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Journal of Microscopy, Vol. 248, Pt 3 2012, pp. 234–244
doi: 10.1111/j.1365-2818.2012.03668.x
Received 4 April 2012; accepted 28 August 2012
Second harmonic generation imaging of the deep shade plant
Selaginella erythropus using multifunctional two-photon
laser scanning microscopy
A . H . R E S H A K ∗, † & C . - R . S H E U E ‡
∗ School of Complex Systems, FFPW, CENAKVA, University of South Bohemia in CB, Nove Hrady
37333, Czech Republic
†School of Material Engineering, Malaysia University of Perlis, P.O Box 77, d/a Pejabat Pos Besar,
01007 Kangar, Perlis, Malaysia
‡Department of Life Sciences, National Chung Hsing University, 250, Kuo Kuang Rd, Taichung
402, Taiwan
Key words. Chloroplast, multifunctional two-photon laser scanning
microscopy (MF-2PLSM), second harmonic generation (SHG), Selaginella
erythropus, two-photon excitation fluorescence (TPEF).
Summary
Background: Multifunctional two-photon laser scanning
microscopy provides attractive advantages over conventional
two-photon laser scanning microscopy. For the first
time, simultaneous measurement of the second harmonic
generation (SHG) signals in the forward and backward
directions and two photon excitation fluorescence were
achieved from the deep shade plant Selaginella erythropus.
Results: These measurements show that the S. erythropus
leaves produce high SHG signals in both directions and
the SHG signals strongly depend on the laser’s status of
polarization and the orientation of the dipole moment in
the molecules that interact with the laser light. The novelty
of this work is (1) uncovering the unusual structure of
S. erythropus leaves, including diverse chloroplasts, various
cell types and micromophology, which are consistent with
observations from general electron microscopy; and (2) using
the multifunctional two-photon laser scanning microscopy
by combining three platforms of laser scanning microscopy,
fluorescence microscopy, harmonic generation microscopy
and polarizing microscopy for detecting the SHG signals in
the forward and backward directions, as well as two photon
excitation fluorescence.
Conclusions: With the multifunctional two-photon laser
scanning microscopy, one can use noninvasive SHG imaging
to reveal the true architecture of the sample, without
photodamage or photobleaching, by utilizing the fact that the
Correspondence to: A. H. Reshak, Tel: +420 777729583; fax: +420–386 361255;
e-mail: [email protected]; and C. R. Sheue, Tel/fax: +886 422857395; e-mail:
[email protected]
SHG is known to leave no energy deposition on the interacting
matter because of the SHG virtual energy conservation
characteristic.
Introduction
Nonlinear optical effects, such as two-photon (Denk et al.,
1990) and three-photon (Wokosin et al., 1996; Maiti et al.,
1997; Schrader et al., 1997; Tuer et al., 2008) fluorescence,
significantly improve depth resolution and reduce the
background noise. Nonlinear optical techniques have been
used to develop a new generation of optical microscopes with
novel capabilities. These new capabilities include the ability
to use near-infrared light to induce absorption and enhance
fluorescence from fluorophores that absorb in the ultraviolet
region. Other capabilities of nonlinear microscopes include
improving spatial and temporal resolution without the use of
pinholes or slits for spatial filtering, improving signal strength
for deeper penetration into thick and highly scattering tissue
and confining photobleaching to the focal volume (Denk
et al., 1990). The invention of nonlinear laser microscopy
has opened new opportunities for noninvasive examination of
the structure and functioning of living cells and tissues (Denk
et al., 1990).
Among different multiphoton implementations (Zumbusch
et al., 1999; Zipfel et al., 2003), second harmonic generation
(SHG) imaging (Roth & Freund, 1980; Freund et al., 1986;
Campagnola et al., 2001; Yeh et al., 2002; Campagnola
& Lowe, 2003; Cox et al., 2003) is particularly suitable
for investigating noncentrosymmetric structures. SHG is a
nonlinear optical process that occurs only at the focal point of
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a laser beam (Shen, 1989). The application of SHG imaging
of cellular structure and functioning is quite new and notable
(Campagnola & Loew, 2003). Advances in mode-locked lasers
[instead of a continuous wave, mode-locked lasers, which emit
short pulses in the range of nanoseconds to femtoseconds (fs)]
makes SHG imaging of cells possible, because lower intensities
can be used (Reshak et al., 2009). Using chiral chromophores,
chiral SHG imaging can be applied to otherwise impossible
symmetric structures (Yan et al., 2006).
