5. Experimental
4. Experimental
Each experiment was performed with the aim of retrieving the maximum of
information regarding each lens. Unsuccessful examinations are still considered
a result and valuable to this project.
Samples 1 and 2 (See details in Appendix B) were received first, with instructions
not to damage or contaminate them. They were contained in BSS and all
surfaces they came in contact with were throughly cleaned with Isapropanol
solution and then rinsed with distilled water. They were handled with a plastic
tweezers, to cause minimal damage, and were cleaned in the same way. Sterile
gloves were used during all the experiments. After non-destructive testing these
samples were sent back to Italy.
Shortly after, samples 3, 4 and 5 (see details in Appendix B) arrived. These
came with permission to conduct destructive testing. All non-destructive tests
were performed first with the same care as samples 1 and 2. While still in
possession of samples 3, 4 and 5, samples 1 and 2 arrived back from italy, this
time with permission to conduct destructive testing.
4.1 Non-Destructive Testing
The following non-destructive analyses were performed on the samples available
at the time of testing.
4.1.1 Microscopy
Apparatus:
49
5. Experimental
An Olympus optical microscope was used, model: bx60m with a TIC-1280E
colous video camera connected to sufficient software on the computer to capture
bitmap images. This microscope can be seen in Figure 4.1 below.
Samples available:
Sample 1, 2, 3 and 5. Sample 4 was a special interest lens due to its nature of
retrieval. No unnecessary risks of contamination were being performed at this
time.
Procedure:
-
Glass slides and a plastic tweezers were cleaned as mentioned earlier.
Each sample was taken out of the BSS and placed on a slide.
-
Both the anterior and posterior sides of each sample were then viewed
using different magnifications.
50
5. Experimental
-
Any images of interest were captured using the camera and saved as
bitmap images for future scrutiny.
4.1.2 Metrology
Apparatus:
A Shadow graft made by Baty International using Quadra check 2000, shown in
Figure 4.2, was used to measure the samples. Quadra check is an automatic
measurement instrument which is connected to a light sensitive probe.
The
sample is moveable in the x and y plane (horizontal and vertical direction)
relative to the probe. The probe senses and records the shadow created by the
sample and automatically measures the distance until the next light change i.e.
the end of the shadow.
The machine is calibrated in the z-direction (distance
from the sample to the back surface) to give correct measurements.
An Auto cad drawing package was also used.
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5. Experimental
Samples available:
Sample 1, 2, 3 and 5. Sample 4 was a special interest lens due to its nature of
retrieval.
No unnecessary risks of contamination were being performed on
sample 4 at the time in question.
As a result it was not involved in this
procedure.
Procedure:
-
Glass slides and a plastic tweezers were cleaned as outlined above.
Each sample was taken out of the BSS and placed on a slide.
-
Each slide with sample was then, in turn, placed in front of the light source
on the designated spot to project an enlarged view of the IOL on the back
surface.
-
The length from haptic was measured, then the diameter of the optic using
the light prob. The results were read from the LED display on the Quadra
check.
-
The curvature of the IOL was then measured by taking three points along
the shadow of the curve and recording their x and y positions.
-
The three x and y values were then input into a drawing package which
determined the best fit curve and subsequently, the radius.
4.1.3 Optical Measurements
Focal length is the distance from the lens to the image plane when parallel rays
of light impinge upon the lens.
Apparatus:
The Apparatus used included a metal bar with a small hole at the end of it and an
adjustable height, a light source from far away which creates parallel rays of light,
a ruler and a sheet of white paper. This set-up is illustrated in Figure 4.3.
52
5. Experimental
Samples available:
Samples 1, 2, 3 and 5.
No unnecessary risks of contamination were being
performed on sample 4 at the time in question.
Procedure:
-
The lenses were taken from solution and blotted dry.
-
They were in turn placed at the end of the bar with the lens optic over the
hole.
-
The bar and lens arrangement was then held under beams of parallel light
and the height adjusted until a sharp spot of light could be seen on the
sheet of paper.
-
The height of the lens from the sheet of paper was then recorded, this is
the focal length, f.
-
Using this value and applying it to equations (1) and (2) in section 3.1 of
the theoretical analysis, values for D, the dioptric power, were calculated.
53
5. Experimental
4.1.4 Atomic Force Microscopy
AFM is a branch of Scanning Probe Microscopy (SPM).
It produces images by
scanning a surface using a probe mounted on a cantilever that is driven by
piezoelectric ceramic materials, and gives information about the outermost
surface of the sample. Deflections in the very small spring-cantilever occur due
to probe-sample interaction forces as features are encountered on the sample
surface
during scanning, giving very accurate height measurements of any
irregularities or deposits. A signal is then collected and processed to produce a
3D image of the sample surface. AFM uses the forces that occur when two
objects are brought within nanometres of each other. In contact mode the probe
obtains a repulsive force.
