Quantitative characterization of adhesion and stiffness of corneal lens of Drosophila melanogaster using atomic force microscopy

journal of the mechanical behavior of biomedical materials 53 (2016) 161–173

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Research Paper

Quantitative characterization of adhesion and stiffness of corneal lens of Drosophila melanogaster using atomic force microscopy A.L. Lavanya Devia,b, Upendra Nongthombac,n, M.S. Bobjib,nn a

Centre for Nanoscience and Engineering, Indian Institute of Science, Bangalore, Karnataka 560012, India Department of Mechanical Engineering, Indian Institute of Science, Bangalore, Karnataka 560012, India c Department of Molecular Reproduction and Development Genetics, Indian Institute of Science, Bangalore, Karnataka 560012, India b

art i cle i nfo

ab st rac t

Article history:

Atomic force Microscopy (AFM) has become a versatile tool in biology due to its advantage

Received 6 February 2015

of high-resolution imaging of biological samples close to their native condition. Apart from

Received in revised form

imaging, AFM can also measure the local mechanical properties of the surfaces. In this

26 July 2015

study, we explore the possibility of using AFM to quantify the rough eye phenotype of

Accepted 7 August 2015

Drosophila melanogaster through mechanical properties. We have measured adhesion force,

Available online 17 August 2015

stiffness and elastic modulus of the corneal lens using AFM. Various parameters affecting

Keywords:

these measurements like cantilever stiffness and tip geometry are systematically studied

Atomic force microscope

and the measurement procedures are standardized. Results show that the mean adhesion

Adhesion

force of the ommatidial surface varies from 36 nN to 16 nN based on the location. The

Stiffness

mean stiffness is 48375 N/m, and the elastic modulus is 3.470.05 GPa (95% confidence

Drosophila melanogaster

level) at the center of ommatidia. These properties are found to be different in corneal lens

Corneal lens

of eye expressing human mutant tau gene (mutant). The adhesion force, stiffness and

Rough eye quantification

elastic modulus are decreased in the mutant. We conclude that the measurement of surface and mechanical properties of D. melanogaster using AFM can be used for quantitative evaluation of ‘rough eye’ surface. & 2015 Elsevier Ltd. All rights reserved.

1.

Introduction

Atomic force microscope (AFM) has emerged as an important tool for imaging at high resolution in biology (Czajkowsky

n

et al., 2000; Santos and Castanho, 2004; Kumar et al., 2005; Kasas et al., 1997; Dufrêne, 2002; Morris et al., 2009). AFM uses the interaction force between a sharp probe and the sample to obtain three-dimensional image of the surfaces.

Corresponding author. Tel: þ91 80 22933258; fax: þ91 80 23600999. Corresponding author. Tel.: þ91 80 2293 3233; fax: þ91 80 23600648. E-mail addresses: [email protected], [email protected] (U. Nongthomba), [email protected] (M.S. Bobji). nn

http://dx.doi.org/10.1016/j.jmbbm.2015.08.015 1751-6161/& 2015 Elsevier Ltd. All rights reserved.

162

journal of the mechanical behavior of biomedical materials 53 (2016) 161 –173

Main advantage of AFM is that it provides valuable information about the biological samples in their native condition with minimal sample preparation. This has enabled extensive use of AFM in imaging various biological samples at different length scales, starting from molecules like DNA (Lyubchenko et al., 2011; Kalle and Strappe, 2012) and protein, microbes like virus (Gaczynska and Osmulski, 2009) and bacteria (Dufrêne 2002), to various cells and tissues (Lombardo et al., 2006; Kreplak et al., 2008; Graham et al., 2010). Comparatively, fewer studies have been reported on the quantitative measurements of local mechanical properties, like elastic modulus and stiffness of various biological samples (Gaboriaud and Dufrêne, 2007; Grant, 2011; Müller and Dufrêne, 2011; Kasas et al., 2013; Kahn et al., 2013). High variability in the measurement of mechanical properties (Wagner et al., 2011; Roy et al., 2014) is the main reason for not using AFM as a routine technique in biology. This variability in measurements arises due to complexities of the biological sample. Most of the biological samples are inhomogeneous in nature (Gabriel et al., 1996; Miyazaki and Hayashi, 1999), with spatial variation in material composition and geometry. The variability resulting from these can be addressed only by making a large number of measurements at spatially different regions and statistical analysis of the measured data (Sokolov et al., 2013). Ability to make fast and numerous measurements makes AFM an ideal measurement tool for studying biological samples. However, there are many measurement parameters that need to be standardized before AFM measurements become repeatable. Some of these parameters, like choosing the correct probes, sample preparation and spatial location and distribution of the measurements are addressed in this study using the corneal lens of fruit fly (Drosophila melanogaster). D. melanogaster is a powerful genetic model used extensively in unraveling various genetic pathways and understanding the human disease conditions (Chan and Bonini, 2000; Bonini, 2000; Bier, 2005). The compound eye of Drosophila is used widely as an experimental system for identifying mechanisms controlled by different genes (Muqit and Feany, 2002; Sang and Jackson, 2005; Kumar, 2012), and the effect of mutation in those genes (Borras et al., 2003; Vo et al., 2014). Each compound eye is made up of uniform hexagonal packing of 800 ommatidial units, forming an array known as ‘Neuro-crystalline lattice’. Each ommatidium consists of an outer transparent corneal lens and an inner cellular retina. The retina comprises of 18 cells, including cone cells and pigment cells, which secrete the corneal lens (Kumar, 2012; Charlton-Perkins and Cook, 2010). Genetic modifications, expressed in the eye using tissue specific UAS-Gal4 system (Duffy, 2002; Muqit and Feany, 2002), affect the cellular layer. Consequently, the secreted corneal lens layer gets modified and appears as a typical ‘rough eye’ phenotype (Lu and Vogel, 2009; Iijima-Ando and Iijima, 2010; Gistelinck et al., 2012; Prüßing et al., 2013). Quantitative measurements of the rough eye can be used for assessing phenotypic effects of genetic variations (IijimaAndo and Iijima, 2010) Various geometric parameters such as size (He et al., 2014) or volume (Ambegaokar and Jackson, 2010) of the rough eye phenotype have been used as a quantifiable parameter for this purpose. In this study, we