Second harmonic imaging microscopy (SHIM) is based on a
nonlinear optical effect known as SHG (Barzda et al., 2004;
Barzda et al., 2005; Greenhalgh et al., 2006). SHIM has
been established as a viable microscope imaging contrast
mechanism for visualization of cell and tissue structure and
function. SHIM using SHG as a probe is shown to produce
high-resolution images of transparent biological specimens
(Campagnola & Loew, 2003). A second harmonic microscope
obtains contrasts from variations in a specimen’s ability
to generate second harmonic light from the incident light
whereas a conventional optical microscope obtains its contrast
by detecting variations in optical density, path length or
refractive index of the specimen. SHG requires intense laser
light to pass through a material with a noncentrosymmetric
molecular structure (Reshak et al., 2009). Second harmonic
light emerging from SHG material is exactly half the
wavelength (frequency doubled) of the light entering the
material (Reshak et al., 2009). The alternative technique,
two-photon-excited fluorescence (TPEF) is also a two-photon
process. TPEF involves some energy loss during relaxation
from the excited state, whereas SHG is energy conserving.
Advances in the developments of SHIM have provided
researchers with novel means by which noninvasive
visualization of nonbiological and biological specimens can be
achieved with high penetration and high spatial resolution,
and is known to leave no energy deposition on the interacting
matter because of SHIM’s virtual energy conservation
characteristic (Gao et al., 2006). That is, the emitted SHG
photon energy is the same as the total absorbed excitation
photon energy. The inhomogeneity inherent to most biological
specimen, and in particular, to the internal structure of
various cells, leads to high quality SHG images without
any preconditioning such as labelling or staining that might
induce undesirable effects in the living cell (Reshak, 2009).
Historically, resolution in fluorescence optical microscopy has
been limited by the Rayleigh criterion. The Rayleigh criterion
states that two images are just resolved when the principal
maximum (of the Fraunhofer diffraction pattern) of one image
coincides with the first minimum of the other (Born & Wolf,
1980). Techniques with better resolution than the Rayleigh
criterion have recently been established, among which is
harmonic excitation light microscopy (Frohn et al., 2000).
The novelty of this work is the application of these
techniques to reveal the structure of plant tissue. In particular,
for the first time the deep shade plant Selaginella erythropus
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Journal of Microscopy 235
is investigated by means of the multifunctional-two-photon
laser scanning microscopy (MF-2PLSM), which the first
author established by combining three platforms of laser
scanning microscopy: fluorescence microscopy, harmonic
generation microscopy and polarizing microscopy. MF2PLSM provides attractive advantages over conventional
fluorescence microscopy for revealing the true architecture
of the samples that can not produce autofluorescence without
labelling or staining, which might induce undesirable effects
in the living cell. Reconstruction of complementary images by
eliminating the angle dependence of images, when using linear
polarized laser, helps maximize the SHG signals and hence
improves the brightness and the sharpness of the features in
SHG images of samples. This technique will provide biologists
and medical researchers another useful visualization tool for
exploring the nature of living cells.
The study organism, S. erythropus, is an unusual plant
growing in the low light understory of tropical rain forests. A
giant chloroplast, termed a bizonoplast, was first discovered in
this plant (Sheue et al., 2007). The bizonoplast is characterized
by unique dimorphic ultrastructure differentiating the
chloroplast into upper and lower zones. However, the
leaves (viz. microphyll) of S. erythropus also contain typical
chloroplasts. Novel patterns of silica bodies on leaf surface
of this plant have also been observed (Sheue et al., 2006).
Baseline studies of the leaf structure of this plant from general
electron microscopy contrast with MF-2PLSM, revealing the
advantages of these new nonlinear techniques to better
understand this deep shade plant noninvasively.
