Evaluation of sample preparation methods for single cell quantitative elemental imaging using proton or synchrotron radiation focused beams

Perrin, Laura; Carmona, Asunción; Roudeau, Stéphane; Ortega, Richard

doi:10.1039/c5ja00303b

Cited by 85 publications

(115 citation statements)

References 34 publications

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“…Matsuyama and others scanned the cryoimmobilized cells in the frozen hydrated state, and compared them to the chemically fixed and dehydrated cells scanned in a separated experiment. In our work, we conducted a side‐by‐side comparison between chemically fixed cells and plunge frozen cells, with both samples subsequently dehydrated and scanned by XFM under ambient temperature, with results similar to those reported by Perrin et al ., . This comparison revealed significant loss of K and Cl in chemically fixed cells, elevated level and altered distribution of Ca, loss of P to a certain extent and some possible difference in Zn distribution, compared to plunge frozen and freeze dried cells.…”

Section: Discussionmentioning

confidence: 76%

“…Si 3 N 4 windows with cells grown on them were carefully detached from the cell culture dish and mounted to the tweezers supplied with a FEI (Hillsboro, Oregon, USA) Vitrobot Mark IV plunge freezer. Cells were then washed respectively with one of the following wash buffers: TG buffer, ammonium acetate buffer (Amac buffer: 100 mM ammonium acetate in H 2 O, osmolarity 200 mOsm L –1 ) as has been used in electron microprobe (Wroblewski & Wroblewski, ) and synchrotron (Perrin et al ., ) microanalysis studies, D‐PBS buffer, or complete culture medium for two consecutive times. After all excess liquid on the back of the window (noncell side) and between tweezers was carefully blotted away, the tweezers with window were mounted onto the Vitrobot plunge freezer for rapid freezing following manufacturer's instructions.…”

Section: Methodsmentioning

confidence: 99%

“…There are many critical factors to be considered while applying XFM to investigate the elemental distribution and quantification of cultured mammalian cells. Sample preparation is one of the most important steps (Perrin et al, 2015). One common preparation approach involves aldehyde-based chemical fixation followed by dehydration, whereas another involves rapid freezing-based fixation (cryoimmobilization), followed by imaging in the frozen hydrated state or with dehydrated, room-temperature specimens.…”

Section: Introductionmentioning

confidence: 99%

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Preserving elemental content in adherent mammalian cells for analysis by synchrotron‐based x‐ray fluorescence microscopy

Jin¹,

Paunesku

Lai

et al. 2016

Journal of Microscopy

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SummaryTrace metals play important roles in biological function, and x‐ray fluorescence microscopy (XFM) provides a way to quantitatively image their distribution within cells. The faithfulness of these measurements is dependent on proper sample preparation. Using mouse embryonic fibroblast NIH/3T3 cells as an example, we compare various approaches to the preparation of adherent mammalian cells for XFM imaging under ambient temperature. Direct side‐by‐side comparison shows that plunge‐freezing‐based cryoimmobilization provides more faithful preservation than conventional chemical fixation for most biologically important elements including P, S, Cl, K, Fe, Cu, Zn and possibly Ca in adherent mammalian cells. Although cells rinsed with fresh media had a great deal of extracellular background signal for Cl and Ca, this approach maintained cells at the best possible physiological status before rapid freezing and it does not interfere with XFM analysis of other elements. If chemical fixation has to be chosen, the combination of 3% paraformaldehyde and 1.5 % glutaraldehyde preserves S, Fe, Cu and Zn better than either fixative alone. When chemically fixed cells were subjected to a variety of dehydration processes, air drying was proved to be more suitable than other drying methods such as graded ethanol dehydration and freeze drying. This first detailed comparison for x‐ray fluorescence microscopy shows how detailed quantitative conclusions can be affected by the choice of cell preparation method.

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Section: Discussionmentioning

confidence: 76%

Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Preserving elemental content in adherent mammalian cells for analysis by synchrotron‐based x‐ray fluorescence microscopy

Jin¹,

Paunesku

Lai

et al. 2016

Journal of Microscopy

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“…It can be hypothesized a decrease of uranium content from the cytoplasm during the different steps of the chemical preparation. This observation could be documented by a lesser binding to the macromolecules within the cytoplasm (Bresson et al, ; Frelon, Mounicou, Lobinski, Gilbin, & Simon, ; Ortega et al, ; Perrin, Carmona, Roudeau, & Ortega, ). In addition, we have previously shown from in vivo experiments i using SIMS microscopy that uranium is heterogeneously distributed in the nephron and localized mainly in cell nuclei of proximal convoluted tubules (Poisson et al, ; Tessier et al, ).…”

Section: Discussionmentioning

confidence: 99%

Intracellular uranium distribution: Comparison of cryogenic fixation versus chemical fixation methods for SIMS analysis

Suhard

Tessier

Manens

et al. 2018

Microscopy Res & Technique

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Localization of uranium within cells is mandatory for the comprehension of its cellular mechanism of toxicity. Secondary Ion Mass Spectrometry (SIMS) has recently shown its interest to detect and localize uranium at very low levels within the cells. This technique requires a specific sample preparation similar to the one used for Transmission Electronic Microscopy, achieved by implementing different chemical treatments to preserve as much as possible the living configuration uranium distribution into the observed sample. This study aims to compare the bioaccumulation sites of uranium within liver or kidney cells after chemical fixation and cryomethods preparations of the samples: SIMS analysis of theses samples show the localization of uranium soluble forms in the cell cytoplasm and nucleus with a more homogenous distribution when using cryopreparation probably due to the diffusible portion of uranium inside the cytoplasm.

