Atomic force microscopy reveals new biomarkers for monitoring subcellular changes in oxidative injury: neuroprotective effects of quercetin at the nanoscale

Oxidative stress is a process involved in the pathogenesis of many diseases, including atherosclerosis, hypertension, diabetes, Alzheimer’s disease etc. The biomarkers for assessing the degree of oxidative stress have been attracting much interest because of their potential clinical relevance in understanding cellular effects of free radicals and evaluation of the efficacy of drug treatment. Here, an interdisciplinary approach using atomic force microscopy (AFM) and cellular and biological molecular methods were used to obtain new potential biomarkers for monitoring oxidative stress condition. Biological methods confirmed the oxidative damage of investigated P19 neurons and revealed the underlying mechanism of quercetin protective action. AFM was employed to evaluate morphological (roughness) and nanomechanical (elasticity) properties that may be specific biomarkers for oxidative stress-induced cytoskeletal reorganization manifested by changes in the lateral dimension and height of neuronal somas. The morphological and nanomechanical analysis of neurons showed the strong mutual correlation between changes in cell membrane elasticity and neuroprotective effects of quercetin. Our findings indicate that AFM is a highly valuable tool for biomedical applications, detection and clarifying of drug-induced changes at the nanoscale and emphasize the potential of AFM approach in the development of novel therapeutic strategies directed against oxidative stress-induced neurodegeneration.


Introduction
Oxidative stress, which occurs when the cellular antioxidant defence is insufficient to keep the levels of reactive oxygen species (ROS) below a toxic threshold, represents a common mechanism of neuronal death in a variety of neuropathologies, including neurodegenerative diseases such as Alzheimer's disease and Parkinson disease [1]. The brain is particularly vulnerable to oxidative stress due to its large oxygen consumption, high content of polyunsaturated fatty acids, accumulation of redox-reactive transient metal ions and limited endogenous antioxidant protection [2].
At physiological levels ROS act as signalling molecules, but when present in excess they may induce an oxidative stress response and trigger cell death by modulating redox-sensitive signalling pathways and gene expression. Among different ROS molecules, H 2 O 2 is considered as the key target in neuroprotection as is one of the most abundant ROS in aerobic organisms. Moreover, it can be converted to more toxic species of which hydroxyl radical is particularly dangerous [3]. The mechanism of the H 2 O 2 -mediated signalling relies on the oxidation of redox-sensitive thiol groups in cysteine residues of different target enzymes and transcription factors, thereby modulating their functions [4]. At concentrations above the physiological threshold, H 2 O 2 can change activities of different signalling cascades, such as pathways of mitogen-activated protein kinases (MAPK) and protein kinase B (PKB/Akt). This ultimately triggers specific nuclear or cytoplasmic response, often ending in cell death [5,6]. In addition to modulation of intracellular transduction pathways, increased levels of ROS may induce damage to all biological macromolecules that further exacerbates neuronal death [7].
Quercetin, a plant-derived polyphenolic nutraceutical, possesses a wide spectrum of healthpromoting effects mainly attributed to its strong antioxidative capacity. Both in vitro and in vivo quercetin is effective against various oxidants and other neurotoxic molecules that induce oxidative stress and mimic the pathological hallmarks of neurodegenerative diseases [8,9]. In vitro, quercetin exerts its neuroprotective effects acting as a potent direct radical scavenger and a metal chelator but is also able to downregulate redox-sensitive signalling [10,11]. Due to low bioavailability, the expected concentrations of quercetin in the brain tissue are below those that are required for direct antioxidant activity. Furthermore, concentrations of endogenous antioxidants greatly surpass levels of quercetin in the brain. Hence, it is suggested that the neuroprotective effects of quercetin in vivo are not achieved through direct ROS scavenging. [9] Instead, it seems that the modulation of intracellular signalling pathways could represent a primary mode of quercetin action in vivo [12,13]. In support of this assumption, it is shown that structural characteristics of quercetin molecule involved in neuroprotective action differ from those that provide free radical scavenging [14].
