Hammer, Christian M.

Schäffer, Tilman E.

English

Medical Hypothesis, Discovery & Innovation (MEHDI) Journals

 
 
Med Hypothesis Discov Innov Ophthalmol. 2015; 4(1)  
 
 
 
Editorial 
Medical Hypothesis, Discovery &Innovation  
Ophthalmology Journal 
   
 
 
Atomic Force Microscopy Crosslinks Interdisciplinary Eye 
Research 
Christian M. Hammer 1,2 & Tilman E. Schäffer 3  
 
1 Department of Anatomy II, Friedrich-Alexander-University, Erlangen, Germany 
2 Department of Ophthalmology, Friedrich-Alexander-University, Erlangen, Germany 
3 Department of Applied Physics and LISA+, Eberhard-Karls-University, Tübingen, Germany 
 
KEY WORDS 
Atomic Force Microscopy; Eye Disease; Interdisciplinary Eye Research 
©2014, Med Hypothesis Discov Innov Ophthalmol. 
This is an open-access article distributed under the terms of the Creative Commons Attribution Non-Commercial 3.0 
License (CC BY-NC 3.0), which allows users to read, copy, distribute and make derivative works for non-commercial 
purposes from the material, as long as the author of the original work is cited properly. 
 
Correspondence to: 
Christian M. Hammer, PhD, Department of Anatomy II, University of Erlangen-Nürnberg, Universitätsstraße 19, 91054 Erlangen, Germany, 
E-Mail: c.m.hammer@t-online.de; Phone: +49(0)9131 85 22681
 
Over the past decades, it has always been a pleasure to 
see how the field of medical research unites with other 
areas of scientific expertise for the sake of medical 
progress. From these connections, groundbreaking 
beneficial devices and applications like computed axial 
tomography (CAT) or magnetic resonance imaging (MRI) 
have sprung (1, 2). 
In the ophthalmology departments and eye clinics, it has 
as well become almost impossible to imagine life without 
optical coherence tomography (OCT), surgical lasers and 
a variety of other techniques. Their constant refinements 
and upgrades by medical and natural scientists working 
together make them even more effective and 
indispensable in ophthalmologic practice. 
In the examples given here, medical engineers and 
physicists play the main roles, working hand in hand with 
physicians and medical researchers, to invent new 
diagnostic tools and therapeutic strategies. 
For the ophthalmological research, giant leaps forward 
are often accomplished when physics departments or 
facilities and institutions of classical eye-related research 
share promising projects and common hypotheses. 
Ocular biomechanics is a wonderful example of a 
research area defined by both biomedical and physical 
aspects of normal and pathologically altered ocular 
tissues. 
In the past, uniaxial and biaxial stress-strain 
measurements were used to biomechanically 
characterize the viscoelasticity of the posterior sclera 
near the optic nerve head (3,4). This region, called 
peripapillary sclera, is assumed to be of critical 
importance in the development of glaucoma. As a matter 
of fact, nearby tissue, the lamina cribrosa, is actually 
 
 
Med Hypothesis Discov Innov Ophthalmol. 2015; 4(1)  
 
2 ATOMIC FORCE MICROSCOPY  
subjected to the most pronounced morphological 
alterations associated with glaucoma-induced blindness. 
For at least one glaucoma subtype, the so-called pseudo-
exfoliation (PEX) glaucoma, theories postulate a 
preceding history of defective connective tissue 
metabolism, manifesting itself as PEX syndrome (5). 
There is a growing body of evidence that this may render 
the lamina cribrosa structurally weakened and, hence, 
susceptible to pathological outward distortion of the 
optic disc, driven by the always present intraocular 
pressure (optic nerve head cupping). As the lamina 
cribrosa looks like a porous and elastic plate scaffolding 
for the retinal ganglion cell axons when they leave the 
eyeball, it is easy to imagine that its excessive and 
prolonged distortion might impart severe stress on these 
cells. This may damage and ultimately kill the retinal 
ganglion cells and result in blindness. The problem with 
the lamina cribrosa is, however, that it is very difficult to 
dissect, isolate and to subject to conclusive stress-strain 
measurements. Thus, in order to test whether the 
connective tissue of the lamina cribrosa is really 
weakened in PEX syndrome, alternative approaches had 
to be found. An elegant solution was to employ atomic 
force microscopy (AFM) to measure the Young’s modulus 
of the lamina cribrosa tissue beams (6). Essentially, an 
atomic force microscope can be used to image a sample’s 
surface topography and to characterize a sample’s 
biomechanical properties. Central element of an atomic 
force microscope is a cantilever endowed with a 
cantilever tip (looking similar to a record player needle). 
A laser beam is reflected from the back of the cantilever 
and detected by a position-sensitive photodiode. If the 
cantilever tip is scanned across a sample, the cantilever 
will be deflected to a higher or lower degree, depending 
on the local sample height (Fig. 1A). The higher the 
sample at a defined location, the larger the cantilever 
deflection and, hence, the laser beam displacement on 
the diode. From this, the surface topography of the 
sample can be computed. Furthermore, AFM can be used 
to indent tissue on a microscopic scale with the 
cantilever tip (Fig. 1B). If the tissue sample is 
comparatively soft, pushing the tip into the sample will 
easily indent it, while the cantilever deflection will be 
small. Stiffer tissues, however, will cause the cantilever 
to deflect substantially, while tissue indentation is 
decreased. The indentation force is calculated from the 
deflection using the spring constant of the cantilever. 
From the force vs. indentation relationship, the local 
Young’s modulus can be determined by fitting the Hertz-
model (6, 7). Reiterating this procedure within the region 
of interest in a grid pattern is called force mapping, 
which gives an image of sample stiffness (quantified as 
Young’s modulus). 
 