Material and methods
Laser sources and imaging system
The schematic of the MF-2PLSM is shown in Figure 1. This
MF-2PLSM consists of an inverted i-mic 2 microscope (TillPhotonics, Grafelfing, Germany), equipped with Ti:sapphire
femtosecond laser with a tuning range of 690–820 nm. The
laser is a Tsunami 3941-M3B pumped by a Millennia-V,
5W solid-state pump laser (Spectra-Physics, Mountain View,
CA, USA). The Tsunami laser was used to generate linearly
polarized pulses at 810 nm, 20 mW and 100 fs pulse width
at frequency of 80 MHz, for fluorescence excitation and SHG.
Thus, in general to maximize the signal (fluorescence emission
and SHG), short pulses should be used and average laser
power should be kept low to prevent heating of the sample
as well as unwanted one-photon absorption and to reduce
the risk of highly nonlinear photodamage (Denk et al., 1995).
A beam expander was used to fill the back aperture of the
objective and λ/2 plate was used to control or maximize
the status of the laser’s polarization at the sample. The
excitation light was directed onto a pair of galvanometer
XY scanners (Yanus; Till-Photonics). The scanned excitation
light was focused onto the sample through the microscope
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objective to scan the sample in the x–y direction at the focal
plane. The stage of the microscope is driven by a computer
controlled motor to take the sample to different z positions
following each x–y scan. The scanning mirrors are metal
coated (silver) with a good thermal resistance (Diaspro, 2001).
Further components from the set-up in Figure 1 are: the
dichroic mirrors [DM-1: Q565LP for TPEF (for the materials
which produce autofluorescence) (Fig. 1b]; emission filter
EF-1: red glass 665 nm; DM-2: Omega 475DCLP for SHG
(Fig. 1c); interference blue emission filter EF-2 (405 nm;
Fig. 1d); photomultipliers: Hamamatsu R6357; objective 1:
Olympus uplanApo/IR 60×/1.20 water immersion; objective
2: Zeiss 40×/1.2W korr or Olympus uplanFLN 10×/0.3). The
Laser power was maintained to be 20 mW at the Tsunami
aperture and 5 mW at the sample. Additional infrared beam
block filters BF (HQ700SP-2p 58398) were placed in front of
each photomultiplier to ensure that illumination light was
effectively suppressed and only TPEF or SHG signals were
recorded. For SHG imaging, optical filtering is achieved with
an interference filter centred on the expected SHG frequency
(Fig. 1d) configurations of the photomultipliers were identical
for both SHG and TPEF imaging. This set-up will enable
the simultaneous measurement of SHG in the forward and
backward directions as well as TPEF (Barzda et al., 2004).
The signals from the photomultipliers are reconstructed by a
computer into images. Images were obtained in stacks stepping
along the z-axis with 0.5 μm steps. Preliminary imaging of
the sample has been performed with a scan rate of 0.25 s−1
(512 × 512 pixels) and signal-to-noise ratio is about 20
dB. The lateral resolution is about 270 nm and the axial
resolution is 973 nm using Olympus uplanApo/IR 60×/1.20
water immersion objective. The microscopy is controlled via a
standard high-end Pentium-4 PC and linked to the electronic
control system via an ultrafast interface.
Material
Fig. 1. Design of the MF-TPSLM in this study. (a) The experimental setup using two objectives for collecting the forward SHG signals (objective
Zeiss 40×/1.2 water immersion or objective Olympus uplanFLN 10×/0.3)
and the backward SHG signals (objective Olympus uplanApo/IR 60×/1.2
water immersion or objective Olympus uplanFLN 10×/0.3). The third
objective is for collecting the TPEF (objective Olympus uplanApo/IR
60×/1.2 water immersion). (b) Dichroic mirror Q565LP for TPEF. (c)
Dichroic mirror Omega 475CLP for SHG. (d) Emission filter 1 for SHG.
