J. Biomedical Science and Engineering, 2009, 2, 363-365
doi: 10.4236/jbise.2009.25052 Published Online September 2009 (http://www.SciRP.org/journal/jbise/
Published Online September 2009 in SciRes.http://www.scirp.org/journal/jbise
Layered-resolved autofluorescence imaging of
photoreceptors using two-photon excitation
Ling-Ling Zhao, Jun-Le Qu*, Dan-Ni Chen, Han-Ben Niu
Key Laboratory of Optoelectronic Devices and Systems of Ministry of Education, Institute of Optoelectronics, Shenzhen University,
Shenzhen, China.
Email: jlqu@szu.edu.cn
Received 21 April 2009; revised 15 May 2009; accepted 19 May 2009.
In this paper, we present our investigation on
the morphological and autofluorescence char-
acteristics of the cones and rods using two-
photon excitation with a femtosecond Ti: sap-
phire laser. The results show that the micro-
structures of the photoreceptor layers can be
visualized at submicron level without any stain-
ing or slicing. The morphology and spatial dis-
tribution of the cones and rods can be resolved
by autofluorescence imaging. The autofluores-
cence in the photoreceptor outer segments is
much stronger than other layers, but suscepti-
ble to light-induced damage.
Keywords: Two-Photon Excitation; Photoreceptor;
Retina; Autofluorescence
There are two basic types of photoreceptors, rods and
cones, which are of different shapes and involved in dif-
ferent visual functions. Rods are highly sensitive to the
weak and faint light, which are used for vision under
dark-dim conditions at night. Cones are the basis of our
color perceptions since they have different wavelength
sensitivity and the consequent pathways of connectivity
to the brain. The dysfunction of photoreceptors is one of
most important factors of ocular fundus diseases, such as
retinitis pigmentosa (RP), age-related macular degenera-
tion (AMD), glaucoma, and so forth. [1,2] Retinitis pig-
mentosa is the leading cause of inherited blindness,
which is characterized by progressive loss of visual
function related to death of rods then cones, and leads to
the breakdown of the photoreceptor outer segment disc
membranes. Patients with retinitis pigmentosa (RP)
show various symptoms. The onset is often gradual and
insidious, and many patients fail to recognize the mani-
festations of this condition until it has progressed sig-
nificantly. When patients do report symptoms, they
commonly include difficulty with night vision (nyctalo-
pia) as well as loss of peripheral vision. In addition,
age-related macular degeneration (AMD) is a slowly
progressive disease that is related to the abnormal accu-
mulation of lipofuscin, which is located in the retinal
pigment epithelium (RPE) cells and is a byproduct of
incomplete digestion of photoreceptor outer segment
disc membranes. Generally, the people who suffer from
AMD have an early abnormal condition and experience
minor visual loss. For many of these people, macular
degeneration will not progress to a more serious condi-
tion. But for the others, macular degeneration may lead
to severe loss of visual acuity (or centralvision). [3]
Fortunately, with the advent of near infrared femto-
second laser, multiphoton excitation fluorescence mi-
croscopy, which is based on simultaneous absorption of
two or more photons, has become a novel and powerful
tool in biomedical imaging. [4] It enables the possibili-
ties to investigate the autofluoresecence and spatial dis-
tribution of photoreceptors at early stage of ocular dis-
eases, since it can increase the penetrability through the
thick biological tissue and provide high-resolution im-
aging of endogenous fluorophores in biological tissue.
[5,6] And also, Multiphoton microscopy can facilitate
the simultaneous excitation of different endogenous
fluorophores in biological specimens at submicron level,
which benefits to the identification and evaluation of the
morphological and autofluorescence characteristics of
the photoreceptors. Several endogenous fluorophores
exist in the photoreceptors, e.g., all-trans-retinol, NAD
(P) H, A2-PE, FAD etc, which are all related to the me-
tabolism and visual cycle. [7,8,9,10] Therefore, mul-
tiphoton microscopy can be used to investigate the auto-
fluorescence characteristics of photoreceptors.
In this study, layered-resolved autofluorescence im-
aging using two-photon excitation has been performed,
which can provide not only structural, but also func-
tional information about the different layers of photore-
ceptors, particularly photoreceptor inner and outer seg-
ments which are vital functional layers in the photore-
ceptors and significant for the early diagnosis of the
retinal diseases.
364 L. L. Zhao et al. / J. Biomedical Science and Engineering 2 (2009) 363-365
SciRes Copyright © 2009 JBiSE
2.1. Materials
Fresh porcine eyes are supplied by the local slaughter-
house and extracted immediately after slaughtering
within one hour. The eyeballs are transported to the lab
in PBS (PH 7.16)/normal saline solution and kept in a
freezing box (0-4). The eyes are kept in the PBS (PH
7.16)/normal saline solution for less than 30 minutes
before experiment. A 6mm-diameter retina-RPE-choroid-
sclera complex near the macula (both the rods and cones
are in existence) is trephined and the neurosensory retina
is peeled off gingerly. The side that connects with the
RPE cells is kept down to contact the slip of a specially
designed chamber. All the specimens are freshly pre-
pared without fixing, slicing and labeling.
