ORIGINAL PAPER

Measurements of elastic modulus for human anterior lenscapsule with atomic force microscopy: the effect of loadingforce

Konstantinos T. Tsaousis • Panagiotis G. Karagiannidis • Nikolaos Kopsachilis •

Chrysanthos Symeonidis • Ioannis T. Tsinopoulos • Varvara Karagkiozaki •

Lampros P. Lamprogiannis • Stergios Logothetidis

Received: 27 June 2013 / Accepted: 14 August 2013 / Published online: 15 September 2013

� Springer Science+Business Media Dordrecht 2013

Abstract The purpose of the study was to appraise

the effect of loading force magnitude on the determi-

nation of the elastic modulus of the anterior lens

capsule through atomic force microscopy. Four human

anterior lens capsules taken during phacoemulsifica-

tion cataract surgery were studied, free of epithelial

cells, with atomic force microscopy. For the experi-

ment, five different indentation loading forces were

applied to near areas of the specimen. Experimental

data was exported and analyzed according to the Hertz

model to obtain the Young’s modulus with regards to

the elastic behavior of the material. Force–distance

curves were acquired by applying a load of 2, 5, 10, 20

and 30 nN. When examining the results it was evident

that determination of Young’s modulus of the anterior

lens capsule is dependent on the loading force

concerning the examined range. Loading forces of

10 and 20 nN led to results without significant

difference (p [ 0.05) and more reproducible (coeffi-

cients of variation 12.4 and 11.7 %, respectively).

Keywords Anterior lens capsule � Atomic

force microscopy � Mechanical properties �Young’s modulus

Introduction

Atomic force microscopy (AFM) or scanning force

microscopy is a versatile tool that is widely used for

analyzing the surface of a rigid material to the level of

the nanoscale and for measuring the mechanical

properties of organic and inorganic materials by

mechanically probing their surface [1, 2]. Especially

in ophthalmology, AFM has been used for many

purposes such as imaging and the study of sclera and

cornea [3–5], intraocular lenses models [6, 7], contact

lens studies [8, 9] and investigation of eye-related

molecules [10–13].

Several researchers used AFM to focus on the study

of the human lens capsule and nonhuman primate lens

capsule [14, 15]. Determination of the lens capsule

elastic (Young’s) modulus is of particular interest. An

emerging problem is that different methodologies are

followed by each laboratory and this diversity does not

The work has been partially presented at the XXX Congress of

the European Society for Cataract and Refractive Surgery

(ESCRS) 8–12 September 2012, Milan, Italy.

K. T. Tsaousis (&) � C. Symeonidis � I. T. Tsinopoulos

2nd Department of Ophthalmology, Medical School,

Aristotle University of Thessaloniki, ‘‘Papageorgiou’’

General Hospital, 56429 Thessalonıki, Greece

e-mail: konstantinos.tsaousis@gmail.com

K. T. Tsaousis � P. G. Karagiannidis � V. Karagkiozaki �L. P. Lamprogiannis � S. Logothetidis

Nanomedicine Group, Laboratory for ‘‘Thin Films—

Nanosystems & Nanometrology (LTFN)’’ Physics

Department, Aristotle University of Thessaloniki,

Thessalonıki, Greece

N. Kopsachilis

Moorfields Eye Hospital, London, UK

123

Int Ophthalmol (2014) 34:519–523

DOI 10.1007/s10792-013-9846-z

allow the direct comparison of experiments. Finding

the most advantageous amount of loading force in

force curve measurements is an important topic which

is not extensively explored in previous studies. The

study of biomechanical properties of biological tissues

with tools of nanotechnology seems quite promising

and requires the establishment of a ‘universal toolbox’

that can be widely adopted.

The purpose of the present study was to explore the

effect of the amount of indentation force on the

determination of Young’s modulus of the human

anterior lens capsule through AFM.

Materials and methods

Lens capsule isolation

The anterior lens capsules were obtained from patients

without any ocular pathology (other than cataract)

after continuous curvilinear capsulorrhexis during

phacoemulsification cataract surgery. Methods of

securing human tissue were humane, included suitable

consent and approvals, complied with the Declaration

of Helsinki, and were approved by the local ethics

committee.

The surface morphology and roughness of the

samples were investigated by AFM using a SOLVER

P47 Scanning Probe Microscope (NT-MDT), in semi-

contact mode, in which the cantilever tip oscillates

above the sample surface and briefly touches the

surface at its lowest oscillation point (Fig. 1). All

measurements were performed with silicon cantilevers

(10 nm nominal curvature of the tip) at ambient

conditions. A reference force–distance curve was

obtained from the sapphire substrate in order to get

the cantilever’s response.

Parameters

Measurements were conducted using a cantilever

approach and a maximal indentation force of 2, 5,

10, 20 and 30 nN (arbitrary selected values). The

deflection signal (DFL) in nA detected at the photo-

diode due to deflection of the cantilever was recorded

as a function of piezoelectric displacement. These

recordings were repeated five times per sample, i.e.,

five consecutive measurements for each loading force

obtained from near areas of each specimen. All

experiments were performed at room temperature.

