We have proposed and developed a new hybrid microscopy system using a soft x-ray microscope and a fluorescence microscope imaging the same biological cells at the nearly same time. Combining the powerful advantages such as high spatial resolution of the soft x-ray microscope and the accurate organelle identification of the fluorescence microscope, we can observe fine structures of the cellular organelles in live hydrated biological cells in situ. Staining the cells with several fluorescent dyes such as Mito-tracker, Phalloidin, and DAPI, the soft x-ray images of the cells have been directly compared with the fluorescent images and the cellular organelles such as mitochondria, actin filaments, and chromosomes in the soft x-ray images have been clearly identified. Since the soft x-ray microscope has higher spatial resolution than that of the fluorescence microscope, not only shape of the cellular organelles but also the fine structures of the cellular organelles of the live biological cells have been clearly observed for the first time.
X-ray flash imaging by contact microscopy with a highly intense laser-plasma x-ray source was achieved for the
observation of wet biological cells. The exposure time to obtain a single x-ray image was about 600 ps as determined by
the pulse duration of the driving laser pulse. The x-ray flash imaging makes it possible to capture an x-ray image of
living biological cells without any artificial treatment such as staining, fixation, freezing, and so on. The biological cells
were cultivated directly on the surface of the silicon nitride membranes, which are used for the x-ray microscope. Before
exposing the cells to x-rays they were observed by a conventional fluorescent microscope as reference, since the
fluorescent microscopes can visualize specific organelles stained with fluorescent dye. Comparing the x-ray images with
the fluorescent images of the exact same cells, each cellular organelle observed in the x-ray images was identified one by
one and actin filaments and mitochondria were clearly identified in the x-ray images.
Ultra thin gold films having a thickness of 20-30 nm are favorable laser plasma targets for a soft x-ray microscopy, because the ultra thin films emit intense soft x-rays at the wavelength of water window region from the rear side with respect to the surface irradiated with short pulse laser. 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. In addition, the microscope system can be designed to be compact when the specimen holder and x-ray source are combined in one piece. In the present study, the biological specimen holder combined with a gold ultra thin film plasma target has been developed for a contact-type
soft x-ray microscope. This x-ray microscope system needs not any x-ray optics such as a condenser and/or an objective optics which causes a decrease in x-ray photons for imaging. Specimen holder equipped with the plasma target keeps biological specimens at wet condition in vacuum. In this study, x-ray images of hydrated living cells (MH-S mouse alveolar macrophage cell line) have been obtained successfully by use of the newly developed specimen holder. These
experimental results reveal that the soft x-ray image can be taken safely. Specimen holder combined with plasma x-ray source will be a key component of a compact soft x-ray microscope using in a laboratory.
Observation of soft x-ray emissions from laser produced plasmas using ultra thin film targets has been carried out. Au
ultra thin films deposited on silicon nitride membranes were irradiated with a high contrast Nd:glass laser pulses. The
spectral properties of emitted soft x-rays were monitored with an x-ray spectrograph from the membrane side. The
observed emission intensities had a clear dependence on the Au film thickness. The results suggest that most of the laser
energy irradiated is absorbed by the Au films and few of the energy goes into the silicon nitride membranes, which
means an efficient laser energy deposition to the ultra thin Au film target and a high energy conversion rate from laser
to x-rays.
This paper gives an overview of recent progress of laser-driven plasma x-ray lasers in Japan Atomic Energy Agency (JAEA). Fully spatial coherent plasma x-ray laser (XRL) at 13.9 nm with 0.1 Hz repetition rate has been developed using new driver laser system TOPAZ, and the succeeding optimization of the pumping condition has realized more efficient generation of the coherent x-ray pulse. The 0.1 Hz XRL is now routinely used in the wide variety of the
application experiments: The highlights of these applications are the study of fluctuation in the atomic structure of ferroelectric substances under the phase transition using the double XRL probe beam technique and the construction of new x-ray laser interferometer to observe nano-scale dynamics of materials.
We review our recent progress in the development of transient x-ray lasers and of their application to plasma diagnostic. The first observation of C-ray laser emission at the new PHELIX-GSI facility is reported. This TCE X-ray laser will be a promising tool for heavy-ion spectroscopy. We then present the main results obtained at the LULU-CPA facility with a compact high-resolution X-UV imaging device. This device was used to investigate the spatial source structure of the Ni-like silver transient X-ray laser under different pumping conditions. The key-role of the width of the background laser pulse on the shape of the emitting aperture is demonstrated. Finally the imaging device was used as an interference microscope for interferometry probing of a laser-produced plasma. We describe this experiment performed at APRC-JAERI.
