Emissive europium complexes that stain the cell walls of healthy plant cells, pollen tubes and roots

Article abstract of DOI:10.1039/c3ra45426f

The cell-staining behaviour of a set of five emissive europium complexes has been studied in Nicotiana tabacum BY-2 cells and pollen tubes, Nicotiana benthamiana plant leaves and in the root hairs of the wild-type plant Arabidopsis thaliana (Columbia). The cell walls were stained selectively, notably in the tobacco BY-2 cells, by the complex [EuL¹] that contains one azathiaxanthone chromophore. Internalisation only occurred in cells that had been deliberately permeabilised or were dying. No uptake was observed within healthy plant leaves. In root hairs, the cell wall was strongly stained as well as the mitochondria, revealed by time-lapsed microscopy that showed the tumbling of the mitochondria in the living tissue, confirmed by co-localisation studies. In pollen tubes, the cell wall was also stained; rapid bursting of the pollen tip occurred following incubation with [Eu.L¹], triggered by excitation with 405 nm laser light. Such behaviour is consistent with local perturbation of cell wall permeability and integrity, associated with the reactivity of the chromophore triplet excited state.

Full text of DOI:10.1039/c3ra45426f

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Emissive europium complexes that stain the cell
walls of healthy plant cells, pollen tubes and roots†
Cite this: RSC Adv., 2014, 4, 9356
Antony J. Palmer,ab Susan H. Ford,ab Stephen J. Butler,a Timothy J. Hawkins,b
b
a
a
a
*
Patrick J. Hussey, Robert Pal, James W. Walton and David Parker
The cell-staining behaviour of a set of five emissive europium complexes has been studied in Nicotiana
tabacum BY-2 cells and pollen tubes, Nicotiana benthamiana plant leaves and in the root hairs of the
wild-type plant Arabidopsis thaliana (Columbia). The cell walls were stained selectively, notably in the
tobacco BY-2 cells, by the complex [EuL1] that contains one azathiaxanthone chromophore.
Internalisation only occurred in cells that had been deliberately permeabilised or were dying. No
uptake was observed within healthy plant leaves. In root hairs, the cell wall was strongly stained as
well as the mitochondria, revealed by time-lapsed microscopy that showed the tumbling of the
mitochondria in the living tissue, confirmed by co-localisation studies. In pollen tubes, the cell wall
was also stained; rapid bursting of the pollen tip occurred following incubation with [Eu.L1],
triggered by excitation with 405 nm laser light. Such behaviour is consistent with local perturbation
of cell wall permeability and integrity, associated with the reactivity of the chromophore triplet
excited state.
Received 27th September 2013
Accepted 15th November 2013
DOI: 10.1039/c3ra45426f
www.rsc.org/advances
lived (orders of ns) endogenous uorescence and light
scattering.
Introduction
There is a range of organelles in plant cells, in common with
all eukaryotes, that form an assortment of functionally different
compartments. The large structures of the endoplasmic retic-
ulum, Golgi apparatus, and vacuoles have all been well char-
acterized with scanning electron microscopy. However, a
number of small, dynamic compartments exist, such as pre-
vacuolar compartments and early endosomes that had been
identied but could not be characterised until recently, when
specic markers for them were found.3 Transgenic tobacco BY-2
cells and Arabidopsis plants expressing GFP fusion proteins
were devised that localised to these compartments.4 These
markers have the usual drawbacks, as discussed above, but are
an established tool in studying dynamic plant structures.5
Further compartments are likely to exist that are involved in the
secretory, endocytotic and exocytotic pathways but remain
undiscovered. These will require the development of novel
luminescent markers.
Signicant developments have occurred in lanthanide
probe chemistry over the last two decades,6,7 notably in their
interactions with biosystems.8 Lanthanides are found only in
trace amounts in natural systems and normally serve no active
role in biology, meaning that there is no interference from
endogenous signals.9 The use of well-designed lanthanide
complexes as probes for spectroscopy10 or microscopy11 can be
advantageous due to their unique properties. They exhibit
long-lived emission, allowing for sensitive time-gated detec-
tion12 (well-dened, narrow peaks in the emission spectrum
Luminescence imaging in plants presents unique challenges as
staining is complicated by endogenous autouorescence and
the impermeability of the cell wall to exogenous protein-based
uorophores, labeled antibodies and many low MW probes.
