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Imaging of Caenorhabditis elegans neurons by
second harmonic generation and two-photon
excitation fluorescence
Article in Journal of Biomedical Optics · March 2005
DOI: 10.1117/1.1886729 · Source: PubMed
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Journal of Biomedical Optics 10(2), 024015 (March/April 2005)
Imaging of Caenorhabditis elegans neurons by secondharmonic generation and two-photon excitation
fluorescence
George Filippidis
Christos Kouloumentas
Foundation of Research and Technology-Hellas
Institute of Electronic Structure and Laser
P.O. Box 1527
Heraklion, Greece 71110
E-mail: filip@iesl.forth.gr
Giannis Voglis
Foundation of Research and Technology-Hellas
Institute of Molecular Biology and Biotechnology
Vassilika Vouton
Heraklion, Greece 71110
Fotini Zacharopoulou
Theodore G. Papazoglou
Abstract. Second-harmonic generation (SHG) and two-photon excitation fluorescence (TPEF) are relatively new and promising tools for
the detailed imaging of biological samples and processes at the microscopic level. By exploiting these nonlinear phenomena phototoxicity and photobleaching effects on the specimens are reduced dramatically. The main target of this work was the development of a
compact inexpensive and reliable experimental apparatus for nonlinear microscopy measurements. Femtosecond laser pulses were utilized for excitation. We achieved high-resolution imaging and mapping of Caenorhabditis elegans (C. elegans) neurons and muscular
structures of the pharynx, at the microscopic level by performing SHG
and TPEF measurements. By detecting nonlinear phenomena such as
SHG and TPEF it is feasible to extract valuable information concerning
the structure and the function of nematode neurons. © 2005 Society of
Foundation of Research and Technology-Hellas
Institute of Electronic Structure and Laser
P.O. Box 1527
Heraklion, Greece 71110
Photo-Optical Instrumentation Engineers. [DOI: 10.1117/1.1886729]
Nektarios Tavernarakis
Paper 04075 received May 10, 2004; revised manuscript received Oct. 8 2004;
accepted for publication Oct. 12, 2004; published online Mar. 30, 2005.
Foundation of Research and Technology-Hellas
Institute of Molecular Biology and Biotechnology
Vassilika Vouton
Heraklion, Greece 71110
Keywords: second-harmonic generation; two-photon excitation fluorescence; imaging; Caenorhabditis elegans; microscopy; touch receptor neurons.
1 Introduction
Nonlinear phenomena have proven to be powerful tools in
biological imaging. Molecular excitation by the absorption of
two or more photons can be advantageous for specific imaging applications over standard fluorescence microscopy,
which is based on the absorption of a single photon. Such
applications are the two-photon1 and three-photon2 excitation
fluorescence microscopy ~TPEF and 3PEF respectively!, or
more generally, multiphoton excitation fluorescence
microscopy.3 Second-harmonic generation ~SHG! has also
emerged as a powerful contrast mechanism in nonlinear microscopy. It has been demonstrated that its combination with
TPEF in a single microscope can be very advantageous, since
they provide complementary information about several biological processes.4,5 The information provided by the two contrast techniques can be differentiated based on the fundamentally different phenomena underlying TPEF and SHG.6 While
TPEF relies on nonlinear absorption of the incident light and
fluorescence emission, SHG relies on nonlinear scattering and
does not involve an excited state, hence the first is not a
coherent process, whereas the second one is. In SHG, light of
the fundamental frequency v is converted by nonlinear materials into light at exactly twice that frequency, 2v. An indicated and reliable solution for the collection and the waveAddress all correspondence to George Filippidis, IESL, FORTH, Vassilika Vouton, Heraklion, Crete 71110 Greece; Tel: ++30 2810 391323; Fax: ++30 2810
391318; E-mail: filip@iesl.forth.gr
Journal of Biomedical Optics
length separation of the low-intensity signals ~SHG and
TPEF! is the combination of lock-in detection with a monochromator. This configuration was followed for the detection
of our signals, while other detection schemes, such as single
photon counting, are also feasible in collecting very weak
signals.
Both TPEF and SHG exhibit intrinsic three-dimensionality
and ability to section deep within biological tissues, due to
their nonlinear nature. They both have significant efficiency
only at extremely high incident light intensities, and therefore
arise only from a well-defined volume around the focal center
of the incident light beam. In both TPEF and SHG, the wavelength of the fundamental incident light lies in the infrared
~IR! spectrum region, thus suffering less scattering and absorption inside the biological samples and exhibiting larger
penetration depths. SHG and TPEF microscopy methodologies do not exhibit higher resolution compared to confocal
one-photon microscopy.7 However, as far as the axial direction is concerned, the excitation of the biological specimen in
SHG and TPEF microscopy is confined in a small region
around the focal plane, due to the quadratic dependence of
SHG and TPEF intensities upon the excitation photon flux.
‘‘Out-of-focal-plane’’ photobleaching and phototoxicity are
thus dramatically reduced, permitting higher possibilities
of survival in the biological specimen during in vivo
experiments.