explore the possibility of using AFM to quantify the rough eye phenotype through mechanical properties. The aim is to exploit quick and easy AFM measurements to find differences in phenotypes resulting from various genetic modifications for large scale screening. Structure and geometry of the corneal lens simplify AFM measurements. The lens is acellular, and does not contain organelles like nucleus. This results in reduced spatial variation in the geometry as well as mechanical properties. The thickness of the lens layer is about 5–7 mm, and thus can be safely probed in AFM without having to consider the effect of the substrate on the force measurements (Dimitriadis et al., 2002). Though, AFM based imaging of the nipple structures of (Anderson and Gaimari, 2003) Drosophila and its mutants have been done (Kryuchkov et al., 2011), this is the first study on measuring the mechanical properties of the lens layer. Through a statistically large number of AFM measurements on the corneal lens, it is shown that the tau mutant (human mutant tau V337M) with a rough eye phenotype has lower adhesion force and stiffness.

2.

Methods and analysis

2.1.

Flies and sample preparation

Wild type fly, Canton-S and GMR-Gal4, a driver for specific expression of gene in retinal tissue were obtained from the Bloomington stock center. Human mutant Tau disease model, UAS-TauV337M was obtained from Prof. Mel Feany (Wittmann et al., 2001). All flies were maintained in standard cornmeal–agar–yeast media, at 23 1C. Mutant flies expressing human tau were obtained by crossing GMR-Gal4 with UASTauV337M. Progenies were transferred to 29 1C at larval stage to enhance the expression of GMR-Gal4. One day after eclosion flies were etherized with diethyl ether. The head was removed from the fly using a sharp scalpel and cut into two halves. By holding the head cuticle, the internal soft tissues, including brain and retina, were removed completely using a tungsten needle. Right eyes of the female flies were used for all the experiments. Corneal lens layer from the flies were cut into rectangular samples of about 300  500 mm2 size to make it flat. The flattened lens layer was then placed on a silicon wafer of size 1 cm  1 cm with a drop of water. As the water evaporates, the corneal lens layer adheres to the wafer surface. This procedure is preferred over mounting of samples on glass slides with double stick tape (Anderson and Gaimari, 2003; Kryuchkov et al., 2011) to avoid any effect of the soft substrate in modulus measurements.

2.2.

Atomic force microscopy

2.2.1.

Instrumentation

Atomic force microscope from Bruker (Dimension Icon) equipped with ScanAsyst for optimizing the imaging parameters was used for all the measurements. Force spectroscopy curves were obtained at the desired location after imaging with tapping mode. Probes made of Silicon Nitride, silicon and diamond, mounted on cantilevers of different

journal of the mechanical behavior of biomedical materials 53 (2016) 161 –173

Fig. 1 – Typical force spectroscopy curves with inset showing the raw data (in volts). stiffness was used. Since the stiffness of the cantilever determine the maximum force that can be applied as well as the sensitivity of the force measured, different cantilevers with stiffness ranging from 0.1 N/m to 419 N/m were used. Corneal lens turned out to be hard and stiff, and to prevent damage to the probe during measurement a diamond probe mounted on stainless steel cantilever was used. Deflection sensitivity of all the cantilevers was obtained from the force spectroscopy curves obtained from a stiff and hard substrate. Single crystal silicon wafer was used as the substrate in all cases, except for the diamond probe for which a sapphire substrate was used. The stiffness of the cantilevers was obtained using thermal tune method (Hutter and Bechhoefer, 1993). Deflection sensitivity and stiffness of the cantilevers were obtained before each set of measurements. Deflection sensitivity was found to vary up to 15% on the same cantilever on different days, and was found to be dependent on the exact location of the laser spot aligned on the cantilever.

2.2.2.

