1. Science High School Advanced Research Program 10 June - 2 August 2013, Los Angeles, USA
Funduscopic Imaging of Fluorescence in
Retinal Ganglion Cells
Tiffany Cheng1
, Andrew Weitz2
and James D. Weiland2
1
Diamond Bar High School, Diamond Bar, CA, USA
2
Department of Biomedical Engineering, University of Southern California, Los
Angeles, CA, USA
Abstract
In order to save researchers’ time and rodents’ lives, the need for an imaging system
that will identify fluorescent retinal ganglion cells has emerged. This study aims to
create a funduscope (adapted from the design of Schejter and colleagues). The
endoscope-based system will enable its user to image in vivo fluorescence images in
retina. Rodents are injected with adeno-associated viral vectors that encode a
genetically encoded calcium indicator. The virus spreads in the eye and infects the
retinal ganglion cells, causing them to become fluorescent green. The funduscope will
detect if the virus has infected the retinal ganglion cells, which it sometimes fails to do if
the virus has leaked. The fundus system will provide high-quality, high-resolution
fluorescence images of the retina. This new system could serve as a basic tool for non-
invasive in vivo retinal imaging for researchers.
Keywords: calcium imaging, viral vectors, topical endoscopy
Introduction
Retinitis pigmentosa (RP) is one of many causes of blindness. It causes deterioration of
the photoreceptor cells in the retina that perceive light. This retinal degeneration causes
a decline in vision that eventually leads to blindness. RP affects nearly 2 million people
in the world [1]. However, new developments in technology provide the resources to
restore partial vision. The retinal prosthesis is an advanced system that acts to bypass
photoreceptor cells, using multielectrode arrays (MEAs). MEAs are placed near the
retina and are connected to an external camera. This camera photographs a patient’s
surroundings and transmits the images to the electrodes. The electrodes then stimulate
retinal ganglion cells (RGCs) and create artificial images for the visually impaired. The
new Argus II Retinal Prosthesis System enables subjects to perform better at tasks such
as object localization, motion discrimination, and discrimination of oriented gratings.

2. Science High School Advanced Research Program 10 June - 2 August 2013, Los Angeles, USA
All of the Argus II patients were able to perceive light when their systems were
activated [2].
Although the Argus II allowed patients to see light, the patients could not always
recognize complex images. When the electrodes were activated, the MEAs did not just
target RGCs in their areas; they also stimulated axon bundles of other RGCs, causing
patients to perceive streaks. Animal studies have provided insight to benefit the retinal
prosthesis system. By injecting rodent eyes with adeno-associated viral (AAV) vectors
encoding for fluorescent calcium indicators, researchers can study which cells are
activated during stimulation. Unfortunately, this virus will sometimes leak out of the
eye during injection and will not infect the RGCs, preventing them from becoming
fluorescent [3]. Because these experiments are performed in vitro, the researchers
cannot detect the lack of fluorescence until sacrificing the animal. Thus, the ability to
image retinas in vivo is widely desired; rodents would not have to be used in the in vitro
experiment if the AAV did not infect their retinas.
The current study describes an imaging system for identifying fluorescent RGCs
expressing genetically encoded calcium indicator using an adaptation of the fundus
system presented by Schejter, Paques, Guyomar, et al. [4-6]. The funduscope utilizes an
illuminating endoscope placed on the cornea of a rodent to image the fundus. The
system enables researchers to image retinas in vivo prior to their experiments. The
fluorescence can be seen through the funduscope to determine if the AAV has infected
the RGCs.
Materials and Methods
Imaging of the retina can be performed using an endoscope-based system introduced by
Schejter, Paques, Guyomar, et al. [4-6]. An endoscope with a 3 mm outer diameter
otoscope and crescent-shaped illumination (Tele Otoscope BERCI 1218 AA, Karl Storz
Endoscopy, El Segundo, USA; Figure 2) is placed in front of a digital camera (D5100
with a Nikkor 80-200mm f/2.8 AF-D lens; Figure 3). The mercury arc lamp (Nikon)
produces the light needed for the endoscope. The bulb (BulbAmerica, New York, USA)
light is transferred into a fiber optic using a microscope objective (Zeiss) and a fiber
launch (ThorLabs, New Jersey, USA). The fiber optic is then connected to the
endoscope through an adaptor. A 470 nm excitation filter (MF469-35, Thorlabs, New
Jersey, USA) is placed in an adaptor and a 535 nm emission filter (MF525-39, Thorlabs,
New Jersey, USA) is placed in front of the camera lens to obtain fluorescence images.
Both of these have custom holders to attach them to their respective places. The custom
holders will allow the filters to be easily removed, which then enables brightfield
imaging.

