Tracking Ophthalmic Drugs in the Eye Using Confocal Fluorescence Microscopy
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Tracking Ophthalmic Drugs in the Eye Using Confocal Fluorescence
Microscopy
K.K. Buttenschön*a, J.M. Girkina, D. Dalyb
a
CfAI, Department of Physics, Durham University, South Road, Durham DH1 3LE, UK;
b
Lein Applied Diagnostics, Reading Enterprise Centre, Whiteknights Rd, Reading RG6 6BU, UK
ABSTRACT
We report on the development of a non-invasive instrument based on scanning confocal microscopy for tracking
inherently fluorescent drugs and measuring spatial features in the anterior chamber of the eye. The new instrument
incorporates all features of the initial instrument1 with the addition of fluorescence detection from within the anterior
chamber of the eye. We have measured the diffusion of Fluorescein with high time resolution within a cuvette, an
artificial eye and ex vivo porcine eyes. Results are be presented that demonstrate the capability of the instrument to
accurately measure the concentration and the location of the fluorescent drug over a given period of time along the
optical axis of the eye with an axial resolution of under 200 µm and temporal resolution of < 1s. We show that the
instrument has high sensitivity and can measure concentrations of < 1µM/L of compounds having a quantum yield as
low as 0.01 with high specificity for the compound of interest over competing background signals. The role of the
instrument in assessing the efficiency of any inherently fluorescent ophthalmic drug as well as monitoring other
medication that might produce fluorescent compounds in the eye will be discussed. We furthermore believe that the
instrument might also be capable of monitoring certain bodily processes which have an impact on the compounds present
in the eye.
Keywords: Confocal microscopy, fluorescence microscopy, ocular pharmacokinetics, drug diffusion, aqueous humour
1. INTRODUCTION
The group of diseases commonly denominated as glaucoma is the largest cause of blindness in the western world today.2
Although several factors such as genetic predisposition, weakness of the optic nerve, severe myopia and thinness of the
cornea have been identified to increase the risk of developing glaucoma,3 one of the main risk factors is still considered
to be elevated intra-ocular pressure (IOP). Therefore the available therapies for open angle glaucoma aim to lower the
IOP in the anterior chamber of the eye by using various pharmaceuticals, laser trabeculoplasty or trabeculectomy. The
two latter options are not desirable since they create a severe disturbance of the eye, leaving pharmaceutical intervention
as the method of choice. However, although the drug diffusion through the cornea, sclera and other tissues in the eye has
been thoroughly studied,4 not much is known about the distribution and diffusion of medication in the anterior chamber
of the eye. Currently, the flow of a specific drug in the eye is being determined by administering the medicine to animal
eyes and then sacrificing the eyes at different time intervals throughout the trial. The concentration of the drug in specific
parts of the eye can then be determined.
Unfortunately, many animal eyes have to be sacrificed in each trial to determine the characteristics of the specific drug
which is a high cost and material factor as well as a complicated surgical procedure. Furthermore this does not enable the
researcher to understand the complete diffusion properties of the drug since there are big time gaps between eye
sacrifices. Therefore the need for a technology that is able to monitor the diffusion of the drug non-invasively in near real
time is high.
The method we have developed allows for non-invasive measurements at discreet intervals over long periods of time and
therefore reduces the number of animal eyes that have to be sacrificed to zero. Other instruments such as e.g. the
ordinary fluorescence microscope, Langham’s fluorometric apparatus,5 Brubaker’s ocular fluorophotometer6as well as
modifications thereof7, 8 and the FluoroTron9 have been developed which would be suitable to tackle the same problem,
but as far as we are aware none of them have yet been used for the purpose of tracking ophthalmic medication in the eye.