Force spectroscopy

Force spectroscopy curves represent the force produced as a result of interaction between the AFM probe mounted on a cantilever and the sample over a distance. The force is obtained from the deflection response of the cantilever to the interaction and its stiffness. A typical force spectroscopy curve measured on the surface of the corneal lens of D. melanogaster is shown in Fig. 1. The tip is brought into contact with the sample surface by moving it with a constant velocity, and is allowed to deform the sample. This forms the ‘trace’ portion of the force spectroscopy curve as shown in Fig. 1. After the tip penetrates by a predetermined value, tip is moved back to its original position at constant velocity. The resulting interaction is captured by the ‘retrace’ portion of the force spectroscopy curve. The trace and retrace portions of the force spectroscopy curves thus contain the information about the interaction between the tip and the sample, from which the adhesion and the elastic modulus of the sample are obtained. As the tip approaches the sample surface, long range van der Waals interaction results in an attractive force between them.

163

This force increases rapidly as the distance between the tip and the sample decreases. When the rate of change of this force with distance equals the stiffness of the cantilever (Pethica and Sutton, 1988; Israelachivili, 2011) the tip suddenly jumps into contact with the sample, resulting in a snap-in (Fig. 1). Once the tip comes in contact with the sample, the interaction becomes repulsive, and the force–displacement (F–d) response characterized by the stiffness of the interaction ðKÞ can be interpreted from contact mechanics. The deformation of the sample will be elastic for very small penetrations, and elastic–plastic at higher penetrations. However, while retracting the initial recovery of the deformation is invariably elastic, and this fact is used to obtain elastic modulus using appropriate models (Oliverand Pharr, 1992) from the measured stiffness ðKu Þ. If there is no plastic deformation in the sample, then the approach curve and the retract curve will coincide with each other. While retracting the tip, due to the attractive interaction, the cantilever experiences a tensile force before the tip is completely moved away from the surface. When the tensile force from cantilever matches the adhesive force ðFA Þ between the tip and the sample, the tip suddenly snaps-out of the sample. The magnitude of this pull-off force gives the adhesive interaction between the tip and the sample (Fig. 1). Force spectroscopy curves were obtained at desired locations on the ommatidia after imaging in tapping mode. An array of points, typically 5  5, was defined on the image and the measurements were automated to be obtained one after another. Approach and retraction of the cantilever were carried out at a speed of the 2 μm/s to obtain a trace and retrace portion of the curve. The maximum force applied by the probe to the sample was adjusted by setting a trigger threshold for the cantilever deflection. Displacement of the piezoelectric drive carrying the cantilever and the corresponding deflection of the cantilever measured by the photodiode was recorded. The data obtained were analyzed using custom developed programs in Matlab to obtain the adhesion and stiffness. Typical raw data from the force spectroscopy experiment consisting of voltage applied to the piezoelectric drive (volts) versus measured cantilever deflection (volts) is shown in Fig. 1 (inset). The voltage applied can be converted to displacement by multiplying with a sensitivity parameter supplied by the manufacturer. The force experienced by the AFM probe can be obtained from the deflection signal by multiplying with the deflection sensitivity and cantilever stiffness ðKc Þ. Deflection sensitivity depends on the alignment of the laser on the cantilever and the exact distance between the cantilever and the detector, and therefore has to be measured. Though nominal cantilever stiffness was provided from the manufacturer of the probes, the measured values were found to vary up to 60%. Hence, these values were measured before the start of the each measurement cycle. Contact mode and Peak-force Quantitative nanomechanical mapping (QNM) mode are used for imaging the local topography of the corneal lens. The parameters like proportionality gain and integral gain settings are adjusted according to the scan size, sample lines and scan rate in contact mode. Whereas in the Peak-force QNM mode these settings are self-adjusted. The Peak-force QNM mode obtains force– distance curves (FD curves) at each pixel of the image at high

164

journal of the mechanical behavior of biomedical materials 53 (2016) 161 –173

Fig. 2 – Force–displacement curve for different Kc in N/m: (a) 0.12, (b) 0.44, (c) 3.8, (d) 52.48, (e) 242.66 and (f) 419.

FA ¼ Kc δmin ;

ð1Þ

where δmin is the minimum deflection of the cantilever measured in the force spectroscopy curve. Thus, for a given AFM sensitivity, a cantilever of lower stiffness will give more accurate adhesion force measurement. Alternatively, if the stiffness is very high then δmin will be smaller than the noise in the deflection measurement and would not be visible in the Force spectroscopy curve. Since magnitude of the adhesion force ðFA Þ of the corneal surface of the D. melanogaster is unknown, cantilevers with varying stiffness (Kc ) were used. Typical force curve obtained from six different cantilevers of stiffness varying from 0.1 to 419 N/m are shown in Fig. 2. For a low-stiffness cantilever, since the cantilever deflection is large even for a small repulsive force, the penetration of the tip into the sample will be negligible, and the adhesion force measured will be affected by any thin layer of contaminant on the surface. On the other hand, high stiffness cantilevers will not even allow the measurement of the adhesion, as can be seen in Fig. 2f. Even though a value can be obtained from Eq. (1), the measured adhesion value would be affected by the measurement errors like thermal drift and instrument noise. Hence,

0.12

0.44

3.80

758±452.6

n=30 n=70 n=219 42.1±18.8

50 40 30 20 10 0

n=200

1441.7±1187.8

1000

965.4±356.5

n=104 n=115

35.1±21.5

Adhesion force and surface energy measurement

The maximum tensile force experienced by the AFM cantilever with the sample was used as a measure of adhesion force. The accuracy with which this force can be measured depends not only on the sensitivity of the photodetector of the AFM but also on the stiffness of the cantilever since,

2000

37.7±19.8

2.2.3.

