Soft X-ray Contact Microscopy Research Articles

Nanometer-Resolution Imaging of Living Cells Using Soft X-ray Contact Microscopy

Soft X-ray microscopy is a powerful technique for imaging cells with nanometer resolution in their native state without chemical fixation, staining, or sectioning. The studies performed in several laboratories have demonstrated the potential of applying this technique for imaging the internal structures of intact cells. However, it is currently used mainly on synchrotrons with restricted access. Moreover, the operation of these instruments and the associated sample-preparation protocols require interdisciplinary and highly specialized personnel, limiting their wide application in practice. This is why soft X-ray microscopy is not commonly used in biological laboratories as an imaging tool. Thus, a laboratory-based and user-friendly soft X-ray contact microscope would facilitate the work of biologists. A compact, desk-top laboratory setup for soft X-ray contact microscopy (SXCM) based on a laser-plasma soft X-ray source, which can be used in any biological laboratory, together with several applications for biological imaging, are described. Moreover, the perspectives of the correlation of SXCM with other super-resolution imaging techniques based on the current literature are discussed.

Adhesion of Triple-Negative Breast Cancer Cells under Fluorescent and Soft X-ray Contact Microscopy.

Understanding cancer cell adhesion could help to diminish tumor progression and metastasis. Adhesion mechanisms are currently the main therapeutic target of TNBC-resistant cells. This work shows the distribution and size of adhesive complexes determined with a common fluorescence microscopy technique and soft X-ray contact microscopy (SXCM). The results presented here demonstrate the potential of applying SXCM for imaging cell protrusions with high resolution when the cells are still alive in a physiological buffer. The possibility to observe the internal components of cells at a pristine and hydrated state with nanometer resolution distinguishes SXCM from the other more commonly used techniques for cell imaging. Thus, SXCM can be a promising technique for investigating the adhesion and organization of the actin cytoskeleton in cancer cells.

Imaging of Cell Structures Using Optimized Soft X-ray Contact Microscopy

This work is to study the relationship between the exposure conditions and the quality of cell imaging with soft X-ray contact microscopy (SXCM). It is a crucial step in the efficient visualization of cell structures. Three different human cell lines: DU145 prostate carcinoma cells, HCC38 breast cancer cells, and Poietics mesenchymal stem cells were used to establish the optimal exposure conditions in SXCM. The image quality depended on the soft X-ray (SXR) absorbed energy and photoresist development conditions. At lower SXR energy (200 or 400 SXR pulses), sharp cell edges, membrane projections, and cell–cell connections were visible. In contrast, higher energy (600 or 800 SXR pulses) allowed observation of the cytoskeleton and the nucleus in a cell type-dependent manner (the influence of cell thickness and internal complexity was noted).

X-ray imaging of bio/medical samples using laser-plasma-based X-ray sources and LiF detector

This contribution to ECPD2019 is dedicated to the memory of Anatoly Faenov. During a period of approximately thirteen years 1994–2006, Anatoly and his wife Tatiana Pikuz (simply “Tania” for friends), accepting the frequent invitations of the National Institute for Nuclear Physics (INFN) and of the Italian National Agency for New Technologies, Energy and Sustainable Economic Development (ENEA), cooperated with many Italian research laboratories dedicated to EUV and soft X-ray generation, spread in different towns (L'Aquila, Frascati, Milano, Padova, Pisa, Roma, etc.). In spite of the fact that they could stay in Italy only about one or two months per year, their activity was so intense that more than 50 peer- reviewed publications were generated from their experimental and theoretical work (just considering only the results obtained at L'Aquila and Tor Vergata—Rome Universities and at the ENEA Research Center of Frascati), without mentioning the cultural atmosphere that they stimulated in the field of Science and Humanity. The numerous experimental spectra obtained at ENEA by means of their spherically bent mica spectrometers, together with the corresponding theoretical simulations performed in Moscow, allowed to study the changing role of different excitations mechanisms for various plasma conditions, and to characterize at best the ENEA laser-plasma source for different applications: polychromatic and monochromatic micro-radiography of dried biological samples at 1 keV, soft X-ray contact microscopy (SXCM) of living cells in the water-window spectral region, spectroscopy of hollow atoms, etc. In this memorial paper, the main results of biological samples imaging on lithium fluoride (LiF) detectors, obtained with the ENEA and Tor Vergata University laser-plasma sources, are presented. In particular, the improvement of the micro-radiography and of the SXCM techniques obtained after moving from photoresist detectors and photographic films to lithium fluoride (LiF) detectors are discussed, for both dried and wet biological samples.

