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protocol Preparation of cells for assessing ultrastructural localization of nanoparticles with transmission electron microscopy 1 1 2 1 Amanda M Schrand , John J Schlager , Liming Dai & Saber M Hussain 1 2 AFRL/711 HPW/RHPB, Wright-Patterson Air Force Base, Dayton, Ohio, USA. Department of Chemical Engineering, Case Western Reserve University, Cleveland, Ohio, USA. Correspondence should be addressed to S.M.H. (saber.hussain@wpafb.af.mil). Published online 25 March 2010; doi:10.1038/nprot.2010.2 otocolsWe describe the use of transmission electron microscopy (teM) for cellular ultrastructural examination of nanoparticle (np)- exposed biomaterials. preparation and imaging of electron-transparent thin cell sections with teM provides excellent spatial resolution (~1 nm), which is required to track these elusive materials. this protocol provides a step-by-step method for the natureprmass-basis dosing of cultured cells with nps, and the process of fixing, dehydrating, staining, resin embedding, ultramicrotome / sectioning and subsequently visualizing np uptake and translocation to specific intracellular locations with teM. In order to m o c avoid potential artifacts, some technical challenges are addressed. Based on our results, this procedure can be used to elucidate . e r the intracellular fate of nps, facilitating the development of biosensors and therapeutics, and provide a critical component for u t a understanding np toxicity. this protocol takes ~1 week. n . w w w / / : p t t IntroDuctIon h The recent development of engineered nanoparticles (NPs) has while sacrificing the nano-sized vision area. In this case, the rela- p u attracted the attention of many individuals from diverse scientific tionship between certain organelles, which are usually sized from o r disciplines, who are rapidly pursuing a plethora of exciting and new submicrons to microns, and NPs internalized into those organelles G g applications. Concurrently, the implications for potential long-term 22–24 n (e.g., endosomes and lysosomes) can be explored . In addition, i health and environmental applications are being addressed by vari- the serial sectioning capabilities of confocal microscopy can be used h s i 1–4 l ous working groups, although at a considerably slower pace . In to identify more qualitatively the uptake of NP agglomerates into b u our studies, we focus on the NP toxicity-associated bioeffects that certain organelles in living cells. However, there is still insufficient P produce acute dose-dependent decreases in viability and alterations resolution ( > 100 nm) to examine individual NPs. e r u in cell function (e.g., membrane leakage, mitochondrial damage, To gain the resolution required to view individual NPs t a reactive oxygen species (ROS) generation, cytokine production, ( < 100 nm), electron microscopy is typically carried out. However, N 0 5–17 1 up- or down-regulation of genes and so on) . Certainly, long term fragile biological samples such as cultured cells used in mechanis- 0 or chronic effects will require much further investigation. For an tic studies require dehydration, heavy-metal staining and electron 2 © overview of recent bioeffect achievements, challenges and current transparency for the sample to withstand the vacuum conditions understanding of NP behavior at the bio-interface, see reviews by and generate appropriate signal contrast to form an image. Further our group18,19 and others20,21. limitations of TEM include time consuming and toxic sample In addition to measuring biochemical cell alterations after preparation, difficulties in distinguishing low-contrast nano-sized exposure to NPs, a variety of microscopic methods, ranging from materials from cellular background features (e.g., cytoplasmic simple light microscopy to more complex electron microscopy, can granules), production of two-dimensional black and white images be used to determine the uptake and intracellular localization of NPs and difficulty in drawing statistical conclusions. At present, stere- inside cells. For example, we have used an ultrahigh resolution imaging ological principles are being utilized to quantify the spatial distri- system, which attaches to a standard research-grade inverted bution of immunogold and other NPs based on their localization microscope for the examination of NP interactions and possible throughout the cells and tissues for statistical evaluation25. These 7,8,13–15,18 