Jean Michel

Boron analysis and boron imaging in biological materials for Boron Neutron Capture Therapy (BNCT)

Boron Neutron Capture Therapy (BNCT) is based on the ability of the stable isotope 10B to capture neutrons, which leads to a nuclear reaction producing an alpha- and a 7Li-particle, both having a high biological effectiveness and a very short range in tissue, being limited to approximately one cell diameter. This opens the possibility for a highly selective cancer therapy. BNCT strongly depends on the selective uptake of 10B in tumor cells and on its distribution inside the cells. The chemical properties of boron and the need to discriminate different isotopes make the investigation of the concentration and distribution of 10B a challenging task. The most advanced techniques to measure and image boron are described, both invasive and non-invasive. The most promising approach for further investigation will be the complementary use of the different techniques to obtain the information that is mandatory for the future of this innovative treatment modality.

Subcellular localization of boron in cultured melanoma cells by electron energy-loss spectroscopy of freeze-dried cryosections

Boron neutron capture therapy (BNCT) is based on the ability of the non‐radioactive isotope 10B to capture thermal neutrons and to disintegrate instantaneously. This reaction opens a way to selectively destroy tumour cells after specific uptake of 10B. In this paper, a method based on electron energy‐loss spectroscopy is presented for detecting and quantifying boron in freeze‐dried cryosections of human melanoma cells. A practical detection limit of around 6 mmol kg−1 in 0.1‐µm2 areas is estimated using specimens prepared from standard boron solutions. Preliminary results of boron mapping in the spectrum‐imaging acquisition mode reveal boron penetration and probably spot‐like accumulation within melanoma cells when exposed to culture medium containing sodium borocaptate.

Burkholderia phytofirmans PsJN reduces impact of freezing temperatures on photosynthesis in Arabidopsis thaliana

Several plant growth-promoting rhizobacteria (PGPR) are known to improve plant tolerance to multiple stresses, including low temperatures. However, mechanisms underlying this protection are still poorly understood. The aim of this study was to evaluate the role of the endophytic PGPR, Burkholderia phytofirmans strain PsJN (Bp PsJN), on Arabidopsis thaliana cold tolerance using photosynthesis parameters as physiological markers. Under standard conditions, our results indicated that Bp PsJN inoculation led to growth promotion of Arabidopsis plants without significant modification on photosynthesis parameters and chloroplast organization. However, bacterial colonization induced a cell wall strengthening in the mesophyll. Impact of inoculation modes (either on seeds or by soil irrigation) and their effects overnight at 0, -1, or -3°C, were investigated by following photosystem II (PSII) activity and gas exchanges. Following low temperatures stress, a decrease of photosynthesis parameters was observed. In addition, during three consecutive nights or days at -1°C, PSII activity was monitored. Pigment contents, RuBisCO protein abundance, expression of several genes including RbcS, RbcL, CBF1, CBF2, CBF3, ICE1, COR15a, and COR78 were evaluated at the end of exposure. To assess the impact of the bacteria on cell ultrastructure under low temperatures, microscopic observations were achieved. Results indicated that freezing treatment induced significant changes in PSII activity as early as the first cold day, whereas the same impact on PSII activity was observed only during the third cold night. The significant effects conferred by PsJN were differential accumulation of pigments, and reduced expression of RbcL and COR78. Microscopical observations showed an alteration/disorganization in A. thaliana leaf mesophyll cells independently of the freezing treatments. The presence of bacteria during the three successive nights or days did not significantly improved A. thaliana responses but prevented the plasmalemma disruption under freezing stress.

Burkholderia phytofirmans PsJN Confers Grapevine Resistance against Botrytis cinerea via a Direct Antimicrobial Effect Combined with a Better Resource Mobilization

Plant innate immunity serves as a surveillance system by providing the first line of powerful weapons to fight against pathogen attacks. Beneficial microorganisms and Microbial-Associated Molecular Patterns might act as signals to trigger this immunity. Burkholderia phytofirmans PsJN, a highly efficient plant beneficial endophytic bacterium, promotes growth in a wide variety of plants including grapevine. Further, the bacterium induces plant resistance against abiotic and biotic stresses. However, no study has deciphered triggered-mechanisms during the tripartite interaction between grapevine, B. phytofirmans PsJN and Botrytis cinerea. Herein, we showed that in contrast with classical rhizobacteria, which are restricted in the root system and act through ISR, B. phytofirmans PsJN is able to migrate until aerial part and forms at leaves surface a biofilm around B. cinerea mycelium to restrict the pathogen. Nevertheless, considering the endophytic level of PsJN in leaves, the plant protection efficacy of B. phytofirmans PsJN could not be explained solely by its direct antifungal effect. Deeper investigations showed a callose deposition, H2O2 production and primed expression of PR1, PR2, PR5, and JAZ only in bacterized-plantlets after pathogen challenge. The presence of PsJN modulated changes in leaf carbohydrate metabolism including gene expression, sugar levels, and chlorophyll fluorescence imaging after Botrytis challenge. Our findings indicated that protection induced by B. phytofirmans PsJN was multifaceted and relied on a direct antifungal effect, priming of defense mechanisms as well as the mobilization of carbon sources in grapevine leaf tissues.

