a.

b.

c.

Figure 2. TappingMode in saline solution images of a fresh, out-of-the-box, commercially available contact lens. (a) 47µm, (b)10µm, (c) 4µm scans.

Advantages of AFM• Highest resolution available:
AFM’s lateral resolution allows
imaging and measurement offeatures on the order of a few
nanometers; the vertical(height) resolution is <1Å.
• Quantitative 3-D surface
maps: AFM images canbe computer-rendered withany tilt or rotational angle,
and provide accurate
measurements in all three
dimensions on featuresof interest.
• Operation in liquid:
AFM can characterize
contact lenses in their nativeliquid environment.
• Non-destructive: Whether
in air or in liquid, the AFM
characterizes the sample
without damage.
• Material properties
characterization: The AFM
combines several techniques
in a single instrument.
On and near the surface,
topography, adhesion,
viscoelasticity, hardness,friction, and other properties
can be revealed – again,
with nanometer resolution.

oscillation amplitude at each lateral
data point. The key advantage of
TappingMode is the elimination of thelateral shear forces present in contact
mode, which, on many specimens, candamage the structure being imaged.
TappingMode AFM can be conductedin an air or liquid environment. The
images and measurements in thisapplication note demonstrate that many
contact lenses, even hydrogel contactlenses with high water content, can beimaged with TappingMode AFM in air
or liquid. TappingMode also facilitates
concurrent “phase imaging,“ which
also provides information on material
properties. All images in this article
were generated using the Digital
Instruments NanoScope Controllers
with MultiMode and Dimension™
AFMs, offered by Veeco Instruments Inc.
®

®

Comparison of AFM with
Other Techniques
The fact that AFM senses small
chemical or mechanical forces point-bypoint by directly contacting the natural
sample surface distinguishes it fromother surface analysis techniques.
TappingMode AFM complements
and improves upon other types of
microscopy. Three key advantages of

AFM over conventional microscopic
techniques are, (1) surfaces can beanalyzed with nanometer-level resolution
in three dimensions, (2) the analysis
can be performed in ambient air or in
liquids, and (3) sample preparations
and imaging environments known togenerate artifacts are eliminated (e.g.,dehydration, fixation, freezing, staining,coating, etc). Table 1 summarizes themain differences between AFM andother conventional imaging andprofiling techniques. Compared tostylus profilometry, AFM provides higher
lateral resolution (by two orders ofmagnitude), without sample damage
due to high contact forces (AFM
imaging forces are more than threeorders of magnitude smaller thanthose of stylus profilometry). Opticalprofilometry provides high verticalresolution (0.1nm), but its lateral
resolution is relatively poor.

Examples of AFM Applications
for Contact Lenses
Characterization of surface
finish quality
Figure 2 shows three TappingMode
AFM images of a brand newcommercial soft contact lens undersaline. The prominent linear feature that

Atomic Force Microscopy

Stylus and Optical
Profilometry

Scanning Electron andConfocal Microscopies

Resolution

Lateral resolution down to 1 nm scan
range of up to 120µm. Verticalresolution to less than 1Å. Verticalrange up to 7µm.

Lateral resolutions subnanometer. Vertical resolutions
down to 1Å.

Lateral resolution for confocal
microscopy down to 170nm;
vertical resolution to ~500nm. SEM
resolution down to 2nm. Scan
range usually on the order of 1mm.

Environment

Many different environmental
conditions are permissible during
analysis including nearly alltransparent liquids and a rangeof temperatures (-40°C to 220°C),
without loss of resolution.

Analysis normally confined toair-dried samples.

Environment usually limited toambient air or vacuum chamber.
Image distortions or loss ofresolution can occur in liquids.

SamplePreparation

No sample preparation; true in-situ
analysis of untreated sample.
Dynamic processes can be monitored
on single lens.

Little or no sample preparation.

Sample preparations are time
consuming and/or cause artifacts(e.g., dehydrations, stainings, andcoatings). Ex-situ analysis only.

