The experiments on the nano-imaging beamline ID16A were performed in the frame of ESRF proposals LS2430, LS2303, and LS2765.

<ol>
	<li>Lewis, D. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intracellular+synchrotron+nanoimaging+and+DNA+damage/genotoxicity+screening+of+novel+lanthanide-coated+nanovectors.">Intracellular synchrotron nanoimaging and DNA damage/genotoxicity screening of novel lanthanide-coated nanovectors.</a> Nanomedicine. 5 (10), 1547-1557 (2010).</li><li>Fus, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+intracellular+localization+of+osmocenyl-tamoxifen+derivatives+in+hormone-independent+breast+cancer+cells+revealed+by+2D+and+3D+nano+X-ray+fluorescence+imaging.">The intracellular localization of osmocenyl-tamoxifen derivatives in hormone-independent breast cancer cells revealed by 2D and 3D nano X-ray fluorescence imaging.</a> Angwendte Chemie. , (2019).</li><li>Janssens, K., Adams, F., Rindby, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Microscopic X-Ray Fluorescence Analysis. , (2000).</li><li>Carmona, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Uranium+exposure+of+human+dopaminergic+cells+results+in+low+cytotoxicity,+accumulation+within+sub-cytoplasmic+regions,+and+down+regulation+of+MAO-B.">Uranium exposure of human dopaminergic cells results in low cytotoxicity, accumulation within sub-cytoplasmic regions, and down regulation of MAO-B.</a> Neurotoxicology. 68, 177-188 (2018).</li><li>Leapman, R. 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The authors have no conflicts of interest.

Cryo-electron microscopy (cryo-EM) won the 2017 Nobel Prize in chemistry and as such the development made by J. Dubochet on vitrification of biological material for the high-resolution structure determination of biomolecules in solution25. As reported by Dubochet in his Nobel lecture "Knowing how to vitrify a droplet of water is one thing, preparing a biological sample for biological observation is another"25. Cryopreparation steps are now considered the standard technique to mitigate radiation dose damage and study cells close to their native state. The preparation remains tedious, however. This is because electron microscopy, due to its unsurpassed spatial resolution, is sensitive to any ultrastructural artifact that occurs during the sample preparation. The synchrotron cryonanoprobes are now approaching similar difficulties going down to spatial resolutions as low as 13 nm in the high energy X-ray range26. Hard X-ray microscopy can analyze entire cells while electron microscopy suffers from the poor penetration depth of electrons enabling only very thin cell slices to be observed.
Monolayers of cells are thin enough so that by plunge-freezing in liquid ethane, the required cooling rates for water vitrification are attained. In theory, cooling rates as high as 108 K/s are possible using high-pressure freezing27 which allows vitrification of specimens too thick for plunge freezing. A cooling rate of 105 K/s, required to allow full vitrification of the sample at ambient pressure28, is reached reproducibly using the automatic plunge-freezing machine and parameters presented here. This allows a researcher to vitrify thin biological specimens (&#60;10 &#181;m) such as a monolayer of cells12,13,14,15,29,30 by plunge-freezing in liquid ethane.
An important challenge with this protocol is to also preserve as much as possible the chemical integrity of the intracellular content to provide reliable elemental distributions within the cell in 2D or 3D. As published elsewhere2,16,17,31, in the case of elemental imaging at the subcellular level, the analysis of frozen hydrated cells should be considered. Otherwise, the combination of plunge-freezing and freeze-drying of cells can be used for room temperature analysis. For the latter, the amorphous ice is removed through the process of sublimation, while the bound water molecules are removed through the process of desorption. This process may be far from ideal compared to frozen hydrated samples due to the possible alteration of the cellular membranes and the morphology of some subcellular structures32. Also, for speciation studies, the water extraction may lead to metal speciation artifacts. Still, it has been successful and the best alternative to frozen hydrated samples for elemental imaging at sub-100 nm levels2,16,17,18,20,33,34,35,36.
As it has been reported37, the quality of cryopreserved cellular preparations can be evaluated through the potassium-to-sodium K/Na ratio. Unfortunately, it cannot yet be determined with the hard X-ray nanoprobe used here, due to the low energy cut-off of the silicon drift detector used to detect the X-ray fluorescence photons of the elements (E &#8805; 1.3 keV magnesium). Indeed, a high K/Na ratio (&#62;10) that can be measured using TOF-SIMS, EPMA, or nuclear microprobe PIXE16,37 is indicative of the preserved chemical integrity of the cell compared to the expected K/Na of 25 in a living cell37. This can be supported by a concomitant low Cl/K ratio38. Still, imperfect vitrification, particularly if the speed of sample cooling is too low, can lead to the formation of large ice crystals that can damage cell membranes and organelles, consequently altering the distribution of chemical elements. Although there is no routine procedure to monitor this potential damage and impact on the intracellular distribution, the above elemental ratios and the possibility to image the cell at high resolution using X-ray phase contrast or cryo-soft X-ray microscopy can be the best approaches to support good preservation of intracellular compartments with concomitant preservation of the elemental integrity. The combination of these techniques and the use of newly developed cryocorrelative fluorescence optical microscopes will help assess to what extent this damage occurs and affects the intracellular elemental distribution.
Overall, a detailed and comprehensive protocol to prepare cellular samples for synchrotron X-ray fluorescence nano-analysis is presented. It is a good starting point for the research community, helping to solve the difficult issue of how to prepare appropriate cellular samples for 2D and 3D elemental imaging at (cryo) hard X-ray nanoprobes. These approaches can be merged with optical fluorescence and electron microscopy capabilities for in-depth correlative chemical and structural imaging of cells.