The impermeability of cell walls, due to their cellulose rich
composition, provides a challenge if protein based stains, such
as labelled antibodies, or other large molecules are used.
Autouorescence arises from a variety of plant biomolecules;
the main contributor is chlorophyll, which absorbs in the blue
region of the visible spectrum with a high molar extinction
coefficient, and emits above 600 nm.
Several approaches have been developed to reduce auto-
uorescence, such as the use of methacrylate when xing
cells.1 However, this is only suitable for static, xed samples
and not for the dynamic study of living cells. Alternatively, it is
possible to use stains that do not absorb and emit in the same
region as the endogenous chromophores responsible for the
autouorescence and to use a long pass lter set, e.g. a LP450
lter, for excitation.2 The use of time-resolved microscopy
with longer-lived (orders of ms) probes offers much scope for
selective probe observation as time-gating removes short-
aDepartment of Chemistry, Durham University, South Road, Durham, DH1 3LE, UK.
E-mail: david.parker@dur.ac.uk
bSchool of Biological Sciences, Durham University, South Road, Durham, DH1 3LE, UK
† Electronic supplementary information (ESI) available. See DOI:
10.1039/c3ra45426f
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Scheme 1 Structure of the Eu complexes examined in this study.
allow for more complex analysis), and have large Stokes' shis,
of hundreds of nanometres. Several examples exhibit dynamic
responses to local conditions such as pH or bicarbonate,13
allowing the measurement of these parameters within partic-
ular organelles of mammalian cells. The cell-uptake and
localisation behaviour of several series of complexes have been
characterised, allowing structure-localisation correlations to
be made, based on the nature and linkage mode of the sensi-
tising moiety, as it is this moiety that primarily controls their
uptake (most oen by macropinocytosis), egress, and transport
behaviour.14–19
In this work, we have set out to examine the staining
behaviour of a set of ve lanthanide probes (Scheme 1),
amenable to excitation at 355 nm (Nd:YAG), 365 nm (UV-LED)
or 405 nm (diode laser), for microscopy studies in Nicotiana
tabacum BY-2 cells and pollen tubes, Nicotiana benthamiana
plant leaves and in the root hairs of the wild type plant Ara-
bidopsis thaliana. The Bright Yellow 2 (BY-2) cells have become
the most widely used plant cell culture, due to their desirable
characteristics of high growth rate and homogeneity. They are
easy to culture and maintain indenitely, relatively simple to
manipulate genetically, and offer a powerful tool in plant
molecular biology due to the ease of transformation to form
stable transgenic lines.20 A benet of BY-2 cells is that when
grown in the dark they do not produce chlorophyll, which
greatly mitigates against autouorescence. Furthermore, it is
easy to make protoplasts from a BY-2 cell culture by using a
cocktail of enzymes to degrade the cell wall, which can be
used to help a dye penetrate the cell. They are the most widely
used model plant cell line and a set of non-perturbing dyes
that can be added to the live cells to co-stain and conrm
localization is desirable, while allowing for study of dynamic
processes.
Results and discussion
Probe synthesis
The europium probes examined (Scheme 1) possess either an
azathiaxanthone sensitising moiety or an aralkynylpyridyl
moiety.21,23,24 The latter pair of complexes are very bright (B ¼
3 ꢀ f ¼ 30 mMꢁ1 cmꢁ1) and can be excited at 355 or 365 nm. It
is known that azathiaxanthones have absorption maxima that
extend to over 400 nm, making them better suited for use with
common microscopy equipment and also of potential use in
two photon excitation studies.21 Notwithstanding their longer
absorption wavelength, the azathiaxanthones possess a smaller
singlet–triplet energy gap than the azaxanthones.22 As a result,
azathiaxanthones triplet energies are in the range suitable for
sensitisation of Eu3+, although Tb3+ sensitisation is very inef-
cient, and sensitive to oxygen and temperature variation.
