Low uptake of silica nanoparticles in Caco-2 intestinal epithelial barriers

• Dong Ye,
• Mattia Bramini,
• Delyan R. Hristov,
• Sha Wan,
• Anna Salvati,
• Christoffer Åberg and
• Kenneth A. Dawson

Beilstein J. Nanotechnol. 2017, 8, 1396–1406, doi:10.3762/bjnano.8.141

• ][50]. Lysosomal staining showed that some of the (few) internalised nanoparticles were found in the lysosomes, and this was more evident for the 50 nm SiO2-NPs in Caco-2 barriers cultured for 4 days compared to those cultured for 21 days. Transmission electron microscopy (TEM) imaging was used to

Evaluation of quantum dot conjugated antibodies for immunofluorescent labelling of cellular targets

• Jennifer E. Francis,
• David Mason and
• Raphaël Lévy

Beilstein J. Nanotechnol. 2017, 8, 1238–1249, doi:10.3762/bjnano.8.125

• cytosolic structures: The vast majority of studies using Qdots show tubulin staining [6][12][16][18][20], therefore we sought to label this abundant cytosolic protein in order to have a positive control for our labelling protocol. After incubation with an anti-tubulin primary antibody and a Qdot 625-Ab, we

• considers them unsuitable in their current form "… because of the lack of reproducibility of the experiments" when using these materials [35]. Interestingly, in all of our Qdot 625 images, even when the staining was non-specific, there was no labelling within the nucleus (Figure 2–6). This suggested that

• primary anti-GFP antibody and Qdot 625-Ab. Both approaches yielded homogenous labelling in the cytosol, with the Qdot signal being excluded from the nucleus (Figure 7). There was also little non-specific Qdot 625 staining in the non-transfected cells (Figures S16 and S17 in Supporting Information File 1

Nano-engineered skin mesenchymal stem cells: potential vehicles for tumour-targeted quantum-dot delivery

• Liga Saulite,
• Dominyka Dapkute,
• Karlis Pleiko,
• Ineta Popena,
• Simona Steponkiene,
• Ricardas Rotomskis and
• Una Riekstina

Beilstein J. Nanotechnol. 2017, 8, 1218–1230, doi:10.3762/bjnano.8.123

• proliferation of MSCs. The differentiation of MSCs into adipocytes, chondrocytes and osteocytes was not affected by the presence of QDs (Figure 5). Quantification assays for Alcian Blue staining and Alizarin Red S staining confirmed that QDs did not influence chondrogenesis and osteogenesis of skin MSCs (Figure

• S staining. The cells were washed with 1 mL of PBS and fixed with 4% paraformaldehyde (PFA) at room temperature for 30 min. After fixation, the cells were washed two times with distilled water and stained with a 2% Alizarin Red S solution in water (pH adjusted to 4.2 with a 0.1% solution of NH4OH

• evaluated using Oil Red O staining. Cells were washed with PBS and fixed with 4% formaldehyde for 30 min at room temperature. After fixation, cells were washed with distilled water. Prior to staining, cells were incubated for 5 min at room temperature with 60% isopropanol and subsequently stained with 180

Silicon microgrooves for contact guidance of human aortic endothelial cells

• Sara Fernández-Castillejo,
• Pilar Formentín,
• Úrsula Catalán,
• Josep Pallarès,
• Lluís F. Marsal and
• Rosa Solà

Beilstein J. Nanotechnol. 2017, 8, 675–681, doi:10.3762/bjnano.8.72

• ), as described further below. Staining on actin and nuclei and fluorescence confocal microscopy HAECs were cultured on the functionalized substrates for 2 and 7 days. After cell culture experiments, culture media were removed and cells were washed twice with PBS at 37 °C and afterwards fixed as

Innovations from the “ivory tower”: Wilhelm Barthlott and the paradigm shift in surface science

• Christoph Neinhuis

Beilstein J. Nanotechnol. 2017, 8, 394–402, doi:10.3762/bjnano.8.41

• same properties as the lotus leaves. Figure 10 shows one of these early attempts. The plate has two sides, one smooth and one covered with PTFE particles. After contaminating both sides with toner from a photocopier and the red staining powder Sudan III, the plate was briefly rinsed with water. While

Comparison of four methods for the biofunctionalization of gold nanorods by the introduction of sulfhydryl groups to antibodies

