Name: preparing adherent cells for x-ray fluorescence imaging by chemical fixation
Uploaded: 2015-03-12T13:00:00
Description: Watch this Scientific Journal Video about Preparing Adherent Cells for X-ray Fluorescence Imaging by Chemical Fixation at JoVE.com

The authors acknowledge Stefan Vogt for his assistance in the fitting of the representative data shown in this paper, and helpful discussions. The authors also acknowledge Chris Jacobsen for his support to Q. J. 
Use of the Advanced Photon Source, beamlines 2-ID-E and 8-BM-B, at Argonne National Laboratory was supported by the U. S. Department of Energy, Office of Science, Office of Basic Energy Sciences, under Contract No. DE-AC02-06CH11357.

1. Preparation of Instruments, Substrates, Culture Media and Dishes
<ol>
	<li>Handling of Silicon Nitride (Si3N4) windows.
	<ol>
		<li>Open the capsule slightly by squeezing one end containing the window in such a way as to not squeeze the window itself, while rotating the other end of the capsule with the other hand.</li>
		<li>Check a pair of reverse, fine-tipped tweezers under stereomicroscope to make sure there are no adhesive, gaps or bends at the tip. Otherwise, the windows may be broken by ...

X-ray fluorescence imaging is useful in many fields, including geosciences, materials science, and chemical biology26-34. Advances in synchrotron X-rays, and their focusing, have produced very high-intensity beams. Focused X-ray beams sufficient to excite the endogenous metals in cells now exist, producing signal that can be measured with currently available silicon drift detector technology. And studying the chemical biology of metals in the cell is one application in particular that makes use of many of the ...

X-ray fluorescence imaging allows for both the identity and quantity of elements present in a sample to be spatially resolved. Incident X-rays, of an energy selected to be greater than the electron binding energy of the heaviest element of interest, overcome the binding energy of inner-shell electrons to the nucleus1. This creates a &#8216;hole&#8217; in the electron shell. As higher-energy electrons fall down into these holes, fluorescent X-rays are emitted whose wavelength is dependent on the energy separation of those orbitals. Since the energy spacing of the orbitals is characteristic of a given element, the X-ray fluorescence emission also has characteristic wavelengths, dependent on the element. It is this emission at a characteristic wavelength that allows the identification of the elements present. Calibration of the fluorescence intensity allows the quantitation of the elements present.
X-ray fluorescence microscopy (XFM) has become increasingly utilized, partly due to the development of very brilliant X-ray synchrotron sources, such as those at Spring-8 in Japan, the European Radiation Synchrotron Facility (ESRF) in France, and the Advanced Photon Source (APS) in the US2. These sources provide very high intensity X-ray beams. At the same time, improvements in X-ray optics, such as zone plate technology, allowed the focusing of these beams to sub-micron spots, albeit rather inefficiently3. With very high-intensity beams, even a relatively small amount of light that can be focused is sufficient to excite the endogenous metals in cells, producing signal that can be measured with currently available detector technology. Thus, studying the chemical biology of metals in the cell is one application in particular that makes use of many of the recent developments in this technique4-10.
There are many critical factors to be considered while applying XFM to investigate the elemental distribution and quantification of cultured mammalian cells or other biological samples. Firstly, the sample needs to be kept intact, both structurally and with respect to its elemental composition, in order for the measurement to be meaningful. Secondly, the sample must also be preserved in some way so that it is hardy to the radiation damage that can be caused by a focused X-ray beam. One way that a sample can meet both of these criteria at once is to be rapidly frozen into a vitreous, amorphous ice11,12. Rapid freezing is often achieved through various cryopreservation techniques such as plunge freezing or high pressure freezing13-16. It is generally accepted that cryopreservation preserves overall cellular architecture and chemical compositions in biological samples as close to native state as possible. Chemical fixation, on the other hand, due to the slow and selective penetration of fixatives into cells and tissues as well as subsequent changes in membrane permeability, may allow various cellular ions especially the diffusible ions such as Cl, Ca and K to be leached, lost or relocated, thus rendering investigation of these elements suboptimal17-19. Despite the clear advantage of cryo-fixation over chemical fixation in general, for adherent mammalian cells in particular, cryopreservation has various limitations20-23. The most obvious one is that not every research lab has easy access to cryopreservation instruments. Most current high pressure freezers or even plunge freezers are costly and owned only by a subset of cryo facilities, which may be far from where cells are incubated. The benefit of cryopreservation might be traded for the disadvantage of travel stress placed on the cells. Thus, while cryopreservation is surely the most rigorous way to preserve samples for X-ray fluorescence analysis, it is certainly not the most accessible to all researchers under all circumstances; nor is it always essential&#160;&#8212; if the metals of interest are tightly bound to fixable macromolecules, and the resolution at which the sample will be imaged is greater than the damage to the ultra-microstructure that might occur during drying. Mindful of the caveats24, chemical fixation and drying may be a suitable choice.
Other factors in a successful X-ray fluorescence imaging experiment include proper analysis. X-ray fluorescence imaging is fundamentally X-ray fluorescence emission spectroscopy combined with raster-scanning to provide spatial resolution. The X-ray fluorescence emission spectra collected contain a combination of overlapping emission peaks, background, and the elastic and inelastic scattering peaks of the incident beam. Software that enables the de-convolution of these contributions, and the fitting of the emission peaks, has been a critical development to this field25. Also, the development and commercial distribution of thin-film standards of known composition, used to calibrate fluorescence intensity relative to material quantity, has also been very important.
This protocol provides a description of the preparation of adherent cells by chemical fixation and air drying. A vital step in this process is the growth of the cells on the silicon nitride windows, which often do not adhere well, making gentle rinsing in a particular fashion key to success.