Furthermore the instruments that are available take a relatively long time to acquire a measurement due to slow imageacquiring and –building processes or do not have the necessary resolution and depth discrimination to accurately
determine the diffusion characteristics of the drug. The instrument presented in this paper (hereafter called the F410) has
the advantage that no sectioned images are acquired although a one-dimensional depth profile through the anterior
chamber of the eye is obtained. This allows for much faster data acquisition and on-the-fly data analysis after each scan,
reducing the amount of saved data to only those scans that meet the selection criteria and a much smaller file size.
Furthermore the instrument consists of the combination of a confocal reflectance with a confocal fluorescence
microscope, thus increasing both resolution and depth discrimination.
In addition, the instruments currently available on the market are not as cost-effective as the F410 and thus not as easily
attainable for many smaller institutions and potentially for home monitoring systems.
2. METHODOLOGY
2.1 Instrumentation
The F410 is shown in figure 1. The fibre coupled laser (1, λ=405nm) expands through a 70:30 beam splitter. The
transmitted beam is incident on a photo diode which monitors the laser power (2). The reflected beam is reflected again
off a dichroic beam splitter (Comar Optics) and subsequently passes through a pellicle beam splitter (8:92). The beam is
then collimated by a doublet lens and focused onto the sample by a second doublet lens (3, CVI Melles Griot). This lens
is mounted on a scanning stage (SMAC) with a travel of 10 mm which scans the focus at 40 mms-1 along the optical axis
of the eye through the anterior chamber. Fluorescent light is excited in the focus of the beam in the sample (4) and passes
back through the scanning lens, the collimating lens, the pellicle beam splitter where a part of the beam is diverted onto a
webcam to help the operator to monitor the correct alignment of the eye, and the dichroic mirror. The beam then passes
through a 50 µm confocal pinhole onto the photo-multiplier tube (PMT) (5, Hamamatsu Photonics). The beam incident
on the sample passes through an optical chopper (Thorlabs) which modulates the beam at a specific frequency to increase
the signal to noise ratio of the returned fluorescent light. The returned signal recorded by the PMT is coupled into a lock-
in amplifier (FEMTO) which rejects any signal that has not been modulated and therefore does not come from the
sample. Further rejection of out-of-focus light takes place at the confocal pinhole, making the system specificity very
high.
Figure 1. Schematic diagram of the F410. 1) Fibre coupled laser. 2) Laser power monitoring photo diode. 3) Scanning
lens. 4) Sample. 5) Confocal fluorescence detector. 6) Confocal reflection detector. 7) Eye position monitoring
camera.Since the F410 is derived from a confocal microscope it has very simple and cost-effective optics. It combines confocal
reflection and fluorescence detection in a single instrument. However rather than recording a three dimensional image
out of two-dimensional image slices which require complicated scanning mechanisms and high computing power, it
records a one-dimensional depth profile along the optical axis of the eye. In a later version of the instrument this will be
advanced to include x-y-z scanning, thus recording a three dimensional depth profile of the whole anterior chamber.
However this will still require less computing power than commercially available instruments since no image will be
generated. Furthermore, the position of the fluorescence in the eye can be determined with accuracy precision of 5 µm
and a resolution of under 200 µm. The position is determined by the coupling of the closed-loop encoder in the scanning
stage with the data acquisition via an A/D converter (Measurement Computing). The recording of a data point is
triggered by the incoming pulses from the stage encoder which are divided such that a measurement is taken every 20
µm. The control of the instrument and data acquisition as well as the data analysis is achieved by a custom written
python programme. The data is analysed on-the-fly after each individual scan to ensure that only such data that meets the
selection criteria is saved on the hard disk, thus reducing the space required for data storage. This system also has the
advantage that the data can be displayed almost simultaneously with the scan thus allowing for the operator to get an
impression of the drug dispersion immediately. The data is also saved for later analysis where the operator can switch
between different display modes that allow for a better understanding of the distribution of the drug in the eye over time.
The available display modes are both two-dimensional and three-dimensional in nature so that the distribution and
concentration of the drug at any single point in time at any chosen position as well as the whole image of the drug
diffusion and concentration over time and position can be viewed.