3. Science High School Advanced Research Program 10 June - 2 August 2013, Los Angeles, USA
Figure 2- Karl Storz Tele Otoscope
http://epc.karlstorz.com/epc/Starter.jsp?locale=EN&practiceArea=PED&product=1218AA&sid=SID-
2E1B6B2A-D730E4F6
Figure 3- Nikon 5100 Digital SLR Camera and 80-200 mm lens
http://shop.nikonusa.com/store/nikonusa/pd/productID.230054600
http://photocrati.com/wp-content/uploads/2009/04/nikon-80-200mm-f-28d-ed-af-nikkor.jpg
Endoscope
Fiber
optic
Fiber launch
with objective
470 nm
excitation filter
in adaptor
Collimated light
Light source
(Mercury Arc
Lamp)
535 nm
emission
filter in
adaptor
Lens
Camera
Figure 1- Funduscope set up

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Results
The funduscope presented many choices for components. The parts came in different
sizes or configurations that had to all fit together to obtain high-quality images. The
endoscope itself came in two different forms. There was the one used in this device
(1218 AA, Karl Storz) and another model (67208BA, Karl Storz). Both provided high-
quality imaging. However, the 67208BA endoscopewould emit light at a 30º angle
rather than straight ahead (See Figure 4). The user would have to hold the endoscope at
an angle or mount it at an angle that would facilitate imaging. Thus, we chose to use the
1218 AA model.
The filters for the endoscope and camera were chosen based off of the wavelength of
the fluorescent calcium indicator. Since a green fluorescent protein (GFP) was used, the
filter for the camera had to pass 535 nm light (see Figure 5). The endoscope filter allows
blue light at 470 nm to pass through and be absorbed by the GFP expressed by rodent
ganglion cells (see Figure 6).
Another aspect of the funduscope that could change in design is the fiber launch and
fiber optic. The fiber launch collects collimated light from the mercury arc lamp. The
light then passes through the microscope objective in the fiber launch into the fiber
optic. Fiber optics have different specifications that can cause problems in the study if
not addressed. For example, there are single mode fibers and multimode fibers. Single
mode fibers are best used for single rays of light. Their cores are skinnier and can
therefore maintain light for a longer distance than multimode optical fibers (see Figure
7). Single mode fibers also have a higher precision and are easier to focus. Multimode
fibers, on the other hand, can hold more light because of their larger cores. For this
study, a multimode fiber was used because of its ability to acquire a larger amount of
light. Also, the arc lamp light source uses white light rather than just a single
wavelength of light. Even after choosing a certain fiber type, there are still more
implications to consider such as the numerical aperture and core size. The numerical
aperture is the angle at which a fiber optic can accept light. The lower the numerical
aperture, the tighter the focus. However, the overall light that is transmitted through the
fiber would be less with a smaller numerical aperture. Also, the amount of power that is
transmitted through the fiber has to be measured. If there is too much light, the core of
the fiber could decay and blacken overtime which would minimize its effectiveness.
The arc lamp was another component to consider. The studies by Paques, Guyomar, et
al. [5-6] used a xenon arc lamp, while Schejter, et al [4] used a mercury arc lamp to
provide the light. Mercury bulbs produced poorer color renditions than xenon bulbs.
However, mercury bulbs operate at lower powers and have a longer lamp life. Xenon
arc lamps are brighter than mercury lamps between certain wavelengths (see Figure 8).
For this study, a mercury arc lamp was used because of its sustainability and
availability.

