J. Phys. Chem. B 2009, 113, 3669–3675
3669
Mechanical Properties of Langmuir-Blodgett Films of a Discogen-DNA Complex by Atomic Force Microscopy† Alpana Nayak‡ and K. A. Suresh*,§ Raman Research Institute, SadashiVanagar, Bangalore 560 080, India, and Centre for Liquid Crystal Research, P.B.No.1329, Jalahalli, Bangalore 560 013, India ReceiVed: July 25, 2008; ReVised Manuscript ReceiVed: December 15, 2008
We have studied the mechanical properties of films of a novel ionic discogen, pyridinium tethered with hexaalkoxytriphenylene (PyTp) and its complex with DNA (PyTp-DNA) using atomic force microscope (AFM). The PyTp and PyTp-DNA complex monolayer films were first formed at air-water interface and then transferred onto silicon substrates by Langmuir-Blodgett (LB) technique. For the mechanical properties, particularly to obtain elastic modulus, we have carried out nanoindentation measurements on the LB films of PyTp and also PyTp-DNA complex. The load versus indentation curves from the nanoindentation measurements were analyzed quantitatively using Hertz model. Our analysis yields Young’s modulus values of 54 and 160 MPa for the PyTp and PyTp-DNA complex films, respectively. In addition, the LB films were imaged in the tapping mode AFM to obtain topography and phase images simultaneously. The energy dissipation maps were constructed from the phase images to determine qualitatively the variation in stiffness on the film surfaces. We find that the complex film exhibits a nonuniform surface with varying stiffness while the pure film exhibits a uniform surface. 1. Introduction Discotic molecules have drawn lot of attention for their intriguing supramolecular architectures.1 In addition, because of their potential applications in devices2 such as light emitting diodes, photovoltaic solar cells, field-effect transistors,and gas sensors, researchers have designed and synthesized several types of discogens (discotic mesogens). Compared to the conventional discogens, ionic discogens are even more interesting owing to the presence of ions as charge carriers. The ionic discogens find application in designing anisotropic ion-conductive materials.3,4 The cationic discogen molecules can be complexed with negatively charged biological molecules like DNA. There are several reports on the studies of aliphatic cationic lipid-DNA complexes, which are primarily stimulated by nonviral gene delivery.5,6 There are also efforts for using lipid-DNA complexes for organic electronics,7 biosensors,8 biomedical applications9 and optical applications.10 Although aliphatic lipid-DNA complexes are known for decades, the discogen-DNA complexes have been studied recently,11,12 and they are expected to have some novel applications. The successful long-term performance and reliability of these materials in practical devices are usually limited by their mechanical properties. Hence, measurements of the mechanical properties of such materials are of paramount importance. The conventional techniques to measure the mechanical properties are generally restricted to macroscopic length scale, whereas the current trend in miniaturization of products and devices pose the need for characterization of materials at nanoscale. One of the convenient and reliable techniques for the precise measurement of mechanical properties of nanostructures is †
Part of the “PGG(Pierre-Gilles de Gennes) Memorial Issue”. * To whom correspondence should be addressed. E-mail: suresh@ clcr.res.in. Tel.: +91-80-28382924. Fax: +91-80-28382044. ‡ Raman Research Institute. § Centre for Liquid Crystal Research.