2.2 Calibration
The F410 has been built to measure the concentration of pharmaceutical drugs in the eye. However, drugs might have a
small quantum yield, therefore the initial calibration of the instrument was performed using Fluorescein sodium salt
(Sigma-Aldrich) in micro-filtered water (pH 7) as it has an excellent quantum yield (slightly reduced by the pH of the
water) and is a well known and well defined reference standard for fluorometric measurements. At a later stage, the
instrument will be calibrated with the same method as described for Fluorescein for inherently fluorescent
pharmaceutical compounds used for treating glaucoma, of which there are several. All initial measurements reported
here, both in a cuvette (1cm path length), a home-build artificial eye and ex vivo porcine eyes, were taken using
Fluorescein.
To calibrate the F410 for measurements in the cuvette, a series of known Fluorescein concentrations in pure water were
prepared. The signals measured from these concentrations yielded the calibration curves for all Fluorescein
measurements that were taken using a cuvette as sample. (The instrument also had to be calibrated for loss of signal with
increasing scanning depth as light is absorbed and scattered in the layers before the focus as the focus is scanned deeper
into the sample. This was achieved by using a derivation of Beer’s Law. The Beer-Lambert Law is given by10
A = σ ⋅l ⋅n = α ⋅l , (1)
where A is the absorbance, ε is the extinction coefficient, l the path length, c the concentration and α the absorption cross
section. Since
T = exp( − A) , (2)
the intensity loss of the fluorescence signal follows an exponential trend. However, in the F410 the loss of signal with
depth does not follow Beer’s law exactly. It does instead follow a triple exponential trend of the form
I = I 0 ⋅ exp(( −σ 402 + σ 510 + σ C ) ⋅ x ) , (3)
which shows that the intensity of the fluorescent light is attenuated with depth x by the absorption of excitation light, re-
absorption of emission light and scattering of both excitation and emission light. σ402 and σ510 are the extinction
coefficients for the excitation and the emission light respectively. σC is the concentration dependent scattering
coefficient.
The concentration and depth calibration were then combined and used in a look-up table approach to determine the
concentration of unknown measurements at any depth in the sample by converting the signal from the unknown
measurement to a known concentration using the calibration curves.To calibrate the instrument for measurements within the artificial eye, the same method as described above was used.
Known quantities of Fluorescein were inserted into the anterior chamber of the artificial eye and calibration curves
curve
recorded. The concentration
ation of unknown measurements could then be determined using these
th calibration curves.
For measurements in ex vivo porcine eyes, the F410 had to be calibrated differently since no known concentrations of
Fluorescein can be created in the aqueous humour due to the unknown volume of aqueous in the anterior chamber of the
respective sample eye. Therefore the aqueous humour was extracted completely from the eye after measuring the
background fluorescence signal with the instrument. The anterior chamber was then filled with a known concentration of
Fluorescein and measured with the F410 to create the concentration calibration curves. The depth calibration was then
performed in a similar way as described above. Therefore, unknown
nknown concentrations of Fluorescein that were added to the
aqueous humour of ex vivo porcine eyes could be measured and determined with the F410.
a b
c d
e f
Figure 2. A series of traces taken of a drop of Fluorescein dispersing in a cuvette. The traces were recorded at a time
interval of 5 seconds (e to f: 10 seconds).
seconds). Trace 2a shows the cuvette before the insertion of the Fluorescein. 2b-d
show the drop falling
ng through the focus and saturating the detector in 2c. In 2e, the drop has been reflected off the
bottom wall of the cuvette, and in 2e the Fluorescein has reached a more equal distribution.3. DATA
As mentioned above, different types of measurements were taken. As the first and easiest sample the distribution of
Fluorescein in a cuvette filled with water was measured. An unknown volume of an unknown concentration of
Fluorescein was dropped into the water filled cuvette using a pipette. Figure 2 shows a series
series of scans taken during the
distribution of the drop in the cuvette with a time interval of 5 s (2e – 2f: 10 s). The blue trace shows the reflection from
the cuvette wall with the anterior and the posterior surface of the front wall of the cuvette (t ( = 580 ± 5 µm) resolved
clearly. The green trace shows the fluorescence signal recorded from the drop of Fluorescein falling through the cuvette.