Europium complexes with integral azathiaxanthone sensitisers
possess dual emission: short-lived uorescence around 440 nm
(s 1.7 ns, f between 40 and 50%), accompanied by the
characteristic long-lived europium emission in the range 580 to
710 nm.22
The complex [Eu.L2b +
] has not been reported previously,
whilst the synthesis and characterisation of the other complexes
studied have been described.21,23,24 The photophysical proper-
ties of [Eu.L2b]+ (Table 1) are very similar to those of [Eu.L2a]+,
with the exception of its absorbance spectral prole. The
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Table 1 Photophysical data for [Eu.L2a +
Paper
]
and [Eu.L2b +
] (H2O, 295 K), compared to the complexes used in this study
Complex
labs/nm
3/Mꢁ1 cmꢁ1
s/ms, (D2O)
s/ms, (H2O)
q
fem/%
[Eu.L1(H2O)]3+
[Eu.L2a (H2O)]+
[Eu.L2b(H2O)]+
[Eu.L3(H2O)]ꢁ
[Eu.L4]ꢁ
384
328
368
380
330
328
12 800
5800
6200
4070
60 400
56 500
0.90
1.70
1.76
0.44
0.65
0.63
1.0
1.1
1.1
1.0
0
5.0
10
8.3
1.2
49a
50a
0.59b(0.63)
1.30
0.30b(0.29)
1.04
[Eu.L5]
1.31
1.07
0
a
b
In methanol solution. Values in parenthesis refer to pH 8, values quoted refer to pH 3.5.
absorption spectrum of [Eu.L2b]+ (Fig. 1) shows two maxima at the complex (20 min.), uptake of every dye under investigation
310 nm (3 ¼ 12 000 Mꢁ1 cmꢁ1) and 368 nm (3 ¼ 6200 Mꢁ1 was observed, with staining within the nucleus and cytoplasm.
cmꢁ1), attributed to transitions with dominant n-p* and p–p* These conditions are known to destabilize cell membranes, and
character. The emission spectra of [Eu.L2b]+ and [Eu.L2a]+were therefore as such conditions necessarily perturb cell homeostasis
measured following excitation at their absorption maxima, (Fig too much, further investigations were not conducted.
1). The form of the europium spectral emission (580 to 710 nm)
Protoplasts were made from the BY-2 cell culture, to assess
is very similar, indicating that the symmetry and coordination whether the cell wall or plasma membrane was key to blocking
environment about Eu3+ is the same in each case. This is entry. Aer the cell wall was degraded overnight, [Eu.L1] was
conrmed by the measured hydration number (q) values of 1.1 incubated with the protoplasts for 20 min and they were
for each complex. A second observation is the increased uo- imaged. Spherical cells were observed that did not stain with
rescence centred at 440 nm for the azathiaxanthone analogue. Calcouor (which binds to cellulose and so would stain the cell
The uorescence emission makes up 37% of the total observed wall if it had not been digested), signalling that the proto-
emission, with a lifetime of 1.7 ns. The signicant amount of plasting procedure was successful. The dye did not stain or
uorescence suggests that the radiative rate constant for uo- penetrate the protoplast cell membrane, although it was clearly
rescence emission is greater for the azathiaxanthone complex visible in the medium around the cell. Furthermore, the dye
compared to the azaxanthone analogue.22a
MitoTracker was still able to penetrate and label the mito-
chondria of protoplasts, suggesting that the cells were behaving
‘normally’, and were not abnormally impermeable.
Staining of tobacco BY-2 cells
Although it was obvious that the compounds were not
entering living cells, it can be seen from the images that there is a
strong signal outlining the cell. A co-localisation experiment
using [Eu.L1] and the amphiphilic styryl dye, FM4-64, (an estab-
lished membrane stain)25 was undertaken (Fig. 2). In dead or
dying cells, the europium complex also stains the shrunken
cytoplasm and also the cell wall, which remains rigidly in place.
This shows that the complexes are an effective stain for the
outline of living cells. In a co-stain with MitoTracker-Green, the
mitochondria are clearly labelled, and [Eu.L1] is able to outline
the cell and give a clear picture of each individual cell and its
shape. No time dependent (10 min–24 h) localization or change
in image brightness was observed throughout these experiments.