The material used here is S. erythropus, a deep shade plant
native to South America. This plant was originally collected
from Singapore Botanic Gardens and grown in the laboratory
in a deep shade environment. General electron microscopy
was applied to semithin sections of a leaf prepared by
standard TEM methods (Sheue et al., 2007). In addition,
a tabletop microscope (TM3000, Hitachi, Japan) gave leaf
surface images. This plant was moved to a dark location for
two weeks before the investigation of MF-2PLSM to eliminate
starch grains from the chloroplasts. To apply MF-2PLSM, a leaf
was detached with watchmakers forceps from a darkened part
of the plant. The leaf was mounted between two cover slips
in water and the edges of the smaller cover slip were sealed
to the lower larger cover slip by means of nail varnish. The
paired cover slips were placed on the stage of a Till-Photonics
microscope and illuminated with a Titanium sapphire laser
at 810 nm (linearly polarized laser), 5 mW and 100 fs pulse
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Fig. 2. Morphology, leaf structure and chloroplast ultrastructure of Selaginella erythropus. (a) Shoots showing the anisophyllous and dorsiventral structure
with two rows of small dorsal leaves and two rows of large ventral leaves on each branch of its stem. (b) A transverse section of a branch showing the
arrangement of leaves around the central stem axis with dorsal leaves (on the top) and ventral leaves (below). Here we apply the terms ‘ventral side’ to
the lower surfaces and ‘dorsal side’ to the upper surfaces of both types of leaf. (c) Silica bodies on the ventral side of a ventral leaf. (d) Ventral leaf cross
section near the vein area showing the internal leaf structure, chloroplasts and silica bodies (arrows). (e) TEM view of a giant cup-shaped chloroplast
(bizonoplast) located in a dorsal epidermal cell and characterized by a unique dimorphic ultrastructure differentiating the chloroplast into upper and
lower zones. (f) TEM view of a typical chloroplast in a ventral epidermal cell. Bp, bizonoplast; Cm, chloroplast of a mesophyll cell; Cv, chloroplast of a
ventral epidermal cell; CW, cell wall; DE, dorsal epidermal cell; DL, dorsal leaf; DLDS, dorsal leaf dorsal side; DLVS, dorsal leaf ventral side; LZ, lower zone;
M, mesophyll; Mi, mitochondrion; S, starch grain; St, stoma; UZ, upper zone; V, vein; Va, Vacuole, VE, ventral epidermal cell; VL, ventral leaf; VLDS,
ventral leaf dorsal side; VLVS, ventral leaf ventral side.
width. The objectives were aligned relative to one another and
focused on the sample. A set of images was captured.
Results
Selaginella erythropus is anisophyllous, with two rows of small
dorsal leaves and two rows of large ventral leaves on each
branch of its stem (Fig. 2a). The arrangement of these leaves is
prominently dorsiventral, with a stem located between dorsal
leaves on the top and ventral leaves beneath (Fig. 2b). Because
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Journal of Microscopy the axis (viz. stem) is in the middle and the leaf structure of both
types is basically the same relative to the vertical direction,
here we apply the terms ‘ventral side’ to the lower surfaces
and ‘dorsal side’ to the upper surfaces of both types of leaf
rather than the common terms ‘abaxial and adaxial sides’,
which with this unique foliar arrangement are not helpful.
The dorsal sides of both dorsal and ventral leaves are green
(Fig. 2b). The ventral side of the dorsal leaf, which cannot be
easily viewed from either the dorsal or ventral surface of the
shoot, is green except for a red margin, whereas the ventral side
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Fig. 3. Backward SHG signal of the ventral side of the dorsal leaf from randomly orientated linearly polarized laser. (a) Before adjusting the direction
of polarization of the linearly polarized laser. (b) After maximally orientating the direction of the linearly polarized laser to the orientation of the dipole
moment in the molecules. The backward SHG signal was collected by the objective Olympus uplanFLN 10×/0.3. The double arrow shows the direction of
the polarization of the 810 nm laser beam. The procedure in Figure 3(b) for orientating the polarization of the laser was applied also to produce Figs 4–6. V,
vein.
of the ventral leaf is deep red. Silica bodies appear as conical
protrusions from the epidermal cell walls on both surfaces of
dorsal and ventral leaves as previously reported by Sheue et al.
(2006). Silica bodies forming a single row on a single ventral
epidermal cell of ventral leaves are the most evident silica body
pattern (Fig. 2c). Stomata are aggregated in a band along the
vein on the dorsal side of dorsal leaves and the ventral side of
ventral leaves (Fig. 2d).
