Immobilisation of living bacteria for AFM imaging under physiological conditions

Ultramicroscopy 110 (2010) 1349–1357

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Immobilisation of living bacteria for AFM imaging under physiological conditions Rikke Louise Meyer a,b,n, Xingfei Zhou a,1, Lone Tang a,b, Ayyoob Arpanaei a,2, Peter Kingshott a, Flemming Besenbacher a,c a

Interdisciplinary Nanoscience Center (iNANO), Aarhus University, DK-8000 Aarhus C, Denmark Department of Biological Sciences, Aarhus University, DK-8000 Aarhus C, Denmark c Department of Physics and Astronomy, Aarhus University, DK-8000 Aarhus C, Denmark b

a r t i c l e in fo

abstract

Article history: Received 30 October 2009 Received in revised form 18 May 2010 Accepted 23 June 2010

Atomic force microscopy (AFM) holds great potential for studying the nanoscale surface structures of living cells, and to measure their interactions with abiotic surfaces, other cells, or specific biomolecules. However, the application of AFM in microbiology is challenging due to the difficulty of immobilising bacterial cells to a flat surface without changing the cell surface properties or cell viability. We have performed an extensive and thorough study of how to functionalise surfaces in order to immobilise living bacteria for AFM studies in liquid environments. Our aim was to develop a scheme which allows bacterial cells to be immobilised to a flat surface with sufficient strength to avoid detachment during the AFM scanning, and without affecting cell surface chemistry, structure, and viability. We compare and evaluate published methods, and present a new, reproducible, and generally applicable scheme for immobilising bacteria cells for an AFM imaging. Bacterial cells were immobilised to modified glass surfaces by physical confinement of cells in microwells, physisorption to positively charged surfaces, covalent binding to amine- or carboxylterminated surfaces, and adsorption to surfaces coated with highly adhesive polyphenolic proteins originating from the mussel Mytilus edulis. Living cells could be immobilised with all of these approaches, but many cells detached when immobilised by electrostatic interactions and imaged in buffers like PBS or MOPS. Cells were more firmly attached when immobilised by covalent binding, although some cells still detached during AFM imaging. The most successful method revealed was immobilisation by polyphenolic proteins, which facilitated firm immobilisation of the cells. Furthermore, the cell viability was not affected by this immobilisation scheme, and adhesive proteins thus provide a fast, reproducible, and generally applicable scheme for immobilising living bacteria for an AFM imaging. & 2010 Elsevier B.V. All rights reserved.

Keywords: Immobilisation Gelatin Polyethyleneimine Cell-Tak Poly-L-lysine

1. Introduction Since its invention two decades ago, atomic force microscopy (AFM) has become a widely used technique for imaging the surfaces of materials, coatings, biomolecules and entire cells at length scale ranging from nanometer to microns. The advantage

n Corresponding author at: Interdisciplinary Nanoscience Center (iNANO), Aarhus University, DK-8000 Aarhus C, Denmark. Tel.: +45 6020 2794; fax: + 45 8942 2722. E-mail address: [email protected] (R. Louise Meyer). 1 Present address: Department of Physics, School of Science, Ningbo University, Ningbo, China. 2 Present address: Industrial and Environmental Biotechnology Department, National Institute of Genetic Engineering and Biotechnology, P.O. Box: 14965/161, Tehran, Iran.

0304-3991/$ - see front matter & 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.ultramic.2010.06.010

of an AFM over many other high-resolution imaging techniques, such as an electron microscopy, is that cells and biomolecules can be imaged under in situ physiological conditions i.e. in liquid, without any staining. Imaging living cells with nanoscale resolution is an exciting leap forward in the field of microscopy, as it enables detailed studies of dynamics events, such as cell growth [1,2] and morphological responses to external stimuli or stresses [3]. As the design of AFM instruments have improved to accommodate the handling of biological samples, application of AFM in life science areas has increased dramatically [4]. Although first introduced as an imaging technique, the power of an AFM as an analytical tool for measuring intermolecular interaction forces with high sensitivity has become evident. For example, chemical force microscopy can resolve the chemical properties and interactions of living bacteria with nanoscale resolution, and this method has for instance been used to study the