2 internalization into live cells . Frequently, surface-active NPs studies rely on χ analysis between treatment groups or within a sin- in agglomerated structures are observed using this method. How- gle group to determine differences in uptake amount or localization ever, the exact localization of individual NPs inside of the cells to specific intracellular compartments. The purpose of these studies was not determined owing to resolution limitations (~150 nm) is being able to carry out these relative quantification techniques and the inability to carry out serial sectioning to distinguish mem- in the TEM in an unbiased manner. brane-bound versus internalized NPs. Other limitations of live cell The use of lower voltage imaging with scanning TEM (STEM) imaging with light microscopy include organelle autofluorescence, in a standard scanning electron microscope (SEM) may generate subtle changes in brightness with NPs found inside or outside greater contrast without the use of heavy-metal stains, but requires of cells and short observation times, which can alter the image an electron-transparent sample; hence a protocol involving embed- interpretation. Fluorescent microscopy suffers many of the same ding and thin sectioning is presented. Hydrated samples can be limitations as light microscopy and is limited to NPs that will emit viewed under high vacuum conditions in the SEM if specialized light upon excitation. However, fluorescent microscopy is still a capsules (e.g., from Quantomix, Rehovot, Israel) are used. However, very valuable technique to observe illuminated internalized NPs we found that heavy-metal staining was still required to generate | | | 744 VOL.5 NO.4 2010 nature protocols protocol | Figure 1 Cellular structures, possible mechanisms of nanoparticle (NP) uptake and some potential NP physicochemical uptake factors. R V Receptor (R), vacuole (V), mitochondria (M), cytoplasmic granules (CG), CG NP uptake based on size, shape, cytoskeleton (C), rough endoplasmic reticulum (RER), smooth endoplasmic M composition, charge, reticulum (SER), nucleus (N), golgi (G), endosome (E) and lysosome (L). SER surface Steps in endocytic uptake (1–4) or nonendocytic mechanism. coating/ surface RER N chemistry, cell type, C 4 cellular energy L requirements and so on. sufficient contrast and that only high atomic number NPs were E 1 readily detectable. More recently, de Jonge et al.25 have unveiled Intracellular 3 G 2 Endocytic uptake a new STEM-based technique for imaging whole cells in liquid by a microfluidic device with electron-transparent windows26. Extracellular Other groups have used field emission SEM (FESEM) to directly Non-endocytic entry mechanism observe nuclear morphologies, including nuclear envelope struc- tures, with immunolabeling or freeze fracture techniques27–29. versus non-differentiated cells and cells with shorter versus longer otocols 54 Although alternative electron microscopy and spectroscopic tech- doubling times . These factors and more should be taken into con- niques are continuously being developed to combat the artifacts sideration when choosing a cell line for NP research. However, no natureprgenerated during the extensive sample preparation (e.g., fixation, one particular cell type is currently favored for NP-uptake studies. / dehydration and heavy-metal staining) required for high vacuum, Further, the cell line chosen for toxicity studies should be based m o c high-voltage electron beam energy imaging of thin sections in upon the potential target organ or application. . e r TEM, there are no true comparative methods. Therefore, TEM u t a is routinely used by many groups to determine the uptake and General characterization techniques for NPs. For the NPs in these n . w localization of NPs inside cells, as well as provide clues to the studies, characterization was accomplished with TEM for size w 30–37 38 and morphology, inductively coupled plasma–optical emission w uptake mechanism whether it is endocytic or not (Fig. 1). / / : In TEM, an electron beam is transmitted through samples spectroscopy for purity, dynamic light scattering (DLS) for p t t h typically < 100 nm thick to generate a bright-field (BF) image con- hydrodynamic radius in solution and zeta potential to estimate p taining information about the internal structure of the sample. charge in solution. The lack of sufficient pre-exposure charac- u o In addition to TEM BF imaging, TEM cryo-microscopy, tomo- terization details in