Dorsal and ventral leaves of S. erythropus are six cells thick
in the vein region, with leaf thickness gradually reduced
to two layers (the upper and lower epidermis) towards
the margin (Fig. 2d). The outer tangential cell wall of
ventral epidermal cells is very thick with multiple layered
ultrastructure (Figs 2d and e). Chloroplasts are found in
dorsal epidermal cells, mesophylls and ventral epidermis,
including guard cells in leaves of S. erythropus. However,
the size and number of chloroplasts vary between these
tissues (Table 1). Bizonoplasts, giant cup-shaped unique
chloroplasts with dimorphic ultrastructural organization in
a single chloroplast, are located in dorsal epidermal cells:
the upper zone is occupied by numerous layers of two to
four stacked thylakoid membranes, whereas the lower zone
contains both unstacked stromal thylakoids and thylakoid
lamellae stacked in normal grana structures oriented in
different directions (Fig. 2e). The chloroplasts in other tissues
(such as mesophyll, ventral epidermis) are normal chloroplasts
and are smaller. These observations viewed by LM, SEM and
TEM provide a substantial basis of comparison for the results
from SHG signals.
Figure 3 shows the backward direction SHG signal of the
ventral side of the dorsal leaf before (Fig. 3a) and after (Fig. 3b)
maximizing the polarization of the linearly polarized laser in
the orientation of the dipole moment in the molecules. The
orientation of the laser’s polarization is illustrated by double
arrows in Figure 3. These images were collected using the
objective Olympus uplanFLN 10×/0.3. Figure 3(a) shows the
weak signal of the SHG before the orientation of the laser’s
polarization parallel to the orientation of the dipole moment
in the molecules; then after slowly changing the polarization’s
direction of the laser beam, the SHG signals significantly
increased to reach the maximum value, as it is illustrated
by Figure 3(b).
Figures 4 and 5 show the simultaneously acquired forward
and backward SHG images of the dorsal and ventral surfaces of
a dorsal leaf. The forward SHG signal was collected with a Zeiss
40×/1.2 water immersion objective and the backward SHG
signal was collected using the objective Olympus uplanApo/IR
60×/1.2 water immersion. These figures of the forward
(Figs 4a, c and 5a, c, e) and backward images (Figs 4b, d
and 5b, d, f) are almost identical except that the backward
images usually have slightly higher contrast. Figure 4 shows
the images of the dorsal epidermal cells with a stomata band
along the middle part. The area of this stomata band is slightly
curved, leading to different focal planes under a microscope.
The outlines of the dorsal epidermal cells, stomata and guard
cells surrounding stomata can be recognized easily with SHG
signals. The bizonoplasts in dorsal epidermal cells are revealed
as much bigger than the chloroplasts in the mesophyll cells
and guard cells (Figs 4a and b). Compared to a single giant
chloroplast in a dorsal epidermal cell, there are three to five
chloroplasts per mesophyll cell and four chloroplasts per guard
cell (confirmed by confocal scanning light microscopy, data
not shown). Scanning to a deeper position of these dorsal
epidermal cells reveals numerous vacuole-like vesicles in each
cell (Figs 4c and d). Whether these signals are derived from
vacuoles or other organelles needs further investigation.
SHG images from the ventral epidermis show that its outer
tangential cell walls have very strong signals with multiple
layered dark curve patterns (Figs 5a and b). Compared to
isodiametric dorsal epidermal cells, ventral epidermal cells are
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239
Fig. 4. SHG signals of the dorsal side of the dorsal leaf (polarization as in Fig. 3b). (a, c) Forward SHG signals collected by the objective Zeiss 40×/1.2,
water immersion, these are slightly different focal planes. (b, d) Backward SHG signals collected by the objective Olympus uplanApo/IR 60×/1.2, water
immersion. These figures show giant bizonoplasts in the dorsal epidermal cells, and smaller chloroplasts in mesophyll cells and guard cells. Bp, bizonoplast;
Cg, chloroplast in a guard cell; Cm, chloroplast in a mesophyll cell; Cv, chloroplast in a ventral epidermal cell; DE, dorsal epidermal cell; St, stoma. These
figures are slightly different focal planes.
much elongated with smaller bead-like chloroplasts arranged
as chains (Figs 5c and d). There are three to five disc-shaped
chloroplasts in a mesophyll cell, with median size (Figs 5e
and f).