AFM can also be conducted in non contact mode,
when the probe is a few nanometres away, causing an attractive force. The
forces are usually in the nano-Newton range.
AFM can be utilised in air, liquid
or a vacuum (Russell et al., 2004).
One of the methods for measuring the deflection of the cantilever is the use of a
laser beam, reflected from the back of the spring cantilever. The motion of the
probe is sensed by the displacement of the reflected beam on a photodetector
seen in Figure 4.4.
Apparatus:
The AFM images reported in this study were obtained using a TopoMetrix
Explorer Scanning Probe Microscope (ThermoMicroscope-VEECO), seen in
Figure 4.5, in contact mode in the BSS solution. The tip used was silicon nitride
with a 4micron base, 4micron long and a tip of radius <50nm. This tip was
attached to a 200micron long cantilever. The tip and lever can be seen in Figure
4.6.
The raw data collected was processed using horizontal and vertical
levelling, left shadowing and equalization of the colour.
54
5. Experimental
Samples available:
Sample 2. Due to funding and financial restrictions only one sample could be
analysed.
Procedure:
55
5. Experimental
-
The sample was taken straight from the BSS solution and held between
two glass slides under the microscope (There was a glass slide to the left
and right of the sample holding it in place and restricting it from moving).
-
The BSS solution was then poured over the lens so that it covered it
completely.
-
A microscope connected to a video camera showed views of the
cantilever and tip, in order to find the correct positioning on the sample.
-
Four random spots were analysed on the posterior side of the lens in
contact mode, meaning the tip is in physical contact with the sample.
-
The probe moves across the surface in the x-y plane and the movement in
the z plane as a response to the probe-sample interaction is stored on the
computer system to generate a 3D representation of the surface.
4.1.5 UV Analysis
UV light coming from the sun occurs at a wavelength below the visible spectrum
(<400nm). These are harmful rays and can cause damage to the inner eye. The
natural lens aborbs most of these rays before they reach the retina. This natural
protection is removed along with the cataract lens. All IOLs, therefore, contain
UV absorption properties.
This experiment analyses how well the IOL UV
blockers work by graphing their percentage absorbance of UV light.
If the
harmful rays are absorbed by the IOL they do not reach the inner workings of the
eye.
Apparatus:
The Cary-100 spectrometer was used with a tungsten filament lamp and a
deuterium lamp, which operate in different wavelenghts.
the IOL sample.
A quartz cuvette held
Quartz does not absorb UV light, providing an accurate
measure of UV absorption of the samples. The Cary-100, seen in Figure 4.7, is
a dobule beam, recording spectrophotometer controlled by a computer operating
under Windows2000, and running Varian’s WinUV software.
56
5. Experimental
Samples Available:
Sample 1, 2, 3 and 5. Sample 4 was a special interest lens due to its nature of
retrieval. No unnecessary risks of contamination were being performed on
sample 4 at the time in question.
As a result it was not involved in this
procedure.
Procedure:
-
Each sample, in turn, was placed inside the quartz cuvette, positioned so
the light beam would strike it at 90° the optic surface. Due to the tacky
nature of the hydrated IOLs, they stuck firmly to the side of the cuvette.
-
The spectrometer was run at wavelengths between 250nm and 600nm for
each sample and graphs plotting wavelength versus percentage
absorption were recorded on the software.
4.1.6 Swell test
57
5. Experimental
A swell test tells how much water the lens absorbs or takes in. As two of the
samples used were hydrophobic any water taken on indicated a polymer gap
caused by manufacturing and could be the cause of glistenings or voids.
Apparatus:
Sartorious enclosed electronic weighing scales with accuracy to 5 decimal places
seen in Figure 4.8.
Samples Available:
Sample 3, 4 and 5. Sample 1 and 2 were in Italy for analysis at the time of this
procedure.
Procedure:
-
Sample 3 and 5 were firstly weighed in the fully hydrated state.
-
All samples were left to dry out for a few days.
-
They were then weighed in the fully dehydrated state.
58
5. Experimental
-
The samples were placed in distilled water for one hour, taken out using
the tweezers, and the excess water was removed by dabbing them with
tissue. Again, they were weighed and the weights recorded.
-
They were placed back in distilled water and left for a further 23 hours.
They were again dabbed dry and weighed. These final values were
recorded.
-
Using equation (3) and (4) in section 3.2, theoretical analysis, the
Equilibrium water contents(EWCs) were calculated.