employing an atomic force microscope (AFM). An AFM can provide direct spatial mapping of surface topography, heterogeneity, and elasticity with nanoscale resolution.13 Compared with other tools, AFM can be used to probe local surface elastic properties of soft systems by indentation method with precise control of applied force, down to a few nano-newtons.14 In addition, the phase shift measurements in tapping mode AFM can be used to qualitatively determine the material surface properties, such as stiffness and viscoelasticity.15 The combination of nanoindentation and phase shift measurements with AFM is an useful technique for studying the mechanical properties of film surfaces at nanoscale.16 In this article, we report the mechanical properties of Langmuir-Blodgett films of a novel ionic discogen, pyridinium tethered with hexaalkoxytriphenylene (PyTp) as well as its complex with DNA (PyTp-DNA) using an AFM. To measure local elasticity of these films, nanoscale indentation was performed in the contact mode AFM. Hertz contact mechanics model is commonly used to describe the indentation of a nondeformable indenter into a deformable elastic surface.17 We have used the Hertz model for quantitative analysis of the force-distance curves obtained in the indentation measurements and calculated the values of Young’s elastic modulus, which is essentially a material property useful for device applications. In addition, both PyTp and PyTp-DNA complex films were imaged in the tapping mode AFM and simultaneous topography and phase images were acquired. The phase images were used to construct the energy dissipation maps for qualitative characterization of stiffness variation on the film surfaces. 2. Experimental Section The material PyTp was synthesized by Sandeep Kumar and Santanu Kumar Pal.18 It is a discotic mesogen exhibiting columnar mesophase with the phase sequence: solid, columnar phase (Col), 83.7 °C; Col, isotropic, 95 °C. The monolayer film
10.1021/jp806600g CCC: $40.75 2009 American Chemical Society Published on Web 02/16/2009
3670 J. Phys. Chem. B, Vol. 113, No. 12, 2009 of PyTp was prepared at air-water (A-W) interface in a Langmuir trough (NIMA, model: 611M). To obtain PyTp-DNA complex monolayer film at the A-W interface, we formed a PyTp monolayer on an aqueous subphase containing 10-8 M concentration of DNA. We used double stranded DNA sodium salt (from Salmon testes) with an approximate molecular weight of 1.3 × 106 and ∼2000 bp (Sigma). The PyTp monolayer and the PyTp-DNA complex monolayer films were transferred at a target surface pressure of 35 mN/m onto polished silicon substrates by Langmuir-Blodgett (LB) technique. Although there are several techniques to obtain thin films, LB technique is commonly employed as it is a convenient approach to obtain well-ordered films.19 We have used both hydrophilic and hydrophobic silicon substrates for LB film deposition. To obtain hydrophilic surfaces, we treated polished silicon wafers for about 5 min in hot piranha solution (mixture of concentrated H2SO4 and H2O2 in 3:1 ratio) which were then rinsed in ultrapure deionized water and later dried. For hydrophobic surfaces, freshly prepared hydrophilic silicon substrates were dipped in hexamethyldisilazane for 12 h and then rinsed with HPLC grade chloroform.20 To estimate the molar ratio between DNA and PyTp in the complex LB film, Fourier transform infrared (FTIR) spectroscopy was carried out using a Shimadzu 8400 FTIR spectrometer. For the mechanical property measurements, we have carried out nanoindentation and phase imaging on these LB films using a multimode AFM (model: PicoPlus, Molecular Imaging). All the measurements were carried out at room temperature (∼25 °C). To measure the elastic properties of the films, AFM was used in contact mode to perform nanoscale indentation. In this technique, AFM was used as a depth sensing instrument. Instead of scanning with the tip laterally across the sample surface, the tip was positioned above the surface and moved vertically down. The cantilever deflection, as measured with the optical lever detection system, was plotted as a function of the vertical motion of the piezoelectric scanner to produce a force-distance curve. The advantage of using AFM as a nanoindenter was that we could have fine control on applied load, down to a fraction of nano-newton. In addition, it was possible to observe the deformation (elastic or plastic) by imaging the area before and after indentation. We have maintained the depth of indentation to be small to avoid any plastic deformation. The measurements were carried out inside an environmental chamber in which dry nitrogen gas was circulated to avoid capillary condensation of water at the contact point between the tip and surface. V-Shaped silicon nitride cantilevers with a tip radius of 15 nm (Nanosensors) were used. The spring constant of the cantilevers were in the range of 0.06 to 0.10 N/m, as determined by measuring the free resonance frequency in air using ThermalK software (Molecular Imaging). Freshly cleaned silicon wafer was used as a hard nondeformable reference substrate for photodetector sensitivity calibration. In addition, the topography and phase images of the films were obtained simultaneously using tapping mode AFM (TMAFM). Here, the cantilever oscillates close to its resonance frequency and the tip makes contact with the sample surface only for a short duration (∼10-7 s) in each oscillation cycle. As the tip approaches the sample, the amplitude and phase angle of the oscillating cantilever change due to the tip-sample interaction. The changes in the phase angle produces the phase image which can provide significantly more contrast than the topography image. We have used super sharp probes made up of single-beam silicon cantilevers with a spring constant of 47 N/m and a tip radius typically of 2 nm. The cantilever was
Nayak and Suresh
Figure 1. Surface pressure (π)-area per molecule (Am) isotherms for pure PyTp (dashed line) and PyTp-DNA complex (continuous line) films. The complex film was obtained with a subphase containing 10-8 M concentration of DNA in ultrapure water. Inset shows the chemical structure of pyridinium tethered with hexaalkoxytriphenylene (PyTp) molecule.