Because the simultaneously acquired forward and
backward SHG images are very similar (see supplementary
figures), here we show only the backward SHG images
of the dorsal and ventral surfaces of the ventral leaf
(Fig. 6). These images were collected using the objective
Olympus uplanApo/IR 60×/1.2 with water immersion. The
isodiametric dorsal epidermal cells (Fig. 6a) and oblong
mesophyll cells (Fig. 6b) shown in the top view in a ventral leaf
are similar to those observed in a dorsal leaf. Silica bodies on the
ventral side of a ventral leaf also emit strong signals (Figure 6d)
matching the results observed by SEM in Figure 2(c). The
smallest chloroplasts in the leaves of S. erythropus were
observed in the trichomes of the leaf margin near the basal part
(Figs 6c and e), note that trichomes along the leaf margin have
smaller chloroplasts (arrows) see Figure 6(c). Ventral leaves
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Journal of Microscopy (Figs 6e and f) and dorsal leaves (Figs 4 and 5) are similar in
the patterns of size and arrangements of chloroplasts in dorsal
epidermal cells, mesophyll cells and ventral epidermal cells.
Discussion
This study reveals high SHG signals in the leaves of
S. erythropus originating from micromophology, cell walls,
cell contents and chloroplasts. Various categories of size and
number of chloroplasts can be recognized from the leaves
of S. erythropus (Table 1), in strong agreement with the
observations of Sheue et al. (2007). These diverse chloroplasts
include (1) large cuplike chloroplasts, bizonoplasts, in the
dorsal epidermal cells; (2) disk-shaped chloroplasts in the
mesophyll; (3) elongated or beadlike chloroplasts arranged
as a chain in the elongated, ventral epidermal cells;
(4) trichome chloroplasts; and (5) stomatal chloroplasts. In
terms of ultrastructure, only the bizonoplasts have dimorphic
ultrastructure: the upper zone is occupied by numerous layers
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Fig. 5. SHG signals of the ventral side of the dorsal leaf (polarization as in Fig. 3b). (a, c, e) Forward SHG signals collected by the objective Zeiss 40×/1.2,
water immersion. (b, d, f) Backward SHG signals collected by the objective Olympus uplanApo/IR 60×/1.2, water immersion. (a, b) The top of ventral
epidermal cells with outer tangential cell walls and some chloroplasts. (c, d) Chloroplasts in ventral epidermal cells, beadlike, arranged in chains.
(e, f) Chloroplasts in mesophyll cells, which are larger than those in ventral epidermal cells. Cm, chloroplast in a mesophyll cell; Cv, chloroplast in a
ventral epidermal cell; CW, cell wall; M, mesophyll; VE, ventral epidermal cell.
of two to four stacked thylakoid membranes whereas the
lower zone contains both unstacked stromal thylakoids and
thylakoid lamellae stacked in normal grana. The other types
of chloroplasts in the leaves of S. erythropus are typical
chloroplasts with grana and stoma thylakoid membranes
mingled together. These features of chloroplasts observed
from S. erythropus are consistent with previous findings that
many shade plants have large chloroplasts with numerous
thylakoids per granum (Nasrulhaq-Boyce & Duckett, 1991;
Sarafis, 1998). The SHG signal is not as effective as TEM
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241
Fig. 6. Backward SHG signals of both sides of the ventral leaf collected by the objective Olympus uplanApo/IR 60×/1.2, water immersion (polarization
as in Fig. 3b). (a) Isodiametric dorsal epidermal cells. (b) Oblong mesophyll cells near leaf tip. (c) Lower magnification of the ventral side near middle
and basal parts showing abundant chloroplasts in tissues. Note that trichomes along the leaf margin have smaller chloroplasts (arrows). (d) Ventral
epidermal cells near vein area with stomata. Silica bodies can be observed on the elongated ventral epidermal cells, but not on the stomatal band along
the vein. There are four chloroplasts in each guard cell. (e) The area near the basal part of the leaf margin with trichomes (labelled with T). Chloroplasts in
mesophyll cells, ventral epidermal cells and trichomes can be distinguished by size. (f) The beadlike chloroplasts arranged as chains in ventral epidermal
cells are smaller than those in mesophyll cells. Cg, chloroplast in a guard cell; Cm, chloroplast in a mesophyll cell; Ct, chloroplast in a trichome; Cv,
chloroplast in a ventral epidermal cell; DE, dorsal epidermal cell; M, mesophyll cell; Si, silica bodies; St, stoma; T, trichome; VE, ventral epidermal cell.