Adhesion Force (nN)

3000

speed. From each FD curve, adhesion and elastic modulus values are calculated in real time by the control software supplied with the AFM. The software uses the DMT model to obtain modulus and a user settable tip radius. This surface map of adhesion and modulus gives visually the spatial variation in properties.

52.48 242.66 419

Fig. 3 – Adhesion force measurement with different Kc mean with standard deviation is shown. n is the number of force spectroscopy curves obtained on the ommatidial surface. for proper adhesion measurement of biological sample, it is important to choose the appropriate stiffness of the cantilever. The mean value of adhesion force obtained from each cantilever is shown in Fig. 3. For each cantilever, the measurements were carried out on at least three different ommatidia of eyes from three different individual flies. The measurements were carried out in an array with the minimum distance of at least 350 nm apart. The number of force spectroscopy curves ðnÞ used to obtain these values ranged from 30 to 200; the exact numbers are mentioned in Fig. 3. The measured data have a high standard deviation of about 50% of the mean values. Error bars represented in all the subsequent figures indicate 95% confidence level on the measured mean value. From Fig. 3, it is seen that the measured adhesion force is independent of Kc o3.8 N/m. Thus, the cantilevers with Kc of 0.12, 0.44 and 3.8 N/m are found to be appropriate as they deflect sufficiently to capture the adhesion force of the sample in the order of a few tens of nN. Adhesion force values obtained by high stiffness cantilevers are not reliable as can be seen from the high level of scatter in the

measurement and the dependency of measurement on stiffness. Although, the cantilever with Kc ¼40 N/m is capable of capturing the adhesion force of the sample, the deflection of the cantilever is very small to be distinguished from the base line error. Therefore, to study the adhesion property for the corneal lens of D. melanogaster, the force spectroscopy curves can be obtained with a cantilever stiffness of about 3.8 N/m or less. All other reported values of adhesion in this study have been obtained with a cantilever stiffness of 3.8 N/m. The experiments were carried out in a controlled environment of 22 1C and relative humidity of 45%. The measured adhesion force depends on the surface energy of the sample (γ s ), and the tip (γ t ) as well as the geometry of the contact. Typically, it is assumed that the apex of the tip is spherical, defined by a finite radius ðRÞ. For a sphere interacting with a flat surface, the adhesion force lies between two limits defined by JKR and DMT models (Johnson et al., 1971; Derjaguin et al., 1975). JKR model assumes that there is no adhesive interaction outside the contact region, while the DMT model assumes that there is no adhesion within the contact. These simplifying assumptions, along with the assumption that the adhesion is a surface force, enable a closed-form solution of F ¼ 32πRγ and 2πRγ for JKR and DMT models respectively. Applicability of either of these extremes to a given configuration depends on the reduced elastic modulus, En of the sample and the tip, which can be determined from the Tabor parameter ðmÞ (Greenwood, 1997). For the ommatida, using the measured modulus (Section 3.1.2) the Tabor parameter was obtained as 3.45, which is greater than 1, and hence these measurements are characterized by JKR model (Maugis, 2000). Using JKR model the relative surface energy (Δγ) can be obtained from the pull-off force ðFadh Þ as Δγ ¼

2 Fadh ; 3πR

ð2Þ

with R as the radius of the nipple structure (Johnson et al., 1971). To validate the methodology used here, we measured the adhesion force on a test sample of poly dimethyl siloxane (PDMS) supplied by Bruker. The measured adhesion forces were 11.1570.15, 19.7173.5, 22.171.4 nN using cantilever of stiffness less than 3.8 N/m. Using Eq. (2) the surface energy obtained was 0.03 J/m2, which is comparable to 0.029 J/m2 obtained by contact angle measurement (Yang et al., 2009).

2.2.4.

Stiffness and elastic modulus measurement

Elastic Modulus of the substrate can be obtained from the retrace portion of the force spectroscopy curve, if geometry of the contact is known (Oliver and Pharr, 1992; Pethica and Sutton, 1988). Assuming that the initial portion of the unloading (retrace) curve is elastic (Stilwell and Tabor, 1961; Oliver and Pharr, 1992), the reduced elastic modulus can be obtained as En ¼

Ks ; 2a

ð3Þ

where Ks is the slope of the Load-Penetration curve and a is the contact radius. Force spectroscopy curve obtained from AFM (Fig. 1) gives the force (F) as a function of displacement (d) of the base of the cantilever. The actual penetration of the

Unloading Stiffness, Ku (N/m)

journal of the mechanical behavior of biomedical materials 53 (2016) 161 –173

n=115 258.59

300

165

n=40 232.32

200

100 n=30 0.13

n=91 0.31

n=253 2.57

0.44

3.80

n=47 51.42

0 0.12

52.48 242.66 419.00

Fig. 4 – Measured Ku with different Kc .