Inside the cell quercetin accumulates in the nucleus, at distinct loci, where may affect transcription and/or activity of numerous transcription factors [15]. For example, induction of NF-E2-related factor-2 (Nrf2) transcription factor pathway, which drives the expression of antioxidant genes and increases endogenous antioxidant defence, is one of the well-recognized mechanisms of quercetin action that greatly contributes to neuroprotection [9,16]. Quercetin also may act inhibitory on a number of kinases signalling pathways including Akt/PKB, extracellular signal-regulated protein kinases (ERK) 1/2 and c-Jun N-terminal kinase (JNK) [12,17,18]. The stimulatory effects on the same kinases are also demonstrated, leading to the expression of survival and defensive genes [11,19]. Hence, the exact mechanism of neuroprotective effects of quercetin remains puzzling, particularly when considering modulation of intracellular signalling pathways.
Physiological functions of a cell are closely related to its morphological characteristics [20,21].
When pathological toxin-and drug-induced molecular changes occur inside the cell, its overall morphology usually changes as well. Recent studies have shown that the nanomechanical behaviour of the cell plays an important role in maintaining cellular physiological functions and also presents a novel biomarker for indicating different cell states [22]. In the present study, we implemented a novel experimental approach, combining molecular biology with atomic force microscopy (AFM) as an advanced tool, to obtain information about drug-induced neuronal changes during oxidative stress. The field of AFM applications in neuronal research is developing. AFM is primarily used for quantitative imaging of surface topography. In non-imaging mode, AFM provides spatially resolved maps of the nanomechanical characteristics usually reported as cell elasticity [23,24]. Both imaging and non-imaging AFM modes can bring specific information about neuronal membranes and cytoskeleton architecture. Structural and nanomechanical properties of neurons are highly affected by environmental conditions that can change the organization of their microtubules, actin filaments and neurofilaments [24,25]. AFM was already employed for detecting nanoscopic changes resulting from oxidative damage in the plasma membrane of glioblastoma cells [26].
The aim of the present study was to combine capabilities of AFM with molecular biology tools to better understand cellular and molecular consequences of H 2 O 2 -induced oxidative injury in P19 neurons and to investigate molecular mechanisms of quercetin-mediated neuroprotection that falls beyond its antioxidant activity. For the first time, we used AFM to reveal subtle changes of neuronal membrane topography and nanomechanical properties induced by drug treatment during the oxidative insult.
For induction of neuronal differentiation, exponentially growing P19 cells (1x10 6 ) were seeded into nonadhesive bacteriological-grade Petri dishes (10 cm) containing 10 ml of DMEM medium supplemented with 5% FBS, 2 mM L-glutamine, antibiotics and 1µM ATRA (induction medium). Embryonal bodies of P19 cells were formed in 1-2 days. After 48 h, the old medium was replaced with the fresh ATRA-containing medium and aggregated cultures were grown for two more days. After the four-day of ATRA treatment, P19 embryonal bodies were harvested, washed with PBS, trypsinized, collected by centrifugation (200 g, 5 minutes), and resuspended in growth medium. For optimal neuronal differentiation single cells at a density of 10 5 cells/cm 2 were plated onto 96-well plates or 35 mm Petri culture dishes (Cell + , Sarstedt, Newton, NC, USA and NUNC, Roskilde, Denmark), and grown in growth medium for two more days. Finally, the growth medium was replaced with serum-free medium containing DMEM supplemented with insulin, transferrin, selenium and ethanolamine solution (ITS-X, Gibco), 2 mM L-glutamine and antibiotics (neuron-specific medium), and cells were grown for additional 2 days in the presence of 10 μM mitotic inhibitor cytosinearabinofuranoside (AraC) to inhibit proliferation of non-neuronal cells. Complete neuronal maturation was confirmed with monoclonal anti-tubulin β-III mouse IgG, clone TU-20 conjugated with Alexa Fluor®488 (Millipore, Temecula, CA). Differentiated cells expressing neuronal marker β-III tubulin were visualized by fluorescence microscopy (data not shown). Completely differentiated P19 neurons exert morphological, neurochemical and electrophysiological properties resembling neurons from the mammalian brain, and represent an established model for pharmacological studies. [27,28] Drug treatment In all experiments, P19 neurons were treated 8 days after the initiation of differentiation procedure (DIV8).
Each batch of cultured cells was divided into control and drug-treated groups. For inducing oxidative damage, P19 neurons were incubated with 1.5 mM H 2 O 2 in the neuron-specific medium for 24 hours, alone or in the presence of various concentrations of quercetin that failed to affect the viability of P19 neurons when applied alone. [28] To examine the effects of quercetin on kinase signalling pathways, P19 neurons were pretreated with UO126 or wortmannin for 60 min and then exposed to H 2 O 2 , inhibitor and 150 µM quercetin for additional 24 hours.

