Micron 60 (2014) 1–4

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Identifying bacterial fragments on morphologically similar substrate using UAFM A. Bhattacharya, S. Banerjee ∗ Surface Physics Division, Saha Institute of Nuclear Physics, 1/AF Bidhannagar, Kolkata 700 64, India

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Article history: Received 11 October 2013 Received in revised form 19 December 2013 Accepted 20 December 2013 Available online 30 December 2013 Keywords: Ultrasonic AFM Elasticity Bacteria

a b s t r a c t The ultrasonic atomic force microscopy (UAFM) can be used effectively to map the elasticity of a surface. Using this technique we have demonstrated that biological fragments on a substrate can be easily identified which is otherwise difficult using only an AFM image. We have shown that AFM image can falsely interpret the surface morphological features on the substrate. We have taken the bacteria Pseudomonas sp. as a case study to demonstrate that UAFM technique is a powerful tool to study biological samples and differentiate morphological features on the substrate. © 2013 Elsevier Ltd. All rights reserved.

1. Introduction Atomic force microscopy (AFM) has been extensively used for imaging biological cells (Alonso and Goldmann, 2003; Kasas et al., 1997; Müller and Dufrêne, 2008; Shahin and Barrera, 2008). AFM image simply represents only surface topographical map, but sometime, surface elasticity map is useful to distinguish distribution of soft and hard matter on the sample surface. Differentiation of soft and hard regions on the sample surface for biological sample is very important. Some of the modified techniques of AFM used to obtain the local elastic properties (Kuznetsova et al., 2007) are force modulation microscopy (Maivald et al., 1991), force versus distance curve (Heuberger et al., 1995; Zammaretti and Ubbin, 2003) nanoindentation (Joyce and Houston, 1991) and scanning micro-deformation microscopy (Vairac and Cretin, 1996). In these techniques, one generally gets only a local information at that point of measurement. To obtain an elasticity map of any surface using these techniques is very cumbersome. Now it is well established that contact resonance imaging technique where the cantilever (tip) or the sample is vibrated at ultrasonic frequencies in contact mode (some time with overtone excitation of cantilever) (Rabe and Arnold, 1994; Burnham et al., 1996; Oulevey et al., 1996; Yamanaka and Nakano, 1996; Rabe et al., 2000, 2002; Dupas et al., 2001; Hurley et al., 2003, 2007; Rabe, 2006) allows one to obtain a complete elasticity map of the sample surface. We have also shown

recently (Banerjee et al., 2005, 2007, 2009) that, a complete elasticity map can be obtained by similar contact resonance imaging techniques of the AFM: (1) the atomic force acoustic microscope (AFAM) (Banerjee et al., 2005, 2007) and (2) the ultrasonic atomic force microscope (UAFM) (Banerjee et al., 2005, 2009). In AFAM the sample is vibrated with an ultrasonic transducer attached beneath the sample substrate and in UAFM the cantilever tip is vibrated at ultrasonic frequencies for both the modes and the change in amplitude or frequency of vibration of the cantilever can be monitored to get the elasticity map of the surface. Both these techniques probe the elasticity of the sample at nanoscale (Banerjee et al., 2005, 2007, 2009). Banerjee et al. (2005) have compared the UAFM and AFAM techniques in one of their paper and showed both techniques yield similar results. In this manuscript we shall show that the UAFM technique can be effectively used to map soft biological samples also. This technique can be used to differentiate soft cells from hard cells and surfaces. It has been generally found that malignant cancer cells are softer than the normal cells (Sokolov, 2007; Cross et al., 2007) and malaria affected red blood cells are harder than the normal red blood cells (Hosseini and Feng, 2012). Hence, UAFM technique can be used to identify such elastic properties of biological cells and thus can be used as good microscopic bio-sensor tool. In this paper we have used bacteria Pseudomonas sp. to demonstrate the effectiveness of the UAFM technique. 2. Experimental

∗ Corresponding author. Tel.: +91 33 23375346x3328; fax: +91 3323374637. E-mail addresses: [email protected], [email protected] (S. Banerjee). 0968-4328/$ – see front matter © 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.micron.2013.12.010

The Pseudomonas sp. bacterial cells were immobilized on an anodized Ti substrate. The details. of the preparation technique

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Fig. 1. Amplitude of vibration of the cantilever when in contact with hard and soft surface.