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interactions between the bacterial cell surface and antimicrobial drugs [5–7]. In microbiology, an AFM imaging has provided detailed structural information about the bacterial cell surface [8,9]. Many studies have dealt with dried bacteria, as sample preparation in this case is easy, and the resolution obtained when imaging in air is in general higher than when imaging is carried out in liquid environments [10]. However, the most exciting applications of AFM in microbiology lie when the imaging of living cells is carried under in liquid environments, where interactions between bacteria and their surroundings can be studied. The cell–surface interactions involved in bacterial adhesion, cell–cell interactions occurring during biofilm formation, and cell–host interactions responsible for infections are just a few examples. A prerequisite for an AFM analysis of bacteria is the immobilisation of the cells to a flat substrate, and the failure to immobilise cells sufficiently to withstand the lateral force exerted by the cantilever during scanning, continues to be a challenge. This is particularly problematic when imaging bacteria, as the size and shape of bacterial cells only provide a small contact area between the cell and the underlying substrate. Bacteria are often immobilised by letting a drop of cell culture dry out on a surface before re-hydrating and imaging in water or buffer [11,12]. However, even transient drying of cells may affect cell viability or trigger a biological response that alters the cell surface before the AFM imaging. The most commonly used immobilisation methods that do not require drying are physical entrapment in a porous membrane [13] or immobilisation by electrostatic interactions to surfaces that have been coated with positively charged substance, such as polyethyleneimine (PEI) [14], poly-L-lysine [11], or gelatine [15]. Physical entrapment in membranes is only suitable for coccoid cells, and there are indications of mechanical stress being exerted by the entrapment process [16]. While electrostatic immobilisation does not exert mechanical stress, the strength of the interaction is weakened at high ionic strength [17], making imaging most successful in distilled water, which exerts osmotic stress on the cells. It has been shown that even rinsing cells in distilled water prior to imaging in buffer can destabilise extracellular structures, such as the bacterial capsule [18]. Currently no method exists for immobilising living bacteria for AFM studies under physiological conditions, which can be applied to cells with varying size, shape, and surface properties. A robust, reproducible scheme that exerts minimal chemical or mechanical stress during sample preparation and the subsequent imaging is thus needed to exploit the full potential of an AFM in microbiology. Here, we provide an overview of existing approaches to immobilise living bacteria for an AFM analysis under physiological conditions, and present a new, easy, and generally applicable method for immobilisation of bacteria based on highly adhesive polyphenolic proteins.

2. Materials and methods 2.1. Cell cultures Staphylococcus sciuri (DSM 20345) was used as a Gram positive, coccid model organism to evaluate the different immobilisation techniques. Members of the S. sciuri group are widespread and can be isolated from animals and animal products [19–21]. They form biofilms and constitute up to 4.3% of coagulase-negative Staphylococci isolated from clinical samples and are frequently found in wound infections [22,23]. Immobilisation to Cell-TakTM was further evaluated for cells with other cell morphologies and surface properties. The bacteria used for this purpose were the Gram positive Bacillus subtilis (DSM 10), the

Gram negative Escherichia coli (DSM 498), and Mycobacterium sp. (donated by Dr. K. Finster, Aarhus University), which is Gram positive, but has a characteristic hydrophobic cell wall rich in mycolic acids. Cell suspensions were prepared by inoculating cells from agar plates into 50 mL 5 g/l Tryptic Soy Broth and growing them overnight at room temperature, with shaking at 120 rpm. Cells were harvested by centrifugation at 5000g for 5 min, washed three times in PBS, and finally resuspended in PBS or deionised water (depending on the subsequent immobilisation technique). For all immobilisation techniques, we found it important to wash the cells thoroughly, and to not resuspend them to an optical density at 600 nm of more than 0.5.

