Atomic force microscopy and optical microscopy - Formatex Research ...

Current Research, Technology and Education Topics in Applied Microbiology and Microbial Biotechnology A. Méndez-Vilas (Ed.) _______________________________________________________________________________________

Atomic force microscopy and optical microscopy: suitable tools for the study of the initial stages of biofilm formation Patricia Schilardi1, Carolina Diaz1, Constanza Flores1, Florencia Alvarez1, and Mónica Fernández Lorenzo de Mele1,2 1

Instituto de Investigaciones Fisicoquímicas Teóricas y Aplicadas (INIFTA), Facultad de Ciencias Exactas, UNLP – CONICET, C.C. 16 Suc. 4 (1900) La Plata, Argentina 2 Facultad de Ingeniería, UNLP, 1 esq. 47 (1900) La Plata, Argentina Biofilms are communities of microorganisms consisting in a biologically active aggregate of cells immersed in extracellular polymeric substances (EPS) and attached on a live or dead surface. Atomic force microscopy (AFM) and optical microscopy (OM), including epifluorescence microscopy (EM) have been proved to be suitable tools in order to follow the initial stages of biofilm development. AFM is a local imaging technique based on repulsive forces between the sample and a tip probe, allowing imaging and quantification of surface topographic features as well as biological elements at the nanometer scale. Changes in cell morphology, orientation, size, flagellar direction, bacterial organization and distribution, and EPS production can be detected by AFM, without sample pretreatment, frequently needed in other microscopic techniques. In this chapter we describe the use of AFM to study the formation of Pseudomonas biofilms (considered models in case of motile bacteria) on substrates with different morphology, roughness and chemical composition. The submicroscale images were complemented with the whole landscape of the biofilm obtained by using OM including EM. Keywords biofilm; atomic force microscopy; epifluorescence microscopy; bacterial adhesion; Pseudomonas

1. Introduction Biofilms are communities of microorganisms consisting in a biologically active aggregate of cells immersed in a matrix of extra-cellular polymeric substances (EPS) and attached on biotic or abiotic surfaces. Due to the crucial implications in industry, environment and medicine there had been an increasing interest in understanding and controlling those factors that affect the growth and development of these communities. Biofilms affect heat exchangers, filters, piping, etc. because they induce biocorrosion and biofouling, producing damages on metallic surfaces and the efficiency loss in industrial set-up [1, 2]. In the case of human health, a number of microbial infections are associated with surface colonization not only on live surfaces (sinusitis, pulmonary infection in cystic fibrosis patients, periodontitis, etc. [3] but also on medical implants (contact lenses, dental implants, intravascular catheters, urinary stents) etc. [3, 4]. However, biofilms have also useful applications in bioremediation [5] of different environments (microorganisms degrade and convert pollutants into less toxic forms) and biolixiviation (bacteria can efficiently dissolve minerals used in industry, to obtain copper and gold). Planktonic cells are able to attach on the surfaces and form biofilms through a process that include several steps [1, 3]: (1) formation of a conditioning film, by adsorption of molecules that promote cell attachment; (2) transport and adhesion of planktonic bacteria which move on the surface and form the primary 2-D assemblage, (3) formation of micro-colonies, by bacterial growth and EPS production; (4), formation of a full biofilm, by spreading of microcolonies to reach biofilm maturation; (5) detachment of biofilm patches to begin the cycle in another environment. The initial stages of biofilm formation, i.e. steps (1) and (2) (Figure 1), are the key points for the further colonization of the surface. These steps are influenced by different factors, such as nutrient availability, surface properties of substrates and cells, etc. In particular, physical chemical properties of the surfaces such as topography, wetting properties and chemical functionality are important in cell attachment and growth.

