W. Richard Bowen and Nidal Hilal 4

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214 7. MICRO/NANOENgINEERINg ANd AFM FOR CELLULAR SENSINg and appropriate physiological conditions (medium temperature and pH). Although advances in cell immobilisation and AFM imaging modes now permit soft, live cells to be imaged, the process remains far from trivial [74]. For living cell imaging, an environmental control chamber with suitable medium is essential to keep cells alive and viable. When imaging in contact mode, cantilevers with the lowest spring constants (0.003– 0.06 N m �1 ) operating with a small loading force (�1 nN) are preferred. Following these basic considerations, it is possible to successfully image live cells. Figure 7.9(a)–(f) show height and error images of live 3T3 cells on a fibronectin-coated glass slide and a fibronectin-coated PDMS microgrooved substrate. A well-spread cell morphology with fibrous cytoskeleton structures can be seen clearly in Figure 7.9(a) in comparison to the restrained morphology of the cell on a flat PDMS substrate (Figure 7.9(c) and (d)). This can be further demonstrated by quantitative analysis of the cell height and its projecting area. It should be noted that cells tend to detach from the PDMS substrates during imaging. All of these observations demonstrate that 3T3 fibroblasts have much weaker adhesion to soft PDMS substrates. On microstructured PDMS structures, cells tend to position their nucleus in the trough of the channel, close to one edge and mostly attach filopodia to the upper surfaces (Figure 7.9(e) and (f)). This gives rise to a preferred cell alignment along the channel length, as discussed earlier. Thus, AFM imaging provides a quantitative and high resolution approach to studying the behaviour of living cells. 7.3.2 Elasticity Measurement of living Cells On a soft sample surface, an approaching tip with controlled force will cause an indentation. Although this is often problematic for AFM imaging of living cells, it is a way to measure and map the elastic properties of the cell structure. For a force–distance measurement, the deflection of the cantilever is plotted as function of the z-separation of the probe and the sample. On a stiff sample (e.g. glass), the deflection is proportional to the probe sample separation. However, on the soft sample, the movement of the tip will be less than that of the sample, and the difference is the indention of sample (Figure 7.10(a)). In order to be able to extract material properties from an AFM force– distance curve, we must interpret the deflection using an appropriate model. As a starting point, it is reasonable to consider an AFM force– distance curve as arising from a conical tip indenting a planar surface. This was first considered by Hertz in 1882 [75] (equation (7.1)) and later generalised by Sneddon [76]. F � � 2 2 E ⋅ ⋅ π 2 1 � v ( ) ⋅ ( �) tan (7.1)
Slow [μm] (a) Slow [μm] (c) Slow [μm] (e) 100 50 0 0 50 Fast [μm] 80 60 40 20 0 0 50 100 20 Fast [μm] 0 0 20 Fast [μm] 40 7.3 AFM IN CELL MEASUREMENT 215 718 nm 0 nm 2.774 μm 0 μm 7.749 nm 0 μm This model has subsequently been applied to AFM data by many researchers [77, 78] and modified to account for a number of tip geometries [79]. In order to use the Hertz model to calculate the Young’s modulus (E) of a sample, several experimental parameters have to be known: the applied force (F), indentation depth (�), semi-opening Slow [μm] (b) Slow [μm] Slow [μm] (f) 100 50 0 0 50 Fast [μm] 80 60 40 20 0 0 50 40 20 Fast [μm] 0 0 20 40 Fast [μm] 428.7 mV 0 mV 2.038 V 0 V 1.375 V FIgurE 7.9 AFM images of living cells on different substrates, glass (a and b), flat PDMS (c and d) and a microgrooved PDMS substrate (e and f). The grooves in (e) and (f) have a 12.5 �m period and are 1 �m deep. All the substrates were coated with fibronectin. (d) 0 V
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Butterworth-Heinemann is an imprint
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x Preface in their work. Hence, it
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xii Preface them from hostile condi
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xiv About thE Editors Professor Nid
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xvi List of Contributors Dr Daniel
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2 1. BAsIC PRINCIPLEs OF ATOMIC FOR
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20 1. BAsIC PRINCIPLEs OF ATOMIC FO
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32 2. MEASUREMENT OF PARTICLE ANd S
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82 3. QUANTIFICATION OF PARTICLE-BU
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100 3. QUANTIFICATION OF PARTICLE-B
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C H A P T E R 4 Investigating Membr
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4.2 THE RANgE OF POssIbILITIEs FOR
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4.2 THE RANgE OF POssIbILITIEs FOR
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4.3 CORREsPONdENCE bETwEEN sURFACE
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4.3 CORREsPONdENCE bETwEEN sURFACE
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4.4 IMAgINg IN LIqUId ANd THE dETER
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4.4 IMAgINg IN LIqUId ANd THE dETER
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4.5 EFFECTs OF sURFACE ROUgHNEss ON
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4.6 ‘vIsUALIsATION’ OF THE REjE
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4.7 THE UsE OF AFM IN MEMbRANE dEvE
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Dfractional 0.18 0.16 0.14 0.12 0.1
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4.8 CHARACTERIsATION OF METAL sURFA
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At pH 5.5, dissolution began to occ
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REFERENCEs 137 [4] W.R. Bowen, N. H
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C H A P T E R 5 AFM and Development
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5.2 MEAsUREMENT OF ADHEsION OF COLL
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5.2 MEAsUREMENT OF ADHEsION OF COLL
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The antibacterial activity of initi
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5.3 MODIFICATION OF MEMBRANEs 147 t
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5.3 MODIFICATION OF MEMBRANEs 149 B
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Force (nN m -1 ) Loading force (mN
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5.3 MODIFICATION OF MEMBRANEs 153 p
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Adhesion force (mN m -1 ) 10 8 6 4
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Adhesion force (mN m -1 ) 20 15 10
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Adhesion force (mN m -1 ) 10 8 6 4
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5.3 MODIFICATION OF MEMBRANEs 161 F
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264 9. APPLICATION OF ATOMIC FORCE
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276 10. FuTuRE PRosPECTs most AFM s
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278 10. FuTuRE PRosPECTs targeted d
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A Actin stress fiber, 197 Adhesion,
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Natural organic matter, 140 Negativ
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W. Richard Bowen and Nidal Hilal 4

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