Trypan blue exclusion assay
The viability of P19 neurons in the presence of 1.5 mM H 2 O 2 was analysed by trypan blue exclusion assay.
The method is based on the principle that healthy cells effectively exclude the dye from their cytoplasm, while those with damaged membranes lose this ability and appear blue. Following treatment, culture mediums with floating cells were collected in a centrifuge tube. Attached cells were trypsinized for 5 min, resuspended and pooled with the corresponding medium. Samples were centrifuged at 250 g for 5 min; pellets were resuspended in 250 µl of neuron-specific medium and incubated for 5 min in the presence of 0.4% trypan blue solution. The ratio of trypan blue stained nuclei over the total number of cells was used to determine the percentage of cell death. For each examined group at least 500 neurons were counted by two different investigators.

MTT assay
Effects of inhibitors of Akt and ERK1/2 signalling, wortmannin and UO126, respectively, on the neuroprotective effect of quercetin, were determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. MTT assay was also employed to determine effects of different concentrations of H 2 O 2 on the viability of P19 neurons. Estimation of viability is based on the ability of P19 neurons to cleave dissolved MTT into an insoluble formazan product by cleavage of the tetrazolium ring by dehydrogenase enzymes. Briefly, P19 neurons were seeded on 96-well micro-plates and were incubated for 24 hours with H 2 O 2 , quercetin and inhibitors. At the end of treatment schedule, the medium was removed, and the cells were incubated with 40 μl of MTT solution (0.5 mg/ml final) for 3 h at 37°C. Precipitated formazan was dissolved by adding 160 μl of dimethyl sulfoxide (DMSO). Optical densities of coloured solutions in each well were determined by an automatic ELISA reader at 570 nm. The data were analysed after blank subtraction from all absorbance readings and viability of P19 neurons was calculated according to the following equitation: % cytotoxicity = [A control -A treatment ]/A control x 100.

Measurement of reduced glutathione
Reduced glutathione (GSH) is one of the major non-enzymatic intracellular antioxidant defence mechanisms. Changes in the intracellular level of GSH were monitored by using a GSH-Glo TM Glutathione Assay (Promega, Madison, WI, USA) based on the conversion of a luciferin derivative into luciferin by glutathione S-transferase (GST) in the presence of GSH. Hence, the signal generated in a coupled reaction with luciferase is proportional to the amount of GSH present. According to the manufacturer's instruction, following treatment, the medium was removed and 100 μl of GSH-Glo TM reagent was added per well. After a 30-minute incubation, 100 μl of luciferin detection reagent was further added and following 15-minute incubation emitted light was measured in the luminometer (Fluoroskan Ascent FL, Thermo Scientific).

Determination of Bcl-2, Bax, p53 and GAPDH mRNA levels by semi-quantitative RT-PCR
Expressions of Bcl-2, Bax, p53 and GAPDH mRNA were examined by semiquantitative RT-PCR analysis according to the method previously described by Jazvinšćak Jembrek and co-workers. [28] cDNAs were amplified and analysed during two consecutive cycles in the log phase of PCR reactions. PCR primers, annealing temperatures, and numbers of cycles are shown in Table 1. The reactions were performed in a Perkin Elmer 9600 thermocycler. Amplified products (10 µl) were electrophoretically separated on a 1.5% agarose gel and stained with ethidium bromide (0.5µg/ml) for 20 minutes. Optical densities of detected bands were analysed using ImageJ NIH software 1.0. Expression of housekeeping gene TATA-binding box protein (TBP) mRNA was used as an internal standard for normalization.

AFM measurements
All cell imaging and force mapping measurements were obtained on the JPK NanoWizard® ULTRA Speed and JPK NanoWizard® 4 AFM system coupled to the Nikon Eclipse TE2000-U inverted optical microscope.
where y i are vertical deviations height profiles from the mean plane, while RMS Roughness, R q , is the root mean square average of the profile heights over the evaluation length defined as: The Young's modulus was determined using the Hertz model for conical indenters.

Statistical analysis
Statistical analysis of the data was carried out using GraphPad Software (San Diego, CA). All values are represented as mean ± SEM from at least three independent experiments. Comparisons between group means were evaluated by one-way ANOVA, and when statistically significant, post hoc analysis with Dunnett's multiple comparison test or Tukey's test followed. The P values less than 0.05 were considered statistically significant.