4.1.7 Scanning Electron Microscopy (SEM)
SEM operates by creating a voltage between a conductive sample and a
filament, causing an electron emission from the filament to the sample. The
electrons are guided to sample by a series of electromagnetic lenses in the
electron column. This occurs in a vacuum environment. The resolution of the
image is determined by the beam current and spot size (size of area of the
sample being viewed). Electrons interact with the sample and are emitted back
from the sample as a signal used for investigations of surface morphology.
Once these electrons escape from the sample surface they are detected by a
scintillater-photomultiplier detector. The SEM image formed is the result of the
intensity of the electron emission from the sample. The sample must be
conductive to interact with the electrons (Russell et al., 2004).
Apparatus:
The Joel 5600 Scanning Electron Miscoscope, seen in Figure 4.9, with
compatible image capturing software.
59
5. Experimental
Samples available: Sample 1 and 2.
Procedure:
-
Sample 1 was blotted dry and left uncoated to see if any images could be
obtained without causing damage to the lens.
-
It was placed in the SEM chamber on a tungsten disc and viewed with a
low accelerated voltage of 3kV.
4.2 Destructive Testing
The following destructive analyses were performed on the samples available at
the time of testing.
4.2.1 Microscopy
The samples may have harboured dirt deposits or dead proteins while examined
by microscopy before. By soaking the samples in disinfectant all these
unwanted residues were removed. This experiment was considered destructive
60
5. Experimental
because it is not known if the fluid removes any deposits which may be of
interest.
Apparatus:
An Olympus optical microscope was used, model: bx60m with a TIC-1280E
colour video camera connected to sufficient software on the computer to capture
bitmap images. This microscope can be seen in Figure 4.1 above.
Samples Available:
Sample 3, 4 and 5. Sample 1 and 2 were in Italy for analysis at the time of this
procedure.
Procedure:
-
The IOLs were soaked overnight in undiluted Milton sterilising fluid which
contains sodium chloride. They were then rinsed with distilled water and
viewed as explained earlier in section 4.1.1.
4.2.2 Microhardness
Shore hardness is used to determine the relative hardness of soft materials,
usually plastic or rubber. It is a measure of the resistance of material to
indentation by a 3 spring-loaded indenter. The scale provides an empirical
hardness value that does not correlate to other properties or fundamental
characteristics. The Shore hardness is measured with an apparatus known as a
durometer and as a result is also known as “Durometer hardness” (Matweb,
2005).
Apparatus:
Zwick Roell Shore D handheld microhardness tester as seen in Figure 4.10.
61
5. Experimental
Samples available:
Sample 3, 4 and 5.
Procedure:
-
The specimen’s were tested in the hydrated state. The tip of the tester,
the surface used and the tweezers were all cleaned as previously
described.
-
The samples were in turn placed on the cleaned, hard, flat surface.
-
The indenter for the instrument was then pressed into the sample making
sure it was parallel to the surface.
-
The hardness was read within one second of firm contact with the
specimen, and each sample was checked to ensure the indenter did not
penetrate it completely.
4.2.3 Scanning Electron Microscopy (SEM)
SEM is discussed in section 4.1.8.
Apparatus:
An Edwards Sputter Coater S150B seen in Figure 4.11 and a Jool JSM-840
electron microscope seen in figure 4.12.
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5. Experimental
Samples available:
Sample 3, 4 and 5. Sample 1 and 2 were in Italy for analysis at the time of this
procedure.
Procedure:
-
After soaking in Milton and subsequently transferred to distilled water the
samples were left out of the solution for a number of days to dry.
-
The posterior side of each of the samples were sputter coated with gold to
make them conductive and placed in the SEM chamber.
-
A very low 3kV accelerated voltage was used to view the samples.
4.2.4 Differential Scanning Calorimetry (DSC)
DSC measures the amount of energy absorbed or released by a sample when it
is heated or cooled. It provides data on endothermic (heat absorption) and
endothermic (heat evolution) processes. Thermal analysis may be defined as
the measurement of the physical and chemical properties of materials as a
function of temperature and time. The different thermal properties obtained from
DSC analysis include the glass transition temperature and solid-state reactions
including nucleation temperatures, crystallisation termperatures and endothermic
or melting reactions (Anderson Materials Evaluation Inc., 2004).
Apparatus:
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5. Experimental
DSC 10 Differential Scanning Calorimetry made by TA instruments, seen in
Figure 4.13.
Aluminium pans and crimper.
Liquid nitrogen and nitrogen gas.
Samples Available:
All
Procedure:
-
The samples were dry after being left out of solution for some days. A
small piece was cut from each.
-
The sample was placed in an aluminium pan and covered with a lid,
Figure 4.14. It was then closed by pressing in a crimper, seen in Figure
4.15. An emptyreference pan was also prepared.
64
5. Experimental
-
The crimped pans were then placed in the appropriate spots in the furnace
shown in Figures 4.16 and 4.17.