5. Science High School Advanced Research Program 10 June - 2 August 2013, Los Angeles, USA
Figure 4- Tele Otoscope Angles (1218 AA vs. 67208 BA)
The 1218 AA has a 0º angle of light while the 67208 BA has a 30º angle of light. The 1218 AA has a
diameter of 3 mm and a length of 6 mm while the 67208 BA has a diameter of 2.7 mm and a length of
7 mm. They both use crescent-shaped illumination to emit light.
http://epc.karlstorz.com/epc/Starter.jsp?locale=EN&practiceArea=PED&product=1218AA&sid=SID-
2E1B6B2A-D730E4F6
http://epc.karlstorz.com/epc/Starter.jsp?locale=EN&practiceArea=VET-S&product=67208BA&sid=SID-
2E1B6B2A-D730E4F6
Figure 5- 469 nm excitation filter spectrum
http://www.thorlabs.com/images/popupimages/MF469-35.jpg
Figure 6- 525 nm emission filter spectrum
http://www.thorlabs.com/images/popupimages/MF525-39.jpg

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Figure 7- single mode vs. mulimode fiber core
Single mode fibers have a smaller core that is used for single rays of light. Because of its smaller core,
single mode fibers carry light at a longer distance, but at a smaller intensity than the multimode fibers.
The multimode fibers can carry light at its original intensity and hold a larger amount of light.
http://www.rp-photonics.com/img/fiber_core.png
Figure 8- Mercury (red) vs. Xenon (blue) arc lamp spectrum
The emission spectrum typical of mercury-vapor lamps is less even than the xenon lamp. It exhibits a
number of strong peaks and valleys throughout the visible range, with the strongest peak in the yellow
region. The red end of the mercury-vapor spectrum also tends to be low compared to the blue end
(450nm-500nm), visible as a cool white.
http://www.christiedigital.com/TechPapers/Christie-Lamp-Technology-Mercury-vs-Xenon-Technical-
Guide.pdf
Discussion
The funduscope is a low-cost means of obtaining in vivo acquisition of fluorescence
fundus images. Nevertheless, there are commercially available systems that provide
images with favorable resolution (compared, for example, to the Micron IV of Phoenix
Research Labs).
If used with rodents, the system would potentially be able to produce results similar to
those of Schejter, et al [4] (see Figure 9). The in vitro images compare well to slightly
blurred in vivo images. This endoscope-based system could be improved using more
expensive products that present higher quality images. For instance, the use of a xenon
arc lamp may change the quality because it emits a greater amount of light and provides
better color renditions of images. A different multimode fiber with a smaller numerical
aperture could produce a more focused light though with less intensity. Another option

7. Science High School Advanced Research Program 10 June - 2 August 2013, Los Angeles, USA
for the fiber would be to decrease the core size which could also create a focused light.
More methods can be used to project the light from arc lamps into an optical fiber, such
as a custom-made adaptor or cable.
The ability to image rodent retinas using an easily accessible and simple technique
should facilitate the development of retinal prosthetics [4]. The funduscope may also
provide a noninvasive means of tracking progression of certain diseases, such as
retinitis pigmentosa, or monitoring infected GFP cells. The system could enable
progress in retinal imaging and the further development of knowledge in translational
vision technology.
Figure 9- In vivo vs. in vitro fluorescence imaging [4]
In vivo imaging pinpoints the GRP infected cells similar to blurred in vitro imaging.
Acknowledgements
We would like to thank Windsong Trust for its generous financial support of SHSARP.
Also, thanks to Dr. James D. Weiland for providing the resources for this project. Thank
you to Dr. Andrew Weitz for his guidance and help in researching.
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8. Science High School Advanced Research Program 10 June - 2 August 2013, Los Angeles, USA
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