<table border="0" cellpadding="0" cellspacing="0" width="924">
	<tbody>
		<tr height="231">
			<td height="231" style="height:231px;width:216px;">silicon nitride windows</td>
			<td style="width:423px;">Silson Ltd/J B J Business Park/Northampton Rd, Northampton NN7 3DW, United Kingdom</td>
			<td style="width:119px;">No part numbers available. Order by size. Membrane size: 1.5 mm x 1.5 mm.&#160; Thickness 500 nm.&#160; Frame size: 5 mm x 5 mm.&#160; Frame thickness: 200 &#181;m</td>
			<td style="width:167px;">Alternate source: SPI Supplies / Structure Probe, Inc.West Chester, PA</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">reverse tweezers</td>
			<td style="width:423px;">Electron Microscopy Sciences, P.O. Box 550, 1560 Industry Road, Hatfield, PA 19440, Tel: 215-412-8400, Toll Free: 800-523-5874, Fax: 215-412-8450</td>
			<td style="width:119px;">78520-5X</td>
			<td style="width:167px;">EMS 5X, NC &#8211; Ultra Fine Tweezers</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">rubber grid mat</td>
			<td style="width:423px;">Electron Microscopy Sciences, P.O. Box 550, 1560 Industry Road, Hatfield, PA 19440, Tel: 215-412-8400, Toll Free: 800-523-5874, Fax: 215-412-8450</td>
			<td style="width:119px;">71170</td>
			<td style="width:167px;">Round Grid Mat</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">acetic acid</td>
			<td style="width:423px;">Sigma-Aldrich, 3050 Spruce St., St. Louis, MO 63103, Tel: 800-325-3010, Fax: 800-325-5052</td>
			<td style="width:119px;">338826</td>
			<td style="width:167px;">trace metals grade concentrated acetic acid</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">PIPES buffer</td>
			<td style="width:423px;">Sigma-Aldrich, 3050 Spruce St., St. Louis, MO 63103, Tel: 800-325-3010, Fax: 800-325-5052</td>
			<td style="width:119px;">P6757</td>
			<td style="width:167px;">solid PIPES buffer</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">formaldehyde stock solution</td>
			<td style="width:423px;">Electron Microscopy Sciences, P.O. Box 550, 1560 Industry Road, Hatfield, PA 19440, Tel: 215-412-8400, Toll Free: 800-523-5874, Fax: 215-412-8450</td>
			<td style="width:119px;">RT 17113</td>
			<td style="width:167px;">10 x 10mL ampules of 20% aqueous paraformaldehyde</td>
		</tr>
	</tbody>
</table>

preparing adherent cells for x-ray fluorescence imaging by chemical fixation

X-ray fluorescence imaging allows us to non-destructively measure the spatial distribution and concentration of multiple elements simultaneously over large or small sample areas. It has been applied in many areas of science, including materials science, geoscience, studying works of cultural heritage, and in chemical biology. In the case of chemical biology, for example, visualizing the metal distributions within cells allows us to study both naturally-occurring metal ions in the cells, as well as exogenously-introduced metals such as drugs and nanoparticles. Due to the fully hydrated nature of nearly all biological samples, cryo-fixation followed by imaging under cryogenic temperature represents the ideal imaging modality currently available. However, under the circumstances that such a combination is not easily accessible or practical, aldehyde based chemical fixation remains useful and sometimes inevitable. This article describes in as much detail as possible in the preparation of adherent mammalian cells by chemical fixation for X-ray fluorescent imaging.