2.2. Immobilisation of bacteria by physical confinement Glass microscope slides were cleaned by sonication in acetone, alcohol, and deionised water for 10 min each. This cleaning method was employed for all microscope slides before any of the surface modification methods applied in this study. The principle of immobilisation of bacteria by physical confinement was the random capture of cells in microwells made by colloid lithography (Fig. 1a). A colloidal monolayer mask was assembled on the substrate by electrostatic self assembly. This approach included positive charging of the substrate surface by an electrostatic deposition of sequential layers of polyelectrolytes: (1) poly(diallyldimethylammonium chloride) (PDDA, MW 200,000–350,000, Sigma Aldrich), (2) poly(sodium 4-styrenesulfonate) (PSS, MW 70,000, Sigma Aldrich), and (3) aluminium chloride hydroxide (ACH, Reheis). A solution of 3 mm sulphate-modified polystyrene colloids (IDC) was deposited on the surface, and a dispersed colloidal monolayer was obtained after drying with compressed nitrogen. Thin gold films (about 600–700 nm) were then deposited by electron-beam evaporation. Finally, tape was gently placed on the thin gold films, and the dispersed colloids on the glass substrate were removed by pulling off the tape, exposing the micro-wells once colloids were removed [24,25]. Bacteria were immobilised in the microwells by placing a drop of S. sciuri in PBS on the slide and incubating for 20 min before washing gently with PBS. Samples were kept submerged in PBS until AFM imaging was performed in PBS.

2.3. Immobilisation of bacteria by physicochemical interactions and covalent binding of cells 2.3.1. Preparation of surfaces Glass surfaces were modified with either carboxylic acid or amine groups before immobilising cells to the surface with physisorption or covalent binding (Table 1, Fig. 1b–e). Five different approaches were used to coat glass surfaces with amine groups. Surfaces were either treated by N-(2-aminoethyl)-3-aminopropyltrimethoxy-silane (EDS, Sigma) or 3-aminopropyltriethoxysilane (APTES, Sigma) to form self assembled monolayers, or coated with polyethyleneimine (PEI, Sigma), poly-L-lysine (PLL, Sigma) or gelatin (Sigma). EDS was added by placing slides in 1% (v/v) solution of an EDS containing 94% methanol, 1% acetic acid, and 4% distilled water for 30 min, shaking occasionally (a modified method from Vermette et al. [26]). EDS-coated slides were then washed three times with methanol, heated for 1 min at 1201C, washed with deionised water, and dried under a nitrogen stream. APTES was applied by an immersion of slides in 5% APTES in ethanol for 1 h at room temperature. Slides were then washed with ethanol and distilled water before drying under a nitrogen stream.

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Fig. 1. Schematic representation of the immobilisation methods employed. (a) physical confinement by capture in microwells, (b) attractive electrostatic interactions, (c) covalent binding to amine-functionalised surfaces by EDC–NHS, (d) covalent binding to carboxyl-functionalised surfaces by EDC–NHS, (e) covalent binding to aminefunctionalised surfaces by glutaraldehyde, and (f) attachment to polyphenolic adhesive protein from the mussel Mytilus edulis.

Table 1 Methods used for chemical modification of glass surfaces. Coating

Chemical group

Used in immobilisation by physisorption

Used in covalent immobilisation

EDS APTES PEI PLL Gelatin PolysineTM slides PAA

amine amine amine amine mixed amine COOH

X X X X X X

X

Coating with polyethyleneimine (PEI) was obtained by immersing PAA-coated glass slides (see below) for 15 min in PBS with 1 mg/ml 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC, Sigma) and 0.1 mg/ml N-hydroxysuccinimide (NHS, Sigma), and then transferring slides to a solution of 1 mg/ml PEI in PBS for 30 min, while shaking occasionally. Finally, PEIcoated slides were rinsed with PBS and deionised water before drying under a nitrogen stream. Two procedures were used to coat glass slides with poly-Llysine. In the first procedure, PAA-coated glass slides (see below) were immersed in PBS with 1 mg/ml EDC and 0.1 mg/ml NHS for 15 min. Slides were then transferred to 1% (w/v) PLL in PBS and incubated for 30 min, while shaking and finally washed with PBS, and then deionised water. In the second procedure, PEI-coated surfaces were immersed in 2% (v/v) glutaraldehyde overnight and transferred to 1% (w/v) PLL in PBS for 30 min, while shaking. Finally, slides were washed with PBS and deionised water before drying under stream of nitrogen. Gelatin coated slides were prepared by dissolving 0.5 g gelatin (Sigma) in 100 ml distilled water at 90 1C and cooling down to 60 1C before briefly immersing glass microscope slides in this solution and air drying overnight by standing the slides upright in a laminar flow bench.