• Xuefeng Wang,
• Zhong Mei,
• Yanyan Wang and
• Liang Tang

Beilstein J. Nanotechnol. 2017, 8, 372–380, doi:10.3762/bjnano.8.39

• cationic surfactant bilayer [17][24], because of the replacement of CTAB by thiolated anti-IgG [17][25]. Anti-IgG were detected in thiolation and nanoconjugates with GNRs, but not in free GNRs, by gel electrophoresis followed by Coomassie brilliant blue staining (Figure S1, Supporting Information File 1

“Sticky invasion” – the physical properties of Plantago lanceolata L. seed mucilage

• Agnieszka Kreitschitz,
• Alexander Kovalev and
• Stanislav N. Gorb

Beilstein J. Nanotechnol. 2016, 7, 1918–1927, doi:10.3762/bjnano.7.183

• along a dirt road in the wilderness near Wrocław, Poland. To examine the mucilage composition, staining reactions with 0.1% aqueous solution of ruthenium red (pectin staining), with 0.01% aqueous solution of methylene blue (cellulose staining) and with 0.1% aqueous solution of Direct Red 23 (specific

• analysis, and the P-value was set to P < 0.001 if one of both conditions was not achieved. Results Mucilage composition The mucilage of Plantago lanceolata contained both pectin and cellulose components, which are characteristic of cellulose mucilage. Positive staining with ruthenium red (Figure 1A

• ) revealed the presence of pectins in the mucilage. Staining with methylene blue (Figure 1B) and Direct Red 23 (Figure 1C) revealed the presence of cellulose. The cellulose fibrils kept the mass of pectins in place. The thickness of the mucilage envelope ranged from 350 to 450 μm. Cellulose fibrils were very

Facile fabrication of luminescent organic dots by thermolysis of citric acid in urea melt, and their use for cell staining and polyelectrolyte microcapsule labelling

• Nadezhda M. Zholobak,
• Anton L. Popov,
• Alexander B. Shcherbakov,
• Nelly R. Popova,
• Mykhailo M. Guzyk,
• Valeriy P. Antonovich,
• Alla V. Yegorova,
• Yuliya V. Scrypynets,
• Inna I. Leonenko,
• Alexander Ye. Baranchikov and
• Vladimir K. Ivanov

Beilstein J. Nanotechnol. 2016, 7, 1905–1917, doi:10.3762/bjnano.7.182

• applications of O-dots for alive/fixed cell staining and labelling of layer-by-layer polyelectrolyte microcapsules were evaluated. Keywords: cell culture; citric acid; layer-by-layer (LbL)-microcapsules; luminescence; organic dots (O-dots); staining; toxicity; Introduction Luminescent nanosized semiconductor

• (Supporting Information File 1, Figure S18 and Figure S19). Obviously, this was due to the low concentration of O-dots in microcapsular delivery systems, which was significantly lower than both MTC and Maxmet. Cellular staining by O-dots Results of cell staining are shown in Figure 5 and Figures S20–22

• (Supporting Information File 1) for fixed and pristine (alive) cells, respectively. It is well known that cellular staining with fluorescent carbon dots is typically more effective for pre-fixed cells [52]. Data obtained indicate that the normal intact ST-cells were stained with O-dots (50 μg/mL) diffusely

Low temperature co-fired ceramic packaging of CMOS capacitive sensor chip towards cell viability monitoring

• Niina Halonen,
• Joni Kilpijärvi,
• Maciej Sobocinski,
• Timir Datta-Chaudhuri,
• Antti Hassinen,
• Someshekar B. Prakash,
• Peter Möller,
• Pamela Abshire,
• Sakari Kellokumpu and
• Anita Lloyd Spetz

Beilstein J. Nanotechnol. 2016, 7, 1871–1877, doi:10.3762/bjnano.7.179

• attachment of the cells on the chip, as well as LTCC, was monitored by fixing the cells with 4% paraformaldehyde 24 h after inoculation and staining the cells with a DNA binding dye (Hoechst, 33342) and anti-α-tubulin antibody. Based on the cell morphology shown in Figure 5a–f, the cells attached normally

• cells grow on top of the chip. (d–f) The cells grow on top of LTCC. In (a) and (d), the blue color indicates the cell nuclei stained with a DNA binding dye, Hoechst 33342. In (b) and (e), immunofluorescence staining was performed with anti-α-tubulin antibody and Alexa 488 secondary antibody. The green