-
The thermal program was then set up on the computer. The material
name and temperature information is input. For this experiment the DSC
Ran at 10°C per minute from -50°C to 200°C, isothermal for 1 minute.
-
The calorimeter was set running to burn off any dirt or dust in the
chamber.
65
5. Experimental
-
The sample was then placed in the furnace and Liquid nitrogen was
poured on top of it until the temperature reached -50°C. The computer
turned on the furnace, making sure that the heating rate stayed the same
throughout the experiment and that the two separate pans heated at the
same rate as each other.
-
The furnace heated and the temperature differences between the sample
and reference was monitored so that the DSC can keep the temperatures
the same. If an exothermic phase transition occurs, the temperature of
the sample will tend to surge ahead of the reference. In this case the DSC
does not need to provide as much heat to the sample. If an endothermic
transition occurs, the DSC must supply more heat to the sample. The
electrical power difference between the sample and reference measures
the heat flow in the sample.
-
During the experiment a stream of nitrogen gas was directed inside the
furnace to purge it of oxygen. This prevented oxidation of the sample
during heating.
-
The calorimeter stopped automatically on reaching 200°C producing a
heat flow versus temperature graph. The temperature is also directly
related to the time in minutes.
4.2.5 Calcium Testing and Energy Dispersive X-Ray analysis
The calcium stains described in the literature review were not available in the
University. Along with these stains researchers such as Yong et al., (2004), used
X-Ray analysis on the samples. The X-Ray analyser also proved the presence of
Calcium. The analyser can carry out elemental analysis on spot sizes as small
as 2nm. Along with electron emission used to form a morphological image of a
surface in SEM a number of other signals are emitted as a result of the electron
beam impinging on the surface. Each of these signals carries information about
the sample which provides clues to its composition. X-Ray signals are
frequently used to provide elemental analysis by the addition of an Energy
Dispersive Spectrometer to the SEM system. The interaction between the
electron beam and the sample results in the expulsion of an inner shell election
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5. Experimental
from the atom creating a vacancy that is filled by an outer shell electron. This
jump from outer to inner shell results in a change in energy that produces an XRay. These are then compared against an X-Ray spectrum of the periodic table
of elements.
Apparatus:
The Jool JSM-840 Scanning Electron Microscope seen in Figure 4.12 equipped
with an energy dispersive (EDX) analyser.
Samples available:
Sample 4 and Sample 1.
Procedure:
-
The analysis was run in conjunction with SEM pictures taken as described
in section 4.2.3 above.
-
Sample 4, previously gold coated, was cut in half before being placed in
the chamber. A spot on the inner surface of the sample was analysed
using the X-Ray. The posterior surface was also examined. Unusual
deposits seen previously using SEM were targeted and analysed.
-
After drying for a number of days sample 1 was sputter coated with gold
and analysed using the energy dispersive X-Ray analyser. Sample 1 was
taken straight from the BSS before drying and had never been disinfected
using Milton.
4.2.6 Fourier Transform Infrared Spectroscopy (FTIR)
FTIR is an analytical technique used to identify organic or inorganic materials. It
measures the absorption of various infrared light wavelengths of the material of
interest, and identifies types of chemical bonds in a molecule. These infrared
bands identify specific molecular components and structures. The region
between 1500 – 4000 wave numbers is referred to the fingerprint region.
Absorption bands in this region are highly specific for each material. This
specificity allows a computerised data search to be performed against a
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5. Experimental
reference library to identify the material. FTIR can be applied to identify foreign
materials such as particles and residues.
Two techniques can be used in this analysis, the transmission technique or the
reflection technique. The transmission technique is the most simple of the
infrared spectroscopic techniques. Infrared radiation is passed through a sample
and the transmitted radiation is measured. It is only applicable to thin samples.
The reflection technique requires the sample to be placed on a gold disk. The
IR light hits the surface of the metal and reflects off it. Only the molecular
entities aligned with the electric vector of the incident light will interact with the
light, resulting in an absorption band and providing information about the
macromolecules at the surface (Materials Evaluation and Engineering, 2000).
Apparatus: A Perkin Elmer spectrum 2000 with a FTIR microscope.
Samples available: All.
Procedure:
-
Samples 3, 4 and 5had undergone SEM at this stage so the gold material
on them was scratched off the IOL surface.
-
Each sample was then sectioned and compressed in a dye to obtain a thin
cross section for analysis.
-
The samples were then placed on Potassium Bromide (KBr) solid disk, as
this is mostly invisible to the IR method.
-
The spectrums were obtained using the transmission technique through
the lenses with the FTIR microscope. All spectrums obtained are relative
to the KBr background disk.
68