We have succeeded in developing a laser-pumped x-ray laser with full spatial coherence at 13.9 nm. A highly directed x-ray laser beam with the divergence of 0.2 mrad was generated from the double target experiment, where a seeding light from the first laser medium was amplified in the second medium. The observed divergence is close to the diffraction limited value within a factor of two. The seeding light was amplified in the second medium without refraction influence and the gain coefficient was about 8 cm-1. The gain region of the second medium was far away from the target surface compared with that of the first medium and located in the considerably low density region. From the measurement of visibility, it was found that the spatial coherent length is longer than the beam diameter.
We proposed a method to generate highly spatial coherent x-ray laser, in which high order harmonics was used as a seed light of a laser-produced x-ray amplifier. In this case, the intensity and the spatial coherence of the output x-ray depended on the harmonic conversion efficiency and the spectral and spatial coupling efficiencies between the harmonics and the lasing line. Based on the present x-ray lasers using a transient collisional-excitation (TCE) scheme, we investigated the values of these efficiencies, which were needed to realize a high spatial coherence. For this purpose we constructed a Ti:Sapphire laser system in which the central wavelength and the spectral bandwidth were tunable, and we conducted a preliminary experiment. The neon-like Ti x-ray laser at a wavelength of 32.4 nm was taken as an example, and harmonics at the same wavelength was generated using Ar gas target under the conditions that the central wavelength of 810 nm and the pulse duration of 1 ps. The experimental result showed that the characteristics of the harmonics were good enough to use as a seed light of x-ray lasers.
We have observed lasing on Ne-like 3s-3p line from titanium (32.4 nm), Ni-like 4p-4d line from silver (13.9 nm) and tin (11.9 nm) with the transient collisional excitation (TCE) scheme that uses combinations of a long pre-pulse (approximately ns) and a short main pulse (approximately ps) or a short pre-pulse (approximately ps) and a short main pulse (approximately ps). A gain coefficient of 24cm-1 have been measured for plasma length up to 4mm with silver slab targets and 14cm-1 up to 6 mm with tin slab targets. We have installed a step mirror in the focusing system to generate traveling wave on the target. The traveling speed on the target is measured to be 3.08 cm/s and very close to the traveling speed of light. The traveling wave system improves the gain coefficient to 35cm-1 from 24cm-1 for Ni-like Ag and to 30cm-1 from 14cm-1 for Ni-like Sn. The strong gain saturation has been observed for the Ni-like Ag and Ni-like Sn. The output energy of the N-like Sn x-ray laser is 20 (mu) J. Spatial beam profiles of propagating x-ray lasers through gain plasma have been measured and are indicating localization of very high gain area and x-ray laser refraction.
We have developed a collisional radiative model of electron collisional excited x-ray lasers. We calculate the ion abundance and soft x-ray gain for the 4d-4p transition of Ni-like multiple charged ion, in short pulse laser irradiated plasmas. We combine a detailed model using the atomic data calculated by HULLAC code and the averaged model based on the screened hydrogenic approximation. In order to choose a proper set of the levels to be included in the atomic model, investigation of the model dependence of the gain is carried out. The population in the fine structure levels in 3d94s and 3d94f configuration, autoionizing double excited configurations of Ni-like and Cu-like ions are found to cause approximately factor of 2 difference in the soft x-ray gain. The steady-state gain and its dependence on plasma density and temperature are investigated over elements from z equals 45 to 65. The time dependent calculation for a plasma condition corresponds to a thin Ag foil irradiated by tow short laser pulse shows the transient gain which is 40 times greater than the steady state gain.
An ultra-short pulse CPA laser system for x-ray laser driver has been developed with a combination of Ti:sapphire front end and Nd:glass rod amplifiers. This laser system has two beam outputs and each beam line produces 20J pre pulse and 20J main. This laser system is designed for x-ray laser pumping driver, especially for transient gain scheme. The new transient gain x-ray laser scheme with thin foil metal targets has been proposed. This scheme has higher laser energy efficiency and less x-ray laser refraction effect and makes possible to generate shorter x-ray wavelength with a compact table-top sized laser system. The electron temperatures of plasma heated with a short pre pulse and short main pulse have been calculated with 1D hydrodynamic code and obtained electron temperature higher than 1 keV with 20J laser energy. X-ray laser propagation is also calculated with gain guiding effect.