• color shows the microtubules of the cell cytoskeleton. In (c) and (f), the merged image of the nuclear staining and cytoskeleton are shown. The images were taken with a Zeiss LSM700 confocal microscope with 63× plan-apo immersion objective and appropriate filter sets. Average voltage change from the

Nano- and microstructured materials for in vitro studies of the physiology of vascular cells

• Alexandra M. Greiner,
• Adria Sales,
• Hao Chen,
• Sarah A. Biela,
• Dieter Kaufmann and
• Ralf Kemkemer

Beilstein J. Nanotechnol. 2016, 7, 1620–1641, doi:10.3762/bjnano.7.155

• staining is the most used assay to determine cell survival on a particular substrate and hence the cytotoxicity of a material is assessed [14][209]. Besides this live/dead cell investigation, biochemical proliferation assays, such as EdU or BrdU staining, are frequently applied to determine the cell

On the pathway of cellular uptake: new insight into the interaction between the cell membrane and very small nanoparticles

• Claudia Messerschmidt,
• Daniel Hofmann,
• Anja Kroeger,
• Katharina Landfester,
• Volker Mailänder and
• Ingo Lieberwirth

Beilstein J. Nanotechnol. 2016, 7, 1296–1311, doi:10.3762/bjnano.7.121

• blotting and Hoechst staining to collect data about the cytotoxic effects already indicated by EM. Results Nanoparticle characterization A prerequisite for detailed studies of cell–NP interaction is a thorough characterization of the applied particles. Therefore we determined essential particle properties

• indicator for a necrosis-based cell death. Moreover, the live cell imaging with the detection of a burst event and the Hoechst staining of the cell nucleus points into the direction of necrosis. Despite of this common conclusion, we analyzed the cleavage of Caspase-3 showing no specific activation of major

• -fixed within a few milliseconds at a pressure of 2000 bar under liquid nitrogen using a high-pressure freezer Compact 1 (Wohlwend GmbH, Switzerland). Freeze-substitution was conducted using a Leica EM AFS 2 device (Leica Microsystems, Germany). Here, the substitution/staining medium (acetone p.a., 0.2

Functional diversity of resilin in Arthropoda

• Jan Michels,
• Esther Appel and
• Stanislav N. Gorb

Beilstein J. Nanotechnol. 2016, 7, 1241–1259, doi:10.3762/bjnano.7.115

• by single conventional dyes. Chemical reactions with the Masson and Mallory dyes were mentioned to stain resilin red. Staining of resilin with aqueous solutions of methylene blue and toluidine blue is a common method and can provide good information about the presence and distribution of resilin

• fluorescence microscopy and histological staining revealed structures with large proportions of resilin in the pleural area of the metathorax (Figure 4A–D). These structures stretch dorso-ventrally across the entire pleural area (Figure 4F) and are much larger than comparable structures present in fleas (see

Multiwalled carbon nanotube hybrids as MRI contrast agents

• Nikodem Kuźnik and
• Mateusz M. Tomczyk

Beilstein J. Nanotechnol. 2016, 7, 1086–1103, doi:10.3762/bjnano.7.102

• which component of the hybrid was responsible mainly for the cytotoxicity impact. The classical MTT assay is usually applied for this purpose. However, there have been reports of high measurement error in this method with MWCNT [40], thus Trypan Blue staining of the dead cells could be more reliable

• iron content [18]. On the other hand, mitochondrial staining allowed us to analyze cell pathogenesis. There, again the iron-poorest oMWCNTs left the organelle intact, while others led to shape alteration, from typical tubular to globular forms. A higher concentration, above 300 µg/mL, led to a further

Improved biocompatibility and efficient labeling of neural stem cells with poly(L-lysine)-coated maghemite nanoparticles

• Igor M. Pongrac,
• Marina Dobrivojević,
• Lada Brkić Ahmed,
• Michal Babič,
• Miroslav Šlouf,
• Daniel Horák and
• Srećko Gajović

Beilstein J. Nanotechnol. 2016, 7, 926–936, doi:10.3762/bjnano.7.84

• their cellular uptake, the mechanism of internalization, cytotoxicity, viability and proliferation of neural stem cells, and compared them to the commercially available dextran-coated nanomag®-D-spio nanoparticles. Results: Light microscopy of Prussian blue staining revealed a concentration-dependent