Fig. 1. Transport and adhesion of planktonic bacteria which move on the surface and form the primary 2-D assemblage

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Detailed investigation of bacterial adhesion involved in the developmental process from single sessile bacteria to multicellular biofilm is crucial to elaborate strategies to control biofilm development. For the study of cell attachment and organization, scanning electron microscopy (SEM) is often used. However, sample pretreatments, such as dehydration, deposition of a thin metallic layer on the sample to turn the substrate conductive and high vacuum environment, change biofilm appearances and generate several kinds of artifacts. Atomic force microscopy (AFM) and optical microscopy, including epifluorescence microscopy, have been proved to be suitable tools in order to follow the initial stages of biofilm formation. AFM is used in order to obtain information about morphology and stickiness of biofilms by exploring the aggregation and forces involved during cell attachment. Changes in cell morphology, orientation, size, bacterial organization and EPS production can be detected by AFM. On the other hand, the submicroscale observations should be complemented with the whole landscape of the biofilm that can only be obtained by using optical microscopy. In some cases, epifluorescence microscopy, that implies the use of appropriate dyes that fluorece under ultraviolet light, is needed to evidence viability and biological activity of cells. In this chapter we describe the formation of Pseudomonas biofilms on substrates with different sub-microstructures, roughness and chemical composition. Pseudomonas species comprises a diverse group of bacteria that can be found ubiquitously in several environments. They are frequently used as model species for the study of bacterial attachment and motility [6-18]. Pseudomonas fluorescens (P. fluorescens) are particularly interesting because they participate in both harmful and beneficial processes. Among detrimental effects, P. fluorescens have been found to be involved in human infections [8, 11, 15, 17] and food contamination. In contrast, they have been successfully used as biocontrol species for soil ecosystems [19]. On the other hand, P. aeruginosa is a Gram-negative bacterium and is considered a model not only for biofilm formation but also for pathogenesis [20]. In addition, P. aeruginosa is an opportunistic microorganism that can cause severe, life-threatening infections that causes urinary tract and respiratory system infections as well as a variety of systemic infection in immunosuppressed patients.

2. Atomic force microscopy 2.1 Basic set up and principles of operation AFM is a local imaging technique based on interaction forces between the sample and a sharp tip probe, allowing imaging and quantification of surface topographic features at the nanometer scale [21]. The instrument is also capable to measure interaction forces, which in turn depends on the distance between the tip and the sample, ranging from micro-newtons to pico-newtons.

Photodetector

Contact mode

Laser beam Tip Cantilever

Intermittent mode

(a)

(b)

Fig. 2. (a) Basic set up of AFM (b) Operating modes.

The basic experimental setup consists in a sharp tip probe attached to the free end of a cantilever. The tip is kept very close to the surface (Figure 2a). Attractive or repulsive interactions forces between the probe and the surface will move the cantilever upwards or downwards, producing the bending of the cantilever. A laser beam is reflected from the back side of the cantilever and is detected by a photodetector consisting in four photodiodes. As the tip traces various surface features, its upward and downward movement shifts the beam between upper and lower photodiode components, creating voltage differences which are electronically rendered into height information. In order to scan the sample, the

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tip is displaced in the x- and y- direction by means of piezoelectric actuators. Piezoelectric materials are materials that enlarge or shrink as a voltage gradient is applied to them and, similarly, they produce an electrical potential as response to mechanical deformations. Voltages applied to the x- and y- axes produce a raster scan pattern so that the probe can sense the surface by displacing the sample in those directions. Among the many advantages of AFM, the more important are: (i) higher resolution than other imaging techniques (such as SEM), typically 10 nm, (ii) able of operate in air and in liquids, (iii) able to measure and quantify data in three dimensions, (iv) able to measure conductive and non-conductive surfaces (SEM and scanning tunneling microscopy (STM) need conducting surfaces). However, the following AFM disadvantages, should be pointed out: (i) images artifacts or sample damages due to a incorrect election of the tip, (ii) scan area limited by piezoelectric scanners, (iii) zscan size limited by cantilever and piezoelectric scanners, (iv) time consuming, due to slower scan rate, compared with SEM. High resolution images can be obtained in an easy way when the substrate is relatively robust. However, imaging soft samples, such as biological samples, is more difficult, because the forces exerted by the tip can induce deformation. By combining the appropriate operation mode, probe and operation conditions, useful information about biological samples can be accurately obtained. Imaging samples in fluid is used when is needed to observe biological specimens in their natural or near native, fluid environments or in those cases in which drying can distort the original features of the sample. The fluid can be exchanged during operation, allowing the observation of processes in real time. Thus, this technique allows us to investigate morphological details of cells by the examination of cell surface, organelles and EPM [20] at the nanometer in real time. 2.2 Operation modes AFM can operate in different modes, depending on the characteristics of the sample and the desired information. In AFM contact mode (Figure 2b), also known as repulsive mode, the tip –sample interaction are repulsive forces. The tip is attached to a cantilever with a soft spring constant and scans the surface by “touching” it. The topography is measured by sliding the probe’s tip across the sample surface. Thus, the contact forces cause the bending of the cantilever as response to changes in topography. Contact mode AFM operates both in air and in fluids. Intermittent mode (also known as TappingMode®) of operation (Figure 2b) measures topography by tapping the surface with an oscillating tip. The amplitude of oscillation of the cantilever changes as the forces acting between the sample and the tip changes. Also, these interactions forces cause changes in the resonance amplitude and phase of the cantilever, so that it is possible to image simultaneously topography and phase, allowing the detection of different components of the sample, which will show larger phase contrast. This operation mode, available in air and fluids, eliminates shear forces which can damage soft samples and reduce image resolution. Table 1 shows a comparative landscape of some advantages and disadvantages of contact and intermittent modes of AFM operation. Table 1. Comparative table showing advantages and disadvantages of contact and intermittent modes of AFM operation