An ultra-short pulse CPA laser system for x-ray laser driver has been developed with a combination of Ti:sapphire front end and Nd:glass rod amplifiers. This laser system has two beam outputs and each beam line produces 20J pre pulse and 20J main. This laser system is designed for x-ray laser pumping driver, especially for transient gain scheme. The new transient gain x-ray laser scheme with thin foil metal targets has been proposed. This scheme has higher laser energy efficiency and less x-ray laser refraction effect and makes possible to generate shorter x-ray wavelength with a compact table-top sized laser system. The electron temperatures of plasmas heated with a short pre pulse and short main pulse have been calculated with 1D hydrodynamic code and obtained electron temperature higher than 1 keV and 20 J laser energy. X-ray laser propagation is also calculated with gain guiding effect.
X-ray microscopy inherently possesses characteristics complementary to optical and electron microscopy. Short wavelength x-ray radiation, especially in the so-called 'water window', permits a twenty-fold improvement in spatial resolution over optical microscopy while preserving a depth of field large enough to image whole biological specimens int heir natural state. Whereas electron microscopy can access atomic-scale resolution,this can only be applied to biological and medical specimens at the expense of detrimental preparation procedures that preclude real-time analysis of structural changes in living organisms. We describe progress being made in an x-ray imaging technology that provides high-resolution single frame x-ray images of in-vitro specimens captured in a time sufficiently short that any radiation damage mechanisms to the structure are not recorded. Several different biology and medical research groups find this type of microscopy particularly well-suited to the detailed analysis of sub-cellular features, and to the study of live organisms subjected to various forms of external stimuli. This technology utilizes bright x-ray sources produced by compact pulse laser systems. The incorporation of advanced x-ray optical and electron-optical systems will lead to the development of a compact, real-time x-ray microscope, having a broad range of applications.
Nanosecond flash x-ray microscopy of living biological specimens is demonstrated with subcellular spatial resolution. Single shot images, produced by a compact laser- plasma x-ray source optimized for maximum image contrast, are captured before radiation processes can affect the specimen.
Images of a 130fs laser produced Ta-plasma were obtained with a spherically curved Mica crystal in four narrow spectral bands simultaneously. These are to our knowledge the first published x-ray images of a fs plasma. Also Mica was used for the first time as the imaging crystal. The resolved plasma size in the experiment (50 micrometers (DOT) 90 micrometers ) agrees with ray-tracing calculations and corresponds to the actual source size in our current setup. The four spectral bands of imaging are due to the strong Mica reflectivity in higher Bragg diffraction orders. All contributing bands have an energy higher than 1keV. Several laser shots were accumulated to achieve the required flux on the detector. Source spectroscopy was conducted to verify the source emission and the plasma density during the interaction with the 130fs laser pulse. Based on our experimental data the luminosity of crystal imaging is compared to a pinhole camera.
X-ray images of the various live bacteria, such as Staphylococcus and Streptococcus, and micromolecule such as chromosomal DNA from Escherichis coli, and Lipopolysacchride from Burkholderia cepacia, are obtained with soft x-ray contact microscopy. A compact tabletop type glass laser system is used to produce x-rays from Al, Si, and Au targets. The PMMA photoresists are used to record x-ray images. An AFM (atomic force microscope) is used to reproduce the x-ray images from the developed photoresists. The performance of the 50nm spatial resolutions are achieved and images are able to be discussed on the biological view.
We report progress in two areas of technology relevant to the development of high-resolution x-ray microscopy techniques based on laser plasma x-ray sources for the analysis of wet and dry biological specimens. The approach we discuss involves the use of ultrashort x-ray emission from a laser plasma source. Precision x-ray optics are used to collimate and filter this light onto the specimen. Imaging and image magnification are accomplished with a combination of x-ray and electron-optical systems. We discuss our progress towards establishing a flexible laser-plasma x-ray source for the development of biological imaging and contact microscopy, and progress we have made towards the development of an electro-optical imaging system having high magnification and spatial resolution.
We propose the use of mass-limited, line emitting cryogenic targets for SXPL, which permit a continuous supply of targets without the problem of particulate debris and excessive heating of multilayer optics by an intense x-ray flux in wavelength regions outside the multilayer bandwidth. In preliminary experiments we measured the oxygen line emission in the vicinity of 13 nm. The x-ray emitting plasma was produced by using a laser intensity of 2 X 1012 W/cm2 on the surface of an ice target. From the observed crater on target we can deduce that clusters are also ejected from cryogenic targets.
The development of a compact, affordable, high-resolution x-ray microscope will have a strong impact on the biological and medical sciences. We discuss the potential that pulsed, laser-plasma x-ray sources have to this development. Several approaches to the high-resolution analysis of dried and in-vitro biological specimens with laser-plasma sources are described. We discuss the details of the laser and plasma conditions required for optimum x-ray generation, and the various x-ray optical and x-ray electro-optical imaging systems which could be incorporated into a compact, laser-plasma x-ray microscope.
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