probe (h) into the sample is given by h ¼ d–δ ¼ d

F ; Kc

ð4Þ

where δ is the deflection of the cantilever and Kc is the stiffness of the cantilever. The sample stiffness ðKs Þ can be obtained once the actual penetration is obtained. However, this method was found to produce large scatter as it involves subtraction of two similar numbers. Hence we measured the slope of the retrace curve (Ku ) by least square fitting of a line to the initial portion of the retrace curve and obtained sample stiffness, as shown below: Since the sample ‘spring’ of stiffness, Ks and the cantilever ‘spring’ of stiffness, Kc , are in series, the effective stiffness (Ku ) of the combined spring measured by the F–d curve (Fig. 1) is given by 1 1 1 ¼ þ Ku Ks Kc

ð5Þ

For a sample that is rigid compared to the cantilever (Ks ⪢Kc ) the measured Ku value will be close to the value of the stiffness of the cantilever ðKc Þ, as can be seen from Fig. 2. On the other hand, if the cantilever stiffness is very high, then the sensitivity of the force measurement is reduced below the noise level of the photodetector. Thus, it is important to choose the cantilever stiffness as close to the stiffness of the sample to be measured. This is complicated by the fact that the measured sample stiffness also depends on the actual geometry of the AFM probe. We carried out a systematic measurement of stiffness with various AFM probes by obtaining force spectroscopy curves at various locations. Some of these force spectroscopy curves are shown in Fig. 2. The measured stiffness ðKu Þ obtained from such measurements are shown in Fig. 4. The geometry of the probe was obtained from SEM images (Fig. 6), and the contact radius for different probes varied from 58.45 nm to 360.86 nm. It can be seen from Fig. 4 that for cantilevers of stiffness up to 242.66 N/m, the measured slope is very close to the stiffness of the cantilever. Hence, we decided to use a very stiff cantilever (DNISP-HS from Bruker) for modulus measurement. Since the force encountered will also be very high we decided to use a diamond probe. The diamond probe being very hard, also has an added advantage that the shape of the apex of the probe does not change during the sensitivity calibration. We find that a diamond probe on cantilever of stiffness Kc ¼ 419 N/m to be the most suitable for measuring ommatidia stiffness.

journal of the mechanical behavior of biomedical materials 53 (2016) 161 –173

3.

Result

3.1.

Wild type

3.1.1.

Adhesion force of wild type

The adhesion force measured on the ommatidial surface showed substantial scatter with a standard deviation of 52% of the mean value, in spite of a large number of trials. This clearly indicated that there must be other parameters affecting the measurements, besides cantilever stiffness. First, it was hypothesized that the sample preparation techniques may have a profound influence on the adhesion force measurement as the adhesion force depends on the chemical nature of the surface. To study the influence of moisture content on the sample surface on the adhesion force measurement, samples were prepared in anhydrous environment. For this, the flies were fixed in 70% ethanol, followed by a treatment with 100% ethanol to remove moisture. Adhesion force was found to be about 25 nN. It was also found that there is no significant change in this value over time. For the samples prepared with water, the adhesion force values were higher (57 nN) for the measurements taken just after sample preparation (Fig. 5a), which decreased to about 25 nN after 6 h of exposure to air. It is possible that the water from the atmosphere might have condensed on the ethanol-treated samples, and water might have evaporated from the water-treated samples after reaching an equilibrium concentration. It was found that even in the measurement done after 6 h of mounting, the scatter was about 50% of the mean. On further investigations, it was found that the adhesion force values also depend on the location of the measurement on the ommatidia. Fig. 5b shows the adhesion force values measured at center, intermediate and peripheral region of the ommatidia. Force spectroscopy curves were obtained from these regions of the ommatidial surface using low Kc

80

Adhesion, FA (nN)

Stiffness measurements were carried out with the diamond probe having a tip radius ðRÞ of 100 nm with maximum displacement ðdÞ of 60 nm. The nipple has a radius of about 50 nm (from SEM) (Fig. 7a), and height and pitch of about 30 nm and 200 nm (from AFM) respectively (Fig. 8). Thus, the geometry of the nipple has little effect on the stiffness or elastic modulus measurement as the deformation is spread over a large distance from the point of contact. Since ommatidia radius was very large (17 mm) compared to the pffiffiffiffiffiffi probe tip radius, the contact radius can be given by a ¼ Rh and it is 80 nm. To validate the methodology of measuring elastic modulus, force spectroscopy curves were obtained for a known material (PMMA—Poly methyl methacrylate), using a stiff silicon or stainless steel cantilever with a diamond tip; the elastic modulus was found to be 5.570.7 and 4.970.5 GPa respectively. These values are within the range of values reported previously using AFM indentation (Bhushan, 2008; Schofield et al., 2009). It should be noted that this is the indentation modulus, which is slightly different from the modulus of 3 GPa obtained for PMMA in a tensile test (Gómez and Elices, 2003).