X X X

Functionalisation with carboxylic acid (COOH) groups was obtained by binding polyacrylic acid (PAA) covalently to the EDS amine groups on the previously prepared surfaces using a modified version of the method suggested by Vermette et al. [26]. A solution of 1 mg/ml PAA (MW 250,000, Sigma) in PBS was prepared. EDC and NHS were then added to a final concentration of 1 and 0.1 mg/ml, respectively, and the amine-coated glass slides were submerged in this solution immediately. The beaker was shaken for the first hour, and then kept overnight. Finally, the surfaces were rinsed repeatedly with MilliQ water and dried by a nitrogen stream.

2.3.2. Immobilisation of bacteria by physicochemical interactions S. sciuri suspended in distilled water or PBS were immobilised by electrostatic interactions to the EDS, APTES, PEI, PLL, and gelatin coated slides plus commercial microscope slides chemically modified to resemble poly-L-lysine (PolysineTM, Menzel). A drop (0.1–0.5 mL) of cell suspension was placed on the modified glass substrates and incubated for 20–30 min (without drying out), before rinsing gently with PBS and storing in PBS until an AFM imaging. Cells were imaged first in PBS and thereafter in distilled water.

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2.3.3. Immobilisation of bacteria by covalent binding Covalent binding was achieved in two ways: (1) by linking amine and carboxyl groups with EDC–NHS (Fig. 1c+ d), or (2) by linking amine to amine groups with glutaraldehyde (Fig. 1e). EDC–NHS was used to covalently bind cells to glass slides modified with carboxyl groups (PAA coated slides) and amine groups (EDS, PLL, or PolysineTM). EDC (1 mg/ml) and NHS (0.1 mg/ml) was dissolved in PBS and immediately used for resuspending bacteria that had been washed in PBS and centrifuged to a pellet. A drop of this solution was then placed on the modified glass surfaces and incubated for 30 min before rinsing with PBS. Slides were stored in PBS until an AFM imaging. Covalent binding with glutaraldehyde was obtained by placing slides in a 2% (v/v) glutaraldehyde overnight, and rinsing thoroughly before placing a drop of cells suspended in PBS on the slide and incubating for 30 min. Slides were then gently washed with PBS and stored in PBS before AFM imaging in deionised water, PBS, 20 mM HEPES, and 20 mM MOPS buffers (all pH 7). 2.4. Immobilisation of bacteria with adhesive polyphenolic proteins Cell-TakTM (BD Diagnostics) is a strongly adhesive polyphenolic protein extract from Mytilus edulisi, and we here use the proteins to facilitate a firm contact between bacterial cells and a glass surface (Fig. 1f). These adhesive proteins were applied to a glass surface by mixing 1 ml Cell-TakTM with 28.5 ml 0.1 M sodium bicarbonate and 0.5 ml 1 M NaOH. The solution was immediately transferred to an area of approximately 1 cm2 on a circular coverslip, and left for 20 min at room temperature. The surface was then submerged in deionised water, air-dried, subsequently stored at 4 1C for up to two weeks before immobilisation of bacteria. Cells were immobilised by adding a drop of the cells suspended in PBS to the protein coated surface, incubating for 5 min, and removing non-adhering bacteria by rinsing vigorously with the solution subsequently used for AFM imaging. The surface was immediately placed in a BioCellTM and imaged. In addition to S. sciuri, B. subtilis, E. coli and Mycobacterium sp. were also imaged in 20 mM MOPS. 2.5. AFM imaging Cells immobilised by physical confinement, physisorption, or covalent binding were imaged by an AFM in PBS and in deionised water. Cells immobilised on adsorbed protein layers were imaged in either deionised water, 2 g/l NaCl, PBS, 20 mM HEPES, 20 mM MOPS, or 5 g/l nutrient broth (all pH 7). When interactions between the cantilever tip and the sample surface was too strong to allow use of intermittent contact mode, cells were imaged in contact mode. A NanoWizardII atomic force microscope (JPK Instruments, Germany) combined with an inverted optical microscope (Zeiss Axiovert 200 M, Zeiss, Germany) was used to record AFM images at 256 or 512 pixels per line, scanning at 0.2–2 Hz. Cells were imaged in contact mode using Silicon nitride cantilevers (NP or NP-S, Veeco, USA) with a spring constant of 0.03 N/m, and Silicon cantilevers (CSC38/noAl, Mikromasch, USA) with a spring constant of 0.03 N/m. The Silicon cantilevers were taller and therefore produced less tip artifacts when imaging cells taller than 1.5 mm. Intermittent contact mode imaging was performed with Silicon cantilevers (NSC19/CrAu, Mikromasch, USA) with a spring constant of 0.6 N/m and a resonant frequency of 80 kHz in air (drive frequency in liquid was 42 kHz). Cells were viewed by optical microscopy simultaneously with an AFM imaging to assess whether any cells were detached during scanning.