 
 
Figure 1: A: Simplified schematic of the setup of an atomic force 
microscope (with kind permission of Asylum Research, Santa Barbara, 
CA, USA). The topography of the sample is scanned with a cantilever 
tip and recorded by a computer. Cantilever deflection is measured 
with a laser beam reflected from the cantilever’s back. B: Principle of 
nanoindentation measurement. If the cantilever is pressed upon a 
soft sample (left), it will markedly indent the sample, but deflect only 
moderately. The converse is the case with a stiff sample (right). 
 
 
Med Hypothesis Discov Innov Ophthalmol. 2015; 4(1)  
 
3 ATOMIC FORCE MICROSCOPY  
We applied force mapping to the lamina cribrosa of three 
deceased PEX syndrome patients and three normal 
controls. We were able to selectively image and 
biomechanically test the lamina cribrosa connective 
tissue beams, ignoring pores filled with neural tissue. We 
also analyzed the peripapillary sclera correspondingly. 
This sophisticated method yielded results that were 
consistent with the aforementioned theory. For the first 
time, it was possible to show that the average Young’s 
modules of a PEX group were significantly decreased in 
both the lamina cribrosa and peripapillary sclera. Thus, 
combining the expertise in advanced AFM application 
(Schäffer TE et al.) with that in ophthalmology (Hammer 
CM et al.), allowed us to disentangle a problem that had 
bothered glaucoma specialists for a long time. 
While strengthening this interdisciplinary approach, we 
focused our attention on a very different 
ophthalmological problem related to corneal 
biomechanics -- the keratoconus. Keratoconus is a 
refractive defect of the eye caused by a substantially 
weakened and/or thinned central cornea (8). Due to the 
intraocular pressure, this region bulges outward and 
gives rise to a cone-shaped form of corneal ectasia. This 
defect normally develops progressively, over years, and 
results in an increasing refractive error, difficult to 
correct with glasses or contact lenses. However, if 
diagnosed early, keratoconus progression can be 
arrested and partially reversed by a minimally invasive 
intervention called corneal collagen crosslinking (CXL) (8, 
9). 
In the standard procedure, the corneal epithelium is 
abraded to undo its barrier function. Then, the exposed 
stroma is imbibed with the photosensitizer riboflavin 
(vitamin B2) and subsequently irradiated with UVA light 
(370 nm). This induces the formation of collagen 
crosslinks, which increases the intrinsic biomechanical 
strength of the cornea. In the past, stress-strain 
measurements were utilized successfully to prove the 
CXL-induced augmentation of the corneal Young’s 
modulus (10). However, it has been unclear for a long 
time how deep the crosslinking effect reaches into the 
stroma. Due to the depth-dependent and riboflavin-
enhanced absorption of the UVA radiation scientists 
assumed that effective crosslinking was restricted to the 
anterior stroma. Yet again, stress-strain measurements 
gave first experimental evidence verifying this 
hypothesis. Kohlhaas et al. used a microkeratome to cut 
two sequential stromal lamellae of 200 µm thickness in 
cross-linked and non-cross-linked porcine and human 
eyes (11). Only the anterior lamella was found to display 
a significant increase in corneal strength, whereas the 
posterior lamella showed no difference from the 
controls. From this, it was assumed that effective CXL 
was confined to the anterior 200 µm of the corneal 
stroma. However, only a continuous depth-dependent 
profile of Young’s modulus would have allowed for an 
accurate determination and characterization of the zone 
exhibiting effective CXL. This was exactly the gap we 
were able to fill with a modified version of our AFM force 
mapping technique (7). Several regions of interest were 
placed threaded together, collectively spanning the 
entire depth of the central corneal stroma. 
This way, we were able to generate the first continuous 
depth profile of corneal Young’s modulus after CXL with a 
mechanical probe. In that vein, we verified the findings 
of Kohlhaas et al., localizing the maximum depth of 
effective CXL at 220 µm. This confirmed previous 
estimates of other scientists who predicted the 
maximum depth of the CXL zone to be spotted between 
200 µm and 300 µm (11-13). Moreover, our data 
demonstrated an exponential decrease of Young’s 
modulus with increasing depth, which is in line with 
Schumacher et al. who showed the intensity of UVA light 
to decline exponentially with increasing stromal depth 
(13). 
With the two examples of interdisciplinary eye research 
given, we would like to emphasize the immense potential 
inherent in AFM force mapping and interdisciplinary 
research for the biomedical sciences in general and for 
ophthalmological research in particular. If researchers of 
different areas of expertise continue to find common 
hypotheses and a common language to communicate 
ideas, problems and methodological limitations, chances 
are high for a successful outcome. Besides, we are 
convinced that venturing into an initially unfamiliar 
scientific field and integrating parts of it into one’s own 
research area tremendously enriches and widens the 
academic scope of one’s mindset. This, of course, is not 
only of value for scientific progress, but also for personal 
development. A pivotal prerequisite for this, however, is 
a certain degree of interest and respect for each 
collaborator’s scientific field. Moreover, a mutual 
 
 
Med Hypothesis Discov Innov Ophthalmol. 2015; 4(1)  
 
4 ATOMIC FORCE MICROSCOPY  
account of and tolerance for a collaborator’s partial lack 
of knowledge pertaining to one’s own area of expertise is 
essential. Therefore, a minimum of open-mindedness is 
required, among others, to work hand in hand across the 
scientific disciplines. We, for our part, have found 
interdisciplinary research in the field of ophthalmology 
both scientifically fruitful and intellectually enriching. We 
can only recommend it. 
 
DISCLOSURE 
Conflicts of Interest: None declared. 
 
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Atomic Force Microscopy Crosslinks Interdisciplinary Eye Research

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Atomic Force Microscopy Crosslinks Interdisciplinary Eye Research

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