Biological approach reveals neuroprotective effects of quercetin in H 2 O 2 induced injury
Exposure to H 2 O 2 in a concentration-dependent manner decreased the viability of P19 neurons ( Figure 1A). P19 neurons tolerated relatively high concentrations of quercetin, up to 150 μM ( Figure 1B).
Quercetin applied together with 1.5 mM H 2 O 2 improved survival of P19 neurons indicating the neuroprotective effect of quercetin against oxidative injury. Quercetin action was dose-dependent, and at the highest concentration applied it maintained viability at 87.6 ± 6.7 % of the control group ( Figure 1C).

Quercetin did not improve H 2 O 2 -induced decrease in glutathione (GSH) content
As previously reported, quercetin prevents H 2 O 2 -provoked upregulation of ROS production. [30] Here, we analysed levels of reduced glutathione (GSH), one of the major non-enzymatic intracellular antioxidants, as oxidative stress may alter reservoirs of intracellular antioxidant systems. Exposure to H 2 O 2 depleted GSH to 55.7% of the vehicle-treated group, but in the presence of quercetin intracellular GSH content was not restored. Comparable results were also demonstrated in mouse embryos exposed to H 2 O 2 and quercetin [31]. Detoxification of H 2 O 2 depends predominantly on glutathione peroxidase in a reaction that utilizes two GSH molecules, consequently leading to GSH depletion. In addition, GSH decrease in H 2 O 2 -treated neurons may result from the direct free radical scavenging, and via formation of GSH-conjugates with various electrophilic compounds [32]. However, in P19 neurons a substitution of endogenous antioxidant with an exogenous molecule with strong antioxidative activity probably provided sufficient antioxidative defence and improved survival despite the reduced GSH content. expression [35]. We also analysed expression of GAPDH mRNA. GAPDH is primarily viewed as a metabolic enzyme engaged in glycolysis, but it mediates numerous non-glycolytic functions, including the sensing of oxidative stress and induction of cell death [36,37]. Although H 2 O 2 exposure did not change GAPDH expression, the pro-survival effect of quercetin correlated with pronounced GAPDH up-regulation. Total RNA was extracted and reverse transcribed into cDNA. The obtained cDNA is further amplified using specific primers. Following densitometric quantification, band intensities were normalized to the expression of housekeeping gene TBP. The data are expressed as means ± SEM from 3 independent RT-PCR analyses. # P < 0.05 vs. cont; *P < 0.05, **P < 0.01 vs. 0 group (ONE-way ANOVA and post-hoc Tukey's multiple comparison test). Representative agarose gel electrophoresis is also shown.
H 2 O 2 readily stimulates the conversion of sulfhydryl groups into disulfides and other oxidized species. Hence, it may inhibit or promote disulfide bonding within or between redox-sensitive cytoplasmic proteins involved in translation, glycolysis, cytoskeletal structure and antioxidative defence [38]. Following H 2 O 2 exposure, different cysteine modifications, such as thiol oxidation and S-glutathionylation, were observed in the catalytic site of GAPDH, affecting its structure and function [37,39]. Thus, although we failed to observe H 2 O 2 -related changes in GAPDH expression, it is possible that P19 neurons have experienced a loss of at least some GAPDH functions that were restored by quercetin-induced GAPDH up-regulation. It is known that membrane-associated GAPDH binds to tubulin and regulates bundling of microtubules [36].
Hence, by stimulating microtubule bundling quercetin-mediated GAPDH up-regulation potentially could contribute to the preservation of normal neuronal branching that is disrupted by H 2 O 2 treatment [30].
