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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:3 Percentile:85.61

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.

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

Observation of apoptotic nuclei with 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 (LPSXM) with high spatial and temporal resolution has been developed and it has an ability to observe live hydrated biological specimens. The LPSXM is very powerful tool to study life phenomenon and it has been used to study apoptosis. Apoptosis is the process of programmed cell death to protect an organism by killing abnormal cells. The apoptotic process has been studied using light and electron microscopes and dynamics of nuclear deformation is being clear. However in order to fully understand the mechanism of the apoptosis, detailed structure of the deformed nuclei must be studied carefully. Observing the apoptotic nuclei fine structures of deformed nuclei were revealed and important information was provided to help understanding the mechanism of the apoptosis.

Oral presentation

Development of a soft X-ray microscope with a intense laser-plasma soft X-ray source produced by a high power pulsed laser

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

no journal, , 

Laser-plasma soft X-ray source produced by a high power pulsed laser is extremely bright and very suitable for biological X-ray microscopy to capture an image of living specimens which require a single flash exposure to avoid any damages on the specimens. We have developed a bright soft X-ray source irradiating a highly intense laser pulse generated by a Nd:glass laser system onto a thin foiled gold target. The highest intensity was obtained at the thickness of 400 nm and the photon flux was measured to be about 1.3 $$times$$ 10$$^{15}$$ photon/sr. The photon flux on the specimens was estimated to be about 4.4 $$times$$ 10$$^{5}$$ photons/$$mu$$m$$^{2}$$ and it will give the spatial resolution of about 80 nm. The bright soft X-ray source was combined with a contact soft X-ray microscope and established as a laser plasma soft X-ray microscope. Live biological cells were observed with both of the soft X-ray microscope and a fluorescence microscope at the same time.

Oral presentation

High contrast imaging with a contact soft X-ray microscope controlling water layer thickness around specimens

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

no journal, , 

Soft X-ray microscope using water window X-ray as light sources has been developed as epoch-making technology to observe living hydrated biological cells with high spatial resolution. Controlling the thickness of water layer is critical to observe living biological cells since the absorption of the soft X-ray by the water layer degrade the cell images. It is important to keep the thickness of the water layer at 5 $$mu$$m to obtain clear cell images. We have designed new silicon nitride windows with spacer and groove structure to keep the water layer thickness at 5 $$mu$$m and have succeeded to obtain high resolution cell images.

Oral presentation

In situ observation of inner structures of biological cells with a soft X-ray microscope

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

no journal, , 

Soft X-ray wavelengths between absorption K-edges of Oxygen and Carbon (2.3 nm and 4.4 nm) are so called "water window" and the X-ray were well absorbed by Carbon and less absorbed by water. Soft X-ray microscope using the water window X-ray as the light source has advantage to be able to observe live biological cells without any artifacts and can observe fine structures of cells compared to the light microscope. Combining with bright and short-pulsed laser-plasma soft X-ray the soft X-ray microscope which named a laser-plasma soft X-ray microscope can observe live biological cells in situ without radiation damages. We have generated bright water window soft X-ray irradiating a high power laser with 1053 nm in wavelength, 20 J in pulse energy and 600 ps in pulse duration onto thin foiled gold targets. Cultivating biological cells directly on the PMMA photoresists in situ observation of live biological cells with the laser-plasma soft X-ray microscope has been realized.

Oral presentation

Intense laser-plasma soft-X-ray sources and application for biological X-ray microscopy

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

no journal, , 

Although soft X-ray microscope has an ability to observe live biological cells, in order to avoid radiation damages onto the biological cells the cells had to be frozen and live cell imaging was not realized. Since laser-plasma soft X-ray source was extremely bright and had short pulse duration, it makes possible to capture soft X-ray images of biological cells before the cells damaged. Since the spatial resolution of the soft X-ray microscope depends on the photon flux irradiated onto the samples, it is important to increase brightness of the soft X-ray sources to observe inner structures of biological cells. We have increased the brightness of the soft X-ray source and succeeded to observe inner structures of live biological cells.

