Layered-resolved autofluorescence imaging of photo-receptors using two-photon excitation

doi:10.4236/jbise.2009.25052

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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/

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.

ABSTRACT

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

1. INTRODUCTION

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

2. MATERIALS AND METHODS

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

photoreceptors.

3. RESULTS AND DISCUSSIONS

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)

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

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.

4. CONCLUSIONS

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

5. ACKNOWLEDGMENTS

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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