Quercetin-induced changes in the expression profile of GAPDH was also demonstrated in vivo [40]. The relationship between GAPDH and p53, a transcription factor that regulates expression of stress response genes is also observed. Protein p53 is upregulated and promotes death in response to diverse genotoxic cellular stresses, including oxidative stress [10,41]. In glutamate-induced oxidative stress, GAPDH translocates to the nucleus, enhances p53 expression and activates p53-mediated death pathway [42]. This suggests that quercetin-mediated decrease in p53 expression potentially could disrupt GAPDH-p53 interactions and offer neuroprotection. P19 neurons exposed to high concentration of H 2 O 2 died by caspase-independent apoptosis in combination with necrosis [30]. Cell death mediated by p53 may be caspase-independent [43]. The main effector of caspase-independent death program is an apoptosis-inducing factor (AIF). Overexpression of Bcl-2 (as we found in P19 neurons exposed to quercetin) may prevent AIF release and consequent cell death [44]. Necrosis also may be prevented by Bcl-2 overexpression [45]. In cultured cortical neurons interaction between death-associated protein kinase, 1 (DAPK1) and p53 was identified as a signalling point of convergence of necrosis and apoptosis [46]. They showed that activated DAPK1 phosphorylates p53. This induces expression of pro-apoptotic genes in the nucleus and initiates necrosis in the mitochondrial matrix through interactions with cyclophilin D. In addition, p53 protein can directly promote mitochondrial outer membrane permeabilization (MOMP) to trigger apoptosis [47]. Hence, by preserving the p53 and Bcl-2 expression quercetin might contribute to the survival of P19 neurons at the level of apoptotic and necrotic death events.
To investigate if the neuroprotective effects of quercetin were mediated by the modulation of intracellular signalling, P19 neurons were exposed to H 2 O 2 and quercetin and two selected inhibitors: UO126 (an inhibitor of the Ras/Raf/MEK/ERK signalling pathway) or wortmannin (a covalent inhibitor of phosphoinositide 3-kinases (PI3K) that activates Akt/PKB). As represented in Figure 4, beneficial effects of quercetin on neuronal survival were abrogated in the presence of UO126 and wortmannin. Both inhibitors were applied in a concentration that did not affect viability when applied alone. The obtained results indicate that the neuroprotective action of quercetin was achieved through the modulation of ERK1/2 and PI3K/Akt signalling. The PI3K/Akt and ERK1/2 signalling pathways are critically involved in controlling cell survival in response to extracellular stimuli, including H 2 O 2 -induced oxidative stress [5]. As in our study, activation of PI3K/Akt and MAPK/ERK pathways offered protection against p53-mediated cell death in sympathetic neurons. [48,49] PKB/Akt acts upstream of p53 and suppresses transcriptional activation of p53-responsive genes, [50] while signalling through both Akt and ERK1/2 pathways may be involved in the protection against H 2 O 2 -induced neuronal death through the activation of cyclic AMP regulatory-binding protein (CREB) that promotes transcription of antiapoptotic genes such as Bcl-2 [5,8]. Similarly to our results, quercetin prevented an H 2 O 2 -induced decrease in cell viability by inducing ERK1/2 phosphorylation, [51] and through the activation of the PI3K/Akt pathway [11]. Of note, PI3K could be involved in ERK1/2 activation in primary cortical neurons [5] and neuroblastoma cells [6]. Furthermore, quercetin may offer protection against oxidative injury by activating the Nrf2 pathway as Akt and ERK pathways participate in Nrf2 activation [9,52].  [53,55]. This indicates that fixed P19 neurons represent a good model system to study potential biomarkers of oxidative stress and protective drugs effects at the nanoscale.
In order to more precisely identify morphological differences between treated groups, we introduced a multiple biophysical analysis by employing AFM. First, as represented in Figure 5, isolated control neurons displayed an elongated soma shape ( Figure 5A), whereas neurons exposed to H 2 O 2 have irregularly shaped and degenerated cell bodies ( Figure 5C). The morphological shape of P19 neurons simultaneously treated with quercetin and H 2 O 2 was more regular, better resembling to control neurons ( Figure 5A and 5E), indicating beneficial effects of quercetin on the preservation of neuronal morphology. Hence, with the applied approach using cell volume as one of the biomarkers, we were able to improve detection and recognition of protective effects of quercetin on neuronal morphology.
As evident in Figure 6B, distinct regions of control neurons contain ruffling structures probably consisting of the assembly of diverse membrane proteins and membrane folding. Similar findings of fine ruffling formations have been observed in morphological studies of neuronal growth cone [23,56]. AFM imaging revealed that individual ruffling structures are of various sizes in control neurons. These membrane protrusions were significantly suppressed in H 2 O 2 -exposed neurons ( Figure 6E), probably due to the formation of higher molecular weight components by cross-linking of membrane proteins. [57] The protective effect of quercetin was particularly evident in the cross-section profile of neurons treated with both quercetin and H 2 O 2 whose fine ruffling assemblies showed only minor modifications in comparison with control neurons (Figure 6 H). We also performed surface roughness analysis of the neuronal soma membrane to specifically evaluate changes in membrane surface topography. A membrane roughness is an important parameter in cell studies, also with great potential for medicinal applications as a valuable biomarker. It indicates the deviation of the membrane surface topography from the ideally smooth surface. The roughness parameters described by R a and R q were derived from the raw height images and the height images treated with a quadratic plane fit. Ten neurons were analysed by randomly measuring several areas of each cell surface.