Newly designed synchrotron X-ray fluorescence (SR-XRF) nanoprobes allow visualization of the subcellular distribution of elements in a fully quantitative manner. As an example, this analytical capability allows investigation of the uptake of nanoparticles1 or organometallic molecules such as osmium-based complexes2, providing insight into the intracellular uptake of metal-based molecules with potent anticancer properties. As a multielement technique, SR-XRF3 with a nanoprobe provides a way to simultaneously quantify and localize intracellularly most biologically important elements, including phosphorus, sulfur, potassium, calcium, iron, copper, and zinc. Indeed, the use of hard X-rays provides large penetration depth to image whole frozen-hydrated cells in a label-free fashion. Furthermore, providing access to the K-edge of most elements of interest, the X-ray fluorescence is excited most efficiently. The use of cryogenic approaches allows reduction of radiation damage and optimization of the preservation of the cell structure and elemental distribution.
Most available spatially resolved analytical techniques to study metals in cells are surface techniques requiring very thin and flat sections of cells to be produced. This mainly encompasses scanning transmission electron microscopy with energy-dispersive X-ray analysis (STEM&#8211;EDX), energy-filtered transmission electron microscopy (EF-TEM), and nanoscale secondary ion mass spectrometry (nanoSIMS). The latter cannot be performed on frozen, hydrated cell sections while cryo-analysis can be done with electron microscopy with unsurpassed spatial resolution but poor elemental sensitivity. Particle-induced X-ray emission (PIXE) has allowed the study of elemental distributions in whole cells. It has the advantage of being fully quantitative with a fair elemental sensitivity at the micron scale and even at submicron resolution4, but suffers from radiation damage and lack of cryogenic capabilities to study frozen-hydrated cells. All these analytical techniques complement each another in the elemental imaging of cells, but for all techniques the sample preparation procedure is a crucial step. It should be kept simple to limit possible contamination as well as elemental redistribution and/or leakage to obtain meaningful results. As demonstrated in electron microscopy, a cryogenic workflow, including cryo-immobilization of the cell and cryotransfer to a cryoscanning stage, allows an optimal elemental preservation at subcellular levels as close as possible to the native state5,6,7,8,9,10. This understanding has been successfully implemented into the development of synchrotron cryo-soft X-ray microscopy (e.g., full field microscopes and scanning microscopes) to produce ultrastructural imaging of entire frozen-hydrated cells in 2D or 3D. Various cryogenic workflows were developed11 for soft X-ray microscopes at Beamline 2.1 (XM-2) of the Advanced Light Source at Lawrence Berkeley National Laboratory12, beamline U41-XM at the electron storage ring BESSY II (Germany)13, beamline MISTRAL of the ALBA light source (Spain)14, and at Beamline B24 of the Diamond light source15, among others. A similar workflow was recently shown to be the most reliable preparation and preservation method for intracellular elemental analysis using X-ray microprobes16,17.
Although X-ray nanoprobe techniques are starting to be widely used for cellular elemental analysis, particularly with the advent of cryogenic SR-XRF capabilities, no stepwise protocol has been disseminated so far to the research community. Here, a detailed procedure is provided to prepare cryofixed adherent cells cultured as monolayers on silicon nitride membranes to be analyzed under cryogenic conditions. A freeze-drying step to be applied after the protocol in case the X-ray analysis must be performed at room temperature is also provided. While the proposed protocol has been successfully used with human breast cancer cells MD-MB-2312 and the freeze-drying was demonstrated among others on mouse neurons18,20,21, it can be easily extended to various types of human or animal cells.