AFM operating mode

Contact AFM

Advantages High scan speed, when compared with other operation modes Allows molecular resolution

Disadvantages Distorted images due to lateral forces Distortion of soft samples due to the strong probe-surface interactions.

Rough samples can be scanned. Decreasing of shear forces that can distort the image Intermittent AFM

Higher lateral resolution

Lower scan speed, as compared with contact mode

Less damage of soft specimens

3. Optical microscopy Optical microscopy uses light which is focused by using glass lenses. The resolution of this technique depends on the aperture number of the lenses set up and the wavelength of the light. Typical resolutions are about 200 nm. Higher magnifications need the immersion of lenses in special oils, which, in turn, may decrease the image quality. Traditional optical microscopy uses transmitted light, i.e., the light passes through the sample. Thus, most of the biological specimens analyzed must be supported on transparent substrates (agar, glass, etc.). However, in case of the observations of biofilms formed on different solid substrates non-transparent samples are required. In these cases the use of epifluorescence microscopy, which also provides information about the cell activity, is adequate. The image is formed

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by the emitted light of a fluorescent specimen that has been previously excited by ultraviolet light. Usually, the specimen is stained with a florescent dye that fluoresces after being illuminated with ultraviolet light. The most commonly used fluorescent single dye is acridine orange while commercial viability kits of combined dyes, (such as Live/Dead BacLight® from Invitrogen) are also widely used. Acridine orange is a nucleic acid selective fluorescent cationic dye useful for cell cycle determination. Acridine orange fluoresces either green or red, depending on the nature of the binding reaction with nucleic acid. The color of acridine orange-stained bacteria is frequently used as an indication of the activity of the cell [22]. The Live/Dead kit includes the green fluorescent DNA-binding stain SYTO 9 and the red fluorescent DNA-binding stain propidium iodide (PI), enabling the determination of bacterial viability from the difference in membrane integrity in embedded cells. When used alone, the SYTO-9 stain generally labels all bacteria in a population, whereas propidium iodide penetrates only bacteria with damaged membranes, causing a reduction in the SYTO-9 stain. On the other hand, other dyes which are not fluorescent, like violet crystal and methylene blue, are used in optical microscopy in order to evidence the presence of cells.