2.6. Assessment of cell viability The viability of immobilised cells was assessed by testing the cell membrane permeability using the Live/Dead BacLightTM staining kit (Invitrogen) containing SYTO9 and propidium iodide. S. sciuri was harvested in the mid-exponential growth phase as described above, washed three times in PBS, resuspended in PBS, and immobilised to surfaces as described above by (1) physisorption to PolysineTM slides (Fig. 1b), (2) covalent binding to PolysineTM slides using EDC–NHS (Fig. 1c) or (3) covalent binding to PolysineTM slides using glutaraldehyde (Fig. 1e), or (4) adsorption to Cell-TakTM coated slides (Fig. 1f). Slides were incubated for 4 h in PBS before staining according to the manufacturer’s protocol. Samples were gently rinsed before visualization and image acquisition on a Zeiss Axiovert 200 M epifluorescence microscope.

3. Results and discussion Methods for immobilising bacteria from a suspension on a surface can roughly be divided into four categories: physical confinement, physisorption, covalent binding, and binding via adhesive proteins (Table 2). We were able to immobilise cells in a physically confined space by random capture in 0.5 mm deep and 1.5 mm wide microwells (Fig. 2a and b). However, the success of cell capture was not very reproducible and large areas had to be scanned to locate cells by an AFM. The frequency of capturing cells in the wells may be increased by modifying the chemistry inside wells to combine physical entrapment with electrostatic interactions as demonstrated by Rowan et al. [27]. A general advantage of immobilising bacteria in holes or wells compared to a flat substrate is that the cells do not fully protrude from these holes. Thereby tip artifacts caused by large sample height are minimised [1]. Kasas and Ikai [13] developed a simple and elegant method for physical confinement of spherical cells by filtering a cell suspension through a polycarbonate filter and trapping cells in the filter pores. Unfortunately filtering exerts mechanical stress on the cells [16,28], and only spherical cells with a cell diameter slightly larger than the filter pore size are captured. Although filtering is probably the most reproducible and commonly used immobilisation scheme, it is best suited for Gram-positive coccid bacteria, and less applicable to rod-shaped or filamentous Gram-negative bacteria. Random capture in microwells may exert minimal mechanical stress during immobilisation, but it does not circumvent the problem of not being able to immobilise cells of variable size and shape. That can only occur on a flat substrate. On flat substrates bacteria were easily immobilised by physisorption to chemically modified glass surfaces. As model systems, we have studied cells immobilised to gelatin- and APTES-coated surfaces (Fig. 2c and d). The immobilisation of bacteria to APTES or gelatin coated surfaces is thought to be mediated by a mixture of electrostatic and hydrophobic interactions. Gelatin possesses a number of different chemical groups, and hydrophobic interactions may also play a role here. While imaging of cells immobilised to APTES or gelatin coated surfaces was relatively straight forward in deionised water, all cells detached when imaged in PBS (data not shown). Stukalov et al. [18] also observed detachment of electrostatically immobilised cells during AFM imaging in buffer compared to deionised water. This observation is presumably due to weakening of electrostatic interactions at higher ionic strength [17]. We used Type A gelatin, which has an isoelectric point of 7–9, and therefore carries a net positive charge at lower pH. The net positive charge of gelatin may thus have decreased as the buffer pH approached its