oscillated at a frequency of about 200 Hz below its resonance frequency (∼163 kHz) with a free amplitude in the range of 40 to 55 nm. The images were acquired at a scan rate of 2 Hz. To represent phase shifts, we have adopted the standard convention that assigns a 90° phase shift lag between the excitation and response when the free cantilever is excited at its fundamental resonance.21 Driving frequencies below the free resonance would produce phase shifts between 0° and 90° yielding a repulsive interaction regime, while frequencies above resonance would produce phase shifts between 90° and 180° yielding an attractive interaction regime. In all our measurements, the driving frequencies were below the free resonance producing phase shifts between 0° and 90° in the repulsive interaction regime. 3. Results and Discussion The PyTp monolayer was formed at the air-water interface. The chemical structure of PyTp molecule is shown in Figure 1. On spreading the PyTp molecules at the surface of water, the Br- counterions dissolve into the subphase and a positively charged PyTp monolayer forms at the air-water interface. This provided scope for studying electrostatic interaction of such positively charged monolayer with negatively charged biomolecule like DNA. When a PyTp monolayer was formed on an aqueous subphase containing DNA, the negatively charged phosphate (PO2-) groups of DNA interacted electrostatically with the cationic pyridinium groups in the PyTp monolayer and formed a PyTp-DNA complex monolayer film at the air-water interface. We find that an optimum concentration of DNA in the subphase, that is, 10-8 M, formed the most stable monolayer complex.12 The surface pressure-area per molecule (π-Am) isotherms of the PyTp monolayer, and the PyTp-DNA complex monolayer formed with 10-8 M concentration of DNA in the subphase, are shown in Figure 1. We find that the PyTp-DNA complex monolayer exhibited much higher collapse pressure as compared to that of the pure PyTp monolayer, indicating a better stability of the complex monolayer. We have transferred the pure PyTp film as well as the PyTp-DNA complex film at a target surface pressure of 35 mN/m on silicon substrates by LB technique. In an earlier report, it has been shown that the molecules exhibit an edge-on configuration at this surface pressure.12 Interestingly, the formation of the DNA complex enhanced the transfer efficiency over several tens of layers. We have estimated the molar ratio between DNA and PyTp in the complex LB film using FTIR spectroscopy. Figure 2 shows the FTIR spectrum of a physical
Langmuir-Blodgett Films of a Discogen-DNA Complex
Figure 2. FTIR spectra of mixture of DNA and PyTp in the ratio 3:1 (mol/mol), pure DNA and pure PyTp in the range of 900-1800 cm-1. The peaks at 1173 cm-1 (C-O stretching vibrations in PyTp) and 1695 cm-1 (CdO stretching vibrations in DNA) are deconvoluted into individual absorption bands shown as dotted lines, using Peakfit software. Curves are offset for clarity.
J. Phys. Chem. B, Vol. 113, No. 12, 2009 3671 (transfer ratio ≈ 1). This provides scope for device applications such as field-effect transistors and nucleic acid-based biosensors.8 Hence, mechanical property measurements of such systems become important. For nanoindentation studies we have used LB films with 2 layers, and for phase shift measurements we have used LB films with 1, 2, 5, and 20 layers. All the films with odd number of layers were transferred on hydrophilic silicon substrates, and those with even number of layers were transferred on hydrophobic silicon substrates. In the case of a hydrophilic substrate, the first layer was deposited as the substrate was raised through the subphase. Subsequently, a layer was deposited on each up as well as down traversal of the substrate. Thus, a multilayer structure containing only odd number of layers was produced. However, in the case of a hydrophobic substrate, a layer was deposited as it was first lowered into the subphase. This led to a multilayer film structure containing even number of layers. 3.1. Nanoindentation: Determination of Young’s Modulus. Quantitative measurements of local elasticity were performed on LB films with two layers for PyTp and also for PyTp-DNA complex. The cantilever deflection versus piezo displacement curves were obtained at several regions on these films and were converted into load versus indentation curves for further analysis. The load, F, applied by the cantilever tip to the surface was computed from the cantilever spring constant, kc, using Hooke’s law,
F ) kcd
(1)
The indentation depth, δ, is given by
δ)z-d Figure 3. FTIR spectrum of LB film of PyTp-DNA complex with 19 layers in the range of 900-1800 cm-1. The film was transferred at a target surface pressure of 35 mN/m on a hydrophilic silicon substrate. The peaks at 1173 cm-1 (C-O stretching vibrations in PyTp) and 1695 cm-1 (CdO stretching vibrations in DNA) are deconvoluted into individual absorption bands shown as dotted lines, using Peakfit software.