Materials
Properties

Chemical and mechanical materials
properties including surface adhesion,
hardness, and friction. AFM tip canbe chemically modified in order tomeasure tip/sample interactions.

Not useful for characterizing
chemical and mechanicalsurface properties.

Not useful for characterizing
chemical and mechanicalsurface properties.

SampleDamage

AFM, particularly TappingMode
technique is non-destructive. Tipforces are finely controlled (<10-9 N)

Stylus profilometers exert highforces against sample surface(>10-6 N); can cause damage.

Usually non-destructive, but beam
artifacts and damage can occur
on insulative or hydrated samples
with SEM.

Table 1. Comparison of techniques.

appears in these three images was asurprised finding. The detailed threedimensional structure is visible inprogressively smaller scans, and thefeatures can be measured for their inplane and out-of-plane (vertical) size.
It is not fully known what size and typeof defects or features on the contact
lens surfaces are critical in prompting
unfavorable responses by the eye.
The adhesion and entrapment of protein
and contaminants between the lens andthe cornea are believed responsible forpromoting the growth of bacteria.
To help understand how and where
protein molecules and contaminants
adhere to the lens, the topographyof new and used lens surfaces canbe mapped in great detail with
AFM (Figures 2, 3).
Figure 4 shows a TappingMode AFM
image of a contact lens in saline
solution, which was made using a
diamond-turned mold. The diagonalcross-section reveals the short andlong range variation in height. The
periodicity of the surface grooves
is a 1.5µm (the Digital Instruments
NanoScope software uses a Fast
Fourier Transform (FFT) to make thismeasurement). The grooves resulted
from the manufacturing process. When
combined with clinical studies, this typeof information can help clarify the effectof different size grooves.

Figure 3. TappingMode image of a
1µm x 1µm area of the same type ofcontact lens as in Figure 2, also immersedin saline. The RMS roughness is 3.5nm forthe area shown. A surface defect or pit,
clearly seen in the lower right of theimage, measures 170nm in width and
150nm in depth and, therefore, is large
enough to trap proteins or contaminants.
However, it is too small to be resolved inliquid using conventional techniques.

production cycle, i.e., on the
production molds. AFM is a fast andeasy-to-use tool for imaging the molds
and accurately measuring feature
dimensions. Figure 6a shows a lightmicroscope image of an area on apolystyrene contact lens mold, andFigure 6b is an AFM image taken
within the same area. Unlike optical
microscopy, AFM provides additional
quantitative information about the natureand size of the surface features. Figure
6c shows an AFM image cross-section
and measurements of height, width andangle on some of the features.

Figure 4. TappingMode AFM
measurements on the grooves of ahydrogel lens in saline solution. The
grooves originate from the diamondlathed mold. Section Analysis reveals a
1.5µm periodicity (peak in spectrum) andnanometer-sized peak-to-valley heights ofthe grooves at various locations. The
cross-section can be drawn and analyzed
as often as desired, and in any direction,
because the AFM image, unlike a SEM
micrograph, is a digital object that
includes 3-dimensional measurement
information about the area scanned.
65µm scan.

Figure 5 is an AFM image of anotherhydrogel lens that was made withdiamond-turned molds. The lathegrooves run diagonally from bottom
left to top right. The periodic occurrenceof a defect on the lens surface,approximately every fourth groove,strongly suggests that the origin of thesetypes of defects are traceable back to
the mold lathing process.
One way to improve the production
yield of contact lenses is to detectand characterize defects early in the

Figure 5. TappingMode AFM image ofa hydrogel lens in saline. The grooves ofthe lens surface are faintly visible and run
from lower left to upper right in the image.
Periodic defects, running from top left tolower right in the image, are clearly
detected. Such defects originate on the
diamond-turned mold during the lathing
process and are subsequently transferredto the lens surface. 20µm scan.

a.