The analysed regions of control cells were relatively small (2×2 μm 2 ), but they appeared cone-shaped in cross-section profile, and their surfaces were relatively rough as shown in Table 2. The plane fit was used to subtract the curvature of the neuronal body which would otherwise influence the roughness data ( Table   2). The plane fit more accurately revealed surface roughness. alterations [59] and apoptotic processes [60] but also the organization of membrane components [61]. Due to the aforementioned formation of the higher molecular weight components by the crosslinking of membrane proteins, [57] the surface of H 2 O 2 -damaged membranes became markedly smoother ( Figure 6F) in comparison to the rough surface of control neurons ( Figure 6C). Membranes of neurons treated with both H 2 O 2 and quercetin did not alter significantly, remaining their surface still rough ( Figure 6H). Table 2 summarizes the roughness, height and lateral dimension values determined from the AFM height measurements on the highest domain of the neurons. Table 2. Roughness, height and lateral dimension data from the histograms of the neuronal detail images below We further used AFM to analyse local mechanical properties of specific somatic regions by performing nanomechanical measurements. AFM provides quantitative data of the mechanical properties, as well as the direct relationship between mechanical and structural characteristics of neuronal cells [24]. Force-distance curves were acquired to determine the elastic moduli (Young's moduli) of the different regions ( Figure 7). Hertz's model was used to fit the portions of the force curve and the resulting Young's moduli are summarized in Table 3. As presented in the frequency histogram, significant variations in the elastic (Young's) modulus were found between treated groups (Figures 7 B, E, H in control neurons are homogeneously distributed (brown colour within square in Figure 7B), the addition of H 2 O 2 induced structural and mechanical changes that are reflected in the increased Young modulus value i.e. increased stiffness (white area within square in Figure 7E). The protective role of quercetin was observed in the discrete increase in the neuron stiffness (left side on the bottom square in Figure 7H)  suggest that quercetin offers protection towards certain mechano-chemical processes induced by oxidative environment that affect cytoskeleton and membrane organization in the investigated regions of the neuronal soma. The height topographic image of specific somatic regions in control system (A), H 2 O 2 -exposed neurons (D) and in neurons simultaneously exposed to both quercetin and H 2 O 2 (G) determined by nanomechanical measurements using AFM. The nanomechanical mapping and histogram of Young´ modulus of the specific somatic region in control system (B), H 2 O 2 -exposed neurons (E) and neurons simultaneously exposed to both quercetin and H 2 O 2 (H). Colour-coded frame lines identified zoomed domain of control system (C) H 2 O 2 -exposed neurons (F) and neurons simultaneously exposed to both quercetin and H 2 O 2 (I). higher Young's modulus value. The stiffness (considered as the absence of the cell elasticity) of H 2 0 2 -treated neurons was markedly increased and then reduced almost to half in the presence of quercetin. Finally, we observed a strong correlation between cell stiffness and roughness parameters during H 2 O 2 -induced injury.
As a new biophysical approach in the study of oxidative stress-induced neurodegeneration, we used AFM for revealing protective effects of quercetin at the nanoscale resolution. The obtained results suggest that AFM and QI™ mode are powerful tools to characterize the morphology and resolve stiffness differences of the neuronal cell bodies. If combined with standard cellular and molecular methods, AFM can greatly improve detection and recognition of effects of various drugs and toxins on cellular morphology and physiology using very sensitive biomarkers as roughness and elasticity. In a wider context, a better understanding of these effects can be used for drug screening, and potentially to the development of novel therapeutic strategies. This AFM study represents the first detailed analysis of the protective effects of quercetin on neuronal membrane and cytoskeleton organization and in general, indicates a great potential of AFM in biomedical research. If combined with standard cellular and molecular methods, AFM can greatly improve detection and recognition of effects of various drugs and toxins on cellular morphology and physiology.