft X-ray microscope, which is a combination of a laser plasma soft X-ray source and a contact type soft X-ray microscope, has been developed. Since the laser plasma soft X-ray sources have advantages of high brightness and short pulse duration, they can avoid radiation damage and motion blur affect on the images during acquisition of the soft X-ray images of live biological specimens. Hybrid microscopy, which was combination of a soft X-ray microscope and a fluorescence microscope, enabled the soft X-ray microscopy to identify cellular organelles precisely. Observing the Leydig cells, a single mitochondrion in the live biological cell was imaged for the first time and detailed structure of it was obtained.

35 (Records 1-20 displayed on this page)