• than that of nanomag®-D-spio To evaluate the uptake of nanoparticles by NSCs, Prussian blue staining was used. Both types of nanoparticles were taken up by the NSCs depending on concentration (Figure 2). When the same concentration of nanoparticles (0.2 mg/mL) was used, PLL-γ-Fe2O3-labeled cells were

• ± 1.73)% (4 mg/mL; Figure 3). Similarly to Prussian blue staining, efficient labeling of PLL-γ-Fe2O3 nanoparticles was reached at the considerably lower concentration (0.2 mg/mL) compared with nanomag®-D-spio (4.0 mg/mL). Proliferation and viability To define if the nanoparticle labeling had any negative

Frog tongue surface microstructures: functional and evolutionary patterns

• Thomas Kleinteich and
• Stanislav N. Gorb

Beilstein J. Nanotechnol. 2016, 7, 893–903, doi:10.3762/bjnano.7.81

• frogs with 4% Lugol’s iodine potassium iodide solution before we dissected the tongues. For this purpose, we followed the protocol by Metscher [33] but adjusted the staining duration to two weeks to allow the staining solution to diffuse deep into entire frog specimens. After staining, we dissected the

Atomic force microscopy as analytical tool to study physico-mechanical properties of intestinal cells

• Christa Schimpel,
• Oliver Werzer,
• Eleonore Fröhlich,
• Gerd Leitinger,
• Markus Absenger-Novak,
• Birgit Teubl,
• Andreas Zimmer and
• Eva Roblegg

Beilstein J. Nanotechnol. 2015, 6, 1457–1466, doi:10.3762/bjnano.6.151

• developed brush border (Figure 3A). In contrast, F-actin staining at the apex of M cells was markedly decreased due to a reduced or absent brush border (Figure 3B–D). Elasticity (force-indentation) measurements of Caco-2 cells and M cells Villin is not only involved in the formation and/or regulation of the

• hydrated and flexible cells, measurements were performed in a semi-dry state as demonstrated elsewhere [37][38]. Tetramethylrhodamine (TRITC)-phalloidin staining Visualization of the cytoskeletal F-actin network was performed using TRITC-phalloidin (Invitrogen GmbH, Darmstadt, Germany) in a similar manner

• the intense red F-actin staining. In contrast, M cells show a reduced/absent brush border indicated by a reduced F-actin labeling (B–D) (scale bar = 20 µm). Force–indentation curves and topographical images of a Caco-2 cell (A–C) and a M cell (D–F) classified into peripheral region/cell edge, nuclear

Peptide-equipped tobacco mosaic virus templates for selective and controllable biomineral deposition

• Klara Altintoprak,
• Axel Seidenstücker,
• Alexander Welle,
• Sabine Eiben,
• Petia Atanasova,
• Nina Stitz,
• Alfred Plettl,
• Joachim Bill,
• Hartmut Gliemann,
• Holger Jeske,
• Dirk Rothenstein,
• Fania Geiger and
• Christina Wege

Beilstein J. Nanotechnol. 2015, 6, 1399–1412, doi:10.3762/bjnano.6.145

• dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) (Figure 2a). The efficiency of peptide conjugation was determined by the ratio of the band intensities of modified and nonmodified CPs after Coomassie Blue staining. The binding efficiencies to individual CP subunits were ≈60% for all

• mg−1 cm−1 [94]) . For estimating concentrations of the different biotemplate rods, the band intensities of modified CPs and unmodified CPLys after SDS-PAGE separation and Comassie Blue staining were compared (see below). Electrophoretic analysis The modified CPs were analyzed by denaturing SDS-PAGE

PLGA nanoparticles as a platform for vitamin D-based cancer therapy

• Maria J. Ramalho,
• Joana A. Loureiro,
• Bárbara Gomes,
• Manuela F. Frasco,
• Manuel A. N. Coelho and
• M. Carmo Pereira

Beilstein J. Nanotechnol. 2015, 6, 1306–1318, doi:10.3762/bjnano.6.135

• endo-lysosomal compartments) for 1 h. The cells were washed with PBS and mounted on a glycerol-based medium with DAPI for nuclear staining. Acquisitions were performed with a Leica TCS SP5 II confocal laser scanning microscope (Leica Microsystems, Germany) in emission mode. Untreated cells were also