Figure 7 is a TappingMode AFM imageof the central area of a Poly(methylmethacrylate) (PMMA) button, or blank,
used in the manufacture of some rigid
gas permeable (RGP) lenses. RGP lensmanufacture begins with the formationof such buttons by diamond lathing
PMMA or other polymers. The RMS
surface roughness for this 50µm scan
was 32nm. The pattern of the spiralcan be used to determine the degreeof uniformity of the lathing. Combinedwith knowledge of the “speed andfeed”, the dimensions and spacing of
the radial grooves, and the directionof folding of the lathed material, AFM
can shed light on the direction and thestrength of the shear forces between the
diamond tool and the polymer surface.

b.

Figure 6. (a) Optical micrograph of a contact lens mold.
Approximately 90 x 90µm. (b) AFM top-view image ofthe same area as box in Figure (a). 20µm scan. (c) AFM
cross-sectional plots allow accurate measure-ments ofsurface feature height, width, and angle and can bemade on any cross-section of the AFM image.

c.

c.
b.

a.

c.

b.

Figure 7. TappingMode AFM image of central area on a diamond-lathed PMMA button (blank).
The RMS roughness is 32nm. 50µm scans. Boxed areas in (a) are zoomed and shown in (b) and(c) respectively. Brighter color represents higher features in topography images like these.

Monitoring of
manufacturing processes
Figure 8 shows how TappingMode
AFM images in saline solution revealed
unexpected information about a novel
lens material being developed –information which necessitatedchanging the manufacturing processitself. The figures show 8µm squareareas on two samples of this material
at different stages in the manufacturing
process. The material is a hydrophobicpolymer which is coated with ahydrophilic film and then further
processed. The pits (darker areas) on
the uncoated hydrophobic lens (Figure8a) were an unexpected finding for thelens manufacturer. Close inspection of
the hydrophilic-coated lens (Figure 8b)
reveals that the distribution of the submicron light-colored spots on the coatedlens is similar to the distribution of thepits (dark areas) on the uncoatedhydrophobic starting material (Figure8a). One possible explanation for thisis that the hydrophilic coating filled in
the pits and, upon exposure to water,swelled to form the sub-micron spots.
The manufacturing process was
modified to eliminate the pits in thehydrophobic substrate.

Evaluating performanceof eye care liquids
For evaluating the performance of eye
care products, a single lens can be
repeatedly analyzed with the AFM
under different conditions. Figure 9shows AFM images in liquid of thesame area on a RGP lens front-side
curve. The RGP lens was worn by a 30
year-old female for an extended periodof normal use and storage. Figure 9ashows the RGP lens before cleaning,
imaged in a commercial saline solution.
Notice that most of the particulate
adsorbates line up along the polishingscratches on the lens surface. A
commercial RGP cleaning solution wasthen injected into the saline bath for
10 minutes and then exchanged withseveral washes of saline. The lens was
re-imaged (Figure 9b) and only aportion of the adsorbates had been
removed by the cleaning solution.
The lens was then manually rubbed
with a latex glove while in the cleaning
solution and then rinsed with moresaline. As shown in Figure 9c, nearly
all of the adsorbates were cleaned off
the surface by this treatment. Based onthese results it is clear that AFM can beused to evaluate and understand theperformance differences between
various eye care solutions.

a.

b.

Figure 8. TappingMode AFM images of a(a) hydrophobic silicone hydrogel lens and(b) the same lens material after hydrophilic
coating and further processing. Darker
areas in (a) are defects that AFM images
revealed, and were subsequently eliminatedby modifying certain processing steps.
8µm scans.

a.

c.

b.

Figure 9. Series of AFM images of the same region on a used RGP lens in saline, (a) before cleaning, (b) after
soaking in commercial cleanser, and (c) after soaking in cleanser and rubbing with latex glove. 30µm scans.