Data analysis

The DFL signal versus piezoelectric displacement

recorded during the measurement scans was converted

to force versus indentation depth after accounting for

the cantilever spring constant and its response when

probing a rigid substrate. The force versus indentation

depth relationship was analyzed using the Hertz model

accordingly modified for conical tip:[16]

F ¼ E

1� v2

2 tan a

pd2

where F (N) is the measured applied force, E (N/m2) is

Young’s modulus, m is Poisson’s ratio (0.47) [17], a is

the semi-opening angle of the tip (a = 18�) and d is

the measured indentation depth.

Statistical methods

Dispersion of measurements calculated through coef-

ficient of variation and the mean value was used for

comparison of different sets of measurements. The

NOVA software (NT-MDT Co, Zelenograd, Moscow,

Russia) was used for curve visualization and analysis

while Origin (OriginLab Corporation, Northampton,

MA, USA) and Excel for Windows (Microsoft Cor-

poration, Redmond, WA, USA) were used for statis-

tical analysis.

Results

Four human anterior lens capsules extracted from four

cataract patients (75.25 ± 4.03 years old) were

examined.

Mean values for Young’s modulus of the measure-

ments as well as their variation for each loading force

are presented in Fig. 2. The range (maximum–mini-

mum) of the Young’ modulus was noticeably wider

for the loading force of 2 nN. Mean values of Young’s

modulus were equivalent (p [ 0.05 in all subjects)

only for loading forces of 10 and 20 nN.

The coefficient of variation (standard deviation/

mean) of repeated measurements for the same

520 Int Ophthalmol (2014) 34:519–523

123

Fig. 1 A 5 9 5 lm2 region

of one sample (imaging

mode)

Fig. 2 Mean values (±2

SD) of measurements for

different loading forces in

each subject

Int Ophthalmol (2014) 34:519–523 521

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indentation force was 15.3, 14, 12.4, 11.7 and 18.1 %

for 2, 5, 10, 20 and 30 nN, respectively.

Representative force curves (closer to the mean

value) for the loading forces of 5 and 10 nN are

presented in Fig. 3a, b. Indentation depth in our

measurements was found between 120 and 360 nm

(positively-dependent on the loading force).

Discussion

In the present experimental study, an atomic force

microscope was used to determine Young’s modulus

of elasticity of the human anterior lens capsule. The

maximal applied forces were 2, 5, 10, 20 and 30 nN

and possible variations of measurements were

evaluated.

Following comparison of mean values and coeffi-

cients of variation, we conclude that measurements

with loading forces of 10 and 20 nN are producing the

most reproducible results while measurements with 2

and 5 nN returned significantly larger values. On the

other hand, a loading force of 30 nN resulted in

considerably smaller values of Young’s modulus with

wider variation.

To evade the substrate’s effect on the measure-

ments, the indentation depth ought to be\10 % of the

sample thickness and it would also be better to keep

the measurement above the noise floor of the deflec-

tion measurement and below the peak where non-

linearity turns to be a problem. Given that the human

anterior lens capsule has a thickness of 15–20 lm we

can suppose that the substrate effect was negligible in

Young’s modulus calculation.

An essential subject for future studies could be the

wide measurements of human anterior lens capsules and

the establishment of nomograms regarding the effect of

age and the remaining potential factors. The need for

this investigation is two-fold—firstly the anterior lens

capsule has recently been used for in vitro experiments

concerning the behavior of cultivated cells on the

specific structure [18] and given that it has been

demonstrated that cells respond to the mechanical

properties of the underlying substrate, it is of vital

importance to explore these properties in detail [19–21].

Secondly, the anticipated introduction of femtosecond

lasers in cataract and corneal surgery demands the

knowledge of the entire significant factors that could

affect the effectiveness of the laser intervention.

Hosokawa et al. [22] have already exhibited the use of

a femtosecond laser impulse quantified by AFM in order

to estimate the intercellular and intermolecular breaking

forces between epithelial cell monolayers and strepta-

vidin-coated microspheres and a biotin-coated

Fig. 3 a Force curve with 4.4 nN real loading force. Note the

area under the loading curve that possibly indicates the

development of relatively high adhesion forces between the

probe and the sample. b Force curve with 12.1 nN real loading

force resulting in a perfect matching of loading and unloading

curve demonstrating a totally elastic deformation of the

material. Furthermore, adhesion forces between the tip and the

sample cause a different pattern of contact than in the lower

loading force

522 Int Ophthalmol (2014) 34:519–523

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substrate, respectively, demonstrating possible analo-

gous applications in biologically pertinent circum-

stances as ophthalmic tissues.

In conclusion, to the best of our knowledge, this is

the first report that inspects the effect of loading force

on the determination of Young’s modulus of the

anterior lens capsule with means of AFM, indicating

that a force of 10–20 nN produces comparable results

with better repeatability and could probably be

optimally selected for similar experiments.

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