• harvested and fixed with 70% v/v ethanol. The cells were then stained with a DNA staining solution (0.1% v/v TritonX-100, 20 µg/mL PI and 35 µg/mL of RNase A in PBS) at a cell density of 106 cells/mL. FCM (FACSCalibur, BD Biosciences, CA, USA) was performed by plotting 12,000 gated events per sample. The

Pulmonary surfactant augments cytotoxicity of silica nanoparticles: Studies on an in vitro air–blood barrier model

• Jennifer Y. Kasper,
• Lisa Feiden,
• Maria I. Hermanns,
• Christoph Bantz,
• Michael Maskos,
• Ronald E. Unger and
• C. James Kirkpatrick

Beilstein J. Nanotechnol. 2015, 6, 517–528, doi:10.3762/bjnano.6.54

• quantification method for determination of the number of viable cells is cell staining with crystal violet (CV, purchased from Merck, 1407) [19]. Crystal violet (N-hexamethyl pararosaniline) is a monochromatic dye which stains cell nuclei. After fixation of NP-exposed cells they were incubated with 50 μL/96er

• –plain, which was apically applied to the A549, is depicted in red. ISO-HAS-1 underneath aSNP–plain stimulated A549, (Figure 7e–f) were counterstained (IF) for CD31 (Figure 7f). E-cadherin staining of A549 shows an inconsistent pattern. However, A549 as well as ISO-HAS-1 formed a confluent monolayer. No

• aSNPs displaying different surfaces (–plain, –NH2, –COOH; 5–100 µg/mL, untreated control (uc)). A: Cell viability (mitochondrial activity, MTS, % of untreated control (uc)); B: cell viability (crystal violet staining of nuclei, cell loss measurement, % of uc); C: LDH release (membrane integrity, % of

Hematopoietic and mesenchymal stem cells: polymeric nanoparticle uptake and lineage differentiation

• Ivonne Brüstle,
• Thomas Simmet,
• Gerd Ulrich Nienhaus,
• Katharina Landfester and
• Volker Mailänder

Beilstein J. Nanotechnol. 2015, 6, 383–395, doi:10.3762/bjnano.6.38

• -8 was not significantly altered in the presence of the polymeric nanoparticles (Supporting Information File 1, Figure S4). Differentiation behavior under nanoparticle influence Cytochemical staining of hMSCs For analyzing the particle influence on the differentiation capacity of hMSCs, the cells

• were incubated with 300 µg/mL nanoparticles for 24 h before starting the differentiation by providing the adequate differentiation medium. After 24 days, osteogenic and chondrogenic differentiation was analyzed by detecting alkaline phosphatase activity or methylene blue staining, respectively

• . Detection of adipogenic differentiation was performed after 4 weeks with Oil-Red O staining. In the presence of the nanoparticles, the differentiation into the three investigated lineages was not affected, as determined by cytochemical staining (Figure 5). The detection of the extracellular matrix for the

Comparative evaluation of the impact on endothelial cells induced by different nanoparticle structures and functionalization

• Lisa Landgraf,
• Ines Müller,
• Peter Ernst,
• Miriam Schäfer,
• Christina Rosman,
• Isabel Schick,
• Oskar Köhler,
• Hartmut Oehring,
• Vladimir V. Breus,
• Thomas Basché,
• Carsten Sönnichsen,
• Wolfgang Tremel and
• Ingrid Hilger

Beilstein J. Nanotechnol. 2015, 6, 300–312, doi:10.3762/bjnano.6.28

• -angle diamond knife (Diatome, Biel, Switzerland) in a Leica Ultracut S ultramicrotome was used to make ultrathin sections and then staining with freshly prepared uranyl acetate and lead citrate was performed. The sections were evaluated using a transmission electron microscope EM 902A (Zeiss, Germany

The effect of surface charge on nonspecific uptake and cytotoxicity of CdSe/ZnS core/shell quantum dots

• Vladimir V. Breus,
• Anna Pietuch,
• Marco Tarantola,
• Thomas Basché and
• Andreas Janshoff