<table><tbody><tr><td>Ammonium Acetate solution, BioUltra, for molecular biology, ~5M in H2O</td><td>SIGMA</td><td>09691-250mL</td><td>One can prepare the required solution from high-grade ammonium acetate powder and ultrapure water, pH and osmolarity needs to be adjusted anyway.</td></tr><tr><td>B27 supplement, 50x</td><td>Life Technologies, Invitrogen</td><td>17504-044</td><td>for hippocampal neuron culture</td></tr><tr><td>Dulbecco&amp;rsquo;s Phosphate Buffered Saline, DPBS, ([-] CaCl2, [-] MgCl2)</td><td>GIBCO</td><td>14190-094</td><td>cell culture</td></tr><tr><td>DMEM with Phenol Red/Glutamax I (Medium ATCC modification)</td><td>GIBCO</td><td>21885025</td><td>cell culture</td></tr><tr><td>Dulbecco&amp;rsquo;s modified Eagle medium (DMEM)</td><td>Life Technologies, Invitrogen</td><td>31966-02</td><td>for hippocampal neuron culture</td></tr><tr><td>Dumont Tweezers #5, Straight Self-closing, 0.05x0.01mm Tips, Biology</td><td>World Precision Instrument</td><td>501202</td><td></td></tr><tr><td>Emitech K750X Peltier-Cooled EM Freeze Dryer</td><td>Quorum Technology</td><td>EK3147</td><td></td></tr><tr><td>Ethane N45</td><td>Air Liquid</td><td>p0505s05r0a001</td><td>C2H6 &gt; 99,995 %</td></tr><tr><td>Fetal Bovine Serum, Performance Plus, certified One Shot format, US origin</td><td>GIBCO</td><td>A31604-02</td><td>cell culture</td></tr><tr><td>HBSS 10x</td><td>Life Technologies, Invitrogen</td><td>14185-052</td><td>for hippocampal neuron culture</td></tr><tr><td>Leica GP quick-release forceps</td><td>Leica</td><td>16706435</td><td></td></tr><tr><td>MDA-MB-231 cell line, an epithelial, human adenocarcinoma breast cancer cell</td><td>ATCC</td><td>ATCC HTB-26</td><td>cell culture</td></tr><tr><td>Neurobasal medium</td><td>Life Technologies, Invitrogen</td><td>21103-049</td><td>for hippocampal neuron culture</td></tr><tr><td>Nunc 4-Well Plate</td><td>Thermo Fisher</td><td>176740</td><td>cell culture</td></tr><tr><td>Osmo1 Single-Sample Micro-Osmometer</td><td>Advanced Instruments</td><td>Osmo1</td><td>Alternative can be found at Fisher scientific (Wescor Inc. VAPRO&amp;reg; Vapor Pressure Osmometer)</td></tr><tr><td>Penicillin-Streptomycin</td><td>SIGMA</td><td>P4333</td><td>cell culture</td></tr><tr><td>poly-L-lysine</td><td>SIGMA</td><td>P4707</td><td>Other type of coating can be used that is dependent of the cell type to be cultured on the membrane, other adhesion factors such as fibronectin, collagen, polyornithine can be tested accordingly. Cell can be cultured directly on silicon nitride membrane, but the latter are slightly hydrophobic and adhesion factors are recommended unless the membrane are processed to be hydrophilic (glow plasma discharged).</td></tr><tr><td>Plunge freezing robot Leica EM GP main unit</td><td>Leica</td><td>16706401</td><td>Alternative for automated plunger are the Vitrobot Mark IV (FEI), CryoPlunge 3 (Gatan), MS-002 Rapid Immersion Freezer (EMS). Manual home-made system can be used but an environment-controlled chamber is an asset for plunge-freezing.</td></tr><tr><td>Silicon nitride membrane (Si3N4)</td><td>Silson Ltd.</td><td>SiRN-5.0(o)-200-1.5-500-NoHCl</td><td>The proposed silicon nitride membrane type is optimised for analysis at ID16A ESRF X-ray nanoprobe, The 500 nm thickness of the membrane was chosen being more robust for cellular manipulation and cryofixation detailed within this protocol. Membrane with thickness of 200 nm or below can also be used although quite fragile, and other design of silicon nitride membrane can be purchased (for example TEM compatible membrane...) from Sislon or other company such as Norcada, SPI supplies, Ted Pella, EMS, LabTech, Neyco...</td></tr><tr><td>Trypan blue solution 0.4%</td><td>GIBCO</td><td>15250061</td><td>cell culture</td></tr><tr><td>Trypsin-EDTA, 0.05%</td><td>GIBCO</td><td>25300-054</td><td>cell culture</td></tr><tr><td>Ultratrace Elemental Analysis Grade, Ultrapure Water</td><td>Fisher Chemicals</td><td>W9-1</td><td>MilliQ water can be used but has to be tested for trace element level of contamination using for example ICP-MS analysis.</td></tr><tr><td>Whatman No. 1 filter paper with precut hole</td><td>Leica</td><td>16706440</td><td>Alternative filter paper may be used and must have an outer diameter of 55 mm, the Punch for filter paper system from Leica (ref.16706443) can be used.</td></tr></tbody></table>

cell culture on silicon nitride membranes and cryopreparation for synchrotron x-ray fluorescence nano-analysis