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Since SHG does not come from an absorptive process,
photodamage does not arise intrinsically during SHG microscopy. However, if the incident beam produces simultaneously
two-photon excitation of chromophores in the sample, photobleaching also accompanies SHG. This happens when the
energy of the second-harmonic signal overlaps with an electronic absorption band.8 This is usually the case when the
magnitude of the SHG signal is resonantly enhanced. Because
SHG is a coherent phenomenon, the produced secondharmonic signal is highly directional and propagates forward
in the direction of the fundamental collimated beam, forming
a single lobe. However, when the incident beam is tightly
focused, the SHG radiation pattern exhibits two separate
lobes.9 Mertz and colleagues theoretically described the case
of SHG from an inhomogeneous sample by highly focused
excitation light and concluded that these inhomogeneities can
significantly modify the SHG radiation patterns, and in some
cases, can provoke backward SHG propagation.10–13
Molecular frequency doubling is caused by the nonlinear
dependence of the induced dipolar moment m of the molecule
on the incident optical electric field E. Thus m can be expanded in a Taylor’s series about E50:
1
1
m 5 m o 1 a * E1 b * E * E1 g * E * E * E1...
2
6
~1!
where m o is the permanent dipolar moment of the molecule, a
is the linear polarizability, b is the molecular first hyperpolarizability, which governs in the molecular level SHG, and g is
the second hyperpolarizability which governs among others
multiphoton absorption and 3HG.6,14 Macroscopically the optical response of materials to incident light, or generally electromagnetic radiation, is characterized by the optically induced polarization density, P, which can also be expanded in
a Taylor’s series about E50:
P5 x ~ 1 ! * E1 x ~ 2 ! * E * E1 x ~ 3 ! * E * E * E1...
~2!
where P represents the polarization density vector, and x ( n )
are the nth order optical susceptibility tensors. The first term
describes linear absorption and reflection of light, the second
term describes SHG, sum, and difference frequency generation, and the third term covers multiphoton absorption, thirdharmonic generation, and stimulated Raman processes. The
macroscopic second order susceptibility tensor x ( 2 ) , which is
responsible for SHG, is related to the molecular first hyperpolarizability, b, by:
x ~ 2 ! 5N ^ b &
~3!
where N is the spatial density of molecules, and ^b& represents
an orientational average.15 Equation ~3! implies that only noncentrosymmetric materials have a nonvanishing second order
susceptibility x ( 2 ) , and the coherent summation of their single
molecules’ SHG radiation patterns are not cancelled out, resulting in a highly directional, detectable second-harmonic
signal. The second-harmonic intensity in such media scale
as:15
SHG sig } p 2 t ~ x ~ 2 ! ! 2
Journal of Biomedical Optics
~4!
where p and t are the laser pulse energy and pulse width,
respectively. Combining Eqs. ~3! and ~4!, it is apparent that
the second-harmonic intensity is proportional to N 2 , whereas
TPEF intensity is known to be proportional to N. The quadratic dependence of the second-harmonic intensity on the
spatial density of molecules is somewhat expected, since the
single molecules act as dipole radiators and the total SHG
signal arises from their constructive interference. By contrast
the TPEF is a noncoherent phenomenon, and the radiation of
each fluorescent molecule is independent from the emission of
the neighboring molecules.
Under the symmetry constraints it is obvious that SHG can
mainly be produced at interfaces, where the symmetry breaks,
from metal surfaces, where there is a huge change in the
refractive indices, and from structures that have a high degree
of orientation and organization but lack inversion symmetry,
such as specific crystals. Dyes bounded in cellular membranes
and endogenous arrays of structural proteins can also produce
SHG, which is of significant biological interest.
In 1962, Kleinmann first demonstrated SHG in crystalline
quartz16 and in 1974, Hellwarth first integrated SHG into an
optical microscope to visualize the microscopic crystal structure in polycrystalline ZnSe.17 Freund and colleagues performed one of the first biological SHG imaging experiments
in 1986,18 in a successful effort to study the endogenous collagen structure in a rat tail tendon at approximately 50 mm
resolution. Over the last few years many efforts have been
successful in three-dimensional SHG imaging of endogenous
structural proteins,5,15,19–25 without the addition of fluorescent
dyes, as in the case of TPEF microscopy.4 Structural proteins
that form highly ordered, birefringent arrays such as collagen,
actomyosin complexes, and tubulin, from many animal
sources ~tetra fish, the nematode worm C. elegans, mouse,
and chicken!, produce relatively strong SHG signals. Collagen especially, which has a highly crystalline not centrosymmetric triple-helix structure, produces SHG extremely
effectively.26,27
One of the innovative applications of SHG is its usage as a
highly sensitive monitor of membrane potential.28 –35 When
laser pulses are incident on a membrane they induce membrane bound dipoles making them candidates for SHG. The
observed SHG signal originates only from the asymmetrically
distributed dipoles of the membrane.36 Alterations in the
membrane potential alter the magnitude of the induced dipoles, thus affecting the magnitude of the observed SHG signal. Green fluorescent protein ~GFP! has been used as a SHG
probe in this way32,37 because it undergoes large electron redistribution in the presence of light, and the resulted induced
dipole is affected by the characteristics of the transmembrane
potential. Khatchatouriants and colleagues used GFP as an
SHG probe to monitor alterations of membrane potential in C.