The ability of X-ray fluorescence imaging to provide information about biological samples is contingent upon these samples being prepared in such a way that they are robust to radiation damage on the time-scale of the experiment, and yet their chemical and structural features are well preserved. In viewing the result of a sample that has been prepared as described above and imaged, it is possible to see that there is variation in the elements present &#8212; indicating that fixation preserved these aspects of the cell (<...

Watch this Scientific Journal Video about Preparing Adherent Cells for X-ray Fluorescence Imaging by Chemical Fixation at JoVE.com

Preparing Adherent Cells for X-ray Fluorescence Imaging by Chemical Fixation

Here, we present a protocol on how to determine the quantity and distribution of metals in a sample using synchrotron X-ray fluorescence. We focus on adherent cells, and describe the chemical fixation method to prepare this sample. We then describe how to mount and image the sample using synchrotron X-rays.

X-ray fluorescence imaging allows us to non-destructively measure the spatial distribution and concentration of multiple elements simultaneously over ...

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Chemistry

A Simple and Rapid Protocol for Measuring Neutral Lipids in Algal Cells Using Fluorescence

Algae are considered excellent candidates for renewable fuel sources due to their natural lipid storage capabilities. Robust monitoring of algal fermentation processes and screening for new oil-rich strains requires a fast and reliable protocol for determination of intracellular lipid content. Current practices rely largely on gravimetric methods to determine oil content, techniques developed decades ago that are time consuming and require large sample volumes. In this paper, Nile Red, a fluorescent dye that has been used to identify the presence of lipid bodies in numerous types of organisms, is incorporated into a simple, fast, and reliable protocol for measuring the neutral lipid content of Auxenochlorella protothecoides, a green alga. The method uses ethanol, a relatively mild solvent, to permeabilize the cell membrane before staining and a 96 well micro-plate to increase sample capacity during fluorescence intensity measurements. It has been designed with the specific application of monitoring bioprocess performance. Previously dried samples or live samples from a growing culture can be used in the assay.

A simple protocol to determine the neutral lipid content of algal cells using a Nile Red staining procedure is described. This time-saving technique offers an alternative to traditional gravimetric-based lipid quantification protocols. It has been designed for the specific application of monitoring bioprocess performance.

Algae are considered excellent candidates for renewable fuel sources due to their natural lipid storage capabilities. Robust monitoring of algal ...

X-ray Powder Diffraction in Conservation Science: Towards Routine Crystal Structure Determination of Corrosion Products on Heritage Art Objects

The crystal structure determination and refinement process of corrosion products on historic art objects using laboratory high-resolution X-ray powder diffraction (XRPD) is presented in detail via two case studies.
The first material under investigation was sodium copper formate hydroxide oxide hydrate, Cu4Na4O(HCOO)8(OH)2&#8729;4H2O (sample 1) which forms on soda glass/copper alloy composite historic objects (e.g., enamels) in museum collections, exposed to formaldehyde and formic acid emitted from wooden storage cabinets, adhesives, etc. This degradation phenomenon has recently been characterized as &#34;glass induced metal corrosion&#34;.
For the second case study, thecotrichite, Ca3(CH3COO)3Cl(NO3)2&#8729;6H2O (sample 2), was chosen, which is an efflorescent salt forming needlelike crystallites on tiles and limestone objects which are stored in wooden cabinets and display cases. In this case, the wood acts as source for acetic acid which reacts with soluble chloride and nitrate salts from the artifact or its environment.
The knowledge of the geometrical structure helps conservation science to better understand production and decay reactions and to allow for full quantitative analysis in the frequent case of mixtures.

Modern high resolution X-ray powder diffraction (XRPD) in the laboratory is used as an efficient tool to determine crystal structures of long-known corrosion products on historic objects.

The crystal structure determination and refinement process of corrosion products on historic art objects using laboratory high-resolution X-ray powder ...