The amount of Eu complex internalized was assessed using
ICP-mass spectrometry, using previously reported methods.19
Following an incubation of [Eu.L1] and [Eu.L2b] (25 mM) with BY-
2 cells for 24 h, it was found that the concentration of Eu
associated with the isolated, washed cells was 200 mM and 6 mM
respectively, consistent with the much brighter images
observed with [Eu.L1]. The main difference between these two
azathiaxanthone europium complexes is that [Eu.L1(H2O)]3+ can
bind reversibly at the Eu centre to certain anions and to protein
with displacement of the coordinated water molecule. Such
processes alter the Eu emission spectral ngerprint, although
the changes with this particular complex are less signicant
than with analogous systems that lack the macrocyclic ring N-
methyl group.23 In contrast, the Eu complex of L2b has eight
Several different samples of BY-2 cells were examined and incu-
bated with concentrations of up to 100 mM of [Eu.Ln] (n ¼ 1, 2b, 3
and 5). The azathiaxanthone complexes were studied in more
detail here, as they can be excited at 405 nm on most uorescence
microscopes. Cells from three different wild type lineages (BY2-1,
BY2-2 and BY2-JI), and two transgenic cell lines (GFP-Lifeact and
GFP-TDP6) were examined. Internalisation of the probe was
examined by confocal microscopy and the only occasions that
luminescence from the lanthanide complex was observed
occurred with plasmolysed cells that were dead or in the process
of dying. On all other occasions, only the cell wall was stained
(Fig. 2). Attempts to encourage uptake of [Eu.L1] and [Eu.L3] by
deliberate permeabilisation were undertaken. Using either 2%
DMSO or 1.5% glycerol, added to the cells before incubation with
Fig. 1 Absorption and total emission spectra for [Eu.L2a +
[Eu.L2b +
(red) (295 K, water).
] (blue) and
]
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Fig. 2 Upper: Confocal microscopy images showing the selective staining of the cell wall of tobacco BY-2 cells; left: [Eu.L1] following a
30 minute incubation (red, 50 mM, lexc 355 nm; lem 605–720 nm); centre: the organic dye FM4-64 (green, 100 nM, lexc 488 nm; lem 650–750);
right RGB merge co-localised image showing correspondence (P ¼ 0.91). Lower: left: 10 min incubation (red, 50 mM [Eu.L1]) outlines the cell,
staining the cell wall selectively; right: with 10 min incubation MitoTracker Green (100 nM, lexc 488 nm; lem 505–550 nm) the location of the
mitochondria is revealed and the overlay shows that [Eu.L1] clarifies the location of the cell. Bottom: left: time-gated epifluorescence image (lexc
365/10 nm; lem LP 570 nm; tg 10 ms, tacq 5 ms, 500 acq. image), of tobacco BY-2 cells following incubation with [Eu.L1] (80 mM, 12 h), showing the
external cell wall staining; right: time-gated emission spectrum (lexc 365 (10) nm; tg 10 ms, tacq 6 ms, 1000 avg. cycle) extracted from the same
field of view showing the profile typical of the intact complex. Insert: lifetime decay (sEu ¼ 0.35 ms, lexc 365 (10) nm; lem 620/ꢂ20 nm).
ligand donor atoms and one coordinated water that is not dis- agroinltration26 and in the study of pests. Secondly, root hairs
placed in aqueous media by either anions or protein. Therefore, were examined from Arabidopsis thaliana, the most commonly
the differing extent of Eu uptake is likely to be related to these studied plant27 and an ideal candidate for live cell imaging.28
intrinsic intermolecular binding properties.6e
Aer inltration with a solution of [Eu.L1]3+ (100 mM), no
A second application of such stains could be in cell viability entry into the cells within the leaf tissue was seen, (Fig. 3), and
assays with BY-2 cells. Only dead, dying or chemically the other Eu complexes in this study behaved similarly. The only
compromised cells show luminescence within their cytoplasm, visible luminescence was in the extracellular spaces, which
and so [Eu.L1] and the other complexes under investigation highlighted the ‘mosaic’ patterning of the epidermal cells. This
could be used to identify whether a population of cells is healthy result makes sense intuitively: the leaves are not an absorption
or not. A set of healthy cells will give rise to a luminescent surface and are a vital tissue to the plant, being the site of
outline around each cell, while dead and dying cells will show photosynthesis and transpiration. They are a target for herbi-
signal throughout their cytoplasm and nucleus. Such properties vores and pests and so are adapted to be a barrier to entry.
are similar to the behaviour of common live/dead cell assays, Furthermore, fully-grown cells develop a secondary hydrophobic
e.g. using trypan blue or eosin.
cell wall containing lignin to give extra strength and water-
proong. The fact that these cells are impermeable to the Eu
complexes again raises the possibility of using them as dyes to
assess cell viability. For example, leaves may be made permeable
Studies with plant tissues
The two systems investigated were leaf tissue from Nicotiana under pathogen attack and [Eu.L1]3+ could show the extent of
benthamiana, a relative of tobacco and a model organism used for damage by revealing those cells remaining in a healthy state.