2.2. Methods
The experiment is performed on an inverted confocal
laser scanning microscope (Leica TCS SP2). A mode-
locked femtosecond Ti: sapphire laser (Coherent Mira
900F) is coupled to the microscope for two-photon exci-
tation of the specimen. The Ti: Sapphire laser produces
ultrashort laser pulses with tunable wavelength from
700nm to 980nm, a pulse width of about 120 fs and a
repetition rate of 76 MHz. The excitation wavelength in
this experiment is 800nm. A 63×/NA1.32 oil-immersion
objective is used to focus the excitation light onto the
specimen and collect the autofluorescence from the
Both the rod and cone photoreceptors are consisted of
four parts, i.e., the outer segment, the inner segment, the
nuclear region and the synaptic terminal. The outer seg-
ment is filled with stacks of membranes containing the
visual pigment molecules such as rhodopsins. The inner
segment contains mitochondria, ribosomes and mem-
branes where opsin molecules are assembled and passed
to the outer segment discs. The nuclear region contains
the nucleus of the photoreceptor cell and the synaptic
terminal is responsible for the neurotransmission to sec-
ond order neurons.
Usually rod photoreceptors are slim rod-shaped struc-
tures with their inner and outer segments filling the area
between the larger cones in the subretinal space and
stretching to the pigment epithelium cells. Rod cell bod-
ies make up the remainder of the outer nuclear layer be-
low the cone cell bodies. Cone photoreceptors, on the
other hand, are robust conical-shaped structures that
have their cell bodies situated in a single row right below
the outer limiting membrane and their inner and outer
segments protruding into the subretinal space towards
the pigment epithelium. Apical processes from the pig-
ment epithelium envelope the outer segments of both
rods and cones.
In our study, we obtain the image of rod and cone
photoreceptors nicely aligned along vertical and hori-
zontal directions using two-photon excitation fluores-
cence microscopy and we also identify the fluorophores
in photoreceptor outer segments by analyzing the spec-
trum, intensity, distribution and components of auto-
fluorescence [11]. Figure 1 shows the cross-sectional
autofluorescence image of photoreceptors, in which in-
dividual cones and rods are clearly resolved. The XZ
image of the different photoreceptor layer structures are
distinguished accurately with submicron resolution. The
autofluorescence in the photoreceptor outer segments is
much stronger than other layers, since the outer segment
is abundant in many fluorophores, such as all-trans-
retinol, A2-PE, FAD, etc.
The XY image of the photoreceptor outer segment is
shown in Figure 2. Because the photoreceptor outer
segment is connected with the RPE cells, when the
photoreceptor outer segments is separated from the
Figure 1. Cross-sectional autofluorescence
image of photoreceptors excited by two-
photon excitation. (λTi: Sapphire=800nm).
(a) (b)
(c) (d)
Figure 2. Autofluorescence image of photoreceptor out seg-
ment excited by two-photon excitation. (λTi: Sapphire =800nm).
L. L. Zhao et al. / J. Biomedical Science and Engineering 2 (2009) 363-365 365
SciRes Copyright © 2009
RPE cells, the outer segment at top may be disarranged,
but the pillar-like shape can be observed (Figure 2(a)).
When focusing the imaging plane slightly into the
specimen, we can see the rod is aligned annularly,
round in shape and 2 µm in diameter (Figure 2(b)) and
the center is dark. With focusing the objective further
into the specimen, the autofluorescence of cone outer
segment, which locates in the center, can be detected,
and the size of the cones is becoming larger and larger
(Figure 2(c) and Figure 2(d)), whereas the autofluo-
rescence of rod outer segment weakens.
excitation is quadratically dependent on the intensity, it
is susceptible to photondamage at the focal point. In our
work, the structure and properties of procine eyes are
very similar to human eyes, so the laser power on the
sample is limited to the order of mW (3-4mW), which
accords with ANSI about laser safety criteria for human
eye. Moreover, this in vitro autofluorescence imaging of
photoreceptors can give much detailed structural and
functional information at high spatial resolution, which
can help to more clearly understand the in vivo ocular
fundus autofluorescence images obtained by confocal
scanning laser ophthalmoscopy. And also, two photon
excitation is IR illumination, which can penetrate the
anterior segment of eyes, such as cornea and lens, and
get the whole autofluorescence characteristics of the
living retina, and eliminate the autofluorescence inter-
ference of the anterior segment of eyes by tightly focus
without confocal pinhole. Thus, in vitro autofluores-
cence imaging of photoreceptors using two photon exci-
tation can provide a clue and develop a two-photon laser
scanning ophthalmoscope for in vivo living retina imag-
ing. Although much research remains to be done, it ap-
pears that this technique has great potential to bridge the
gap between clinical examination and invasive biopsy
and thus facilitate the early detection and diagnosis of
ocular diseases. The distribution and intensity of auto-
fluorescence may provide an insight into the sequence of
events that leads to retinal damage and may help eluci-
date pathological mechanisms.
Figure 3 shows the autofluorescence image of photo-
receptor inner segment. The bigger cell is corresponding
to the cone inner segment whose diameter is around 6
µm. Rod inner segment is around the cone and the di-
ameter is about 2µm. Figure 4 is the autofluorescence
image of photoreceptor nuclear region, which contains
the nucleus of the photoreceptor cell.
Our results demonstrate, for the first time, that morpho-
logical and autofluorescence characteristics of different
layers of photoreceptors can be identified by autofluo-
rescence imaging using two-photon excitation. Since the
cross section of two photon excitation is much smaller
than that of single photon excitation, and two photon
This work is supported by the National Natural Science Foundation of
China (NSFC) under contract No. 60627003 and No. 60408011, and is
also supported in part by Guangdong Natural Science Foundation grant
No. 5010500.
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(a) (b)
Figure 4. Autofluorescence image of cone nuclear region (a)
and rod nuclear region (b), excited by wo-photon excitation
(λTi: Sapphire=800nm).
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