mixture of DNA and PyTp in the ratio 3:1 (mol/mol). The absorptivity ratio between DNA and PyTp for this mixture was calculated to be 0.7, which is the ratio between the area under the peak 1695 cm-1 (CdO stretching vibrations in DNA) and the area under the peak 1173 cm-1 (C-O stretching vibrations in PyTp). On the basis of this absorptivity ratio, the molar complexation ratio between DNA and PyTp was estimated to be 4.7, using the peak areas at 1695 and 1173 cm-1 in the spectrum of PyTp-DNA complex LB film (Figure 3). Since each nucleotide has one phosphate group and the molar ratio of the phosphate groups to the CdO groups is 1 in DNA, the negative-to-positive charge ratio was estimated to be 4.7 in the PyTp-DNA complex LB film. In a double-stranded DNA, the distance separating adjacent planes of hydrogen-bonded base pairs is 0.34 nm, close to the π-π stacking distance of about 0.34 nm in triphenylene columns.11 Therefore, for a complete complexation between DNA and discogen, the negative-topositive charge ratio should be 1. However, in literature, discogen-DNA complexes prepared in the bulk11 are reported to exhibit negative-to-positive charge ratios in the range of 1.4-2.0. In the case of some lipid-DNA complexes, even high values like 7.2 have also been reported.22 An interesting feature of our system is that the PyTp-DNA complex can form multilayers on a substrate with high efficiency
(2)
where z is the piezo displacement and d is the deflection of the cantilever. To determine the spring constant, the cantilever was taken away from the sample. Here, the cantilever can be treated as a simple harmonic oscillator with its potential energy equal to half of its thermal energy (by equipartition).23 The cantilever was driven by an AC signal to obtain its resonance frequency. A power spectrum of the AC signal yielded the mean-square amplitude of the cantilever oscillation, which was then used to determine the spring constant using ThermalK software. Figure 4a shows cantilever deflection (d) versus piezo displacement (z) curves obtained for hard reference silicon surface and PyTp film with two layers. For hard reference silicon surface, the slope of the d versus z curve was equal to 1 since silicon is an infinitely stiff surface compared to the cantilever stiffness. Hence, the tip followed the piezo displacement. On the other hand, for the PyTp film, the tip indented the film surface and the cantilever deflection was smaller than the piezo vertical displacement, yielding a corresponding slope less than 1. The difference between the cantilever deflection for the hard silicon surface and that for the soft PyTp film surface gives the indentation depth δ of the tip into the sample surface. Figure 5a shows the d versus z curves obtained similarly for the PyTp-DNA complex film with two layers. Using eqs 1 and 2, the deflection versus displacement curves were transformed into load, F, versus indentation, δ, curves. Typical F versus δ curves obtained for the pure and complex films are shown in Figures 4b and 5 b, respectively. It was observed that for a given load, the depth of indentation was more on pure film than on the complex film. We have carried out indentation for small load
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Figure 4. (a) Cantilever deflection (d) versus piezo displacement (z) curves measured on a hard reference silicon surface (continuous line), and PyTp film surface with two layers (dotted line). The difference δ between the curves is equal to the tip indentation depth. (b) Typical load (F) versus indentation depth (δ) curve obtained for the PyTp film. The continuous line represents the fitting of the curve with the Hertz model yielding reduced elastic modulus E* value of 77.1 ( 1.3 MPa. (c) The E* values plotted for different loads. These E* values were calculated from various F versus δ curves acquired at 20 different positions on the film surface under different loads, yielding an average value of 71.6 ( 3.2 MPa.