Development and characterization of a laser-plasma soft X-ray source for contact microscopy

Abstract In this work, we present a compact laser-produced plasma source of X-rays, developed and characterized for application in soft X-ray contact microscopy (SXCM). The source is based on a double stream gas puff target, irradiated with a commercially available Nd:YAG laser, delivering pulses with energy up to 740 mJ and 4 ns pulse duration at 10 Hz repetition rate. The target is formed by pulsed injection of a stream of high-Z gas (argon) into a cloud of low Z-gas (helium) by using an electromagnetic valve with a double nozzle setup. The source is designed to irradiate specimens, both in vacuum and in helium atmosphere with nanosecond pulses of soft X-rays in the “water-window” spectral range. The source is capable of delivering a photon fluence of about 1.09 × 10 3 photon/μm 2 /pulse at a sample placed in vacuum at a distance of about 20 mm downstream the source. It can also deliver a photon fluence of about 9.31 × 10 2 photons/µm 2 /pulse at a sample placed in a helium atmosphere at the same position. The source design and results of the characterization measurements as well as the optimization of the source are presented and discussed. The source was successfully applied in the preliminary experiments on soft X-ray contact microscopy and images of microstructures and biological specimens with ∼80 nm half-pitch spatial resolution, obtained in helium atmosphere, are presented.

Contact X-ray microscopy of living cells by using LiF crystal as imaging detector.

In this paper, the use of lithium fluoride (LiF) as imaging radiation detector to analyse living cells by single-shot soft X-ray contact microscopy is presented. High resolved X-ray images on LiF of cyanobacterium Leptolyngbya VRUC135, two unicellular microalgae of the genus Chlamydomonas and mouse macrophage cells (line RAW 264.7) have been obtained utilizing X-ray radiation in the water window energy range from a laser plasma source. The used method is based on loading of the samples, the cell suspension, in a special holder where they are in close contact with a LiF crystal solid-state X-ray imaging detector. After exposure and sample removal, the images stored in LiF by the soft X-ray contact microscopy technique are read by an optical microscope in fluorescence mode. The clear image of the mucilaginous sheath the structure of the filamentous Leptolyngbya and the visible nucleolus in the macrophage cells image, are noteworthiness results. The peculiarities of the used X-ray radiation and of the LiF imaging detector allow obtaining images in absorption contrast revealing the internal structures of the investigated samples at high spatial resolution. Moreover, the wide dynamic range of the LiF imaging detector contributes to obtain high-quality images. In particular, we demonstrate that this peculiar characteristic of LiF detector allows enhancing the contrast and reveal details even when they were obscured by a nonuniform stray light.

X‐ray microscopy of plant cells by using LiF crystal as a detector

A lithium fluoride (LiF) crystal has been utilized as a new soft X-ray detector to image different biological samples at a high spatial resolution. This new type of image detector for X-ray microscopy has many interesting properties: high resolution (nanometer scale), permanent storage of images, the ability to clear the image and reuse the LiF crystal, and high contrast with greater dynamic range. Cells of the unicellular green algae Chlamydomonas dysosmos and Chlorella sorokiniana, and pollen grains of Olea europea have been used as biological materials for imaging. The biological samples were imaged on LiF crystals by using the soft X-ray contact microscopy and contact micro-radiography techniques. The laser plasma soft X-ray source was generated using a Nd:YAG/Glass laser focused on a solid target. The X-ray energy range for image acquisition was in the water-window spectral range for single shot contact microscopy of very thin biological samples (single cells) and around 1 keV for multishots microradiography. The main aim of this article is to highlight the possibility of using a LiF crystal as a detector for the biological imaging using soft X-ray radiation and to demonstrate its ability to visualize the microstructure within living cells.