Very little is known about the distribution of metal ions at the subcellular level. However, those chemical elements have essential regulatory functions and their disturbed homeostasis is involved in various diseases. State-of-the-art synchrotron X-ray fluorescence nanoprobes provide the required sensitivity and spatial resolution to elucidate the two-dimensional (2D) and three-dimensional (3D) distribution and concentration of metals inside entire cells at the organelle level. This opens new exciting scientific fields of investigation on the role of metals in the physiopathology of the cell. The cellular preparation is a key and often complex procedure, particularly for basic analysis. Although X-ray fluorescence techniques are now widespread and various preparation methods have been used, very few studies have investigated the preservation of the elemental content of cells at best, and no stepwise detailed protocol for the cryopreparation of adherent cells for X-ray fluorescence nanoprobes has been released so far. This is a description of a protocol that provides the stepwise cellular preparation for fast cryofixation to enable synchrotron X-ray fluorescence nano-analysis of cells in a frozen hydrated state when a cryogenic environment and transfer is available. In case nano-analysis has to be performed at room temperature, an additional procedure for freeze-drying the cryofixed adherent cellular preparation is provided. The proposed protocols have been successfully used in previous works, most recently in studying the 2D and 3D intracellular distribution of an organometallic compound in breast cancer cells.

A typical optical video microscope view of frozen hydrated MDA-MB-231 cells that were sub-cultured onto a poly-L-lysine coated Si3N4 membrane support is shown in Figure 7A. The optical view of the sample in the vacuum chamber was obtained in reflection mode using the dedicated online video microscope of the ID16A beamline of the ESRF22. While electron or soft X-ray microscopy requires the ice layer embedding the cell to be as thin as possible (typically &#60;0.5 &#181;m), hard X-rays (&#62;10 keV) have the advantage of a much higher penetration depth and lower dose deposition. The ice thickness can therefore be larger, typically &#60;10 &#181;m including the cell so that the ice embedding the cell is a few &#181;m in thickness. This can be estimated through the measured X-ray intensity in transmission compared to the intensity without the sample, taking into account the absorption of the 500 nm thick Si3N4 membrane. This ice thickness can be achieved through manual blotting as described in the present protocol. In the Newton rings region, the ice thickness can be even thinner (not measured).
The X-ray fluorescence elemental mapping of the frozen hydrated cell is shown in Figure 7B with the representative distributions of physiological elements such as potassium (K), sulfur (S), and zinc (Zn). These maps represent the elemental areal mass (i.e., elemental projected mass). While not done in the present case, such maps can be normalized through X-ray propagation-based phase contrast imaging that provides the estimation of the sample projected mass23. As reported by many studies, the highly diffusible K ion in cells preserved in their near-native state was assumed to be homogeneously distributed throughout the entire cell23,24,16. As shown in the 2D X-ray fluorescence elemental images in Figure 7B, the tightly bound element S was evenly distributed within the cell, similarly to K, and represents a good estimate of the cellular mass profile. The Zn distribution had a higher signal in the nucleus than in the cytosol and clearly outlined the nucleus. It can be noted that small Zn-enriched regions can be detected at the spatial resolution (50 nm) in the nuclear region.
The existing X-ray nanoprobes or the ones to be built do not necessarily accommodate cryogenic capabilities. In this case, the best alternative to get X-ray fluorescence images of cells at sub-100 nm spatial resolutions is to perform a freeze-drying procedure described in this protocol after plunge-freezing of the cell. Figure 8A shows a typical bright field microscopy view of resultant freeze-dried primary mouse hippocampal neurons directly cultured on the Si3N4 membrane. In this case, if stored in a clean desiccated chamber, the samples can be prepared 1&#8722;2 weeks in advance and be observed with an ordinary upright optical microscope for registration of regions of interest. Care should be taken to prevent exposure to ambient humidity as it may be captured by the freeze-dried sample and lead to damage under the X-ray nanobeam. This procedure was applied successfully to very sensitive cells (i.e., neuronal cells) and even better results were obtained with other more robust types of cells, such as cancer cells. As for plunge-frozen cells, the X-ray fluorescence images of K, S, and Zn on the entire freeze-dried cell display are similar to the ones described above. They are representative of the elemental distributions to be found in various types of freeze-dried cells at 50&#8722;100 nm spatial resolution. While freeze-drying whole cells is an alternative to preserve elemental integrity, it is at the expense of a perfect preservation of the cell morphology16, particularly cell membranes.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/60461/60461fig1.jpg" /> 