Low electric field strength self-organization of anodic TiO2 nanotubes in diethylene glycol electrolyte

Self-organization of TiO2 nanotubes with large interconnecting space in-between the tubes has been demonstrated by the anodization of titanium in diethylene glycol/HF electrolyte containing a desired amount of water. The unique morphological features are a consequence of low electric field strength conditions, leading to the growth of tubes on a low population of nucleation sites. The proposed growth model assumes the presence of a metallic titanium in-between the tube cells that are oxidized/etched, resulting in the generation of an inhomogeneous oxide at the bottom of the nanostructure. The presented work contributes to the research field by the following aspects: (i) the low field strength conditions have been demonstrated to have an impact on the tube spacing, (ii) the water content in the electrolyte allows the precise control of the interconnecting space in-between the tubes, (iii) the tubes separation is controlled by the presence of Ti in-between the tube cells.

EELS Spectrum‐Imaging for Boron Detection in Biological Cryofixed Tissues

Abstract Boron imaging at the subcellular level in tissues is of prime importance to understand boron neutron capture therapy (BNCT) effects and to suggest ways to improve the efficiency of the treatments. In this paper, we present boron imaging in cryofixed biological tissues by electron energy loss spectroscopy (EELS), using the spectrum‐imaging acquisition mode. We performed boron imaging in tissues of mice who received boron compounds. We analysed cryosections of tumours, livers, and kidneys of infused mice. Our results point out the relevance of EELS for boron imaging and, particularly, in order to detect small accumulation areas. The data also point out the limitations of the technique and its complementarity with other imaging techniques. We discuss and illustrate the possibility for misinterpretation of EELS or electron spectroscopic imaging (ESI) data. We particularly focus on the problem of phosphorus for boron imaging. This manuscript is dedicated to Dr. K. Zierold who died in 2004.

Photoluminescence quantum yield of CdSe-ZnS/CdS/ZnS core-multishell quantum dots approaches 100% due to enhancement of charge carrier confinement

Quantum dots (QDs) with the highest possible photoluminescence quantum yields are necessary for modern nanotechnology applications to biosensing and optoelectronics. To date, core-shell QDs are the best. We suggest and demonstrate a novel approach to enhancement of charge-carrier confinement in the core of CdSe QDs by creating a ZnS/CdS/ZnS shell with staggered potential barrier. The CdS interlayer breaks the ZnS-shell structure continuity, which allows combining the benefits of a single ZnS-monolayer inner shell, creating the highest possible confinement potential, with a sufficient overall shell thickness and suitability for common surface modification techniques. This approach allows the preparation of CdSe-ZnS/CdS/ZnS QDs with photoluminescence quantum yields approaching 100% and small photoluminescence peak width.

Targeted nano analysis of water and ions using cryocorrelative light and scanning transmission electron microscopy.

Cryo fluorescence imaging coupled with the cryo-EM technique (cryo-CLEM) avoids chemical fixation and embedding in plastic, and is the gold standard for correlated imaging in a close to native state. This multi-modal approach has not previously included elementary nano analysis or evaluation of water content. We developed a new approach allowing analysis of targeted in situ intracellular ions and water measurements at the nanoscale (EDXS and STEM dark field imaging) within domains identified by examination of specific GFP-tagged proteins. This method allows both water and ions- fundamental to cell biology- to be located and quantified at the subcellular level. We illustrate the potential of this approach by investigating changes in water and ion content in nuclear domains identified by GFP-tagged proteins in cells stressed by Actinomycin D treatment and controls. The resolution of our approach was sufficient to distinguish clumps of condensed chromatin from surrounding nucleoplasm by fluorescence imaging and to perform nano analysis in this targeted compartment.

Shrinkage of freeze-dried cryosections of cells: Investigations by EFTEM and cryo-CLEM

Freeze-drying of cryosections of cells or tissues is considered to be the most efficient preparation for microanalysis purpose related to transmission electron microscopy. It allows the measurements of ions and water contents at the ultrastructural level. However an important drawback is associated to freeze-drying: the shrinkage of the cryosections. The aim of this paper is the investigation of this phenomenon by means of three different methods applied to both hydrated and dehydrated cryosections: direct distance measurements on fiducial points, thickness measurements by energy filtered transmission microscopy (EFTEM) and cryo-correlative light electron microscopy (cryo-CLEM). Measurements in our experimental conditions reveal a lateral shrinkage around 10% but the most important result concerns the lack of differential shrinkage between most of the cellular compartments.

Targeted Nano Analysis of Water and Ions in the Nucleus Using Cryo-Correlative Microscopy

The cell nucleus is a crowded volume in which the concentration of macromolecules is high. These macromolecules sequester most of the water molecules and ions which, together, are very important for stabilization and folding of proteins and nucleic acids. To better understand how the localization and quantity of water and ions vary with nuclear activity, it is necessary to study them simultaneously by using newly developed cell imaging approaches. Some years ago, we showed that dark-field cryo-Scanning Transmission Electron Microscopy (cryo-STEM) allows quantification of the mass percentages of water, dry matter, and elements (among which are ions) in freeze-dried ultrathin sections. To overcome the difficulty of clearly identifying nuclear subcompartments imaged by STEM in ultrathin cryo-sections, we developed a new cryo correlative light and STEM imaging procedure. This combines fluorescence imaging of nuclear GFP-tagged proteins to identify, within cryo ultrathin sections, regions of interest which are then analyzed by STEM for quantification of water and identification and quantification of ions. In this chapter we describe the new setup we have developed to perform this cryo-correlative light and STEM imaging approach, which allows a targeted nano analysis of water and ions in nuclear compartments.