elegans neurons.37
Caenorhabditis elegans is a small ~1-mm! free-living hermaphroditic nematode that completes a life cycle in 2.5 days
at 25 °C. The simple body plan and transparent nature of both
the egg and the cuticle of this nematode have facilitated an
exceptionally detailed developmental characterization of the
animal. The complete sequence of cell divisions and the normal pattern of programmed cell deaths that occur as the fertilized egg develops into the 959-celled adult are known.38
One considerable advantage of the C. elegans system is that it
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Imaging of Caenorhabditis elegans neurons . . .
is the first metazoan for which the genome was sequenced to
completion.39 Investigators can take advantage of genome
data to perform ‘‘reverse genetics,’’ directly knocking out
genes. Mutations can be easily induced and large screens can
be performed to isolate mutants having specific phenotypes.
In addition, a novel method of generating mutant phenocopies, called doublestranded RNA-mediated interference
~RNAi!, enables probable loss-of-function phenotypes to be
rapidly evaluated.40 Another advantage of this system is that
construction of transgenic animals is rapid; DNA injected into
the hermaphrodite gonad concatamerizes and is packaged into
embryos, hundreds of which can be obtained within a few
days of the injection.41 The anatomical characterization and
understanding of neuronal connectivity in C. elegans are unparalleled in the metazoan world. Serial section electron microscopy has identified the pattern of synaptic connections
made by each of the 302 neurons of the animal ~including
5000 chemical synapses, 600 gap junctions, and 2000 neuromuscular junctions!, so that the full ‘‘wiring diagram’’ of the
animal is known.42 Although the overall number of neurons is
small, 118 different neuronal classes, including many neuronal types present in mammals, can be distinguished. Other
animal model systems contain many more neurons of each
class ~there are about 10,000 more neurons in Drosophila
with approximately the same repertoire of neuronal types!.
Overall, the broad range of genetic and molecular techniques
applicable in the C. elegans model system allows a unique
line of investigation into fundamental problems in biology.
The coordinated function of individual C. elegans neurons
leads to characteristic behavioral responses. Although systematic investigations have revealed important information about
the neurons that participate in specific behaviors,43– 45 the molecular and physiological processes underlying neuronal function remain poorly understood.
A low cost, flexible, reliable system was developed for the
nonlinear imaging of the samples in this study. We present a
detailed mapping of the nematode C. elegans in its anterior
and posterior body parts using TPEF and SHG scanning images. Mutants, which express GFP in the pharyngeal muscle
cells, have been imaged using both phenomena. Additionally,
animals that express GFP in the cytoplasm of the six mechanoreceptor neurons, as well as animals expressing GFP bound
to the membrane of these cells, have been investigated. We
focused our research on the posterior part of the nematode,
where two of these six neurons are located, and we ascertained that the SHG signal level, arising from the structural
protein arrays, is also very significant in this region of the
worm.
2 Experimental Apparatus
We used a femtosecond t-pulse laser ~high power femtosecond oscillator from Amplitude Systems! as an excitation
source, in our experiments. This source is a compact diodepumped femtosecond laser oscillator, which delivers a train of
high energy, short duration pulses. The laser material is an
ytterbium doped crystal. Ytterbium belongs to the rare earths
family, and has strong absorption bands in the near-infrared
~940–980 nm depending on the host matrix!. The small size
of the laser permits the whole setup to be extremely flexible.
The average power of the laser was 1 W, the pulse duration
Journal of Biomedical Optics
less than 200 fs and the repetition rate 50 MHz. The laser
emission wavelength was at 1028 nm in order to maintain a
high efficiency on the excitation of GFP molecules. The beam
was directed to a modified optical microscope ~Nikon Eclipse
ME600D! using a suitable pair of mirrors and was focused
tightly onto the sample by an objective lens of high numerical
aperture ~Nikon 50X N.A. 0.8!. The average laser power on
the specimen was 10 mW. A CCD camera ~Sony XC-57CE!
was used for observation of the sample through the objective
and suitable optics. Both an ultrafast laser and tight focusing
are necessary for the realization of the high intensities required for nonlinear phenomena such as SHG and TPEF. Biological samples were placed on standard coverslips that fit
into a motorized xyz translation stage ~Standa 8MT167-100!.
The minimum step of the stages in each direction is 1 mm.
The choice of a motorized stage represents an inexpensive
and reliable solution for the realization of the scanning procedure. Its main advantage is the low cost in comparison with
expensive commercial galvano-mirrors. It is feasible to perform reliable and precise imaging of biological samples and
in vivo measurements in real time by using this inexpensive
scanning configuration.
TPEF signals were collected using a photomultiplier tube
~PMT Hamamatsu R4220! connected to a lock-in amplifier
~SR810 Stanford Research Systems!. The photomultiplier
tube was attached at the position of the eyepiece of the microscope. A short pass filter ~SPF 650 nm CVI! was placed at
the photomultiplier input in order to cut off the reflected laser
light. By using the Labview program ~National Instruments,
Labview 6.1!, which was developed specifically for this application, we were able to control the movement of the step
motors and to record the signals in every step during the scanning procedure. The average accumulation time in every step
was 30 ms.