*** 60

ns ns

40 20 0

n=150

n=63

n=306

n=64

T<3h

T<6h

T>72h

T>700h

50

Adhesion, FA (nN)

166

***

40

***

30 20 10 0

n=234 Center

n=306

n=376

Intermediate Periphery

Fig. 5 – (a) Variation in adhesion with time at intermediate region, (b) Variation of adhesion at different locations on an ommatidial surface. (The unpaired t-test is performed between the groups, *** shows a statistically significant difference in mean with Po0.001, ns shows not significant in mean for P40.05, error bar represents 95% confidence level). (3.8 N/m). Adhesion force was found to be maximum of about 36 nN at the center of the ommaditia, while in the peripheral region it reduced to 16 nN. Unpaired t-test, shows that there was significant variation among all the three different locations. It should be noted that in Fig. 5 and all other subsequent results, the error bars represent 95% confidence interval. The surface energy can be calculated from the measured adhesion force and the geometry of the tip, using Eq. (2). The geometry of the apex of the probe was obtained the Field Emission SEM images at high resolutions. Fig. 6 shows the images of the apex of some of the probes used in our studies. It can be seen that in almost all the cases, the original sharp tip geometry, as provided by the manufacturer, is lost. Only the Diamond tip used had maintained the geometry with a tip radius of about 100 nm. It was found that the apex of the tips becomes blunt during deflection sensitivity calibration. Deflection sensitivity of the AFM cantilevers was obtained by carrying out a force spectroscopy curve on a stiff substrate – silicon. It has been found that even at a moderate displacement of 20 nm after bringing the apex in contact resulted in the fracture of the silicon nitride tip. The damage to the tip apex was inevitable, because the deflection sensitivity is an important parameter that needs to be measured for any quantitative AFM measurement. There are methods of measuring the deflection sensitivity (Slattery et al., 2013; Tourek and Sundararajan, 2010) by avoiding the AFM probes contacting a rigid surface substrate. However, these methods are complex and are not being widely used.

journal of the mechanical behavior of biomedical materials 53 (2016) 161 –173

200nm

200nm

200nm

200nm

200nm

200nm

167

Fig. 6 – Tip geometry after obtaining force–distance curves on ommatidia: (a) top view of Si3N4 probe, (b–e) top view of silicon probe, while (f) shows side view of the diamond tip.

1 µm

Fig. 7 – (a) Schematic of nipple–tip interaction (drawn to scale) and (b) SEM of the nipple array on an ommatidial surface.

If the apex of the probe is flat as can be seen from Fig. 6, then the JKR and DMT models cannot be used to obtain a quantitative measurement of the surface energy from the adhesion force measurements. A more appropriate equation in such a case would be the model of a flat AFM probe versus a flat surface configuration (Israelachivili, 2011) as the radius of ommatidia is very large (17 mm). However, as the ommatidia has further fine nipple structures the interaction is defined by the nipple structure rather than the AFM tip geometry (Fig. 6). The diameter of the nipple structure as measured from SEM is about 100 nm (Fig. 7a), and a schematic of the nipple and tip geometry is shown in Fig. 7b. It should be noted that measuring such a small radius, using AFM tip is error prone as the topography obtained is the convolution of both the sample and the probe geometry (Ernst Meyer et al., 2004), and hence SEM images were used. The local contact configuration is now that of a 50 nm nipple structure, interacting with almost a flat AFM tip.

From the measured adhesion force and tip geometry, the surface energy of the nipple structure calculated from Eq. (2) is about 0.1570.01, 0.1070.01 and 0.0670.01 J/m2 at center, intermediate and periphery of ommatidia respectively. These values depend on the material of the tip, and the relative surface energy can be expressed as Δγ ¼ γ tip þ γ sample γ tip–sample ;

ð6Þ

where γ tip is the surface energy of the tip material (silicon nitride), γ sample is the surface energy of the ommatidia and γ tip–sample is the interfacial energy of the silicon nitride–ommatida interface. Since most of the biological samples contain water, the presence of a thin water layer on their surface is possible. When the interaction occurs between the tip and water, the surface energy Δγ ¼ γ H2 O ½1 þ cos θ reaches a maximum value of about 0.14 N/m. To confirm this observation, local topography and the adhesion map were obtained using Peak-force QNM method as shown in Fig. 8, for a single ommatidia. It can be seen that spatial variation followed an alternating pattern of high and low adhesion force. Also, the basal region (region in between the nipple structures) had higher adhesion force compared to the nipple structure throughout the ommatidial surface. Previous study on chemical mapping of ommatidial surface (Anderson and Gaimari, 2003) of Dipteran flies from RamanAFM imaging showed the presence of different chemical groups on the top and base of the nipple structure. The top portion of the nipple region possesses hydrophobic proteins, while the base portion contains hydrophilic ethers, and

168

journal of the mechanical behavior of biomedical materials 53 (2016) 161 –173

Fig. 8 – Topography and adhesion map of Wild type ommatidia using Kc 3.8 N/m. The magnitude of adhesion exhibits an oscillatory behavior as one move from intermediate region (I) to peripheral (P) region.

therefore the silicon nitride and silicon probes that are hydrophilic measured a higher adhesion force in the basal region.