Oral presentation

Cellular imaging with a laser-plasma soft X-ray microscope

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

no journal, , 

We have developed a laser-plasma soft X-ray microscope composed of an intense short pulsed soft X-ray source and a contact microscopy system in which soft X-rays are irradiating onto biological cells directly cultivated on a recording media, an X-ray photo resist, and succeeded in observing inner structures of living biological cells. Using a fluorescence microscope with the soft X-ray microscope to observe the same biological cells at the same time, accurate identification and high resolved observation of cellular organelles have been achieved such as inner structure of Leydig cells from mouse testis, structural deformation of apoptotic HeLa S3 nuclei and mouse immune cells. In the case of the immune cells we have found structural changes possibly attributed to the activation of immune function.

Oral presentation

Live cell imaging with a soft X-ray microscope using a laser-plasma soft X-ray source

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

no journal, , 

We have developed a laser-plasma soft X-ray microscope composed of an intense short pulsed soft X-ray source and a contact microscopy system in which soft X-rays are irradiating onto biological cells directly cultivated on a recording media, an X-ray photo resist, and succeeded in observing inner structures of living biological cells. Using a fluorescence microscope with the soft X-ray microscope to observe the same biological cells at the same time, accurate identification and high resolved observation of cellular organelles have been achieved such as inner structure of Leydig cells from mouse testis, structural deformation of apoptotic HeLa S3 nuclei and mouse immune cells. In the case of the immune cells we have found structural changes possibly attributed to the activation of immune function.

Oral presentation

Correlative microscopy with a laser-plasma soft X-ray microscope and a fluorescent microscope for biological imaging of live hydrated cells

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

no journal, , 

A laser-plasma soft X-ray microscope which is combination of a highly intense laser-plasma soft X-ray source and contact microscopy has been developed. We have proposed a correlative microscopy with the laser-plasma soft X-ray microscope and a fluorescent microscope that is to observe the same biological cells with the both microscopes at the same time. Live hydrated biological cells and various cellular organelles of them have been observed with the correlative microscopy. Using the information of the cellular organelles obtained with the fluorescence microscope, inner structures obtained with the soft X-ray microscope are exactly identified. Since the spatial resolution of the soft X-ray microscope is much higher than that of the fluorescent microscope, fine structures of the cellular organelles of the live biological cells have been visualized with the correlative microscopy.

Oral presentation

Observation of apoptotic nuclei by a soft X-ray microscope with an intense laser-plasma soft X-ray source

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

no journal, , 

A soft X-ray microscope using a highly intense laser-plasma soft X-ray source (laser-plasma soft X-ray microscope) has an ability to observe live biological cells with a spatial resolution higher than 100 nm in a very short time period. Using the laser plasma soft X-ray microscope inner structures of various live biological cells such as cellular organelles of Leydig cells of mouse testis and structure change caused by immunological functions of immunological cells have been observed. We used the laser-plasma soft X-ray microscope to observe structural deformation of nucleus due to apoptotic process which is one of important life functions. We have found the structural deformation of nuclei from ring structure to necklace and also found important structure inside of the nuclei for the first time.

Oral presentation

Observation of cellular organelles by correlative microscopy with a laser-plasma soft X-ray microscope

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

no journal, , 

A laser-plasma soft X-ray microscope which is combination of a highly intense laser-plasma soft X-ray source and contact microscopy has been developed. We have proposed a correlative microscopy with the laser-plasma soft X-ray microscope and a fluorescent microscope that is to observe the same biological cells with the both microscopes at the same time. Live hydrated biological cells and various cellular organelles of them have been observed with the correlative microscopy. Using the information of the cellular organelles obtained with the fluorescence microscope, inner structures obtained with the soft X-ray microscope are exactly identified. Since the spatial resolution of the soft X-ray microscope is much higher than that of the fluorescent microscope, fine structures of the cellular organelles of the live biological cells have been visualized with the correlative microscopy.

Oral presentation

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

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

no journal, , 

Laser-plasma soft X-ray sources have advantage of brightness and short pulse duration and a soft X-ray microscope using them can observe live biological cells with a high spatial resolution. We have succeeded to observe fine structures of cellular organelles of live biological cells and also structural deformations of apoptotic nuclei.

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