NP-dosing studies is being addressed in r G graphy and other TEM imaging modes (e.g., high-angle annular many different ways. Analysis using ‘dry’ characterization is being g n dark-field imaging, energy-filtered TEM (EF-TEM), electron carried out to determine initial primary size, mono- or poly- i h 39–41 dispersity of the size distribution, length, diameter, surface s energy loss spectroscopy and aberration corrected STEM) can i l b image unstained or stained cells. The sample preparation tech- area, elemental composition/trace impurities, crystallinity, etc. u 42,43 P niques can include freeze substitution and freeze drying with However, once the NPs are introduced into cell-culture media, e r the cells grown directly on Au TEM grids coated with various bio- their surface properties change because of interactions with water u t 44,45 and aqueous solution salts, small organic molecules, proteins a molecules (e.g., l-lysine, fibronectin and laminin) . Subsequent N thinning of frozen samples can be accomplished after embedding in and other cell constituents. These solution interactions can affect 0 1 46 47 NP cellular dynamics by immediate and dynamic surface-modi- 0 resin or with a focused-ion beam instrument . Therefore, nano- 2 materials and cell components, such as organelles, can be clearly fication effects such as material size, surface chemistry and deliv- © imaged by TEM once they have been correctly prepared. Here we ered dose, which are likely different for each NP composition. present our current protocol for streamlining the preparation A clear understanding of NP surface dynamics remains poorly of cells for TEM analysis after dosing with NPs using traditional understood and still is a significant hurdle to overcome in this dehydration, embedding and thin sectioning procedures. The field. In addition to the characterization techniques mentioned information gained from thin sections of cells incubated with above, further microscopic solution characterization such as cryo- NPs is critical for understanding the interaction and underlying electron microscopy or computer modeling on the forces contributing mechanisms involved in their uptake, associated applications and to NP interactions with media components, cell membrane and potential toxicity. intercellular environments (e.g., van der Waals forces, electrostatic double-layer interactions, short-range forces arising from charge, Experimental design steric hindrance, NP dissolution, ion leaching, phase transforma- Cell types. A multitude of cell lines are currently available that can tion and solvent interactions and so on), are being explored21. represent target organs of NP exposure (e.g., lung and skin) or have However, it is not the focus of this protocol to impose a particular clinical relevance (e.g., cancer cells versus normal cells). During set of NP parameters on the experimenter, but rather to provide our studies with multiple cell types, we began to notice several some general handling guidelines for NP-uptake studies. important differences in their innate response to NPs regarding internalization mechanism and degree of inflammatory cascade. NP types. The different types of NPs that we have studied include, We demonstrated that immune cells (e.g., alveolar macrophages) but are not limited to, the following: manganese (Mn), silver (Ag), display differential toxicity to various carbon nanomaterials com- single-walled carbon nanotubes (SWNTs), multi-walled carbon pared with neuroblastoma cells; possibly due to greater NP accu- nanotubes (MWNTs), nanodiamonds (ND), carbon black (CB), mulation10. Other recent studies have revealed similar cell-specific silica (SiO ), aluminum (Al), aluminum oxide (Al O ), titanium 2 2 3 21,48–54 dioxide (TiO ), copper (Cu) and gold (Au). For demonstration trends in toxicity and uptake as well as noting the importance 2 of NP uptake during certain stages of the cell cycle in differentiated purposes, we chose a few different carbon-based NPs (NDs (1), | | | nature protocols VOL.5 NO.4 2010 745 protocol CB (2) and SWNT and MWNT (3)) to demonstrate their uptake a b into neuroblastoma (N2A) cells. 