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Fig. 3 Luminescence observed by confocal microscopy (lexc 405 nm; lem 570–720 nm) from [Eu.L1]3+ infiltrated into a Nicotiana benthamiana
leaf: (a) luminescence fills the extracellular spaces without entering the epidermal cells; (b) at higher magnification, no strong luminescence is
seen apart from that associated with autofluorescence from the stoma at the bottom of the image. Stomata are responsible for the bright spots in
the other two images; (c) strong autofluorescence is seen from the trichome through the centre of the image, and the complex does not enter
the cells.
The root hairs were incubated with up to a 100 mM solution [Eu.L5] did not enter the root hair at all. On the other hand, the
of each Eu complex for 30 minutes before they were imaged. structurally similar very bright complex, [Eu.L4]ꢁ did stain the
Every complex outlined the surface of the root hairs, staining root membrane sufficiently well to allow recording of its time-
the cell walls in a similar manner to that observed with BY-2 gated image, using time-gated epiuorescence microscopy.
cells. With [Eu.L1]3+, luminescence was observed in small Furthermore, the characteristic emission spectral and lifetime
mobile organelles. These were observed in time-lapsed images prole of the complex was obtained in the roots, using time-
(see ESI† for a video) to rapidly tumble within the cytoplasm. By gated spectral imaging techniques. (Fig. 5). Such behaviour
acquiring images at the rate of one per second, it was possible to indicates that the coordination environment at Eu does not
see them being streamed through the cytoplasm towards the tip change upon internalization. The differing behaviour of the Eu
of the root hair. Such behaviour is typical of plant root mito- complexes in root uptake and staining is not readily rational-
chondria, and their high number, size and morphology match ised and further experiments will be needed to understand why
those of mitochondria and root hairs. Co-staining with Mito- the observed uptake and staining proles occur.
Tracker Green lent direct support to this hypothesis (Fig. 4).
Root hairs are known to have a high number of mobile mito-
chondria, in order to generate the ATP required for active
transport of nutrients from the soil.
Preliminary observations with pollen tubes from Nicotiana
tabacum
Luminescence was also observed from larger aggregates
within the root hair. These may be collections of immobile
mitochondria or more-general debris. Using [EuLn] (n ¼ 2b, 3)
no particular sub-cellular compartment was stained, but the
complexes were able to enter the root hairs and label large static
aggregates within the cell. The carbohydrate conjugate complex,
Pollen tubes are fast growing tissues that are very delicate and
susceptible to bursting if their external environment is per-
turbed. Pollen from Nicotiana tabacum was germinated and held
at 21 ꢃC for 2 h. At this point, the medium was changed to that
containing [Eu.L1]3+(100 mM) and returned to 21 ꢃC for a
further hour. Using 405 nm excitation (30% power), the
Fig. 4 Confocal microscopy images showing co-localisation with MitoTracker-Green (100 nM) and [Eu.L1]3+(50 mM, lexc 355 nm). Each row is a
set of images from a time-series taken 15 s apart, following a 30 min. incubation with each dye. The column in red shows Eu emission (605–720
nm); the central images show MitoTracker staining in green (lexc 488 nm, lem 505–550 nm), with the co-localisation of the two dyes in yellow on
the right (P > 0.85). Through these time-points, the small mobile mitochondria can be seen (e.g. on the left hand side) streaming through the
cytoplasm, from right to left (ESI†: a movie based in part on this data is available to view).