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“…50 µL of AGuIX NPs at 100 mM were injected into the tumors and 1h after, tumors were extracted and cryofixed. Tissue sections of 50 µm thickness were deposited onto sample holders specially designed for PIXE analyses and tissue sections were freeze-dried [5]. Experiments were performed at AIFIRA facility (Applications Interdisciplinaires de Faisceaux d'Ions en Région Aquitaine) from CENBG laboratory (Centre d'Etudes Nucléaires de Bordeaux Gradignan).…”

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confidence: 99%

Particle Induced X-ray Emission Imaging of Gadolinium Distribution into Xenograft U87 Human Glioblastoma after AGuIX Nanoparticles Injection

et al. 2016

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In recent years, the use of high-Z nanoparticles (NPs) as potential tumor selective radiosensitizers has been proposed as a breakthrough in cancer radiotherapy (RT). NPs are capable of penetrating the cell and they lead to fewer adverse effects than conventional radiosensitizers [1]. Radiation sensitivity using NPs depends on cell line, irradiation energy, NPs type, size, concentration and distribution. High-Z metallic NPs (Au, Gd, Pt, Ag, Fe, etc.) have been predominantly used since they can generate shortrange photoelectrons or Auger electrons, which can enhance the therapeutic dose locally. It is of great interest to limit the diffusion of metallic NPs only to the tumor volume. So current investigations try to optimize the size of NPs and/or use chelating ligands of high affinity for tumor cells. Also the way that NPs are provided to the tumor, intravenously or by direct injection, will determine their distribution. Among those NPs, Gd-AGuIX (Activation and Guiding of Irradiation by X-ray) are 4 nm size polysiloxane GdNPs that have been proposed to be used for theragnostic as magnetic resonance imaging (MRI) contrast agents and radiotherapy sensitizers [2].The purpose of our work was to determine the quantitative distribution of gadolinium into a xenograft U87 human glioblastoma tumor, after intratumoral injection of AGuIX NPs, using PIXE (Particle Induced X-ray Emission) imaging. PIXE is based on the detection of X-ray emitted by a sample when irradiated with a MeV ion beam, usually protons. This technique offers two important specific capabilities for trace metal analysis: imaging and quantification [3]. PIXE is a multi-elemental technique that allows determining elements with atomic number higher than 11 and that is more sensitive than electron microprobe. To study biological samples PIXE can be simultaneously carried out with RBS (Rutherford Backscattering Spectrometry) a technique which provide the local sample mass. This multimodal micro-spectrometry enables expressing metal content in terms of µg of element per g of dry mass all over the scanned area [4].Female mices were grafted with human glioblastoma cells, U87, in right hind leg. 50 µL of AGuIX NPs at 100 mM were injected into the tumors and 1h after, tumors were extracted and cryofixed. Tissue sections of 50 µm thickness were deposited onto sample holders specially designed for PIXE analyses and tissue sections were freeze-dried [5]. Experiments were performed at AIFIRA facility (Applications Interdisciplinaires de Faisceaux d'Ions en Région Aquitaine) from CENBG laboratory (Centre d'Etudes Nucléaires de Bordeaux Gradignan). PIXE and RBS techniques were performed simultaneously to determine elemental content in tumor sections expressed in µg of element per g of dry mass. A proton beam was focalized at 2 µm size and 500 pA intensity. About 80 consecutive squared analyses of 750 µm side were performed to cover the all tumor slice area, of about 5 mm diameter (Fig. 1). Elemental images were reconstructed to overlay all scanned regions.

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Evaluation of sample preparation methods for single cell quantitative elemental imaging using proton or synchrotron radiation focused beams

Abstract: Sample preparation protocols for single cell quantitative elemental imaging using micro-PIXE or micro-SXRF have been compared and optimized for neuronal cells.

Cited by 85 publications

References 34 publications

Preserving elemental content in adherent mammalian cells for analysis by synchrotron‐based x‐ray fluorescence microscopy

Preserving elemental content in adherent mammalian cells for analysis by synchrotron‐based x‐ray fluorescence microscopy

Intracellular uranium distribution: Comparison of cryogenic fixation versus chemical fixation methods for SIMS analysis

Particle Induced X-ray Emission Imaging of Gadolinium Distribution into Xenograft U87 Human Glioblastoma after AGuIX Nanoparticles Injection

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