Since SHG is a coherent process, most of the signal is
transmitted towards the direction of the fundamental beam.
For thin samples ~such as C. elegans! almost the entire signal
propagates with the laser and was collected and collimated by
the condenser lens ~Nikon N.A. 0.9!. The condenser lens was
properly aligned below the xyz motorized stage. The numerical aperture of the condenser lens must be equal or higher
when compared to the objective lens in order to collect the
whole cone of light. A dichroic mirror ~99% at 45 deg, 450–
550 nm! was used to reflect the transmitted beam. A monochromator ~Digikrom CM110 CVI! was employed in order to
distinguish the SH from the two-photon fluorescence signal
and to provide spectral information. The resolution of the
monochromator was 1 nm. This resolution ~1 nm! was necessary since we want to achieve the best separation between
SHG and TPEF signals in the forward detection scheme. The
detected signals in the forward direction were in sufficiently
high intensities, despite the very thin spectral range under
investigation. A filter ~SPF 700 nm CVI! was used in front of
the monochromator to cut off the residual fundamental laser
light. By using this configuration we were able to record SHG
and TPEF signals in distinct sets of measurements by tuning
the monochromator in different spectral regions. For the detection of the signals a photomultiplier tube ~PMT
Hamamatsu R636-10! connected to the lock-in amplifier was
used. The resolution of our experimental setup is 1 mm, lim-
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Fig. 1 Two-photon excitation fluorescence image from the posterior
part of a C. elegans.
ited by the beam waist of the objective lens and the minimum
step of the xyz translation stage.
3 Sample Preparation
3.1 Nematode Strains and Growth
We followed standard procedures for C. elegans strain maintenance, crosses and other genetic manipulations.46 Nematode
rearing temperature was kept at 20 °C. Before each experiment, young adult animals were anaesthetized by immersing
to 0.5 M of sodium azide ( NaN3 ) , and were subsequently
mounted on glass slides.
4 Results
The target of this study is the development of an inexpensive,
compact, and reliable experimental apparatus for the detailed
imaging and mapping of C. elegans neurons and other structural components, by performing second-harmonic generation
~SHG! and two-photon excitation fluorescence ~TPEF! measurements. The use of an infrared wavelength ~1028 nm! as an
excitation source provides negligible photobleaching and phototoxication to the sample due to the low energy per photon.
The nonlinear nature of the recorded phenomena insures that
the effect will be confined only to the focal region, thus dramatically improving the spatial resolution by minimizing outof-focus phenomena such as fluorescence. So, there is no need
to perform confocal microscopy measurements when nonlinear phenomena such as SHG and TPEF are detected.
Figure 1 depicts the TPEF image from the posterior end of
C. elegans. The signal was captured from above. In this transgenic line, GFP is expressed under the control of the mec-4
promoter in the six mechanoreceptor neurons of the animal.
For the realization of the measurements, due to the limited
quantity of GFP molecules onto the sample, an objective lens
with high numerical aperture ~100X NA 1.25 oil immersion!
was employed for tight focusing. In Fig. 1 the contour of the
worm, the region of intestine, and a neuronal cell can be
clearly seen. The dimensions of the scanning region were
30370 mm2. Two of the six mechanoreceptor neurons are
located in the posterior part ~tail! of the worm usually in difJournal of Biomedical Optics
ferent z positions. The scanning was performed in a specific z
position where the TPEF signal arising from one neuron was
maximum.
The autofluorescence intensity in the tail of C. elegans is
weaker than the TPEF from GFP molecules except from the
region of the gut. The neurons we are imaging in the tail of C.
elegans ~PLML—PLMR! do not overlap with the gut and are
situated more posteriorly. Their somata are situated close to
the tip of the tail away from the gut autofluorescence. The
dimensions of the body and the neuronal axon of a C. elegans
mechanoreceptor neuron are 2 mm and 200 nm respectively.
The dimensions of the region where the TPEF signal from
GFP molecules were recorded are similar ~Fig. 1!. The limitation to the spatial resolution of our setup due to the minimum step of the scanning stage ~1 mm! must also be taken
into account. The high intensity of the signal, the position, and
the dimensions of this region in the tail of the worm, as well
as the reproducibility of the images which were obtained by
performing TPEF imaging to similar specimens, lead us to the
conclusion that the recorded signals came from the GFP molecules which are expressed in fusion with the MEC-4 protein
in the mechanoreceptor neuron of the worm.
Endogenous structural proteins of the worm, such as collagen, are responsible for the detection of the weak autofluorescence signal arising from the contour of C. elegans. On the
other hand, the main contribution in the high signal, which
were recorded from the intestine, comes from the lipid inclusions. Thus, by performing TPEF imaging measurements in
C. elegans, unique and reliable information can be extracted
about the structure and the morphology of specific cell types
in the worm.