3.1.2.

Stiffness of wild type

In order to find out whether the modulus varied spatially along the ommatidial surface, like the adhesion force, the stiffnesses were measured along a diameter of ommatidia. Fig. 9 shows the stiffness measured at different locations of the ommatidia. The inset in Fig. 9 shows the line along which the measurements on the surface of ommatidia were carried out. The measured stiffness (Ku ) varies from 141 N/m to 284 N/m. The measured values of the central region had lesser variability compared to the edges of the ommatidial surface. The measurement at the edges was carried out on a curved surface, and hence may also involve curvature effects since the contact force is normal to the ommatidia surface, while the measured force is along the axis of the apex (Bobji and Biswas, 1999). Therefore, further stiffness measurements were carried out in a region of area 2 mm  2 mm about the center of ommatidia. For the diamond probe, the mean stiffness measured at the center of ommatidia ðn ¼ 488Þ is 223.571 N/m (Fig. 9), which translates to a sample stiffness of 48375 N/m using Eq. (5). The reduced modulus obtained from Eq. (3) is about 3.470.05 GPa. It should be noted that the ommatidia is composed of layers, the external cuticulin layer, granular layer and layers of fibrous material (Perry, 1968). Force spectroscopy measures a combined effective modulus of this composite material. Using Peakforce method, modulus map is obtained, which shows the variation in sample’s elastic modulus at each pixels of the image. It should be noted that Eq. (3) can be obtained from the Hertzian equation,

Fig. 9 – Variation of unloading stiffness Ku (green dot) and ommatidial height (blue triangle) at a typical cross-section.

F ¼ 43En

pffiffiffiffiffiffiffiffi Rh3

ð7Þ

since Ks ¼ dF=dh . Most of the AFM analysis softwares used for biological samples obtain modulus from the DMT model (Maugis, 2000) using the equation: pffiffiffiffiffiffiffiffi ð8Þ F  FA ¼ 43En Rh3 FA for the ommatidia is about 40 nN, which is very small compared to the maximum force F encountered in our force spectroscopy curves ( 5500 nN) and hence the effect of FA can be neglected. Thus, the DMT modulus will be same as that of the modulus obtained by Eq. (4). The DMT modulus map obtained using stainless steel cantilever with a diamond tip is shown in Fig. 10; the map was obtained using Eq. (8). It can be seen that the modulus

journal of the mechanical behavior of biomedical materials 53 (2016) 161 –173

169

Fig. 10 – Height and DMT modulus map of Wild type ommatidia.

varies from center to periphery, and was found to be maximum at the center of the ommatidia. It is not clear why a peripheral ring like structure is seen in the ommatidia, but it has been observed that when the eyes are processed using ethanol, these structures were not present. This observation has been confirmed from SEM images as well. The corresponding variation in modulus seems to be an artifact resulting from the interaction of the diamond probe with the ring like structure.

Mutant

Having established the measurement methodology and standardized the experimental protocol, the adhesion force and stiffness of a mutant with ‘rough eye’ characteristics were measured. Transgenic flies with human mutant tau gene (V337M), which causes Frontotemporal dementia in Human (Iijima-Ando and Iijima, 2010; Hasegawa et al., 1998), was used in this study. Tau protein is known to stabilize microtubules, which are the cytoskeleton of the neuronal system. Overexpression of mutant tau protein is thought to destabilize these cytoskeletal structures and causes degeneration of the cells. In the Drosophila eye, expression of these Mutant tau proteins using GMR-Gal4 results in external ‘Rough eye’ appearance, since the underlying internal structures that secretes the corneal lens are destabilized and degenerated. Variation in properties like adhesion force, surface energy, stiffness and elastic modulus were tested for this mutant condition.

Fig. 11 – SEM image of nipple structures of mutant tau V337M.

50

Adhesion, FA (nN)

3.2.

40

Adhesion force of tau Mutant

From the SEM images it was seen that the nipple structures were bigger (Fig. 11) with a diameter of 22879 nm, compared

***

30

***

20 10 0

3.2.1.

Wildtype Mutant

***

n=233

n=226

Center

n=306

n=164

Intermediate

n=376

n=163

Periphery

Fig. 12 – Variation in adhesion force among wild type and mutant with cantilever 3.8 N/m.