Quantifying X-Ray Fluorescence Data Using MAPS

The quantification of X-ray fluorescence (XRF) microscopy maps by fitting the raw spectra to a known standard is crucial for evaluating chemical composition and elemental distribution within a material. Synchrotron-based XRF has become an integral characterization technique for a variety of research topics, particularly due to its non-destructive nature and its high sensitivity. Today, synchrotrons can acquire fluorescence data at spatial resolutions well below a micron, allowing for the evaluation of compositional variations at the nanoscale. Through proper quantification, it is then possible to obtain an in-depth, high-resolution understanding of elemental segregation, stoichiometric relationships, and clustering behavior.
This article explains how to use the MAPS fitting software developed by Argonne National Laboratory for the quantification of full 2-D XRF maps. We use as an example results from a Cu(In,Ga)Se2 solar cell, taken at the Advanced Photon Source beamline 2-ID-D at Argonne National Laboratory. We show the standard procedure for fitting raw data, demonstrate how to evaluate the quality of a fit and present the typical outputs generated by the program. In addition, we discuss in this manuscript certain software limitations and offer suggestions for how to further correct the data to be numerically accurate and representative of spatially resolved, elemental concentrations.

Here, we demonstrate the use of the X-ray fluorescence fitting software, MAPS, created by Argonne National Laboratory for the quantification of fluorescence microscopy data. The quantified data that results is useful for understanding the elemental distribution and stoichiometric ratios within a sample of interest.

The quantification of X-ray fluorescence (XRF) microscopy maps by fitting the raw spectra to a known standard is crucial for evaluating chemical ...

Microfluidic Chips for In Situ Crystal X-ray Diffraction and In Situ Dynamic Light Scattering for Serial Crystallography

This protocol describes fabricating microfluidic devices with low X-ray background optimized for goniometer based fixed target serial crystallography. The devices are patterned from epoxy glue using soft lithography and are suitable for in situ X-ray diffraction experiments at room temperature. The sample wells are lidded on both sides with polymeric polyimide foil windows that allow diffraction data collection with low X-ray background. This fabrication method is undemanding and inexpensive. After the sourcing of a SU-8 master wafer, all fabrication can be completed outside of a cleanroom in a typical research lab environment. The chip design and fabrication protocol utilize capillary valving to microfluidically split an aqueous reaction into defined nanoliter sized droplets. This loading mechanism avoids the sample loss from channel dead-volume and can easily be performed manually without using pumps or other equipment for fluid actuation. We describe how isolated nanoliter sized drops of protein solution can be monitored in situ by dynamic light scattering to control protein crystal nucleation and growth. After suitable crystals are grown, complete X-ray diffraction datasets can be collected using goniometer based in situ fixed target serial X-ray crystallography at room temperature. The protocol provides custom scripts to process diffraction datasets using a suite of software tools to solve and refine the protein crystal structure. This approach avoids the artefacts possibly induced during cryo-preservation or manual crystal handling in conventional crystallography experiments. We present and compare three protein structures that were solved using small crystals with dimensions of approximately 10-20 &#181;m grown in chip. By crystallizing and diffracting in situ, handling and hence mechanical disturbances of fragile crystals is minimized. The protocol details how to fabricate a custom X-ray transparent microfluidic chip suitable for in situ serial crystallography. As almost every crystal can be used for diffraction data collection, these microfluidic chips are a very efficient crystal delivery method.

Microfluidic Chips for In Situ Crystal X-ray Diffraction and In Situ Dynamic Light Scattering for Serial Crystallography

This protocol describes in detail how to fabricate and operate microfluidic devices for X-ray diffraction data collection at room temperature. Additionally, it describes how to monitor protein crystallization by dynamic light scattering and how to process and analyze obtained diffraction data.

This protocol describes fabricating microfluidic devices with low X-ray background optimized for goniometer based fixed target serial crystallography. The ...

Essential Metal Uptake in Gram-negative Bacteria: X-ray Fluorescence, Radioisotopes, and Cell Fractionation

We demonstrate a scalable method for the separation of the bacterial periplasm from the cytoplasm. This method is used to purify periplasmic protein for the purpose of biophysical characterization, and measure substrate transfer between periplasmic and cytoplasmic compartments. By carefully limiting the time that the periplasm is separated from the cytoplasm, the experimenter can extract the protein of interest and assay each compartment individually for substrate without carry-over contamination between compartments. The extracted protein from fractionation can then be further analyzed for three-dimensional structure determination or substrate-binding profiles. Alternatively, this method can be performed after incubation with a radiotracer to determine total percent uptake, as well as distribution of the tracer (and hence metal transport) across different bacterial compartments. Experimentation with a radiotracer can help differentiate between a physiological substrate and artefactual substrate, such as those caused by mismetallation.
X-ray fluorescence can be used to discover the presence or absence of metal incorporation in a sample, as well as measure changes that may occur in metal incorporation as a product of growth conditions, purification conditions, and/or crystallization conditions. X-ray fluorescence also provides a relative measure of abundance for each metal, which can be used to determine the best metal energy absorption peak to use for anomalous X-ray scattering data collection. Radiometal uptake can be used as a method to validate the physiological nature of a substrate detected by X-ray fluorescence, as well as support the discovery of novel substrates.

