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 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.
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–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.
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 (<10 µm) 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 ≥ 1.3 keV magnesium). Indeed, a high K/Na ratio (>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.
The authors have nothing to disclose.
The experiments on the nano-imaging beamline ID16A were performed in the frame of ESRF proposals LS2430, LS2303, and LS2765.
Ammonium Acetate solution, BioUltra, for molecular biology, ~5M in H2O | SIGMA | 09691-250mL | One can prepare the required solution from high-grade ammonium acetate powder and ultrapure water, pH and osmolarity needs to be adjusted anyway. |
B27 supplement, 50x | Life Technologies, Invitrogen | 17504-044 | for hippocampal neuron culture |
Dulbecco’s Phosphate Buffered Saline, DPBS, ([-] CaCl2, [-] MgCl2) | GIBCO | 14190-094 | cell culture |
DMEM with Phenol Red/Glutamax I (Medium ATCC modification) | GIBCO | 21885025 | cell culture |
Dulbecco’s modified Eagle medium (DMEM) | Life Technologies, Invitrogen | 31966-02 | for hippocampal neuron culture |
Dumont Tweezers #5, Straight Self-closing, 0.05×0.01mm Tips, Biology | World Precision Instrument | 501202 | |
Emitech K750X Peltier-Cooled EM Freeze Dryer | Quorum Technology | EK3147 | |
Ethane N45 | Air Liquid | p0505s05r0a001 | C2H6 > 99,995 % |
Fetal Bovine Serum, Performance Plus, certified One Shot format, US origin | GIBCO | A31604-02 | cell culture |
HBSS 10x | Life Technologies, Invitrogen | 14185-052 | for hippocampal neuron culture |
Leica GP quick-release forceps | Leica | 16706435 | |
MDA-MB-231 cell line, an epithelial, human adenocarcinoma breast cancer cell | ATCC | ATCC HTB-26 | cell culture |
Neurobasal medium | Life Technologies, Invitrogen | 21103-049 | for hippocampal neuron culture |
Nunc 4-Well Plate | Thermo Fisher | 176740 | cell culture |
Osmo1 Single-Sample Micro-Osmometer | Advanced Instruments | Osmo1 | Alternative can be found at Fisher scientific (Wescor Inc. VAPRO® Vapor Pressure Osmometer) |
Penicillin-Streptomycin | SIGMA | P4333 | cell culture |
poly-L-lysine | SIGMA | P4707 | 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). |
Plunge freezing robot Leica EM GP main unit | Leica | 16706401 | 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. |
Silicon nitride membrane (Si3N4) | Silson Ltd. | SiRN-5.0(o)-200-1.5-500-NoHCl | 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… |
Trypan blue solution 0.4% | GIBCO | 15250061 | cell culture |
Trypsin-EDTA, 0.05% | GIBCO | 25300-054 | cell culture |
Ultratrace Elemental Analysis Grade, Ultrapure Water | Fisher Chemicals | W9-1 | MilliQ water can be used but has to be tested for trace element level of contamination using for example ICP-MS analysis. |
Whatman No. 1 filter paper with precut hole | Leica | 16706440 | 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. |