in differentiating the upper zone and lower zone of a
bizonoplast, but it provides strong signals with information on
arrangement, shape and size of the five types of chloroplasts.
The chloroplasts exhibit strong birefringence with large local
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Journal of Microscopy variations, most likely originating from grana, and the stacked
regions of the thylakoid membranes (Garab et al., 2005).
The birefringence is important in fulfilling phase-matching
conditions (Boyd, 1992; Reshak et al., 2008; Reshak, 2009).
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Table 1. Chloroplast diversity in microphyll of Selaginella erythropus.
Cell type
Chloroplast no. per
cell, shape and type
Average length
(μm; N = 10)
Dorsal epidermal cell 1, cup, bizonoplast
26.7 ± 3.5
Mesophyll cell
4∼6, disc, normal chloroplast
8.1 ± 1.5
Ventral epidermal cell4∼9, beadlike, normal chloroplast 7.0 ± 1.0
Guard cell
4, oval, normal chloroplast
8.4 ± 1.1
Trichome
0∼13, disk, normal chloroplast
5.3 ± 0.6
The birefringence is the difference between the extraordinary
and ordinary refraction indices. Generally, materials show
high birefringence (a considerable anisotropy in the linear
optical susceptibility) that favours an important quantity
in second-order susceptibility (determining SHG) because of
better fulfilling of phase-matching conditions, determined by
birefringence (Reshak et al., 2008). SHG is very efficiently
generated in chloroplasts (Chu et al., 2001). Chloroplasts
in celery showed a signal in the SHG image, which did
not colocalize with the autofluorescence of the chlorophyll.
Crystalline starch in starch grains is typically organized with
the crystallites in a radial fashion, yielding a characteristic
cross image in polarized light (Clowes & Juniper, 1968).
This in turn means that SHG image will be orientation
dependent (Cox et al., 2004). The significant SHG seen in
biological materials arises from low local symmetry and the
large nonlinear coefficient typical for biological molecules and
structures (Lukins et al., 2003; Helmchen & Denk, 2005).
The chloroplasts containing starch grains (Chu et al., 2001),
which are strong sources of SHG signals. In this measurement,
the plant was kept in the dark for approximately 2 weeks
to eliminate the starch. From above, one can conclude that
the origin of the high amount of SHG signals which comes
from S. erythropus leaves is attributed to the unusually large
chloroplasts (bizonoplasts) and various categories of size and
number of chloroplasts with numerous and thick unusual
thylakoid membranes, which are very strong sources of SHG
signals. In this study, the chloroplasts in trichomes are the
smallest chloroplasts with relatively weaker SHG signals than
the other chloroplasts in this plant. This result is consistent
with the observation of trichome chloroplasts examined
by TEM (data not shown). The chloroplasts in trichomes
have limited and poorly developed thylakoid membranes.
In addition to the abundant SHG signals derived from
chloroplasts, some cell contents, silica bodies and cell walls also
displayed strong SHG signals. However, we do not know which
structure causes the curve pattern of SHG signals around
ventral epidermal cells in Figures 5(a) and (b). Further study
is needed.
As SHG was established by earlier works (Stoller et al., 2002;
Lukins et al., 2003; Reshak 2009; Reshak et al., 2009), the SHG
signal strongly depends on the laser’s status of polarization
and the orientation of the dipole moment in the molecules
that interact with the laser beam. It is therefore advantageous
to control the laser’s status of polarization, to maximize the
SHG signals.