Study of Multicellular Living Organisms by SXCM (Soft X-Ray Contact Microscopy)

Soft X-ray Contact Microscopy (SXCM) of Caenorhabditis elegans nematodes wit typical length 800 μm and diameter 30 μm has been performed using the PALS laser source of wavelength λ = 1.314 μm and pulse duration τ (FWHM) = 400 ps. Pulsed soft X-rays were generated using molybdenum and gold targets with laser intensities I ≥ 1014 W/cm2. Images have been recorded on PMMA photo resists and analyzed using an atomic force microscope operating in contact mode. Cuticle features and several internal organs have been identified in the SXCM images including lateral field, cuticle annuli, pharynx, and hypodermal and neuronal cell nuclei.

Read-out of soft x-ray contact microscopy microradiographs by focused ion beam/scanning electron microscope

A novel focused ion beam-based technique is presented for the read-out of microradiographs of Caenorhabditis elegans nematodes generated by soft x-ray contact microscopy (SXCM). In previous studies, the read-out was performed by atomic force microscopy (AFM), but in our work SXCM microradiographs were imaged by scanning ion microscopy (SIM) in a focused ion beam/scanning electron microscope (FIB/SEM). It allows an ad libitum selection of a sample region for gross morphologic to nanometric investigations, with a sequence of imaging and cutting. The FIB/SEM is less sensitive to height variation of the relief, and sectioning makes it possible to analyse the sample further. The SXCM can be coupled to SIM in a more efficient and faster way than to AFM. Scanning ion microscopy is the method of choice for the read-out of microradiographs of small multicellular organisms.

Soft X-ray contact microscopy of nematode Caenorhabditis elegans

Soft X-ray Contact Microscopy (SXCM) of Caenorhabditis elegans nematodes with typical length \(\sim 800 \mu\)m and diameter \(\sim 30 \mu\)m has been performed using the PALS laser source of wavelength \(\lambda = 1.314 \mu \)m and pulse duration \(\tau ({\rm FWHM}) = 400\) ps. Pulsed soft X-rays were generated using molybdenum and gold targets with laser intensities \(I \ge 10^{14}\) W/cm2. Images have been recorded on PMMA photo resists and analyzed using an atomic force microscope operating in contact mode. Cuticle features and several internal organs have been identified in the SXCM images including lateral field, cuticle annuli, pharynx, and hypodermal and neuronal cell nuclei.

Microscopy characterization of doped fibers

The use of the Soft X-ray Contact Microscopy technique is discussed as a possible new tool to get information on dopant distribution in the core of single-mode optical fibers with 50 nm spatial resolution.

High resolution microscopy techniques for the analysis of biological samples: a comparison

Performances and advantages of different high-resolution microscopy techniques are compared. These include Soft X-ray Contact Microscopy (SXCM), Atomic Force Microscopy (AFM), Focused Ion Beam (FIB) and Transmission Electron Microscopy (TEM). It is shown that they allow complementary approaches to imaging of biological objects. These techniques have been used to image the same type of cells ( Saccharomyces cerevisiae ) thus providing a common benchmark. In particular it is shown that the novel FIB technique allows easy target cell selection, fast operation, high resolution, 3D imaging and sample manipulation during imaging.

X-ray microscopy of living multicellular organisms with the Prague Asterix Iodine Laser System

Soft X-ray contact microscopy (SXCM) experiments have been performed using the Prague Asterix Iodine Laser System (PALS). Laser wavelength and pulse duration were λ = 1.314 μm and τ (FWHM) = 450 ps, respectively. Pulsed X rays were generated using teflon, gold, and molybdenum targets with laser intensities I ≥ 1014 W/cm2. Experiments have been performed on the nematodes Caenorhabditis elegans. Images were recorded on PMMA photo resists and analyzed using an atomic force microscope operating in contact mode. Our preliminary results indicate the suitability of the SXCM for multicellular specimens.

Soft X-ray contact microscopy and transmission electron microscopy: Comparative study of biological samples

Isolated cellular organelles (mitochondria, chloroplasts) and cultured bacteria were analysed both by soft X-ray contact microscopy (SXCM), and by transmission electron microscopy (TEM) after negative staining. For each sample, a comparison was performed between images obtained with either technique, with the aim of facilitating the interpretation of SXCM images. The validity and the limits of this comparative approach are discussed.