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Table 2 Overview of the methods used for immobilisation of bacteria for AFM analysis in liquid. Method

References

Comments

1. Physical confinement Capture in polycarbonate filters

[3,13,16,38–40] and others.

Random cell capture in microwells

[1], This study

Best suited for coccoid Gram positive cells. Can cause deformation of cells. No chemical modification of cells. Applicable to cells of a specific size range. No deformation of cells. No chemical modification of cells.

2. Physisorption Gelatin coating

[15,41], This study

EDS coating

This study

APTES coating PEI coating Poly-L-lysine or PolysineTM Poly-dopamine

This study [14,42–44] [18,45,46] This study [32]

3. Covalent binding EDC/NHS crosslinking of COOH groups to NH2 groups Glutaraldehyde crosslinking of NH2 groups

[28,47], This study [31,34,48–51] This study

Applicable to cells of different shape and size. No deformation of cells. Cells may become chemically modified if an EDC–NHS or glutaraldehyde is added directly to the cell suspension.

4. Immobilisation with adhesive proteins Adhesive proteins (Cell-TakTM) Poly-dopamine coating Sugar-binding proteins (lectins)

This study [32] [52]

Applicable to cells of different shapes and sizes. No deformation of cells and no chemical modification. Immobilisation with lectins is only applicable to cells containing specific sugars on the cell surface.

isoelectric point. Furthermore, it is not easy to control the tip– sample interactions in PBS. Strong tip–cell interactions and unintentional application of higher force under these conditions may also have exacerbated the problem. Immobilisation by physisorption that involves electrostatic interactions is therefore best suited for imaging at low ionic strength [29]. A much stronger interaction was obtained by covalently linking carboxyl or amine groups on the cell to carboxyl or amine groups on the substrate. The methods used here linked carboxyl groups to amine groups using EDC/NHS (Fig. 1c and d) or amine groups to amine groups using glutaraldehyde (Fig. 1e). Both approaches successfully immobilised cells on amine modified glass surfaces, and AFM imaging could be performed in PBS (Fig. 2e and f), HEPES, MOPS, and deionised water (data not shown). Tip artifacts were observed when imaging in PBS (Fig. 2e and f), as the cells were substantially higher when imaged in PBS (1.5–2 mm) compared to deionised water (1–1.5 mm). Such tip artifacts are not unusual when the cell height exceeds 1.5 mm [30]. Among the amine-functionalised surfaces, commercial PolysineTM glass slides were excellent substrates for covalent binding of bacteria using glutaraldehyde or an EDC–NHS (Fig. 2e and f). Whereas cells rarely detached from this substrate, detachment occurred frequently from EDS-, APTES- and poly-L-lysine-coated surfaces. The difference in cell immobilisation efficiency between PolysineTM and the other substrates may be explained by difference in conformation of the chemical groups, better attachment of amine groups on the commercial slides, or a lower density of amine groups on EDS- and APTES-coated surfaces. This could however not be verified. Covalent binding of bacteria to carboxyl groups on PAA-coated glass slides did not result in successful immobilisation, and all cells detached during AFM imaging (data not shown). The reason for the failed immobilisation is not yet fully understood. It could be caused by low abundance of free amine groups on the cell

Applicable to cells of different shape and size. No deformation of cells. No chemical modification of cells. Immobilisation by electrostatic interactions is less strong at high ionic strength.