(∼1 nN) to avoid any plastic deformation. To ensure that the deformation was elastic, the film surfaces were imaged before and after indentation. In addition, the F versus δ curves were analyzed quantitatively with Hertz model from continuum mechanics of contact to extract the values of Young’s elastic modulus.24 Hertz model is commonly used to describe the indentation of a nondeformable indenter (the AFM tip made of silicon nitride) into an infinitely extended deformable elastic surface (the sample surface).17 This simple model is valid for elastic deformations and does not take into account tip-sample adhesion. Since adhesion forces were negligible in this study (using dry nitrogen atmosphere) and the deformation was elastic, it was reasonable to use this model. The AFM tip shape can be generally modeled by two geometries,14 a conical or a paraboloid indenter. In these
Nayak and Suresh
Figure 5. (a) The d versus z curves measured on a hard reference silicon surface (continuous line), and PyTp-DNA complex film surface with two layers (dotted line). The difference δ between the curves is equal to the tip indentation depth. (b) Typical F versus δ curve obtained for the PyTp-DNA complex film. The curve was fitted with the Hertz model (continuous line), yielding reduced elastic modulus E* value of 214.3 ( 1.8 MPa. (c) The E* values plotted for different loads. These E* values were calculated from various F versus δ curves acquired at 30 different positions on the film surface under different loads, yielding an average value of 212.4 ( 3.0 MPa.
cases, the load versus indentation depth relations are respectively given by
Fcone )
2 tan RE*δ2 π
4 Fparaboloid ) E*R1/2δ3/2 3
(3)
(4)
Here, R is the half-opening angle of a conical tip and R is the radius of curvature of a spherical or paraboloid indenter. E* is the reduced elastic modulus of tip-sample system and is defined by the equation
Langmuir-Blodgett Films of a Discogen-DNA Complex
1 - ν21 1 1 - ν2 ) + E E1 E*
J. Phys. Chem. B, Vol. 113, No. 12, 2009 3673
(5)
where E and E1 are the elastic modulus, and ν and ν1 are the Poisson’s ratios, of the film and tip, respectively. Poisson’s ratio is generally a positive number less than 0.5. The AFM tip made up of single crystal silicon has a bulk elastic modulus25 of 130-160 GPa and ν1 value of 0.27. Assuming E , E1, we can rewrite26 the above equation as
E* ≈
E 1 - ν2
related to the surface stiffness.36 However, these phase shift measurements depend more on the interaction regime than on the tip-sample interactions.37 This makes the direct phase shift measurements less sensitive to the material properties. Martinez et al. have demonstrated that by converting phase shifts into energy dissipation data, the material properties become more sensitive to the tip-sample interactions,21 thereby giving the stiffness variation of the surface. A relationship between the phase shift and the energy dissipation can be obtained38 by considering that in the steady state
Eext ) Eair + Edis
(6)
where E is the Young’s elastic modulus of the film and ν is its Poisson’s ratio. We find that our measured F versus δ curves followed a δ3/2 variation for both the pure and complex film surfaces. Therefore, the curves were fitted using the paraboloid model (equation 4) and E* values were extracted. More than 30 such curves were obtained at different positions on the film surfaces with different applied loads. Figures 4b and 5b represent typical F versus δ curves fitted with paraboloid model to extract E* values. Figures 4c and 5c show the E* values obtained at different positions on the films with different loads. From these plots, the average E* values obtained were 71.6 ( 3.2 MPa and 212.4 ( 3.0 MPa for the pure and complex films, respectively. Using these E* values and assuming a Poisson’s ratio of 0.5 (which is expected for soft materials)14 in equation 6, we have calculated the Young’s elastic modulus, E to be 53.7 ( 2.4 and 159.3 ( 2.3 MPa for the pure PyTp and PyTp-DNA complex film surfaces, respectively. In literature, the mechanical properties of DNA itself have been widely studied using various theoretical models27 and experimental techniques.28,29 Zhou et al. have studied the nanoscale mechanical properties of DNA using an AFM and reported radial Young’s elastic modulus values of the order of 100 MPa for a single DNA molecule.30 In our system, we have obtained Young’s elastic modulus value of ∼160 MPa for the PyTp-DNA complex film which is about three times the value obtained for the pure PyTp film. This indicates that the complexation with DNA makes the film surface much stiffer as compared to the pure film surface. The elastic modulus values of films are important in surface mechanical properties of materials and in understanding the role of interface in defect production.31 3.2. Phase Imaging: Determination of Energy Dissipation. Phase imaging is known to improve the resolution and contrast of images as compared with the topography image.32 For the LB film of PyTp-DNA complex with single layer, the phase image revealed a periodic structure with a periodicity of 36 nm (Supporting Information). This periodic structure may be attributed to DNA bundles aligned in the film deposition direction. Such formation of DNA bundles have been reported in literature.33,34 The nanoindentation technique using an AFM gives local elastic modulus.35 For a qualitative understanding of the variation in elastic properties over a sample surface, the phase imaging in TM-AFM is very useful. In TM-AFM, as the tip oscillates over the sample surface, there is damping in the tip oscillation due to the tip-sample interactions arising from viscoelastic effects. This leads to a phase shift between the phase angle of the excited signal and the phase angle of the cantilever response. Magonov et al. have shown that this phase shift (φ) can be