170

journal of the mechanical behavior of biomedical materials 53 (2016) 161 –173

to the diameter of 9774 nm in the wild type. Correspondingly, the distance between the adjacent nipples was also found to be larger at 335718 nm in mutant and smaller at 224710 in wild type. It was also observed that some of the nipples are probably fused together and appear as elongated rods. These kinds of fused structures are found only in the roughened portion of the mutant eye. Adhesion force obtained from the force spectroscopy was found to be variable depending on the location where the measurements were taken, as in the wild type. Fig. 12 shows the mean adhesion force measured at different locations on mutant ommatidia. The corresponding values of the wild type from Fig. 5b are also shown for comparison. For wild type the adhesion force is minimum in the peripheral region, whereas for the mutants the minimum is observed in the intermediate region. In central and intermediate regions the adhesion force on the mutant is found to be lower than the wild type. However, in the peripheral region, the mutant has higher adhesion force than the wild type. Similarly, the calculated surface energies for mutant ommatidial surface at center, intermediate and peripheral regions are 0.1270.01, 0.0770.01 and 0.0970.01 J/m2 respectively. This clearly indicates that the chemical nature of the ommatidial surface has been modified in the mutant. Fig. 13 shows the topography and adhesion map obtained from peak force QNM mode using a cantilever of stiffness 3.8 N/ m. It can be seen that the nipples were not as uniformly distributed as in the wild type. Fused nipple structures can be clearly seen in the topography image, and these structures were predominantly found in the region where the ommatidia appear rough. Around the fused structures, more basal regions were exposed. Adhesion force is relatively high in the basal region as in the wild type. In mutant, adhesion force measured on the top nipple structure is constant over a 5 μm scan area (Fig. 13),

whereas in wild type the measured adhesion was exhibiting oscillatory variation with distance (Fig. 8).

3.2.2.

Stiffness of tau Mutant

Stiffness properties of wild type are compared with mutant corneal lens using cantilever with Diamond tip (Kc ¼ 419 N/m). Force spectroscopy curves were obtained from freshly prepared samples in the central region of ommatidia with a trigger threshold of 15 nm, reaching a maximum force of up to 5500 nN. The calculated value of mutant sample stiffness from unloading curve was (Fig. 14) 44576 N/m, which was lower than that of wild type 48375 N/m. Similarly, the elastic modulus obtained from Eq. (3) reveals the mutants possess lower elastic modulus of 3.070.05 GPa compared to wild type with 3.470.05 GPa. The topography image and the DMT modulus map obtained using the peak force method in the rough region of the mutant is shown in Fig. 15. The scan size is 30 mm  30 mm, and the number of data points per line scan is 64. From such images, the ommatidal sizes in the rougher regions were found to be smaller by about 30%. The ommatidia are not arranged regularly, and hence the distance between them was found to vary widely. Further, peripheral ring like structure seen in the wild type eyes are not seen. These observations were further confirmed from SEM images. The variation in the modulus with location seems to be lower in the mutant.

4.

Summary

The local mechanical properties adhesion force and stiffness of the corneal lens of D. melanogaster were obtained by careful force spectroscopy measurements. For accurate measurements, it is found that the stiffness of the AFM cantilever should be low, for

Fig. 13 – Topography and adhesion map of Mutant.

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600

4

***

*** Elastic modulus, E (GPa)

Stiffness, N/m

171

400

200

n=488

3

2

1

n=455

0

n=488

n=455

0

Wildtype

Mutant

Wildtype

Mutant

Fig. 14 – Bar graph showing stiffness and elastic modulus of wild-type and mutant.

Fig. 15 – Topography and DMT Modulus map of mutant ommatidia.

adhesion force measurements, while the stiffness should be high for modulus measurements. Thus, the cantilever used to make measurements is to be chosen from a set of preliminary experiments. Adhesion force and modulus measurements were carried out in an automated array, such that a large number of observations could be made to take care of the inherent variations in the biological samples. This enabled the identification of spatial variation of adhesion force and stiffness, overcoming the variation resulting from experimental conditions such as the mounting of samples. The geometry of apex of the AFM cantilever was also found to be different from the usually assumed spherical

geometry. However, the spherical nature of the nipple structure enabled us to use the flat-sphere geometry for obtaining the surface energy. High stiffness of the corneal lens necessitated the use a diamond probe with a tip radius of 100 nm. It is found that for this tip the adhesion force is small, and the DMT and Hertzian models give similar values for the modulus. The adhesion force was found to be 3673, 2471.6, 1671 nN, and therefore the surface energy is 0.1570.01, 0.1070.01 and 0.0670.01 J/m2 at center, intermediate and periphery of the ommatidia. The measured stiffness was 48375 N/m, and the calculated elastic modulus is 3.470.05 GPa.

172

journal of the mechanical behavior of biomedical materials 53 (2016) 161 –173

Establishing the variation in adhesion force and modulus enabled the comparison of the wild type eye with the characteristics of a rough-eye phenotype resulting from the mutant gene V337M. It was found that the adhesion force at the center and intermediate regions of the ommatida were lower in the mutant, while in the peripheral region it was higher. An average measurement without taking care of the spatial variation would have given identical adhesion force values. The change in adhesion force and hence the surface energy clearly indicates the chemical nature of the ommatidial surfaces is different. The modulus of the mutant was found to be 10% lower than the wild-type with 95% confidence level. This study thus establishes that careful experiments exploiting the speed and ease of AFM measurements can give quantitative information about adhesion force and elastic modulus of biological samples. These measurements can be used to differentiate the physical variations resulting from genetic modifications in the eye of D. melanogaster.

Acknowledgments We would like to thank Center for Nanoscience and Engineering, IISc for providing Atomic force microscope. Financial support from the Indian Institute of Science, Department of Biotechnology, Government of India is acknowledged.

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