A protocol for the extraction of a periplasmic transition metal chaperone in the context of its native binding partners, and biophysical characterization of its substrate contents by X-ray fluorescence and radiometal uptake is presented.

We demonstrate a scalable method for the separation of the bacterial periplasm from the cytoplasm. This method is used to purify periplasmic protein for ...

Elemental-sensitive Detection of the Chemistry in Batteries through Soft X-ray Absorption Spectroscopy and Resonant Inelastic X-ray Scattering

Energy storage has become more and more a limiting factor of today's sustainable energy applications, including electric vehicles and green electric grid based on volatile solar and wind sources. The pressing demand of developing high-performance electrochemical energy storage solutions, i.e., batteries, relies on both fundamental understanding and practical developments from both the academy and industry. The formidable challenge of developing successful battery technology stems from the different requirements for different energy-storage applications. Energy density, power, stability, safety, and cost parameters all have to be balanced in batteries to meet the requirements of different applications. Therefore, multiple battery technologies based on different materials and mechanisms need to be developed and optimized. Incisive tools that could directly probe the chemical reactions in various battery materials are becoming critical to advance the field beyond its conventional trial-and-error approach. Here, we present detailed protocols for soft X-ray absorption spectroscopy (sXAS), soft X-ray emission spectroscopy (sXES), and resonant inelastic X-ray scattering (RIXS) experiments, which are inherently elemental-sensitive probes of the transition-metal 3d and anion 2p states in battery compounds. We provide the details on the experimental techniques and demonstrations revealing the key chemical states in battery materials through these soft X-ray spectroscopy techniques.

Here, we present a protocol for typical experiments of soft X-ray absorption spectroscopy (sXAS) and resonant inelastic X-ray scattering (RIXS) with applications in battery material studies.

Energy storage has become more and more a limiting factor of today's sustainable energy applications, including electric vehicles and green electric grid ...

Analysis of Minerals Produced by hFOB 1.19 and Saos-2 Cells Using Transmission Electron Microscopy with Energy Dispersive X-ray Microanalysis

This video presents the use of transmission electron microscopy with energy dispersive X-ray microanalysis (TEM-EDX) to compare the state of minerals in vesicles released by two human bone cell lines: hFOB 1.19 and Saos-2. These cell lines, after treatment with ascorbic acid (AA) and &#946;-glycerophosphate (&#946;-GP), undergo complete osteogenic transdifferentiation from proliferation to mineralization and produce matrix vesicles (MVs) that trigger apatite nucleation in the extracellular matrix (ECM).
Based on Alizarin Red-S (AR-S) staining and analysis of the composition of minerals in cell lysates using ultraviolet (UV) light or in vesicles using TEM imaging followed by EDX quantitation and ion mapping, we can infer that osteosarcoma Saos-2 and osteoblastic hFOB 1.19 cells reveal distinct mineralization profiles. Saos-2 cells mineralize more efficiently than hFOB 1.19 cells and produce larger mineral deposits that are not visible under UV light but are similar to hydroxyapatite (HA) in that they have more Ca and F substitutions.
The results obtained using these techniques allow us to conclude that the process of mineralization differs depending on the cell type. We propose that, at the cellular level, the origin and properties of vesicles predetermine the type of minerals.

We present a protocol to compare the state of minerals in vesicles released by two human bone cell lines: hFOB 1.19 and Saos-2. Their mineralization profiles were analyzed by Alizarin Red-S (AR-S) staining, ultraviolet (UV) light visualization, transmission electron microscopy (TEM) imaging and energy dispersive X-ray microanalysis (EDX).

This video presents the use of transmission electron microscopy with energy dispersive X-ray microanalysis (TEM-EDX) to compare the state of minerals in ...