Figure 1: Typical sample support for X-ray fluorescence nano-analysis. A Si3N4 membrane support in its protective capsule. This type of substrate can be used both for room temperature analysis (plunge-freeze cellular preparation followed by low temperature and low vacuum freeze-drying process) or for cryogenic X-ray fluorescence analysis. <a href="https://www.jove.com/files/ftp_upload/60461/60461fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/60461/60461fig02.jpg" /> 
Figure 2: Schematic view of the silicon nitride windows after cell seeding. The cells are cultured directly onto the poly-L-lysine coated flat surface of the Si3N4 membrane support. Sometimes air bubbles can be trapped in the backside cavity of the Si3N4 membrane support and have to be removed as described in the protocol. <a href="https://www.jove.com/files/ftp_upload/60461/60461fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/60461/60461fig03.jpg" /> 
Figure 3: In-house developed 3D printed cryo-box for long-term storage of plunge frozen Si3N4 membrane supports in liquid nitrogen Dewar. (A) Cryo-box disassembled with the container and the caps (lower part) and (B) the assembled cryo-box with locked caps. The caps can be manipulated with the tweezers, opening or locking by rotation. A detailed plan for 3D printing is available upon request from ESRF ID16A. The design has been made to accommodate silicon nitride TEM grids. <a href="https://www.jove.com/files/ftp_upload/60461/60461fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/60461/60461fig4.jpg" /> 
Figure 4: Blotting of cells cultured on Si3N4. Prior to plunge-freezing the cell monolayer cultured onto a Si3N4 membrane needs to be rinsed in ammonium acetate solution (A) and carefully manually blotted using filter paper (B). <a href="https://www.jove.com/files/ftp_upload/60461/60461fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/60461/60461fig05.jpg" /> 
Figure 5: Automatic plunge-freezing EM-GP machine. (A) The automatic plunge freezer. (B) Environmental chamber with the tweezers locked in. (C) The ethane cup covered with the Leica liquefier connected to an ethane bottle. (D) The plunge-freezing enclosure showing the black cup full of liquified ethane and the cryo-box for further storage in LN2 of the vitrified Si3N4 membranes. <a href="https://www.jove.com/files/ftp_upload/60461/60461fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/60461/60461fig06.jpg" /> 
Figure 6: Sample cryotransfer assembly for freeze-drying procedure. (A) The first brass recipient for Si3N4 membranes is mounted on top of the sample transfer holder provided by the freeze dryer supplier. (B) and (C) show that the second flat brass disc is used as a cover and acts as a cold trap enclosure to be inserted in the vacuum enclosure of the freeze dryer. (D) The full assembly with the spring-loaded transfer rod. (E) The sample holder carrying the vitrified cellular preparation grown on the Si3N4 membrane must be further inserted in the LN2-cooled freeze dryer. All the steps for mounting the assembly are done in LN2 in a Styrofoam box. For clarity, all the images were produced in the absence of LN2. <a href="https://www.jove.com/files/ftp_upload/60461/60461fig6large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/60461/60461fig07.jpg" /> 
Figure 7: Cryo-X-ray fluorescence images of a frozen hydrated cell using hard X-ray nanoprobe. (A) Typical online view in reflection mode using the dedicated optical video microscope of the ESRF ID16A beamline. After manual blotting, a total ice thickness of about 5&#8722;10 &#181;m was achieved that allows a clear view of the frozen hydrated cells. A region with Newton rings indicative of even much thinner ice is noticeable. (B) Representative cryo-X-ray fluorescence cellular distributions of physiological elements potassium (K), sulfur (S), and zinc (Zn). <a href="https://www.jove.com/files/ftp_upload/60461/60461fig7large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 8" class="xfigimg" src="/files/ftp_upload/60461/60461fig08.jpg" /> 
Figure 8: X-ray fluorescence images of a freeze-dried neuronal cell using hard X-ray nanoprobe. (A) Typical bright field microscopy view of resultant freeze-dried primary cortical neuronal cells directly cultured onto the Si3N4 membrane. Scale bar = 200 &#181;m (B) Representative room temperature X-ray fluorescence images of a single freeze-dried hippocampal neuron showing the distributions of physiological elements potassium (K), sulfur (S), and zinc (Zn). Scale bar = 2 &#181;m. <a href="https://www.jove.com/files/ftp_upload/60461/60461fig8large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Cell Culture on Silicon Nitride Membranes and Cryopreparation for Synchrotron X-ray Fluorescence Nano-analysis at JoVE.com