a b c
d e f
g h
Figure 3. Three dimensional traces showing the diffusion of a drop of Fluorescein in a cuvette filled with water. Each
three dimensional graph represents a 35 second long snapshot of the drop diffusion. The snapshots were taken at
intervals of 3 minutes (3a-d)
d) and 5 minutes (3d-h).
(3d a) Before
efore the insertion of the drop. b) The drop is starting
st to
reach the focus of the probing beam. c) – e) The drop of Fluorescein falls through the focus. f) The drop starts
spreading towards the front of the cuvette. g) – h) The drop spreads further as the intensity towards the front of the
cuvette increases
ses and a more even distribution of Fluorescein is reached.
Figure 2a shows a scan just before the Fluorescein is released into the water, no fluorescence is visible. Then the drop is
released and figures 2b-dd show the drop falling through the focus in the
the cuvette, temporarily saturating the detector. In
figure 2e the drop has been reflected off the bottom wall of the cuvette and has started to spread laterally, and figure 2f
shows the cuvette after 30 seconds when the Fluorescein is distributed more evenly
evenly throughout the cuvette.The data can also be displayed in three dimensions for a quicker overview of how the fluorophore diffuses with time. An
example is shown in figure 3, where again a drop of Fluorescein disperses in a water filled cuvette. The depth dept of the
focus in relation to the zero position of the scanning lens is displayed on the x-axis,
x axis, the intensity of the fluorescent signal
is displayed on the y-axis
axis and the progressing time is displayed on the z-axis.
z axis. The intensity is colour-coded
colour for ease of
understanding. Each three dimensional graph represents a 35 second long snapshot of the drop diffusing in the cuvette.
The individual snapshots were taken at an interval of 3 minutes (figure 3a to d) and 5 minutes (figure 3d to h)
respectively.
Figure 4. A scan through an ex vivo porcine eye filled with Fluorescein. A double fluorescence peak is visible since
some of the Fluorescein leaked onto the outside of the cornea when it was administered to the eye.
Figure 3a shows the empty cuvette before the drop of Fluorescein is inserted. Very faint fluorescence from the cuvette
wall is visible at a scanning depth of about 2000 micrometers. This does however not disturb the actual measurement
since the instrument is able to detect the position of the fluorescence
fluorescence within the sample through the simultaneously
recorded reflection trace. In figure 3b, the drop has been inserted and has started to reach the focus of the probing beam.
Figures 3c to e then show how the drop falls through the focus of the beam. In figure
figure 3f, it is starting to disperse slowly
within the cuvette. Finally, in figure 3h, the drop has dispersed almost completely and a more even concentration
throughout the cuvette has been reached.
Furthermore, measurements in ex vivo porcine eyes have been
been taken, first of all for calibration purposes and secondly to
track a drop of Fluorescein administered with a syringe to the anterior chamber. Figure 4 shows a trace through an eye
where the aqueous humour has been replaced completely with Fluorescein.
A double fluorescence peak is visible at the cornea. This is due to the filling process of the eye where some of the
Fluorescein leaked out of the eye and onto the outer surface of the cornea. This shows that the F410 is capable of
detecting the exact location
on of the fluorescence and thus determining its relevance towards the measurement of the
compound of interest.
The instrument is also capable of tracking the diffusion of a drop of Fluorescein that has been administered to the
anterior chamber of the eye as shown in figure 5. The eye was in a holder to prevent too much movement but the apex of
the cornea moved slightly off the optical axis of the instrument when the drop was administered with a syringe (fig. 5a).