Our results support the contention that the collecting
efficiency of SHG signals is highly dependent on the numerical
aperture of the objective (Han et al., 2005; Cox et al., 2004;
Reshak et al., 2009). Higher values of numerical aperture
with immersion medium allow increasingly oblique rays to
enter the objective front lens, by capturing higher order of
diffraction rays from the samples, producing a more highly
resolved image (Reshak et al., 2009). The strength of the SHG
signals significantly depends on the numerical aperture and
the immersion medium of the objective. Also, it is strongly
dependent on the polarization direction of the laser beam. The
sample will produce a strong SH signal when the polarization
direction of the linearly polarized laser is parallel to the
orientation of the dipole moment in the molecules.
SH imaging is especially helpful for biological studies
of living samples. Acquiring fluorescence images with
conventional microscopy leads to photobleaching and
photodamage, whereas the SH imaging process does not.
Because the SHG does not use an absorptive process, the
intense laser field induces a nonlinear polarization in the
molecules resulting in the production of coherent waves, twice
the incident frequency. Moreover the SHG image results from
a few femtoseconds, and is energy conserving process. This is
another advantage of the SH imaging when one needs to work
with sensitive samples. Thereby, one can investigate the true
architecture of the sensitive samples.
Conclusions
This is the first time the deep shade plant S. erythropus has
been investigated by means of the MF-2PLSM established
by combining three platforms of laser scanning microscopy.
The MF-2PLSM offers several advantages for uncovering the
true architecture of the sample and enables simultaneous
measurement of the SHG signals in the forward and backward
directions. The leaves of S. erythropus produce very strong
SHG signals that are attributed to various categories of
size and number of chloroplasts with numerous thylakoid
membranes. Moreover, the leaves are multilayered providing
another reason for the strong SHG signals, which accumulate
from these layers. Cell wall, cell content and big silica
bodies also provide signals. This measurement provides
noninvasive, effective and informative images similar to
paradermal sections of the leaf but without the disadvantages
of photobleaching and photodamage.
In summary, the SHG signals strongly depends on two
objects: the first object is the microscope – the laser’s status
of polarization and the numerical aperture of the objective;
and the second object is the biological materials – the
structure of the materials whether if it is homogenous or
not, or centrosymmetric or non-centrosymmetric, and the
C 2012 The Authors
C 2012 Royal Microscopical Society, 248, 234–244
Journal of Microscopy SECOND HARMONIC GENERATION IMAGING
orientation of the dipole moment in the molecules that interact
with the laser beam. This new emerging microscopy shows
high potential for the study of living samples in biological and
medical research.
Acknowledgements
We would like to thank Prof. V. Sarafis and Singapore
Botanic Gardens for providing the plants, Prof. P. Chesson
for correcting the English and two anonymous referees
for valuable comments on the manuscript. This work was
supported from the program RDI of the Czech Republic,
the project CENAKVA (No. CZ.1.05/2.1.00/01.0024), grant
No. 152/2010/Z of the Grant Agency of the University of
South Bohemia. The School of Material Engineering, Malaysia
University of Perlis, P.O Box 77, d/a Pejabat Pos Besar, 01007
Kangar, Perlis, Malaysia.
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Supporting Information
Additional Supporting Information may be found in the online
version of this paper:
As supplementary files we provide two movies and two
figures:
Movie #1: Ventral leaf ventral surface of Selaginella
erythropus.
Movie #2: This movie shows the facility of multifunctional
microscopy for simultaneously recording TPEF images from
two different channels using two different filter sets. Each set of
images was recorded in the x, y, z directions. The two sets of x, y,
z images were combined together to show the signals collected
by two different filter sets simultaneously from two channels.
Fig. S1. SHG signals of the dorsal side of the ventral leaf
after maximizing the polarization’s direction of the linearly
polarized laser to be in the same orientation of the dipole
moment in the molecules.
Fig. S2. SHG signals of the ventral side of the ventral leaf
(polarization as in Fig 3b).
Please note: Wiley-Blackwell is not responsible for the
content or functionality of any supporting materials supplied
by the authors. Any queries (other than missing material)
should be directed to the corresponding author for the paper.
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C 2012 Royal Microscopical Society, 248, 234–244
Journal of Microscopy