Antonia Lai

Soft x-ray submicron imaging detector based on point defects in LiF

The use of lithium fluoride (LiF) crystals and films as imaging detectors for EUV and soft-x-ray radiation is discussed. The EUV or soft-x-ray radiation can generate stable color centers, emitting in the visible spectral range an intense fluorescence from the exposed areas. The high dynamic response of the material to the received dose and the atomic scale of the color centers make this detector extremely interesting for imaging at a spatial resolution which can be much smaller than the light wavelength. Experimental results of contact microscopy imaging of test meshes demonstrate a resolution of the order of 400nm. This high spatial resolution has been obtained in a wide field of view, up to several mm2. Images obtained on different biological samples, as well as an investigation of a soft x-ray laser beam are presented. The behavior of the generated color centers density as a function of the deposited x-ray dose and the advantages of this new diagnostic technique for both coherent and noncoherent EUV so...

Application of a lidar fluorosensor system to the continuous and remote monitoring of the Southern Ocean and Antarctic Ross Sea: results collected during the XIII and XV Italian oceanographic campaigns

A mobile fluorosensor laboratory, including as main constituent a lidar fluorosensor apparatus for remote measurements at water surface and ancillary instruments dedicated to local analyses, was developed for extensive biooptical monitoring of the sea waters. Continuous remote and local measurements of Chl-a (Chlorophyll a) and CDOM (Chromophoric Dissolved Organic Matter) were performed during the XIII (1997/1998) and XV (1999/2000) marine campaigns in the South-western Ross Sea and along the Southern Ocean transects to New Zealand as part of the Italian Research Programme for Antarctica. Chla and CDOM fluorescence maps of the investigated areas revealed the bio-optical peculiarity of coastal zones and seasonal changes encountered.

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. Microsc. Res. Tech., 2008.

Novel portable high-luminosity monochromatically tunable x-ray microscope

A novel experimental setup for transmission x-ray microscopy is presented. It is based on the use of a point isotropic x- ray source and a single spherical crystal. The x-ray beam intensity is modulated by the object attenuation, then monochromatized and enlarged using a spherical crystal and, lastly, imaged using a detector downstream of the crystal. We demonstrate by ray tracing technique and experimental testing that this system allows microscopy studies with image resolution better than the dimensions of the source, high magnification ratios, and great field of view. Microscopes using this model ca be easily built using different micro x-ray sources, like conventional x-ray tube generators, x-rays emitted by laser generated plasmas or synchrotron radiation. Utilization of spherically bent crystals to obtain high-resolution, large field, monochromatic images in a wide range of Bragg angles is demonstrated for the first time. High quality monochromatic images with high magnification about 15-35 times and spatial resolution over a large field of view were obtained. Some possible applications and preliminary experimental verification of the feasibility of the setup are also presented.

SNOM images of X-ray radiographs at nano-scale stored in a thin layer of lithium fluoride

In this work, we report a method to observe soft X‐ray radiographs at nanoscale of various kind of samples, biological and metallic, stored in a thin layer of lithium fluoride, employing scanning near‐field optical microscopy with an optical resolution that reaches 50 nm. lithium fluoride material works as a novel image detector for X‐ray nano‐radiographs, due to the fact that extreme ultraviolet radiation and soft X‐rays efficiently produce stable point defects emitting optically stimulated visible luminescence in a thin surface layer. The bi‐dimensional distribution of the so‐created defects depends on the local nanostructure of the investigated sample.

Selectivity evaluation of label-free detection of Bacillus spp spores using functionalized SERS substrates (Conference Presentation)

Several bacteria evolve in spore if the environmental conditions get to adverse e.g. for nutrient deprivation. The bacteria of genus Bacillus are Gram-positive aerobic bacteria and they are able to produce endospores (physiological inactive and resistant form), usually dispersed as aerosols. Endospores can survive for long time, until the conditions get back favourable. The genera Bacillus include pathogens, as Bacillus anthracis, used in the past as biological weapon. Prompt, accurate and sensitive detection is crucial for its control as pathogens or bioterrorism attacks. In case of the contamination with spores of B. anthracis, the time is essential to assure success in rescue. So, in this context, the early and fast analytical techniques, that need no or negligible sample preparation, is strongly required. Raman spectroscopy, and in particular Surface Enhanced Raman Spectroscopy (SERS), that can amplify nonlinearly the inherently weak Raman signal by several orders of magnitude, have become recognized and versatile analytical techniques also in microorganisms detection. These techniques can be used as sensitive tools for the detection and classification of biological threats, they can provide the chemical fingerprint of samples without complex and time-consuming pre-treatment samples preparation. Furthermore the development of in-field portable compact Raman platforms allows for using SERS for routine analysis. In the framework of the RAMBO (Rapid Air particle Monitoring against BiOlogical threats) project the feasibility of the SERS technique for the rapid identification and classification of few units of Bacillus spp (B. atrophaeus and B. thuringiensis) spores was investigated. B. atrophaeus and B. thuringiensis are harmless but genetically similar to the deadly B. anthracis. The RAMBO project purpose is the development of an advanced sensor with high performances, capable of detecting few spores or bacilli, with high selectivity and reliability, by means of two sensing techniques: SERS for early warning of bioagents dispersed in air or in water, and Polymerase Chain Reaction (PCR) technique for final recognition and validation. SERS and PCR will work in a microfluidic chip. In order to bind selectively the endospores, specific peptide receptors for B. thuringiensis have been selected to functionalize SERS substrates. To characterize the substrates, with and without spores to assess the effective immobilization of target, microscopy inspections, by optical microscope and Scanning Electron Microscope (SEM), were also carried out. The results show up the poor selectively of these peptides for B. thuringiensis, used as target, compared with the non specific Bacillus control. The performance of the system seems to be quite similar for both of them: the data processing by Principal Component Analysis and the following clustering analysis suggest the presence of indistinct answers for any bound endospore on the surface, and this is confirmed by microscope inspection. It could decrease the discrimination power of the sensor. Despite of such poor receptor selectivity, the SERS spectra of B. thuringiensis endospores show characteristic signals that can be related to DNA fragments or, much more probably, to the peptidoglycan (component of the external coat). This spectral feature could be used to detect the presence of B. thuringiensis endospores.