Figure 2 depicts the spectral distribution of the recorded
signal from the pharynx of the worm. GFP is expressed in the
pharyngeal muscle cells of C. elegans. The signal was collected from below. There is a main peak at 514 nm, which
abruptly reduces as the monochromator setting was changed
by 5– 6 nm around this wavelength. This observation is in
perfect agreement with the spectral distribution, which presents the nonlinear phenomenon of second-harmonic generation. Furthermore, the signal appears at the expected spectral
bandwidth of ;4 nm full width at half maximum ~the laser
fundamental has a FWHM of ;6 nm!. For a Gaussian profile,
the bandwidth of the SHG signal scales as a square root ~1/&!
of the fundamental bandwidth. The inset of Fig. 2 shows the
magnification of the same spectral distribution. The collected
signal exclusively comes from TPEF for l.518 nm. As was
expected, the spectrum of two-photon excitation fluorescence
does not present any abrupt reduction over a few nanometers.
Due to the excitation wavelength ~1028 nm!, the signal at 514
nm comprises both SHG and TPEF. The independently measured SHG signal is at least 30 times stronger than the TPEF
counterpart. Consequently, the dominant contribution at 514
nm comes from SHG signals.
Figure 3~a! represents a TPEF image of the anterior part of
C. elegans. The signal was recorded from below. The monochromator was tuned at 525 nm, so the detection of the signal
was performed in a spectral region where the contribution of
SHG had been excluded. The GFP molecules are expressed in
the cytoplasm of the pharyngeal muscle cells of the worm.
The dimensions of the scanning region were 34366 mm2. The
scanning was obtained in a specific z position where the col-
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Fig. 2 Spectral distribution of the collected signal from a pharynx of a C. elegans.
lected TPEF signal that comes from the pharynx became
maximum. The signal from TPEF originated from the inner
part of the pharynx, where the GFP molecules were located.
The shape and the morphology of the pharynx of the worm
can be observed with satisfactory resolution.
Figure 3~b! shows the same scanning of C. elegans but in
this case the monochromator was tuned at 514 nm. Therefore,
the SHG is the dominant factor in the collected signals. As
already mentioned in the introduction, structures of wellordered protein assemblies, such as collagen and actomyosin
complexes, are efficient SHG sources. We hypothesize that
these endogenous structural protein arrays are the main contributors to the recorded signals based on other studies.5,15,19
In addition, the SHG image @Fig. 3~b!# shows clearly the body
wall muscles and the pharyngeal muscles, indicating that the
detected SHG signal originates mainly from actomyosin complexes and collagen. In Fig. 3~b! the structures of high SHG
intensity in the middle of the body appear to have a thickness
of 3– 4 mm, which corresponds to the real thickness of the
pharyngeal muscles of the worm. This is an indication that the
actomyosin complexes, which mainly form the sarcomeres,
are the main contributors to the SHG recorded signals from
Fig. 3 Two-photon excitation fluorescence image (a) and second-harmonic generation image (b) respectively. Images were recorded from the
forward part of a C. elegans.
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Fig. 4 Two-photon excitation fluorescence image (a) and second-harmonic generation image (b) respectively. Images were obtained from the
posterior part of a C. elegans.
this region. Moreover, the structures of high SHG intensity on
the body edges of the worm @Fig. 3~b!# appear to have a
thickness of 2–3 mm, which corresponds, to the real thickness
of hypodermis of C. elegans. Consequently, collagen, which
is one of the basic ingredients of the hypodermis, and actomyosin, seem to be the main components that contribute in
the detection of the SHG signal from the outline of the nematode. In the strain we used in this experiment the GFP molecules are expressed under the control of the myo-2 promoter.
This promoter is tissue specific and the GFP expression is
limited to the cytoplasm of the pharyngeal muscle cells.
Therefore, the GFP molecules, due to their random orientation
in the pharynx region do not contribute to the SHG signal. It
is worth mentioning that the obtained SHG images are similar
with images from other very recent studies.15 In our work the
use of 1028 nm as the excitation source, instead of a typical
wavelength around the 800 nm, was chosen in order to reduce
more of the photodamage effects onto the specimens, due to
the lower power per photon.
By using TPEF imaging we were able to detect the inner
part of the pharynx due to excitation of the GFP molecules in
pharyngeal muscles. Additionally, it is feasible to image the
pharynx and the outline of the worm by performing SHG
measurements. Thus the two images @TPEF Fig. 3~a!, SHG
Fig. 3~b!# provide complementary information about the biological sample. This is due to the fact that the induced signals
come from different components. The endogenous structural
proteins, especially actomyosin complexes and collagen, are
responsible for the observation of SHG signals. On the other
hand, the diffused GFP molecules are the main contributors
for the detection of TPEF signals.