Beilstein J. Nanotechnol. 2015, 6, 281–292, doi:10.3762/bjnano.6.26

• ) for 48 h. Immunostaining After washing with PBS, fixation was carried out by immersing the cells into a 20 °C cold acetone/methanol mixture (1:1 v/v) for 10 min. Afterwards, the cells were washed three times with PBS, the unspecific binding sites were blocked with FCS, and incubation in staining

• solutions was carried out according to the manufacturer’s recommendation: Alexa Fluor-conjugated IgG1 anti-tubulin (BD Bioscience, Heidelberg, Germany) from mouse was used for labeling microtubules, and 4’,6-diamidino-2-phenylindole (DAPI, Sigma-Aldrich, Seelze) for nucleus and DNA labeling. Staining was

• pellet was redissolved in 1 mL of distilled water. Fluorescence microscopy measurements For the fluorescent staining experiments, an upright Olympus fluorescence microscope Olympus BX51, with a 40× water-immersion objective (Olympus, Tokyo, Japan), equipped with a color camera (3 MP) was used. The

Overview about the localization of nanoparticles in tissue and cellular context by different imaging techniques

• Anja Ostrowski,
• Daniel Nordmeyer,
• Alexander Boreham,
• Cornelia Holzhausen,
• Lars Mundhenk,
• Christina Graf,
• Martina C. Meinke,
• Annika Vogt,
• Sabrina Hadam,
• Jürgen Lademann,
• Eckart Rühl,
• Ulrike Alexiev and
• Achim D. Gruber

Beilstein J. Nanotechnol. 2015, 6, 263–280, doi:10.3762/bjnano.6.25

• -inflammatory effects with rather specific accumulation in inflamed tissues [55][56][57]. Due to their sulfate groups and the specific staining properties of Alcian blue, this method has been used successfully, for example, for the detection of dPGS amine accumulated in Kupffer cells in the liver of mice

• using full-field high-resolution transmission X-ray microscopy combined with a potassium permanganate staining of FFPE-tissue sections of the cerebellum and the liver [145]. Soft X-ray microscopy has been successfully applied for 3D imaging of vitrified cells without any further staining [146]. That

• example, titanium dioxide, SiO2-NP or QD, TEM has been widely used to characterize the morphology and size of NP as well as their location in tissues [28][35][39][113][156][157][158]. It has to be kept in mind, however, that artifacts due to staining with lead citrate and uranyl acetate can easily be

Oxygen-plasma-modified biomimetic nanofibrous scaffolds for enhanced compatibility of cardiovascular implants

• Anna Maria Pappa,
• Varvara Karagkiozaki,
• Silke Krol,
• Spyros Kassavetis,
• Dimitris Konstantinou,
• Charalampos Pitsalidis,
• Lazaros Tzounis,
• Nikos Pliatsikas and
• Stergios Logothetidis

Beilstein J. Nanotechnol. 2015, 6, 254–262, doi:10.3762/bjnano.6.24

• to the reference wavelength of 690 nm. Data (n = 3) were presented as means of O.D. values as well as normalized according to the control and presented as % cell viability. Optical imaging through methylene blue staining: Once fibroblasts were seeded onto either unmodified or surface-modified

Mechanical properties of MDCK II cells exposed to gold nanorods

• Anna Pietuch,
• Bastian Rouven Brückner,
• David Schneider,
• Marco Tarantola,
• Christina Rosman,
• Carsten Sönnichsen and
• Andreas Janshoff

Beilstein J. Nanotechnol. 2015, 6, 223–231, doi:10.3762/bjnano.6.21

• number of CTAB molecules which can, in principle, be released from the particle surface assuming a concentration of 12 μg/mL, we only found a Young’s modulus of E = 6.5 ± 1 kPa. Fluorescence microscopy images (staining of actin and microtubules) as well as AFM images (topography) reveal that the cells

• with PBS. Incubation with staining solution was carried out following the manufacture’s recommendation. For F-actin staining Alexa Fluor 546 Phalloidin (Invitrogen, Darmstadt, Germany) and for microtubules labeling Alexa 488 conjugated mouse anti-β-tubulin (BD Biosciences, Heidelberg, Germany) was used

• . Nucleus staining was carried out with DAPI (4’,6-diamidino-2-phenylindol, 50 ng/mL in PBS) (Sigma-Aldrich, Steinheim, Germany). Dark-field microscopy was carried out with an upright microscope with dark-field condensor (Olympus BX51) equipped with a 40× water immersion objective. AFM imaging and force