1. NDs: The NDs used in our laboratory were generously supplied by NanoCarbon Research Institute in Nagano, Japan and were synthesized according to previously reported deto- 55,56 nation techniques . The NDs had primary sizes of 5.1 ± 1.7 nm with 0.13 wt% Fe and 0.23 wt% Zr impurity content from the detonation container and bead milling. They formed strong aggregates in water of ~158 nm that dra- matically increased in size to 2,180 nm in cell-culture media. The charge measured with zeta potential was ~43 mV. 2. CB: CB NPs used in our research were received from Shell/ Cabot (Boston, MA, USA) and were synthesized using an otocols oil furnace process. As furnace-type CB NPs are made from petroleum feedstocks, CB can contain varying amounts of other elements (e.g., sulfur), up to 1 wt%. The CB NPs had naturepr | / primary sizes of 28.8 ± 8.4 nm and 0.43 wt% sulfur-impurity Figure 2 Issue of nanoparticle (NP) agglomeration and dispersion. m content. Because of van der Waals forces, they readily form Multi-walled carbon nanotubes (MWNTs) added to water. (a) Before o c sonication where the hydrophobic nature causes the MWNTs to aggregate . very strong aggregates in aqueous solutions. For example, CB e r at the surface of water. (b) After sonication where the MWNTs are u t NPs have much larger sizes in water (an average of 396 nm) a temporarily suspended in the water before dilution in cell-culture media. n . than their primary size of ~30 nm. Further, CB analyzed in w DMEM/F-12-dosing cell-culture media (no serum) produced w w / / aggregates of ~2,190 nm. physical size change effecting NP surface and concentration dos- : p t 3. MWNTs and SWNTs: MWNTs were purchased from Tsin- t ing by irreversible agglomeration. To combat agglomeration, we h ghua University (Beijing, China) and SWNTs were received typically employ a brief sonication to disperse materials such as p u from Rice University (Houston, TX, USA). SWNTs and carbon nanotubes (Fig. 2). Although conventional sonication o r MWNTs were synthesized by chemical vapor deposition. The in water baths or with probe-tip sonicators has extensively been G g maximum lengths of both the SWNT and MWNT were not used to disperse NPs, new techniques such as bead-assisted sonic n i as readily calculated owing to bundling and tangling. The disintegration have been demonstrated to break up persistent h s i SWNTs existed in bundles that were greater than 3 µm in l agglomerates of NDs concurrent with surface functionaliza- b u length with bundle diameters of ~25 nm, whereas individual tion57. However, chemical-surface modification achieved through P SWNT diameters were 1–3 nm. The MWNTs were estimated extensive sonication or very high-energy sonic pulses may mark- e r u to be from 0.5 to 40 µm in length with diameters from edly change the NP surface characteristics and should be avoided. t a 9–40 nm. The MWNTs had a residual Fe catalyst content Teeguarden et al.64 provide a further discussion of the influence of N 0 of 0.49 wt%, whereas the SWNT had a residual Fe catalyst NP and cell-culture media characteristics on dosiometry. 1 0 content of 0.26 wt%. The DLS size approximation is based on 2 © a spherical particle assumption for calculation, so the results Cell and NP controls. Control cells that are not dosed with NPs but for SWNT and MWNT solutions would not be meaningful with dosing media only are required for comparison of processed due to NT disparity of diameter and length creating. samples for TEM. In toxicological studies, CB has frequently been The results show high polydispersity reading. For example, used as a negative control and we have employed micron-sized in water the SWNTs had a mean size of ~900 nm and MWNTs cadmium oxide as a positive toxic substance control. However, ~821 nm. The zeta potential of the SWNTs was 50.2 mV these materials must also be considered thoughtfully for applica- compared with − 13.6 for MWNTs. tion and comparison. The heterogeneous nature of different CB samples has been addressed by the Monteiro group and others Additional chemicals or treatments for NP dispersion. Although for significant variability depending upon the acquired source. some newly engineered NPs show enhanced stability in biologi- The US National Bureau of Standards, now called the National cal media57,58, the issue of NP agglomeration in cell-culture media Institutes of Standards and Technology, is currently developing before dosing cells is a well-known phenomenon and there is not suitable standard reference materials (SRM). The most likely SRM a common solution capable of suspending all types of NPs. To candidates include nonactive materials such as Au (10, 30 and 60 examine dispersion in aqueous stock solution medium before nm) or polystyrene (60 and 100 nm) spheres, which have very nar- dosing