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Fig. 5 Upper: transmission (left) and laser scanning confocal microscopy (centre) (lexc 355 nm; lem 605–720 nm, 100 Hz, 4 avg. 1024 ꢀ 1024,
voxel size 120 ꢀ 120 ꢀ 790 nm), time-gated epifluorescence image of proximate root hairs (right) (lexc 365/10 nm; lem LP 570 nm; tg 10 ms, tacq
5
ms, 100 acq. image), of Nicotiana tabacum roots following incubation with [Eu.L4]ꢁ (10 mM, 2 h), showing the external cell wall staining; lower:
lifetime decay (sEu 1.10 ms, lexc 365 (10) nm; lem 620/20 nm) and time-gated emission spectrum (lexc 365 (10) nm; tg 10 ms, tacq 6 ms, 1000 avg.
cycle) showing the profile typical of the intact complex.
membrane of the pollen tubes was stained (Fig. 6), but the likely to burst than shorter ones. This was true both within this
pollen tubes almost immediately burst and discharged their series of experiments and as a general comparison, between
contents from the tip, oen before an image could be captured. longer and shorter tubes that had not been exposed to laser
Blank controls without the Eu complex did not burst, aer excitation. Comparing tubes that burst under the same power of
irradiation for at least 150 seconds with the 405 nm laser at the laser excitation, longer tubes burst in a shorter time, i.e.
same 10 mW power (<250 nJ/voxel). The laser power was required less excitation than the shorter tubes.
reduced to 5% but pollen tubes usually exploded too quickly for
These results are consistent with previous ndings in BY-2
a clear image to be obtained. It was noted that using other using [Eu.L1]3+, whereby excitation with 405 nm also was
wavelengths of laser irradiation (488 or 532 nm) or under white observed to lead to cell death over repeated imaging, while
light illumination, the pollen tubes did not discharge. Fixed excitation at longer wavelengths or in the absence of complex did
pollen tubes showed no uptake of the complex, and tubes not. There are a number of mechanisms that could explain this
incubated with the other two Eu complexes containing the behaviour, a priori, but the most likely explanation relates to a
azathiaxanthone chromophore did not stain the tube at all.
photochemical reaction of the excited chromophore of the Eu
A further systematic series of experiments was undertaken complex when it is localised in the membrane. The triplet excited
with [Eu.L1]3+ in order to assess its ability to stain these tubes. state of the azathiaxanthone is known to possess n-p* character,
Varying concentrations of the europium complex were used and hydrogen atom abstraction reactions have been reported in
(10 to 100 mM), and the incubation time and laser power protic media with the parent system following excitation.29
(lexc 405 nm; 1 to 30%) were also changed. Typically, the
Such a hydrogen atom abstraction from water would
concentration of [Eu.L1]3+ was 50 or 100 mM, the incubation generate a reactive hydroxyl radical that could abstract a
time was between 1 and 3 hours and the laser power varied hydrogen atom from an OH or CH group in the cellulose/pectins
between 8 and 30%. Operating within these limits did not or glycoproteins that make up the cell wall, initiating a fast
appear to affect the likelihood of a pollen tube bursting, when radical chain oxidation process that leads to an irreversible
excited with the 405 nm laser.
change in the stability and permeability of the cell wall. Longer
Out of over 40 pollen tubes for which time lapsed images tubes could be more likely to burst because they are closer to the
were captured, 81% of those incubated had burst within 3– length to which they would usually grow before reaching the
10 frames (4 image average) (i.e. 30–100 seconds of excitation). synergid cells; signals which cause the release of sperm cells
Usually when the tubes burst, the force of the ejecting cyto- may be building up, ready to be triggered by the synergid cells or
plasm moved them out of focus or eld of view, but some their environment. It could be that pressure of the cytoplasm on
sequences were captured that allowed the process to be fol- the membrane increases as the tube gets longer to keep growth
lowed (Fig. 6). It was also observed that longer tubes were more going, making the tubes more likely to burst.
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Fig. 6 Upper: images of a Nicotiana tabacum pollen tube tip immediately following incubation with [Eu.L1] (30 min, 50 mM; lexc 405 nm);
showing: left, fluorescence from the azathiaxanthone chromophore (435–565 nm); left centre: Eu emission (570–720 nm); right centre:
brightfield image; right: overlay of the three images; lower: sequential microscopy images taken at 10 second intervals showing the bursting of a
pollen tube at its tip, following incubation with [Eu.L1] (3 h, 50 mM) and 30–50 seconds after excitation with 405 nm light (10% laser power, 3.5
mW, 100 nJ/voxel).