Cell Culture on Silicon Nitride Membranes and Cryopreparation for Synchrotron X-ray Fluorescence Nano-analysis

Presented here is a protocol for cell culture on silicon nitride membranes and plunge-freezing prior to X-ray fluorescence imaging with a synchrotron cryogenic X-ray nanoprobe. When only room temperature nano-analysis is provided, the frozen samples can be further freeze-dried. These are critical steps to obtain information on the intracellular elemental composition.

Very little is known about the distribution of metal ions at the subcellular level. However, those chemical elements have essential regulatory functions ...

cell-culture-on-silicon-nitride-membranes-cryopreparation-for

Research

JoVE Journal

Biochemistry

9.1K Views.  Université Grenoble Alpes. Nieuw ontworpen synchrotron x-ray fluorescentie nano-probes maken visualisatie van de elementaire verdeling van een monster mogelijk. Het gebruik van cryogene benaderingen is verplicht om x-ray stralingsschade te vertragen. Daarom is een snelle cryofixatie essentieel voor het verkrijgen van bevroren gehydrateerde cellen om het behoud van de celstructuur en de chemische integriteit ervan te optimaliseren.De specificiteit van de cellulaire preparaten voor synchrotronstudies leiden tot de...

Video: Cell Culture on Silicon Nitride Membranes and Cryopreparation for Synchrotron X-ray Fluorescence Nano-analysis

Revealing Dynamic Processes of Materials in Liquids Using Liquid Cell Transmission Electron Microscopy

The recent development for in situ transmission electron microscopy, which allows imaging through liquids with high spatial resolution, has attracted significant interests across the research fields of materials science, physics, chemistry and biology. The key enabling technology is a liquid cell. We fabricate liquid cells with thin viewing windows through a sequential microfabrication process, including silicon nitride membrane deposition, photolithographic patterning, wafer etching, cell bonding, etc. A liquid cell with the dimensions of a regular TEM grid can fit in any standard TEM sample holder. About 100 nanoliters reaction solution is loaded into the reservoirs and about 30 picoliters liquid is drawn into the viewing windows by capillary force. Subsequently, the cell is sealed and loaded into a microscope for in situ imaging. Inside the TEM, the electron beam goes through the thin liquid layer sandwiched between two silicon nitride membranes. Dynamic processes of nanoparticles in liquids, such as nucleation and growth of nanocrystals, diffusion and assembly of nanoparticles, etc., have been imaged in real time with sub-nanometer resolution. We have also applied this method to other research areas, e.g., imaging proteins in water. Liquid cell TEM is poised to play a major role in revealing dynamic processes of materials in their working environments. It may also bring high impact in the study of biological processes in their native environment.

We have developed a self-contained liquid cell, which allows imaging through liquids using a transmission electron microscope. Dynamic processes of nanoparticles in liquids can be revealed in real time with sub-nanometer resolution.

The recent development for in situ transmission electron microscopy, which allows imaging through liquids with high spatial resolution, has attracted ...

Engineering

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.

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 ...

Chemistry

Measurement of Total Calcium in Neurons by Electron Probe X-ray Microanalysis

In this article the tools, techniques, and instruments appropriate for quantitative measurements of intracellular elemental content using the technique known as electron probe microanalysis (EPMA) are described. Intramitochondrial calcium is a particular focus because of the critical role that mitochondrial calcium overload plays in neurodegenerative diseases. The method is based on the analysis of X-rays generated in an electron microscope (EM) by interaction of an electron beam with the specimen. In order to maintain the native distribution of diffusible elements in electron microscopy specimens, EPMA requires &#34;cryofixation&#34; of tissue followed by the preparation of ultrathin cryosections. Rapid freezing of cultured cells or organotypic slice cultures is carried out by plunge freezing in liquid ethane or by slam freezing against a cold metal block, respectively. Cryosections nominally 80 nm thick are cut dry with a diamond knife at ca. -160 &#176;C, mounted on carbon/pioloform-coated copper grids, and cryotransferred into a cryo-EM using a specialized cryospecimen holder. After visual survey and location mapping at &#8804;-160 &#176;C and low electron dose, frozen-hydrated cryosections are freeze-dried at -100 &#176;C for ~30 min. Organelle-level images of dried cryosections are recorded, also at low dose, by means of a slow-scan CCD camera and subcellular regions of interest selected for analysis. X-rays emitted from ROIs by a stationary, focused, high-intensity electron probe are collected by an energy-dispersive X-ray (EDX) spectrometer, processed by associated electronics, and presented as an X-ray spectrum, that is, a plot of X-ray intensity vs. energy. Additional software facilitates: 1) identification of elemental components by their &#34;characteristic&#34; peak energies and fingerprint; and 2) quantitative analysis by extraction of peak areas/background. This paper concludes with two examples that illustrate typical EPMA applications, one in which mitochondrial calcium analysis provided critical insight into mechanisms of excitotoxic injury and another that revealed the basis of ischemia resistance.

This paper describes the application of cryoanalytical electron microscopy to the quantitative measurement of total calcium content and distribution at subcellular resolution in physiologically defined biological specimens.

In this article the tools, techniques, and instruments appropriate for quantitative measurements of intracellular elemental content using the technique ...

Neuroscience

Preparation and Cryo-FIB micromachining of Saccharomyces cerevisiae for Cryo-Electron Tomography

Today, cryo-electron tomography (cryo-ET) is the only technique that can provide near-atomic resolution structural data on macromolecular complexes in situ. Owing to the strong interaction of an electron with the matter, high-resolution cryo-ET studies are limited to specimens with a thickness of less than 200 nm, which restricts the applicability of cryo-ET only to the peripheral regions of a cell. A complex workflow that comprises the preparation of thin cellular cross-sections by cryo-focused ion beam micromachining (cryo-FIBM) was introduced during the last decade to enable the acquisition of cryo-ET data from the interior of larger cells. We present a protocol for the preparation of cellular lamellae from a sample vitrified by plunge freezing utilizing Saccharomyces cerevisiae as a prototypical example of a eukaryotic cell with wide utilization in cellular and molecular biology research. We describe protocols for vitrification of S. cerevisiae into isolated patches of a few cells or a continuous monolayer of the cells on a TEM grid and provide a protocol for lamella preparation by cryo-FIB for these two samples.