Analysis of localization and chemical status of minor elements in a mammalian cell using soft X-ray contact microscopy

We have developed computer-assisted procedures for mapping minor light elements and their chemical bond information from a set of soft X-ray transmission images of biological specimens. Minor light elements such as Ca, S and P play an important role in the biological function and structure in a cell. Contact X-ray microscopy with an electric zooming tube covering wide wavelength range from the K absorption edge of S to the L absorption edge of P is applied to elemental mapping. The procedure of elemental mapping depends basically on the subtraction of the contribution of major elements such as C, O and N in intracellular absorption spectra to find the presence of the absorption edges of minor elements. The results of simulation experiments were presented. Chemical bond imaging utilizes a XANES spectrum corresponding to a specific chemical bond. Since the intensity is relatively low for minor elements, the ratio between absorption at the peak and at the bottom, which represents the chemical bond distribution, becomes small. We developed the computer program for the enhancement of very small ratio value. This procedure was applied to the S-S and S-C bond imaging at the S-K absorption edge.

Nanostructured imaging of biological specimens in vivo with laser plasma X-ray contact microscopy

Soft X-ray contact microscopy (SXCM) enables the study of the ultrastructure of living hydrated specimens, without the need of dehydration or any other chemical pretreatment, by using suitable pulsed X-ray sources such as laser plasmas. The successful imaging of biological specimen requires the development of sensitive photoresist materials for image recording; these should have capabilities of high-resolution lithography and an extended grey scale. A very sensitive photoresist, used for the first time in SXCM, enabled the biological imaging with the specific source in single-pulse experiments in the water window spectral range. This photoresist is an epoxy novolac-based chemically amplified photoresist (EPR), which has been proven capable of resolving subtenth-micron features. The photoresist response was at least two orders of magnitude “faster” than polymethyl methacrylate (PMMA), which is the standard resist used so far in SXCM. Atomic force microscopy (AFM) and scanning electron microscopy of the biological specimen images recorded in the resist clearly showed the flagella of the motile green alga, suggesting a lateral resolution better than 100 nm. The resist was also capable of providing height features, as small as 20 nm, in AFM depth profiles and discriminating the flagella intersection areas.

The use of high energy laser-plasma sources in soft X-ray contact microscopy of living biological samples

In this paper the results of an experiment on soft X-ray contact microscopy using a laser-plasma source are presented. A resolution of 50 nm has been achieved imaging pig sperm cells, while other specimens, such as algae and yeast cells, showed internal details, proving the technique to be a powerful tool for biological investigations. Original biological information has been obtained and the conditions for optimal image formation have been studied.

A numerical study of resolution and contrast in soft X-ray contact microscopy.

We consider the case of soft X-ray contact microscopy using a laser-produced plasma. We model the effects of sample and resist absorption and diffraction as well as the process of isotropic development of the photoresist. Our results indicate that the micrograph resolution depends heavily on the exposure and the sample-to-resist distance. In addition, the contrast of small features depends crucially on the development procedure to the point where information on such features may be destroyed by excessive development. These issues must be kept in mind when interpreting contact microradiographs of high resolution, low contrast objects such as biological structures.

Laser plasma x-ray contact microscopy of living specimens using a chemically amplified epoxy resist

We report on the use of an epoxy based chemically amplified resist, to produce x-ray images of living biological specimens, exposed with laser plasma generated soft x rays, in the water window (2.3–4.4 nm). The photoresist response was at least two orders of magnitude “faster” than polymethyl methacrylate, the standard resist used so far in soft x-ray contact microscopy. Atomic force and scanning electron microscopy of the biological specimen images, recorded in the resist, clearly showed the flagella of the motile green alga, chlamydomonas, suggesting a lateral resolution better than 150 nm. The resist was also capable of providing height features, as small as 20 nm, in atomic force microscope depth profiles and discriminating the flagella intersection areas.

X‐ray contact microscopy using an excimer laser plasma source with different target materials and laser pulse durations

Soft X‐ray contact microscopy (SXCM), using a pulsed X‐ray source, offers the possibility of imaging the ultrastructure of living biological systems at sub‐100 nm resolution. We have developed a table‐top pulsed plasma X‐ray source for this application, generated by a large‐volume XeCl laser, achieving a good conversion efficiency to ‘water‐window’ X‐rays (hν≈280–530 eV).Optimum plasma conditions for SXCM are discussed, including the effect of pulse duration, target material and resist development time on image resolution. Soft X‐ray contact images of Chlamydomonas dysosmos (unicellular alga) and of the cyanobacterium Leptolyngbya are shown.