surface, low abundance of carboxyl groups on the PAA coated substrate, or that PAA was not efficiently immobilised to the glass substrate. AFM analysis of living cells under physiological conditions should ideally be performed on cells that have not been stressed chemically or mechanically. Immobilisation of cells by EDC–NHS inevitably exerts some degree of chemical modification of the cell surface, as EDC–NHS is mixed directly into the cell suspension and may crosslink chemical groups on the cell surface. Activating chemical groups on the glass substrate instead of the cells could provide an alternative approach to covalent linking, which only affects cell surface chemistry on the part of the cell in contact with the substrate. This was explored by activating amine groups on the substrate with glutaraldehyde, followed by removal of unbound glutaraldehyde before addition of cells. Bacteria have previously been immobilised using glutaraldehyde to crosslink amine groups on the cell surface to amine groups on a PEI coated AFM cantilever [31]. However, the authors suspended bacteria directly in glutaraldehyde, leading to cross-linking of proteins on the entire cell surface, which affects cell elasticity, adhesion properties, and viability [14,32,33]. Sullivan [34] modified the approach and activated APTES coated mica with glutaraldehyde for immobilisation of spheroplasts, and this inspired us to use a similar approach to immobilise intact cells to amine-functionalised glass surfaces. This approach allowed successful immobilisation (Fig. 2f), although some cells did detach during AFM imaging, most likely due to the low density of amine groups present on the APTES coated glass, which limits the degree of cross-linking. Highly efficient immobilisation of bacteria to glass was mediated by adhesive proteins without the need for covalent binding. The mussel Mytilus edulis excretes polyphenolic proteins to adhere strongly to surfaces in the marine environment, and the unique properties of these proteins have gained attention as an efficient adhesive in aqueous conditions [35]. An extract of these adhesive proteins are now available as a commercial product,

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Fig. 2. An AFM image of S. sciuri immobilised by random capture in microwells, electrostatic interactions, and covalent binding. (a) Microwells before immobilisation of cells, (b) two cells caught in a microwell (imaged in deionised water), (c) cells immobilised by physisorption to gelatin (imaged in deionised water), (d) cells immobilised by physisorption to APTES (imaged in deionised water), (e) cells immobilised covalently to PolysineTM slides by EDC–NHS (imaged in PBS), (f) cells immobilised covalently to PolysineTM slides by glutaraldehyde (imaged in PBS). Image height is 1 mm in (a), 0.5 mm in (b), and 3 mm in (c–f). Scalebar in all images¼ 5 mm.

Cell-TakTM, and bacteria immobilised on glass surfaces coated with these proteins could easily be imaged in deionised water, NaCl, physiological buffers (PBS, MOPS, and HEPES), and nutrient broth (Fig. 3a–h). Imaging could be performed in contact mode as well as intermittent contact mode (Fig. 3i). Consecutive scans of the same area showed that a 1–2 cells detached during each scan in e.g. MOPS buffer (Fig. 3d–f). Continuous scanning in nutrient broth could be performed with only few cells detaching for the first 3 h (Fig. 3h), after which all cells suddenly detached (data not shown). The detachment after 3 h might be linked to cell growth or turnover of cell membrane proteins that could destabilise the contact between the cell and the substrate. Immobilisation of cells with variable morphology and surface properties was investigated using Cell-TakTM, and it appeared that both Gram positive and Gram negative species could be immobilised and imaged under physiological conditions (Fig. 4a–c). The long rods of B. subtilis and Mycobacterium sp. were not always immobilised sufficiently, and only partial

immobilisation of cells was recognised as distortion of the image rather than detachment during imaging (Fig. 4b and c). A potential draw-back of immobilisation with polyphenolic proteins is the risk of contaminating the cantilever with these proteins during imaging. We did observe tip contamination from time to time, but this contamination may also arise from contact with the cells, and it did not appear to occur more frequently during imaging of cells immobilised by Cell-TakTM compared to other methods. Scanning of Cell-TakTM coated surfaces in the absence of cells did not indicate tip contamination (data not shown). It should be noted that the adhesive properties of the protein is pH dependent, and imaging at low pH may detach the protein. The adhesive properties of the proteins excreted by Mytilus edulis are linked to the presence of the amino acid 3,4-dihydroxyL-phenylalanine (DOPA), and Lee et al. [36] developed a biomimetic adhesive polymer, poly-dopamine, inspired from the composition and properties of these proteins. Kang and Elimelech