can be found in Gopal et al. (2007) and Banerjee et al. (2006). The immobilized bacterial cells were stored in ambient atmosphere for a long period of time. The UAFM measurements were carried out using boron doped silicon cantilever from NT-MDT (Model NSG10) with elastic constant kc ∼ 20 Nm−1 , radius of curvature of the tip ∼10 nm, tip height 10–15 ␮m, aspect ratio 3: 1, cone angle ∼22◦ and free resonance frequency of fo ∼ 320 kHz. The imaging was carried out using a typical normal force FN ∼ 1.5 ␮N. All the measurements were performed in ambient atmosphere. A brief description of the UAFM technique is as follows and is also shown schematically in Fig. 1: Initially the cantilever/tip is brought in contact to the sample surface and the contact resonance frequency is measured (Rabe and Arnold, 1994; Burnham et al., 1996; Oulevey et al., 1996; Yamanaka and Nakano, 1996; Rabe et al., 2000, 2002; Dupas et al., 2001; Hurley et al., 2003, 2007; Rabe, 2006; Banerjee et al., 2005). The contact resonance frequency of the cantilever is generally in ultrasonic range depending on the dimension and elastic constant of the cantilever. The solid curve in Fig. 1 shows a typical contact resonance curve. When the tip comes in contact with a hard region on the surface, the cantilever vibrates with two ends clamped (i.e., the sample side and the cantilever holder side as shown in the inset of Fig. 1) and when the tip is in contact with a soft region on the surface,

then the tip depresses the sample surface as shown in the inset of Fig. 1 and vibrates with only one end clamped (i.e., the cantilever holder side), this leads to lowering of the amplitude of vibration (as depicted in the inset of Fig. 1 the tip depresses the soft sample surface). Thus, we observe a decrease in the amplitude of vibration of the cantilever for softer region than that of the harder region on the sample surface. In Fig. 1 we also show cantilever resonance curve for soft, intermediate and hard elastic sample surfaces and are labeled as A, B and C respectively. Thus, if the frequency of operation is selected to the right of the contact resonance frequency (i.e., higher than the peak resonance frequency) then we will observe a decrease in the amplitude of vibration of the cantilever when the tip moves to the softer region of the sample surface i.e., the softer parts will have low value of amplitude while the harder parts will have higher value of vibrational amplitude. If the frequency of operation is selected to left (i.e., lower than the peak resonance frequency) of the contact resonance frequency, the scenario will be reversed as shown in Fig. 1. Thus, by taking images on both the sides of the contact resonance curve we obtain contrast inversion of the map/images. This we can explain from the contact resonance curve shown in Fig. 1 as follows: If low value of the cantilever vibrational amplitude is designated by darker color shade and high value of vibration by lighter color shade to depict the elasticity map of the sample surface as shown in Fig. 1, then we should observe contrast inversion in the image if the image is obtained on the other side of the resonance curve as shown schematically in Fig. 1. For more detail on this aspect please refer Banerjee et al. (2005, 2007, 2009). We can conclude that the regions in the same image having opposite contrast are of different materials and if we compare the two images taken above and below the resonance curve then similar material will show contrast inversion. 3. Results and discussions In Fig. 2 we obtain AFM images of isolated bacteria which is rod like in shape (Fig. 2(a)) and clusters of the same bacteria (Fig. 2(b)). From the clusters of the bacteria (Fig. 2(b)) we see certain features appearing on the bacterial surface (Wu and Zhou, 2010). This features leads to fragmentation of the bacteria upon aging. In Fig. 3 topographic images of the bacteria has been taken after a period of one year of storage of the bacteria. This period is a long time for allowing the bacteria to die and fragment. We observe topographic image of the fragments which appear as nodules on the surface. However, it is not possible to say confirmatively whether it is a feature of the substrate or bacteria. Thus to distinguish this we have to carry out elastic mapping of the surface.

Fig. 2. Topographic images obtained from AFM of (a) isolated bacteria and (b) clusters of bacteria.

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Fig. 3. Topographical images of bacterial cells observe after about more than a year. We see distinct nodules appearing on the surface of the bacterial cell.

Fig. 4. (a) 2D and (b) 3D topographical image of fragmented bacteria but note the regions marked as X in figure it appears as part of bacterial fragment. Images obtained using UAFM technique for the frequency (c) higher and (d) lower than the contact resonance frequency. The region marked as X is the harder region and not a fragment of the bacteria.

To clear this ambiguity we have imaged the bacterial cell in the UAFM mode and the results are given in Fig. 4. Fig. 4(a) and (b) gives 2D and 3D topographic images of the bacteria l surface on the substrate on which it is immobilized. In here the region marked as X can be falsely interpreted as the bacterial fragment in the topographic images. However, in Fig. 4(c) and (d) the frequency of vibration has been selected above and below the contact resonance frequency for which we obtain contrast inversion for the bacterial fragments and opposite contrast inversion for the region X, i.e., when the bacteria is shown as bright in the gray scale image the other harder region is shown as dark in the same image (see Fig. 4(c)) and vice versa in the other image (see Fig. 4(d)). Thus,

we can conclude this region X is not part of the bacterial surface even though in topography it falsely appears to be a part of the bacteria (Fig. 4(a and b)).

4. Conclusion In conclusion, the UAFM technique helps in distinguishing bacterial surfaces from the substrate features leading to correct identification of the bacteria and its fragments. This technique can be further extended to other biological cell systems. We can also use this technique in medical diagnostics.

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