We investigated the feasibility of detecting different cell
types of C. elegans by performing similar measurements with
two out of the six touch receptor neurons in the posterior end
of the worm. In these experiments, GFP was expressed under
the mec-4 touch-cell specific promoter at high levels and was
localized in the cytoplasm of these neurons. Such high GFP
levels result in significantly enhanced recorded TPEF signals,
Journal of Biomedical Optics
since the detection was performed in a spectral region with
bandwidth of 1 nm. Figure 4~a! depicts a TPEF image from
the posterior part of a sample. The signal was recorded from
below. The monochromator was tuned at 525 nm. The dimensions of the scanning region were 30330 mm2. The scanning
was performed in a specific z position where the TPEF signal
emanates from one neuron was maximum. The recorded signal comes from the cytoplasmic GFP molecules, which are
expressed in the six neuronal cells of the worm. It is obvious
from the collected image @Fig. 4~a!# that in a specific z position, it is possible to detect the precise localization of one of
the two touch receptor neurons which are lying near the tail of
the C. elegans.
Figure 4~b! presents the same scanning of the sample but
in this case the monochromator was tuned at 514 nm ~SHG
measurements!. It is expected that in the posterior part of the
C. elegans the muscle abundance is limited. However, as is
shown in Fig. 4~b!, the SHG enabled us to detect the contour
of the tail. The endogenous structural proteins, especially the
collagen, are the main contributors to the recorded SHG signals. The GFP molecules in the neuronal cell are symmetrically distributed, so the contribution to the observed signal of
SHG can be excluded. This is in good agreement with the
experimental data, since it was not feasible to detect the neuronal cell, as depicted in Fig. 4~b!. The same kind of measurements ~at 514 nm! was performed at various z positions. In all
of these images, it was impossible to locate the neurons ~images are not presented!. From the above mentioned observations, it is once again clear that the two types of images ~SHG
versus TPEF! could provide complementary information. By
obtaining TPEF images in a specific z position we are able to
localize one of the two mechanoreceptor neuronal cells which
are lying in the posterior part of the worm due to the contribution of GFP molecules to the collected signal. By performing the same SHG imaging we obtain information concerning
the cuticle, the contour, and the muscles of the worm due to
the contribution of the structural protein arrays to the recorded
signal.
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5 Conclusions
The combination of SHG and TPEF high-resolution imaging
is a new, very promising technique which is expected to be a
useful and unique tool in various fields of medicine and biology, since it can provide a detailed picture of tissue ~especially the SHG imaging!. However, the main inhibitory reason
for the extended application of this innovative technique to
the biological community is that the commercial two-photon
microscopes are very expensive. In the present study a compact, reliable, flexible, inexpensive ~except for the laser component! experimental apparatus has been developed. A properly modified, common, inexpensive microscope was
employed. A low-cost motorized xyz translation stage was
used for the scanning procedure. The time interval for the
realization of the in vivo measurements in C. elegans was
more than two hours during our experiments. Consequently,
stage scanning was appropriate for obtaining a sufficient number of detailed in vivo images of the specific biological specimens. Moreover, this inexpensive experimental setup is preferable for single point measurements onto the neurons of the
worm, in order to monitor the membrane potential via alterations in SHG signals. These measurements that comprise our
potential main future target could provide valuable and unique
information for the transduction of mechanical signals in the
mechanotranducer neuronal cells of C. elegans. Thus, we developed a system that can be easily used for a variety of
experiments in the field of biology.
By using this system, it is feasible to collect both SHG and
TPEF signals in distinct sets of measurements or simultaneously by detecting SHG images from below and TPEF images from above. We obtained detailed images of the C. elegans body by recording nonlinear phenomena at the
microscopic level. Reliable and valuable information in real
time concerning the structure and the morphology of the C.
elegans were obtained by using this inexpensive prototype
system.
Acknowledgments
7.
8.
9.
10.
11.
12.
13.
14.
15.
16.
17.
18.
19.
20.
21.
This work was supported by the UV Laser Facility operating
at the Foundation of Research and Technology Hellas
~FORTH! under the European Commission ‘‘Improving Human Research Potential’’ program ~HPRI-CT-2001-00139!, by
the Integrated Project ‘‘Molecular Imaging’’ ~LSHG-CT2003-503259!, and by the European Molecular Biology Organization ~EMBO!. N. Tavernakis is an EMBO Young Investigator. We thank G. Vasilakis for his valuable help during the
early experiments.
References
22.
23.
24.
25.
26.
1. W. Denk, J. H. Strickler, and W. W. Webb, ‘‘Two-photon laser scanning fluorescence microscopy,’’ Science 248, 73 ~1990!.
2. S. Maiti, R. M. Williams, J. B. Shear, W. R. Zipfel, and W. W. Webb,
‘‘Measuring serotonin distribution in live cells with three-photon excitation,’’ Science 24, 530–532 ~1997!.
3. C. Xu, W. Zipfel, J. B. Shear, R. M. Williams, and W. W. Webb,
‘‘Multiphoton fluorescence excitation: new spectral windows for biological nonlinear microscopy,’’ Proc. Natl. Acad. Sci. U.S.A. 93,
10763–10768 ~1996!.
4. L. Moreaux, O. Sandre, M. Blanchard-Desce, and J. Mertz, ‘‘Membrane imaging by simultaneous second-harmonic and two-photon microscopy,’’ Opt. Lett. 25, 320–322 ~2000!.