(7)
Here, Eext is the external energy supplied to the cantilever, Eair is the energy dissipated via hydrodynamic viscous interactions with the environment, and Edis is the energy dissipated due to the tip-sample interaction. From this equation, the following expression was proposed by Tamayo and Garcia39 that relates the phase shift angle (φ) to the Edis per cycle:
Edis )
(
πkcA0Asp fAsp sin φ Q f0A0
)
(8)
Here, f and f0 are the excitation (driving) and natural (resonance) frequencies of the cantilever, respectively. Asp and A0 are the set point (tapping) and the free oscillation amplitudes, respectively. Q is the quality factor of the cantilever. Thus, the phase shift measurements can be conveniently transformed into energy dissipation values by means of equation 8. Then these measurements can be used to map variations in material properties like stiffness. Figure 6 shows energy dissipation maps for the pure film with two layers and complex films with one and five layers. The topography images of these films are shown in the Supporting Information. Figure 7 shows the topography, phase image, and energy dissipation map for 20 layers of the complex film. The average values of Edis calculated from these energy dissipation maps and the variation in Edis (∆Edis) values are presented in Table 1. It can be seen that, for the pure film, the average value of Edis was higher compared to that for the complex films. This may be attributed to the larger damping for the pure film due to its soft nature which leads to a larger energy dissipation. This result confirms the nanoindentation result that showed the pure film to be soft as compared with the complex film. For the complex films, the ∆Edis values increase with increasing number of layers. Also, they are high compared to the pure film. Our calculations show that for the pure film, ∆Edis value is small and remains almost the same with increasing number of layers suggesting that for the pure film, the surface stiffness remains uniform irrespective of the number of layers. This may be attributed to the fact that the pure film is composed of wellpacked discotic molecules due to the strong π-π interaction between the cores. This leads to a uniform film surface with the molecules arranged in a two-dimensional columnar structure on the substrate.40 For the complex film, such an ordering may not be possible due to the presence of DNA. The nonuniformity in the surface stiffness for the complex film can be clearly seen for the film with 20 layers. The topography image (Figure 7a) shows well-aligned DNA bundles. The phase image (Figure 7b) shows a variation in phase shift of about 16°. The energy dissipation map (Figure 7c) shows comparatively less Edis values at the regions of
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Figure 6. (a) The energy dissipation (Edis) map for the pure PyTp LB film with two layers constructed for a scan range of 1 × 1 µm2. Tapping mode data: f0 ) 162.8 kHz, f ) 162.5 kHz, A0 ) 54 nm, Asp ) 35 nm, and Q ) 125. (b) The Edis map for PyTp-DNA complex LB film with one layer constructed from the phase image with a scan range of 1 × 1 µm2. Tapping mode data: f0 ) 162.95 kHz, f ) 162.75 kHz, A0 ) 43 nm, Asp ) 31 nm, and Q ) 181. (c) The Edis map for PyTp-DNA complex LB film with five layers constructed from the phase image with a scan range of 1.25 × 1.25 µm2. Tapping mode data: f0 ) 162.94 kHz, f ) 162.73 kHz, A0 ) 42.4 nm, Asp ) 31.2 nm, and Q ) 179.7. Panels a, b, and c were constructed using MATLAB software.
DNA bundles indicating these regions to be stiffer. Thus, with the help of phase imaging, we could continuously map the variation in elastic properties over the film surfaces. On the basis of these results and the results from nanoindentation measurements, we suggest that the pure film is soft and comparatively uniform, whereas the complex film is stiff and becomes more and more nonuniform with increasing number of layers. Nanoindentation measurements can give local elastic modulus values, whereas the phase imaging provides continuous maps of variation in stiffness over a sample surface. Hence, the nanoindentation technique, when used together with phase imaging, provides a better understanding of the mechanical properties of film surfaces. Our system of PyTp and PyTp-DNA complex LB films are of the order of molecular thickness and they are very delicate. To measure the nanoscale mechanical properties, particularly, the Young’s elastic modulus, it is necessary to deform the films elastically under very low loads (