cells, we have used high illumination light microscopy7,8 row size distributions (http://www.nist.gov). Other solutions to and DLS15. Other techniques to examine or modify dispersion test as controls can include additives such as surfactants or surface include the addition of surfactants8,59 and centrifugation30,60–63. coating components separate from the NPs. Other considerations The latter may serve a second role to filter out possible bacterial for toxicity controls can include cells not stained, examination of contaminants30. However, we do not use surfactants or any form the NPs without cells and examination by independent laborato- of centrifugation before dosing to avoid problematic interfer- ries to confirm the results53. However, in NP-uptake studies, the ence with the inherent surface chemistry of the NPs, which would best ‘control’ is to prepare and image cells that have not undergone mask NP surface bioeffects and provide potential cytotoxic effects any experimental treatment but that are handled and prepared in of surfactants on the cells8,59. Centrifugation can force overall exactly the same procedure as the NP-dosed cells. | | | 746 VOL.5 NO.4 2010 nature protocols protocol Length of treatment time. The length of treatment time can and glutaraldehyde include the ability of paraformaldehyde to depend upon the toxicity of the NP, cell type and purpose of the quickly stabilize the protein structure through crosslinking and experiment. For example, low-toxicity NPs may be able to accu- glutaraldehyde to more slowly and permanently fix the cells (more mulate over days without significant changes in cell adherence or crosslinking) for better preservation of tissue for both light (histo- morphology, leading to a sample suitable for further processing logical staining) and electron microscopy78,79. Glutaraldehyde for TEM. For example, quantum dots were used to noninvasively fixation alone may not be sufficient because lipids and other cell label Dictyostelium or HeLa cells for over 12 d without affecting cell components may not be fixed. Although the aldehyde component 65 growth or development . In contrast, highly toxic NPs may lead to is the major preservative of proteins, the later post-fixation with cell rounding and detachment from substrates with highly vacu- osmium tetroxide helps to preserve lipids well and proteins to some olated cytoplasms and may not be ideal for further processing for extent. Fixation artifacts typically include extraction of material, TEM. In cell types that have a greater propensity for NP uptake (i.e., distortion of organelles, displacement of chemical components macrophages, monocytes and neutrophils), shorter time points and anomalous deposits (see Troubleshooting section for potential should be considered (see previous discussion of cell-specific dif- artifacts and remedies). It is beneficial, but not necessary, to use a otocolsferences). In contrast, for experiments designed to elaborate upon rotator during the fixation and embedding procedure to ensure the mechanism of NP entry, accumulation and exit, a time course thorough mixing and penetration. approach from 1 to 24 h or longer can be utilized. For cytotoxicity For cell isolation, most cells are grown adherent and must be naturepranalysis, Lanone et al.53 suggested that the 24 h time point (versus enzymatically or mechanically detached. However, there is not a / 3 h) provided more sensitive data. In agreement with this study, the well-defined speed to centrifuge cells into a pellet for TEM process- m o c Monteiro group recommends toxicity assays continue for at least ing. Although we utilize a centrifugation speed of 1,000g in our . e r 24 h to complete one cell cycle, with 24 h being a common cultured- studies, higher speeds have been reported in the literature with u t 73 a cell doubling time and after 48 h, one finds further decreases in cell good results. For example, Nativo et al. used a speed of 5,000g n . 