5. W. Mohler, A. C. Millard, and P. J. Campagnola, ‘‘Second harmonic
Journal of Biomedical Optics
6.
27.
28.
29.
30.
024015-7
generation imaging of endogenous structural proteins,’’ Methods 29,
97–109 ~2003!.
N. Bloembergen, Nonlinear Optics, World Scientific, Singapore
~1965!.
G. Cox and C. J. R. Sheppard, ‘‘Practical limits of resolution in
confocal and non-linear microscopy,’’ Microsc. Res. Tech. 63, 18 –22
~2004!.
T. F. Heinz, C. K. Chen, D. Richard, and Y. R. Shen, ‘‘Spectroscopy
of molecular monolayers by resonant second-harmonic generation,’’
Phys. Rev. Lett. 48, 478 – 481 ~1982!.
L. Moreaux, O. Sandre, and J. Mertz, ‘‘Membrane imaging by
second-harmonic generation microscopy,’’ J. Opt. Soc. Am. B 17,
1685–1694 ~2000!.
J. Mertz and L. Moreaux, ‘‘Second-harmonic generation by focused
excitation of inhomogeneously distributed scatterers,’’ Opt. Commun.
196, 325–330 ~2001!.
A. T. Yeh, N. Nassif, A. Zoumi, and B. J. Tromberg, ‘‘Selective
corneal imaging combined second-harmonic generation and twophoton excited fluorescence,’’ Opt. Lett. 27, 2082–2084 ~2002!.
A. Zoumi, A. Yeh, and B. J. Tromberg, ‘‘Imaging cells and extracellular matrix in vivo by using second-harmonic generation and twophoton excited fluorescence,’’ Proc. Natl. Acad. Sci. U.S.A. 99,
11014 –11019 ~2002!.
E. Brown, T. McKee, E. diTomaso, A. Pluen, B. Seed, Y. Boucher,
and R. K. Jain, ‘‘Dynamic imaging of collagen and its modulation in
tumors in vivo using second-harmonic generation,’’ Nat. Med. 9,
796 – 800 ~2003!.
L. Moreaux, O. Sandre, S. Charpak, M. Blanchard-Desce, and J.
Mertz, ‘‘Coherent scattering in multi-harmonic light microscopy,’’
Biophys. J. 80, 1568 –1574 ~2001!.
P. J. Campagnola and L. M. Loew, ‘‘Second-harmonic imaging microscopy for visualizing biomolecular arrays in cells, tissues and organisms,’’ Nat. Biotechnol. 21, 1356 –1360 ~2003!.
D. A. Kleinman, ‘‘Nonlinear dielectric polarization in optical media,’’
Phys. Rev. 126, 1977–1979 ~1962!.
R. Hellwarth and P. Christensen, ‘‘Nonlinear optical microscopic examination of structure in polycrystalline ZnSe,’’ Opt. Commun. 12,
318 –322 ~1974!.
I. Freund, M. Deutsch, and A. Sprecher, ‘‘Connective tissue polarity,’’
Biophys. J. 50, 693–712 ~1986!.
P. J. Campagnola, A. C. Millard, M. Terasaki, P. E. Hoppe, C. J.
Malone, and W. A. Mohler, ‘‘Three-dimensional high-resolution
second-harmonic generation imaging of endogenous structural proteins in biological tissues,’’ Biophys. J. 81, 493–508 ~2002!.
P. Stoller, P. M. Celliers, K. M. Reiser, and A. M. Rubenchik, ‘‘Quantitative second-harmonic generation microscopy in collagen,’’ Appl.
Opt. 42, 5209–5219 ~2003!.
G. Cox, E. Kable, A. Jones, I. Fraser, F. Manconi, and M. D. Gorrell,
‘‘3-dimensional imaging of collagen using second harmonic generation,’’ J. Struct. Biol. 141, 53– 62 ~2003!.
Y. Guo, P. P. Ho, H. Savage, D. Harris, P. Sacks, S. Schantz, F. Liu,
N. Zhadin, and R. R. Alfano, ‘‘Second-harmonic tomography of tissues,’’ Opt. Lett. 22, 1323–1325 ~1997!.
E. Brown, T. McKee, E. di Tomaso, A. Pluen, B. Seed, Y. Boucher,
and R. K. Jain, ‘‘Dynamic imaging of collagen and its modulation in
tumors in vivo using second-harmonic generation,’’ Nat. Med. 9,
796 – 801 ~2003!.
S. Roth and I. Freund, ‘‘Second-harmonic generation in collagen,’’ J.
Chem. Phys. 70, 1637–1643 ~1979!.
P. J. Campagnola, H. A. Clark, W. A. Mohler, A. Lewis, and L. M.
Loew, ‘‘Second-harmonic imaging microscopy of living cells,’’ J.
Biomed. Opt. 6, 277–286 ~2001!.
S. Roth and I. Freund, ‘‘Optical second-harmonic scattering in rat-tail
tendon,’’ Biopolymers 20, 1271–1290 ~1981!.