Preparation and Cryo-FIB micromachining of Saccharomyces cerevisiae for Cryo-Electron Tomography

We present a protocol for lamella preparation of plunge frozen biological specimens by cryo-focused ion beam micromachining for high-resolution structural studies of macromolecules in situ with cryo-electron tomography. The presented protocol provides guidelines for the preparation of high-quality lamellae with high reproducibility for structural characterization of macromolecules inside the&#160;Saccharomyces cerevisiae.

Today, cryo-electron tomography (cryo-ET) is the only technique that can provide near-atomic resolution structural data on macromolecular complexes in ...

Nanoscale Characterization of Liquid-Solid Interfaces by Coupling Cryo-Focused Ion Beam Milling with Scanning Electron Microscopy and Spectroscopy

Physical and chemical processes at solid-liquid interfaces play a crucial role in many natural and technological phenomena, including catalysis, solar energy and fuel generation, and electrochemical energy storage. Nanoscale characterization of such interfaces has recently been achieved using cryogenic electron microscopy, thereby providing a new path to advancing our fundamental understanding of interface processes.
This contribution provides a practical guide to mapping the structure and chemistry of solid-liquid interfaces in materials and devices using an integrated cryogenic electron microscopy approach. In this approach, we pair cryogenic sample preparation which allows stabilization of solid-liquid interfaces with cryogenic focused ion beam (cryo-FIB) milling to create cross-sections through these complex buried structures. Cryogenic scanning electron microscopy (cryo-SEM) techniques performed in a dual-beam FIB/SEM enable direct imaging as well as chemical mapping at the nanoscale. We discuss practical challenges, strategies to overcome them, as well as protocols for obtaining optimal results. While we focus in our discussion on interfaces in energy storage devices, the methods outlined are broadly applicable to a range of fields where solid-liquid interface play a key role.

Cryogenic Focused Ion Beam (FIB) and Scanning Electron Microscopy (SEM) techniques can provide key insights into the chemistry and morphology of intact solid-liquid interfaces. Methods for preparing high quality Energy Dispersive X-ray (EDX) spectroscopic maps of such interfaces are detailed, with a focus on energy storage devices.

Physical and chemical processes at solid-liquid interfaces play a crucial role in many natural and technological phenomena, including catalysis, solar ...

Cryo-electron Microscopy Specimen Preparation By Means Of a Focused Ion Beam

Here we present a protocol used to prepare cryo-TEM samples of Aspergillus niger spores, but which can easily be adapted for any number of microorganisms or solutions. We make use of a custom built cryo-transfer station and a modified cryo-SEM preparation chamber2. The spores are taken from a culture, plunge-frozen in a liquid nitrogen slush and observed in the cryo-SEM to select a region of interest. A thin lamella is then extracted using the FIB, attached to a TEM grid and subsequently thinned to electron transparency. The grid is transferred to a cryo-TEM holder and into a TEM for high resolution studies. Thanks to the introduction of a cooled nanomanipulator tip and a cryo-transfer station, this protocol is a straightforward adaptation to cryogenic temperature of the routinely used FIB preparation of TEM samples. As such it has the advantages of requiring a small amount of modifications to existing instruments, setups and procedures; it is easy to implement; it has a broad range of applications, in principle the same as for cryo-TEM sample preparation. One limitation is that it requires skillful handling of the specimens at critical steps to avoid or minimize contaminations.

Cryo Electron Microscopes, either Scanning (SEM) or Transmission (TEM), are widely used for characterization of biological samples or other materials with a high water content1. A SEM/Focused Ion Beam (FIB) is used to identify features of interest in samples and extract a thin, electron-transparent lamella for transfer to a cryo-TEM.

Here we present a protocol used to prepare cryo-TEM samples of Aspergillus niger spores, but which can easily be adapted for any number of microorganisms ...

Biology

Graphene Enclosure of Chemically Fixed Mammalian Cells for Liquid-Phase Electron Microscopy

A protocol is described for investigating the human epidermal growth factor receptor 2 (HER2) in the intact plasma membrane of breast cancer cells using scanning transmission electron microscopy (STEM). Cells of the mammalian breast cancer cell line SKBR3 were grown on silicon microchips with silicon nitride (SiN) windows. Cells were chemically fixed, and HER2 proteins were labeled with quantum dot nanoparticles (QDs), using a two-step biotin-streptavidin binding protocol. The cells were coated with multilayer graphene to maintain a hydrated state, and to protect them from electron beam damage during STEM. To examine the stability of the samples under electron beam irradiation, a dose series experiment was performed. Graphene-coated and non-coated samples were compared. Beam induced damage, in the form of bright artifacts, appeared for some non-coated samples at increased electron dose D, while no artifacts appeared on coated samples.

Presented here is a protocol for labeling membrane proteins in mammalian cells and coating the sample with graphene for liquid-phase scanning transmission electron microscopy. The stability of the samples against the damage caused by radiation can also studied with this protocol.

A protocol is described for investigating the human epidermal growth factor receptor 2 (HER2) in the intact plasma membrane of breast cancer cells using ...