66 w viability . Issues that the researcher can expect to address in stud- and the Monteiro–Riviere group typically uses a very rapid 10 sec w ies > 24 h include cell proliferation, microbial growth and possible spin on a microcentrifuge at 12,500 r.p.m. at 21°C (personal corres- w / / : removal and reapplication of dosing solutions. Therefore, in this pondence). Apart from damage to the cells at high centrifugation p t t h protocol we suggest a dosing period of 24 h or shorter. speeds, there is some concern that centrifugal forces could con- p tribute to unwanted interactions between the NPs and cells. In u o Concentrations of NPs. The calculation of NP dose has been our studies, the cells are grown as a monolayer and thoroughly r G carefully considered, and to date most studies employ a mass-basis washed, leaving very few NPs behind to contribute to sedimenta- g n approach compared with surface area calculations, which may or tion or artificial penetration. Further isolation of the cells in an i h may not accurately represent the interface of the NPs with individual agar pellet may reduce these unintended interactions. For example, s i l cells. Support for the mass-basis approach can be found in a study embedding the cells in 2–3% molten agar before post-fixation with b u 60 P by Limbach et al. , where cerium oxide NPs introduced into human osmium tetroxide may be helpful for cells that are not adherent or e lung fibroblasts reveal a strong dependence of the amount of incor- to aid in transferring the pellet during various steps of the prepara- r u 22–24,59,66,73,80–82 t porated ceria on particle size, whereas NP number density or total- tion process . Once the cells are in the form of a pellet, a N particle surface area showed weaker correlations. Other studies have the replacement of the aqueous portion of the cell with ethanol 0 1 found no correlation between toxicity and either specific surface (dehydration series) helps the resin to efficiently penetrate into the 0 2 53,67,68 © area or equivalent spherical diameter . However, some studies cell. However, both dehydration and resin embedment can contri- in animals show a good correlation between the NP surface area and bute to protein denaturation and lipid solubilization, so exposure 68–71 72,73 inflammatory response . NPs such as Au NPs or quantum dots to solvents and embedding media should be as brief as possible. 74 (QDs) can be expressed in ‘molarity’. In the case of QDs, molarity The advantages of using ethanol for dehydration (compared with refers to an entire quantum dot with concentrations of Cd in 20 µM acetone) include no hardening of the sample and less extraction stock solutions translating to ~38,000 µM (refs. 23,74). The concen- of cellular components. Further, traces of acetone can act as a tration range for dosing in uptake studies can be based upon toxicity radical scavenger and interfere with LR White resin polymerization − 1 data, which for most of our studies is between 0–100 µg ml (refs. 5–19). during curing. A survey of the existing literature demonstrates similar concentra- The main purpose of the resin is to provide a solid support − 1 − 1 tion ranges from 0.001 µg ml up to 400 µg ml (refs. 23,35,75,76) for the cells to facilitate the preparation of ultra-thin sections. 73,77 or alternatively 5–100 nM for Au or ceria NPs . Resins can be products, such as LR White (acrylic), Epon or Spurr’s (epoxy-based), which should be prepared and cured according to Electron microscopy preparation and imaging considerations. the manufacturer’s directions. The benefits of epoxy resin include Each step of the sample preparation process (e.g., fixation, dehydra- minimal extraction of cellular components, good sample infil- tion, resin embedding and so on) can have a great impact on the tration, uniform polymerization without significant shrinkage quality of the resulting sample. In general, fixatives are required artifacts, favorable staining characteristics and stable sections that to stabilize the structure of the cells during the transition from can withstand the intense heat and vacuum of the TEM. However, living, dynamic entities to static, rigid, cross-linked structures so we predominantly use LR White resin, which is a one-part formu- that they can withstand the subsequent dehydration and embed- lation with extremely low viscosity, low extraction rate and lower ding processes. A solution of buffered formaldehyde and glutaral- toxicity in both monomeric and polymerized states compared dehyde (sometimes called Trump’s or Karnovsky’s fixative) along with many epoxy formulations. Therefore, the critical proper- with osmium tetroxide for post-fixation are commonly employed ties of the resin include its viscosity for properly infiltrating the as fixatives. The benefits of using a mixture of paraformaldehyde sample, toxicity of the components as well as stability under the | | | nature protocols VOL.5 NO.4 2010 747
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