E. Georgiou, T. Theodossiou, V. Hovhannisva, K. Politopoulos, G. S.
Rapti, and D. Yova, ‘‘Second and third optical harmonic generation in
type I collagen, by nanosecond laser irradiation, over a broad spectral
region,’’ Opt. Commun. 176, 253–260 ~2000!.
O. Bouevitch, A. Lewis, I. Pinevsky, J. P. Wuskell, and L. M. Loew,
‘‘Probing membrane potential with nonlinear optics,’’ Biophys. J. 65,
672– 679 ~1993!.
I. Ben-Oren, G. Peleg, A. Lewis, B. Minke, and L. Loew, ‘‘Infrared
nonlinear optical measurements of membrane potential in photoreceptor cells,’’ Biophys. J. 71, 1616 –1620 ~1996!.
G. Peleg, A. Lewis, M. Linial, and L. M. Loew, ‘‘Nonlinear optical
measurement of membrane potential around single molecules at seMarch/April 2005
d
Vol. 10(2)
Filippidis et al.
31.
32.
33.
34.
35.
36.
37.
lected cellular sites,’’ Proc. Natl. Acad. Sci. U.S.A. 96, 6700– 6704
~1999!.
P. J. Campagnola, M. de Wei, A. Lewis, and L. M. Loew, ‘‘Highresolution nonlinear optical imaging of live cells by second harmonic
generation,’’ Biophys. J. 77, 3341–3349 ~1999!.
A. Lewis, A. Khatchatouriants, M. Treinin, M. Sheves, G. Peleg, Z.
Chen, O. Bouevitch, Z. Rothman, and L. Loew, ‘‘Second harmonic
generation of biological interfaces: probing membrane proteins and
imaging membrane potential around single molecules,’’ Chem. Phys.
245, 133–143 ~1999!.
L. Moreaux, T. Pons, V. Dambrin, M. Blanchard-Desce, and J. Mertz,
‘‘Electro-optic response of second-harmonic generation membrane
potential sensors,’’ Opt. Lett. 28, 625– 627 ~2003!.
A. C. Millard, L. Jin, A. Lewis, and L. M. Loew, ‘‘Direct measurement of the voltage sensitivity of second-harmonic generation from a
membrane dye in patch-clamped cells,’’ Opt. Lett. 28, 1221–1223
~2003!.
T. Pons, L. Moreaux, O. Mongin, M. Blanchard-Desce, and J. Mertz,
‘‘Mechanisms of membrane potential sensing with second-harmonic
generation microscopy,’’ J. Biomed. Opt. 8, 428 – 431 ~2003!.
J. Y. Huang, Z. Chen, and A. Lewis, ‘‘Second harmonic generation in
purple membrane-poly~vinyl alcohol! films: probing the dipolar characteristics of the bacteriorhodopsin chromophore in bR570 and
M412,’’ J. Phys. C 93, 3314 –3323 ~1989!.
A. Khatchatouriants, A. Lewis, Z. Rothman, L. Loew, and M. Treinin,
‘‘GFP is a selective non-linear optical sensor of electrophysiological
processes in Caenorhabditis elegans,’’ Biophys. J. 79, 2345–2352
~2000!.
Journal of Biomedical Optics
View publication stats
38. J. E. Sulston, E. Schierenberg, J. G. White, and J. N. Thomson, ‘‘The
embryonic cell lineage of the nematode Caenorhabditis elegans,’’
Dev. Biol. 100, 64 –119 ~1983!.
39. The C. elegans Sequencing Consortium, ‘‘Genome sequence of the
nematode C. elegans: a platform for investigating biology,’’ Science
282, 2012–2018 ~1998!.
40. A. Fire, S. Xu, M. K. Montgomery, S. A. Kostas, S. E. Driver, and C.
Mello, ‘‘Potent and specific genetic interference by double-stranded
RNA in Caenorhabditis elegans,’’ Nature (London) 391, 806 – 811
~1998!.
41. C. C. Mello, J. M. Kramer, D. Stinchcomb, and V. Ambros, ‘‘Efficient
gene transfer in C. elegans: extrachromosomal maintenance and integration of transforming sequences,’’ EMBO J. 10, 3959–3970
~1991!.
42. J. G. White, E. Southgate, J. N. Thomson, and S. Brenner, ‘‘The
structure of the nervous system of Caenorhabditis elegans,’’ Proc. R.
Soc. London, Ser. B 314, 1–340 ~1996!.
43. C. I. Bargmann, ‘‘Neurobiology of the Caenorhabditis elegans genome,’’ Science 282, 2028 –2033 ~1998!.
44. C. I. Bargmann and J. M. Kaplan, ‘‘Signal transduction in the Caenorhabditis elegans nervous system,’’ Annu. Rev. Neurosci. 21, 279–
308 ~1998!.
45. C. H. Rankin, ‘‘From gene to identified neuron to behaviour in Caenorhabditis elegans,’’ Nat. Rev. Genet. 3, 622– 630 ~2002!.
46. S. Brenner, ‘‘The genetics of Caenorhabditis elegans,’’ Genetics 77,
71–94 ~1974!.
024015-8
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d
Vol. 10(2)