Characterizing Material
Properties with AFM
Force vs. Distance Measurements
With force vs. distance curves, AFM
can distinguish surface regions ofdifferent hardness and adhesion
characteristics. Briefly, forces appliedon the surface are measured by thedeflection of the cantilever while
approaching and retracting from thesurface. On the left side of the force
vs. distance curve (Figures 10b,c),the steeper the slope, the harder the
sample. An inverted peak, seen in the
middle of the force vs. distance curve,
during retraction of the cantilever(white curve) indicates some adhesion

between the tip and the sample. For
example, Figures 10a-c, representing
an area on a soft contact lens in saline
solution, reveal that the pronounced
surface feature (center of image in 10a)
has a greater stiffness and is moreadhesive to the AFM probe than thesurrounding lens area (10b vs. 10c).
The force vs. distance curves not only
show the difference qualitatively, butalso permit quantitative measurement of
the adhesion force (in this case, 5nN).
It is also possible to control the tipsample interactions by functionalizing
the tip with a selected protein or
chemical group in order to measurespecific chemical interactions between
the tip and sample.

Phase Imaging
Phase imaging gives yet another way
to distinguish between domains withdifferent surface properties on lenses
made of heterogeneous polymers
or on chemically-modified surfaces.
The phase image is generated bymonitoring the phase angle of theoscillating probe relative to the phaseangle of the signal that drives the probe
in TappingMode. Differences in phaseshifts indicate differences in material
properties of the lens. A topographic
AFM image of the same area is also
displayed simultaneously with the phase
image. The usefulness of phase imaging
is illustrated in Figure 11. These images
are from identical areas on the surface

b.

c.

b.
c.

a.

Figure 10. Force vs. distance curves allow characterization of stiffness and adhesion propertiesof lens surface. (a) TappingMode AFM image in saline of a surface feature (center of image) on
a contact lens, 4µm scan. (b) Force vs. distance curve taken on the surface of the central feature.
(c) Force vs. distance curve taken at a region adjacent to the central feature. The force vs.
distance curves reveal that the central feature has a greater stiffness and is more adhesive to the
AFM probe than the surrounding surface material. The surrounding region (c) compiles to the
AFM probe as the probe pushes into the sample (yellow line), and very little adhesion isdetected when pulling the probe away from the surface (white line).

a.

b.

c.

Figure 11. In addition to topographic imaging with TappingMode, phase imaging provides material
property information. Shown are simultaneously acquired (a) TappingMode topographic and (b) phase
AFM images of hydrogel lens with both hydrophilic and hydrophobic domains, and (c) detail of phase
image reveals sub-micron features, including ring-like nanostructures. The phase image reveals different
types of patterns that go undetected in the topographic image (e.g., the fine structure of circulardomains as small as 10nm. Figures (a) and (b), 4µm scans, Figure (c), 1µm scan.
d.

c.

of a soft contact lens made from ahydrogel polymer with domains ofdifferent hydrophilicity. Notice thestriking differences between the
topographic (Figure 11a) and phase(Figure 11b) images. In the topographic
image, randomly dispersed, nodular
features of varying sizes are seen on
a fairly smooth background. However,in the phase image (Figure 11b), a
few of these features appear with someinternal structure while others are notseen at all. In addition, the backgroundappears finely speckled only in thephase image. Such differences between
topography and phase images are due
to material property differences on thelens surface.
Figure 12 is another pair of
topography/phase images captured
on an experimental hydrogel material
in saline solution. This hydrophobic
surface, while covered with a mask,
underwent a surface modification
treatment that created a hydrophilicpattern. The central “cross-like”
region (seen clearly in Figure 12b),
corresponds to hydrophobic regions,while the four outer areas correspondto hydrophilic regions. The topographic
images (Figure 12a,c) show no
significant height difference between
the masked and unmasked regions of

a.

c.

b.

d.

Figure 12. Simultaneously acquired (a) topography and (b) phase AFM images ofsilicone hydrogel in saline solution. The four outer areas were exposed to a sequence ofchemical processing steps. The central cross-like region was masked and so protectedfrom the processing steps and hence retained its hydrophobicity. The intention of theprocessing was to selectively alter the hydrophilicity of some parts, but not all of, thesample surface. In the phase images (b,d), a marked phase shift is clearly seen acrossthe boundaries. However, the hydrophillic and hydrophobic region show no topographic
contrast (a,c). The phase image is clearly providing material property contrast on thiswell-defined experimental hydrogel surface. 50µm scans.