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Fig. 3. S. sciuri immobilised by an attachment to polyphenolic adhesive proteins and imaged in contact mode in (a) deionised water, (b) 2 g/L NaCl, (c) PBS, (d–f) 20 mM MOPS, (g) 20 mM HEPES, and (h) 5 g/L nutrient broth. The images in MOPS (d–f) are three consecutive scans of the same area over a period of 30 min. Arrows indicate detachment of cells. S. sciuri was also imaged in tapping mode in deionised water (i). Cantilevers NP-S were used for images (a, b, d, e, f), cantilever CSC38noAl was used for images (c, g, h), and cantilever CSC19CrAu was used for the tapping mode image (i). The taller CSC cantilevers alleviated the tip artifacts seen when imaging S. sciuri in buffer or NaCl with the NP-S cantilever. All images are 20  20 mm2, and the height is 2 mm.

Fig. 4. E. coli (a), B. subtilis (b), and Mycobacterium sp. (c) imaged by contact mode in 20 mM HEPES after immobilisation by an attachment to polyphenolic adhesive proteins. All images are 20  20 mm2, and the height scale is 2 mm in (a, c) and 1.5 mm in (b). Arrows indicate image distortion by insufficiently immobilised cells.

[32] recently demonstrated that poly-dopamine is also suitable for immobilising bacterial on the tip of an AFM cantilever without affecting cell viability or surface properties [32]. Similar to

immobilisation to Cell-TakTM coated surfaces, this approach appears very promising for providing a generally applicable immobilisation method, which can be used for bacteria of all

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Fig. 5. Live/dead (green/red) staining of S. sciuri immobilised (a) electrostatically, (b) covalently by EDC–NHS, (b) covalently by glutaraldehyde, and (c) by an attachment to polyphenolic adhesive proteins.

shapes and sizes, and does not require exposure to chemicals, distilled water, or drying—all of which may affect cell viability, cell surface properties, or trigger an undesirable biological response. To minimise interaction with the cells, Cell-TakTM was first added to the glass substrate, and unbound proteins were removed by rinsing and drying before bringing cells into contact with the surface. We found no indication that any of the immobilisation techniques used in this study affected cell viability (Fig. 5). Viability was in this case measured as cell membrane integrity, and the result does not exclude that cell physiology changed upon immobilisation. This has previously been shown after immobilisation to for instance Poly-L-lysine coated surfaces [37]. Bacteria – especially those able to form biofilms – will almost certainly respond physiologically to become immobilised on a surface. This biological response must be considered when performing AFM on live cells, especially if the immobilisation technique includes antimicrobial compounds, such as poly-Llysine and glutaraldehyde. The approaches to cell immobilisation evaluated in this study all have different advantages and disadvantages with respect to ease of sample preparation, reproducibility, and applicability to different cell types. No single method provides a one-fits-all approach matching the cell type, substrate type, analysis conditions, and instrumental set-up of any experiment. For example, the combination of AFM with an inverted optical microscope requires a transparent substrate, and therefore rules out immobilisation by physical confinement on polycarbonate filters or silicon wafers. Variability in cell surface charge and desired buffer strength during analysis will affect efficiency of immobilisation by electrostatic interactions. Likewise, the surface density of carboxyl or amine groups on the cell surface will affect whether

EDC–NHS or glutaraldehyde is most appropriate for covalent binding of cells to a substrate. Among the methods tested, we found that immobilisation by adhesive proteins provided the most reproducible approach, while at the same time providing fast and easy sample preparation and high flexibility with regard to the experimental set-up.

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