However once the needle was removed the eye returned to its original position and the drop can clearly be seen in the
front of the anterior chamber at approximately 25 seconds into the snapshot. After 2 minutes the drop then starts to
disperse into the eye (5b). After monitoring for a longer period of time the drop then starts to move out of the focus of
the probing beam and is thus outside the detection range (figures 5c – 12 minutes and figure 5d – 27 minutes). It ishowever noticeable that some of the Fluorescein seems to attach itself to the posterior
posterior cornea of the eye since an
increased intensity of fluorescence is visible at this position.
a b
c d
Figure 5. A series of scans through an ex vivo porcine eye with a drop of Fluorescein administered to the anterior
chamber. 5a) 0 minutes. The drop is being inserted using a syringe which shortly puts the apex of the cornea off
the measuring axis. 5b) 2 minutes. The drop has started to disperse into the eye. 5c) 12 minutes. The drop is
starting to diffuse out of the focus of the beam and thus
thu leaving the detection range. 5d) 27 minutes. Most of the
Fluorescein has left the focus, however some seems to have attached itself to the posterior wall of the cornea since
the fluorescence levels are raised at this position.
4. DISCUSSION AND CONCLUSION
CONCL
We have built an instrument that is capable of accurately detecting the position and concentration of Fluorescein in a
cuvette, an artificial eye and ex vivo porcine eyes. The instrument can detect rapid changes in concentration of under one
second, which is not possible with the currently used animal models and thus will enlarge the understanding of drug
diffusion within the eye. The data is displayed in near real time after each scan to give the operator an impression of the
immediate situation in the eye. The operator can also choose in which way the data is presented to them. Scans are
acquired at currently 4 scans a second
econd which can be increased by using a faster analogue to digital converter. The axial
resolution is currently rather large but can easily be changed for specific applications by exchanging the pinholes in the
system. However increasing the axial resolution
resolution decreases the signal to noise ratio and thus the sensitivity of the
instrument which has to be taken into consideration when changing the settings. The signal of the reflected beam is
currently very low due to aberrations in the system which will be addressed
addressed shortly. We will then be able to increase the
axial resolution of the reflection beam so that the instrument will also be capable of monitoring distance changes within
the anterior chamber of the eye. For the fluorescence detection however the accuracyacy of detection matters more than the
axial resolution since determining the accurate location of the drug in the eye is more crucial than being able to
determine the position of the concentration with extremely high resolution.Since the F410 is capable of monitoring the diffusion of drugs in the eye over long time intervals, it also allows
clinicians to assess the correct dose of the drug for each individual patient by monitoring the actual uptake of the drug
and the individual diffusion properties of the patient’s eye, thus enabling more patient-tailored treatment and making
sure that the clinically effective volume of the drug reaches the target within the eye. Furthermore the optics within the
instrument can easily be miniaturised and thus the F410 can be developed into a handheld device for home care self
monitoring systems.
The instrument can be developed further to include the posterior chamber of the eye into the scanning range, which
would open up new opportunities to monitor drugs for treating age-related macular degeneration (AMD) which is also a
major cause of blindness in the western world. It could be used to assess the feasibility of slow-release patches placed in
the posterior chamber of the eye to help increase the comfort of the treatment for wet AMD which currently is
administered using injections into the posterior chamber every six weeks which causes extreme discomfort to the patient
and also is a source of infection.
We furthermore believe that the instrument can also be applied to other areas of research than the human eye, such as the
measurement of skin conditions and the diffusion of drugs through the skin.
We are therefore convinced that this new instrument will play a leading role in assessing the diffusion of drugs in the
eye, both in the anterior and posterior chambers, as well as other application areas.
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ACKNOWLEDGEMENTS
The authors would like to thank Lein Applied Diagnostics for their CASE-studentship support (K.K. Buttenschön)
and EPSRC for the funding of the project.You can also read