A 3D Cartographic Description of the Cell by Cryo Soft X-ray Tomography

Imaging techniques are fundamental in order to understand cell organization and machinery in biological research and the related fields. Among these techniques, cryo soft X-ray tomography (SXT) allows imaging whole cryo-preserved cells in the water window X-ray energy range (284-543 eV), in which carbon structures have intrinsically higher absorption than water, allowing the 3D reconstruction of the linear absorption coefficient of the material contained in each voxel. Quantitative structural information at the level of whole cells up to 10 &#181;m thick is then achievable this way, with high throughput and spatial resolution down to 25-30 nm half-pitch. Cryo-SXT has proven itself relevant to current biomedical research, providing 3D information on cellular infection processes (virus, bacteria, or parasites), morphological changes due to diseases (such as recessive genetic diseases) and helping us understand drug action at the cellular level, or locating specific structures in the 3D cellular environment. In addition, by taking advantage of the tunable wavelength at synchrotron facilities, spectro-microscopy or its 3D counterpart, spectro-tomography, can also be used to image and quantify specific elements in the cell, such as calcium in biomineralization processes. Cryo-SXT provides complementary information to other biological imaging techniques such as electron microscopy, X-ray fluorescence or visible light fluorescence, and is generally used as a partner method for 2D or 3D correlative imaging at cryogenic conditions in order to link function, location, and morphology.

Here, a protocol describing the sample preparation and data collection steps required in cryo soft X-ray tomography (SXT) to image the ultrastructure of whole cryo-preserved cells at a resolution of 25 nm half pitch, is presented.

Imaging techniques are fundamental in order to understand cell organization and machinery in biological research and the related fields. Among these ...

Sample Preparation by 3D-Correlative Focused Ion Beam Milling for High-Resolution Cryo-Electron Tomography

Cryo-electron tomography (cryo-ET) has become the method of choice for investigating cellular ultrastructure and molecular complexes in their native, frozen-hydrated state. However, cryo-ET requires that samples are thin enough to not scatter or block the incident electron beam. For thick cellular samples, this can be achieved by cryo-focused ion beam (FIB) milling. This protocol describes how to target specific cellular sites during FIB milling using a 3D-correlative approach, which combines three-dimensional fluorescence microscopy data with information from the FIB-scanning electron microscope. Using this technique, rare cellular events and structures can be targeted with high accuracy and visualized at molecular resolution using cryo-transmission electron microscopy (cryo-TEM).

Here, we present a pipeline for 3D-correlative focused ion beam milling on guiding the preparation of cellular samples for cryo-electron tomography. The 3D position of fluorescently tagged proteins of interest is first determined by cryo-fluorescence microscopy, and then targeted for milling. The protocol is suitable for mammalian, yeast, and bacterial cells.

Cryo-electron tomography (cryo-ET) has become the method of choice for investigating cellular ultrastructure and molecular complexes in their native, ...

Direct Synthesis of Gold Nanoparticles for Protein Localization Analysis

Direct Synthesis of EM-Visible Gold Nanoparticles in Cells for Protein Localization Analysis with Well-Preserved Ultrastructure

Analyzing the precise localization of protein molecules in cells with ultrastructural resolution is of great significance for the study of various physiological or pathological processes in all living organisms. Therefore, the development of clonable tags that can be used as electron microscopy probes is of great value, just as fluorescent proteins have played a crucial role in the field of optical imaging. The autonucleation suppression mechanism (ANSM) was recently uncovered, which allows for the specific synthesis of gold nanoparticles (AuNPs) on cysteine-rich tags, such as metallothionein (MT) and antifreeze protein (AFP).
Based on the ANSM, an electron microscopy labeling technology was developed, which enables the specific detection of tagged proteins in prokaryotic and eukaryotic cells with an unprecedented labeling efficiency. This study illustrates a protocol for the detection of MTn (an engineered MT variant lacking aldehyde-reactive residues) fusion proteins in mammalian cells with well-preserved ultrastructure. In this protocol, high-pressure freezing and freeze-substitution fixation were performed using non-aldehyde fixatives (such as tannic acid, uranyl acetate) to preserve near-native ultrastructure and avoid damage to the tag activity caused by aldehyde crosslinking.
A simple one-step rehydration was used prior to the ANSM-based AuNP synthesis. The results showed that the tagged proteins targeted various organelles, including the membranes and the lumen of the endoplasmic reticulum (ER), and mitochondrial matrices were detected with high efficiency and specificity. This research provides biologists with a robust protocol to address an enormous range of biological questions at the single-molecule level in cellular ultrastructural contexts.

Author Spotlight: Direct Synthesis of EM-Visible Gold Nanoparticles in Cells for Protein Localization Analysis with Well-Preserved Ultrastructure  

The present protocol describes a clonable electron microscopy labeling technology for detecting metallothionein-tagged proteins in cells using a novel autonucleation suppression mechanism-based gold nanoparticle synthesis technique.

Cell Culture on Silicon Nitride Membranes and Cryopreparation for Synchrotron X-ray Fluorescence Nano-analysis

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