4. Biofilm analysis 4.1. Influence of chemical composition and wettability of the surface on biofilm formation We report here several investigations made in our laboratory in relation to Pseudomonas adhesion on different substrates [23-26]. Among them we comparatively analysed gold, a non corrodible material which is biocompatible [27] and copper due to its toxic and antibiofouling characteristics associated to its corrosion products. Taken into account that in natural and artificial environments the formation of a conditional layer of adsorbed organic substances is considered a crucial factor for bacterial adhesion and vitality of attached cells [28, 29], the influence of changes in surface chemistry due to different chemical functionality and the formation of nanoparticles coatings is also described as well as the associated effect of changes in wetting properties. The influence of the nature of the non-modified substrates (gold vs. copper) on Pseudomonas biofilm formation and cell vitality is shown in Figure 3. The use of epifluorescence microscopy allows us to distinguish the different activity of the bacteria attached on gold and copper. Bacteria attached on gold can be seen as orange cells after staining with acridine orange, indicating their high vitality (Figure 3a). However, those attached on copper appears as green bacteria which colour is associated with lower activity (Figure 3b). It was assumed that the color of acridine orange-stained bacteria could be used to discriminate between active and inactive cells [30] because it bonds to double stranded DNA and fluoresces green while single stranded RNA bound to acridine orange fluoresces orange. Actively metabolising cells would be expected to have higher RNA content and thus appear orange with acridine orange staining. A more precise analysis of the number of live and dead cells can be made by using combined dyes (Figures 3 c and d). In the case of the highly biocompatible titanium, large amounts of extracellular polymeric substances can be clearly detected beneath the cells (Figure 4a).

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Fig. 3. Epifluorescence images of P. fluorescens attached on gold and copper substrates. Cells stained with acridine orange (a and b): (a) orange (vital) bacteria attached to gold; (b) green bacteria (lower cellular activity) attached on copper. Images obtained after Live/Dead kit treatment (c and d): (c) green fluorescence (viable cells) on gold and (d) red fluorescence (damage of cellular wall of no viable cells) on copper.

Fig. 4. (a). AFM image (contact mode, 15x 15 µm2) of P. aeruginosa biofilm on titanium (4 h of exposure to bacterial culture). Extracellular polymeric substances can be clearly detected beneath the cells. (b) AFM image (contact mode, 10x10 µm2 of P. aeruginosa biofilm formed on a titanium substrate modified with a silver nanoparticles coating after 72 h of exposure to bacterial culture. Aggregates of silver nanoparticles appear as small bright spots. Inset: Zoom (contact mode, high pass filtered, 3.4x3.4 µm2) showing the presence of long flagella (white arrow) and aggregates of nanoparticles (back arrows)

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Fig. 5. Images of P. fluorescens biofilm (18 hs of substrate exposition to culture) on gold with different chemical functionality and wetting properties: (a) control; top image: AFM, contact mode, high pass filtered, 60 x 60 µm2; bottom image: epifluorescence image, acridine orange stained; (b) dodecanethiol-modified gold (terminal group exposed to culture: -CH3; hydrophobic); top image: AFM, contact mode, high pass filtered, 33.8 x 33.8 µm2; bottom image: epifluorescence image, acridine orange stained; (c) mercaptoundecanoic acid-modified gold (terminal group exposed to culture: -COOH; hydrophilic); top image: AFM, contact mode, high pass filtered, 60 x 60 µm2; bottom image: epifluorescence image, acridine orange stained.

Several strategies have been developed in order to avoid or inhibit cell attachment by modifying the chemistry of the substrate surface due to their influence in the early stage of biofilm development. The formation of silver nanoparticle coatings (with biocidal action) [31-34] on titanium substrate shows the reduction in bacterial attachment (Figure 4b). Interestingly, nanoscale elements such as the silver nanoparticles and flagella can be clearly distinguished by AFM observations. Strategies related to surface modification also include the formation of self-assembled monolayers (SAMs) [35-38]. Biofilm formation on chemically modified surfaces and a control without treatment is shown in Figure 5. A dodecanethiol-modified gold with the hydrophobic –CH3 group (Figure 5b) is compared with mercaptoundecanoid acid-modified gold with the hydrophilic –COOH exposed to the culture (Figure 5 c). Higher bacterial density is detected on hydrophobic samples. The submicroscale AFM observations of Figures 5 a, b and c on top are contrasted with the landscape images of the optical microscopy at the bottom of each one. 4.2 Influence of surface roughness and topography on biofilm formation The effect of roughness was analyzed considering the influence of the topography on bacterial adhesion [39, 40]. On account of the relationship between the dimensions of the cells and the features, the width of the trenches was selected so that it was close to the cell diameter. Figure 6 shows AFM images and the respective cross-section of three gold substrates exhibiting very different topographic features: smooth, nanorandom surface (NS) (Figure 6a), submicrostructured surface (SMS) (Figure 6b) and nanostructure surface with ripples (NRS) (Figure 6c).