Characterization of Bacilli Spores by Surface-Enhanced Raman Spectroscopy, a Fast and Reliable Technique for Early Warning of Biological Threats

It is demonstrated that Raman Spectroscopy is a fast and sensitive tool for the detection and classification of molecular species. The vibrational spectrum inherently serves as fingerprint of the chemical composition of each sample a thus makes identification and early warning of threats possible. Also microorganisms in areas susceptible to bacterial contamination can be sensed. However, to increase the sensitivity and selectivity of the technique various solutions have been studied such as Resonant Raman Spectroscopy or Surface-Enhanced Raman Spectroscopy (SERS). In this work we present our results on the application of the SERS technique for the characterization of Bacillus atrophaeus spores, a biological and genetic simulant of the deadly bacterium Bacillus anthracis, already used in terroristic attacks in 2001 against U.S. media and government offices causing the death of five people and the infection of other 22. This work is part of the RAMBO project (Rapid Air particle Monitoring against Biological threats) whose ultimate goal is the development of an advanced sensor with high performances, capable of detecting few spores or bacilli of dangerous species with good selectivity and reliability so as to be used as an early warning sensor. In this context, the SERS technique allows to recognize the characteristics vibrational bands of the spores with a scan duration of only few seconds, on amounts of few tenths of spores or less, demonstrating how it can be considered an effective and fast technique for early warning of biological threats as it has been considered in the RAMBO project.

Rapid and label-free screening and identification of Anthrax simulants by Surface Enhanced Raman Spectroscopy

In the framework of RAMBO (Rapid-Air Monitoring particle against biological threats) project of the European Defense Agency (EDA), the feasibility of an unattended Surface Enhanced Raman Spectroscopy (SERS) sensor for biological threats detection was investigated. Its main goal concern Bacillus anthrax detection, both as vegetative cells and endospores. However since such bacteria are classified in Risk Group 3 (very dangerous microorganism), Bacillus thuringiensis and Bacillus atrophaeus were used as simulants. In order to bind selectively the target bacilli, Phages properly selected were immobilized on an active commercially available SERS substrate (functionalization). The Phages are a type of virus that infect selectively, by means of receptors, specific bacteria. Moreover they can resist on water or air environments without losing their binding capabilities. The sensing surface was characterized by standard micro-Raman equipments to assess the background Raman features. The Raman measurements have been carried out from 10X to 100X of magnification to differentiate between average and local features. Moreover the fast response was acquired by limiting the measure time at less than 1 minute. Samples of vegetative cells and endospores of Bacilli were randomly dispersed on the functionalized SERS substrates. The results obtained are promising: samples with and without bacilli could be distinguished one from the other. This is a step toward the use of SERS as an effective and fast technique for early warning of biological threats.

Detection of Lead in Zea mays by Dual-Energy X-ray Microtomography at the SYRMEP Beamline of the ELETTRA Synchrotron and by Atomic Absorption Spectroscopy

This study is related to the application of the X‐ray dual‐energy microradiography technique together with the atomic absorption spectroscopy (AAS) for the detection of lead on Zea mays stem, ear, root, and leaf samples. To highlight the places with lead intake, the planar radiographs taken with monochromatic X‐ray radiation in absorption regime with photon energy below and above the absorption edge of a given chemical element, respectively, are analyzed and processed. To recognize the biological structures involved in the intake, the dual‐energy images with the lead signal have been compared with the optical images of the same Z. mays stem. The ear, stem, root, and leaf samples have also been analyzed with the AAS technique to measure the exact amount of the hyperaccumulated lead. The AAS measurement revealed that the highest intake occurred in the roots while the lowest in the maize ears and in the leaf. It seems there is a particular mechanism that protects the seeds and the leaves in the intake process. Microsc. Res. Tech., 2010.