Ligand-Mediated Nucleation and Growth of Palladium Metal Nanoparticles

The size, size distribution and stability of colloidal nanoparticles are greatly affected by the presence of capping ligands. Despite the key contribution of capping ligands during the synthesis reaction, their role in regulating the nucleation and growth rates of colloidal nanoparticles is not well understood. In this work, we demonstrate a mechanistic investigation of the role of trioctylphosphine (TOP) in Pd nanoparticles in different solvents (toluene and pyridine) using in situ SAXS and ligand-based kinetic modeling. Our results under different synthetic conditions reveal the overlap of nucleation and growth of Pd nanoparticles during the reaction, which contradicts the LaMer-type nucleation and growth model. The model accounts for the kinetics of Pd-TOP binding for both, the precursor and the particle surface, which is essential to capture the size evolution as well as the concentration of particles in situ. In addition, we illustrate the predictive power of our ligand-based model through designing the synthetic conditions to obtain nanoparticles with desired sizes. The proposed methodology can be applied to other synthesis systems and therefore serves as an effective strategy for predictive synthesis of colloidal nanoparticles.

The main goal of this work is to elucidate the role of capping agents in regulating the size of palladium nanoparticles by combining in situ small angle x-ray scattering (SAXS) and ligand-based kinetic modeling.

The size, size distribution and stability of colloidal nanoparticles are greatly affected by the presence of capping ligands. Despite the key contribution ...

Improving High Viscosity Extrusion of Microcrystals for Time-resolved Serial Femtosecond Crystallography at X-ray Lasers

High-viscosity micro-extrusion injectors have dramatically reduced sample consumption in serial femtosecond crystallographic experiments (SFX) at X-ray free electron lasers (XFELs). A series of experiments using the light-driven proton pump bacteriorhodopsin have further established these injectors as a preferred option to deliver crystals for time-resolved serial femtosecond crystallography (TR-SFX) to resolve structural changes of proteins after photoactivation. To obtain multiple structural snapshots of high quality, it is essential to collect large amounts of data and ensure clearance of crystals between every pump laser pulse. Here, we describe in detail how we optimized the extrusion of bacteriorhodopsin microcrystals for our recent TR-SFX experiments at the Linac Coherent Light Source (LCLS). The goal of the method is to optimize extrusion for a stable and continuous flow while maintaining a high density of crystals to increase the rate at which data can be collected in a TR-SFX experiment. We achieve this goal by preparing lipidic cubic phase with a homogenous distribution of crystals using a novel three-way syringe coupling device followed by adjusting the sample composition based on measurements of the extrusion stability taken with a high-speed camera setup. The methodology can be adapted to optimize the flow of other microcrystals. The setup will be available for users of the new Swiss Free Electron Laser facility.

The success of a time-resolved serial femtosecond crystallography experiment is dependent on efficient sample delivery. Here, we describe protocols to optimize the extrusion of bacteriorhodopsin microcrystals from a high viscosity micro-extrusion injector. The methodology relies on sample homogenization with a novel three-way coupler and visualization with a high-speed camera.

High-viscosity micro-extrusion injectors have dramatically reduced sample consumption in serial femtosecond crystallographic experiments (SFX) at X-ray ...

Biological Samples Preparation for Speciation at Cryogenic Temperature using High-Resolution  X-Ray Absorption Spectroscopy

The study of elements with X-ray absorption spectroscopy (XAS) is of particular interest when studying the role of metals in biological systems. Sample preparation is a key and often complex procedure, particularly for biological samples. Although X-ray speciation techniques are widely used, no detailed protocol has been yet disseminated for users of the technique. Further, chemical state modification is of concern, and cryo-based techniques are recommended to analyze the biological samples in their near-native hydrated state to provide the maximum preservation of chemical integrity of the cells or tissues. Here, we propose a cellular preparation protocol based on cryo-preserved samples. It is demonstrated in a high energy resolution fluorescence detected X-ray absorption spectroscopy study of selenium in cancer cells and a study of iron in phytoplankton. This protocol can be used with other biological samples and other X-ray techniques that can be damaged by irradiation.

This protocol presents a detailed procedure to prepare biological cryosamples for synchrotron-based X-ray absorption spectroscopy experiments. We describe all the steps required to optimize sample preparation and cryopreservation with examples of the protocol with cancer and phytoplankton cells. This method provides a universal standard of sample cryo-preparation.

The study of elements with X-ray absorption spectroscopy (XAS) is of particular interest when studying the role of metals in biological systems. Sample ...