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Fig. 6. (a) Vapour deposited gold substrate (NS, smooth): (I) 12 x 12 µm2 AFM image; (II) cross section. (b) Microstructured gold substrate (SMS); (I) 10 x 10 µm2 AFM image; (II) cross section. (c) Nanostructured gold substrate (NRS): (I) 10 x 10 µm2 AFM image; (II) cross-section .

These substrates were used in order to investigate the influence of the size of the topographic features on the formation of small flat assemblages. These assemblages are the first step in the formation of a flat 2-D biofilm. The length of the pioneer cells as well as their orientation can be measured and the relationship between the length, orientation and frequency (percent of bacteria with similar length) can be obtained from AFM images.[23-25]. During the early stages of biofilm formation on smooth gold surfaces cells assemblages can be observed where bacteria are self-organized forming raft-like structures (Figure 7a). SMS seems to inhibit bacterial attachment (Figure 6b) and growth, (Figure 7b, c and d) at least during the first steps of cell attachment. There, a great number of the bacteria are trapped within the trenches and it is difficult for them to climb up the 150 nm height of their lateral wall to join themselves with other cells (Figure 7c).

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Fig. 7. AFM images showing the influence of the gold substrate topography on the self-organization of P. fluorescens after 1 h of exposure to culture. (a) On a NS surface. A 2-D assemblage can be seen; arrows point out flagella around the assemblage. Highpass filtered, 15x15 µm2, contact mode; (b) On SMS. A great number of bacteria are trapped in the trenches. 40x40 µm2, contact mode; (c) Details of the topography of the substrate and flagella (see arrows). 10x10 µm2, highpass filtered, contact mode; (d) Optical image of large areas of the submicrostructured substrate where many cells are trapped in the trenches (dotted line indicates the direction of the trenches).

Experiments made by Whitehead et al [40] using AFM to determine the ease of bacterial removal from substrata, showed that P. aeruginosa cells were removed more easily from substrata with 0.5 µm features (pits) than Staphylococcus aureous. To explain these facts they suggested that cocci (S. aereous) within the pits had larger cell/surface contact area, whereas the rods (P. aeruginosa) that lay across features had a smaller cell/surface contact area. These results are in agreement with our experiments, which also show a high cell/surface contact area relationship between bacteria and the trenches of SMS surfaces. Preferential bacterial attachment on surface unevenness of threads of intrauterine devices (IUD) was also found. Figure 8 shows bacteria (dyed with crystal violet) which are trapped on the valleys of the surface irregularities.

Fig. 8. P. aeruginosa biofilm on the thread of an intrauterine device. Bacteria were dyed with crystal violet. Cell attachment follows the topography of the substrate.

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Fig. 9. AFM image (30 x 30 µm2) of P. fluorescens attached to gold surface with nano-ripples (NRS). The lines indicate preferential direction of bacterial alignement.

Nanoscale features (NRS) also modify bacterial distribution in the assemblages. Figure 9 shows that there is a preferential orientation of the assemblages towards the direction of the nano-ripples (see Figure 6c).

Summary AFM is a suitable tool to study the effect of the surface properties (chemical composition, wettability, roughness, topography) on cell morphology, orientation, size, bacterial organization, flagellar orientation and EPS production without sample pretreatment, frequently needed in other microscopic techniques. Optical microscopy, including the epifluorescence mode complements submicroscale observations with images of the whole landscape of the biofilm including colonies distribution and giving additional information of bacterial vitality. The formation of Pseudomonas biofilms (model biofilms) was particularly analyzed by both microscopic techniques as an example of the influence of the chemical composition of the substrate (gold, copper, titanium, without and with surface chemical modifications and coatings) and surface submicrostructure (NS, SMS, NRS) on the early stages of biofilm development. Acknowledgments. The support by ANPCyT (PICT 05-33225, PICT 05-32906), UNLP (Projects 11/X532 and 11/I129) and CONICET is gratefully acknowledged

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