Pollen tubes that are not growing, e.g. those that are xed, of a Eu complex bearing an azathiaxanthone chromophore.
rarely burst. This can be explained, as pollen tube growth Bursting at the pollen tip was triggered selectively by excitation
emanates from the tip; this is also the point at which they burst with 405 nm light, allowing controlled pollen release processes
and release their cytoplasm. Membrane and cell wall material is to be contemplated. A similar photochemical reaction may
delivered to the tube tip by vesicles via ‘reverse fountain’ cyto- also explain the permeabilisation of the BY-2 cell wall,
plasmic streaming followed by exocytosis. With vesicles fusing following prolonged excitation in the presence of this partic-
to the cell membrane at the tip of a growing tube, this area of ular complex.
the membrane is weaker and so may be more susceptible to
bursting. In tubes that are not growing, vesicle fusion is less
frequent, so the membrane here is as strong as in the rest of the
Experimental
tube. Tubes bursting from points other than the tip were not
Europium complexes
observed, indicating that the usual strength of the membrane is
The synthesis of the complexes [Eu.Ln] (n ¼ 1, 3, 4, 5) has been
sufficient to resist the effects caused by the presence of the
described earlier21,23,24 together with details of general instru-
excited europium complex.
mentation and chemical methods of analysis.
Conclusions and summary
The development of optical probes that function well for the
analysis of cells in plant cells, cultures and mature tissues, is
less well developed than in the animal or fungal kingdoms.
Here, we have examined the scope and utility of a small set of
emissive europium complexes in three different plant systems.
The azathiaxanthone complex, [Eu.L1]3+, that can be excited at
4-Thiophenoxy-6-methylnicotinic acid. 4-Chloro-6-methyl-
405 nm, was found to be a useful general stain for cell walls in nicotinic acid, (4.6 g, 27 mmol) and thiophenol (3.6 g, 33 mmol)
BY-2 cells, leaves and in plant root hairs. Indeed, it also serves were dissolved in dimethylformamide (50 mL). CuBr (250 mg,
as a means of distinguishing dying, plasmolysed or dead cells 1.7 mmol) and K2CO3 (5.4 g, 40 mmol) were added and the
with compromised permeability. In roots, it could also be used mixture was stirred at 150 ꢃC for 16 h under argon. Aer
to visualize the tumbling of mitochondria in the living root, in allowing the brown solution to cool to 23 ꢃC, H2O (150 mL)
real time. The combined use of time-gated microscopy and was added and excess thiophenol was extracted with Et2O (3 ꢀ
spectral imaging were exemplied in the same system with the 50 mL). The pH was reduced to 4.6 by the addition of AcOH.
bright complex, [Eu.L4]ꢁ.
Upon stirring at 0 ꢃC a pale brown precipitate formed which was
Finally, an unusual means of triggering pollen tube collected by ltration and washed with cold ether to give a pale
discharge has been identied, that requires prior localization yellow solid (1.8 g, 27%); m.p. 142–144 ꢃC, dH (CDCl3) 8.82 (1H,
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s, H6), 7.58 (5H, m, H8,9,10,11), 6.52 (1H, s, H3), 2.29 (3H, s, CH3); (C13H9ONS79Br requires 305.9508); Rf
dC (CDCl3) 166.8 (C7), 160.0 (C4), 156.9 (C5), 154.7 (C6), 149.6 CH2Cl2 : MeOH 2%).
(C8), 135.7 (C3), 130.0 (C2), 129.2 (C9), 119.5 (C10), 109.2 (C11),
22.0 (C1), m/z (HRMSꢁ) 244.0436 [M ꢁ H]ꢁ (C13H10O2NS
requires 244.0438).
¼
0.58 (silica,
2-Methyl-3-azathiaxanthone.
4-Thiophenoxy-6-methyl-
nicotinic acid, (1.80 g, 7.35 mmol) and polyphosphoric acid (50
g) were heated to 120 ꢃC for 16 h under argon. The resulting
brown solution was cooled to room temperature and poured
into ice (250 g). The pH of the yellow solution was adjusted to 7
by addition of KOH pellets. The aqueous solution was then
extracted with CHCl3 (3 ꢀ 100 mL). The organic layers were
combined, dried over MgSO4, ltered and the solvent was
removed under reduced pressure to give a brown solid, which
was puried by column chromatography on silica (CH2Cl2: 0–
5% MeOH in 0.5% increments) to yield a pale yellow solid (1.00
g, 60%), m.p. 174–176 ꢃC, dH (CDCl3) 9.54 (1H, s, H4), 8.56 (1H,
d, J 8.4 Hz, H6), 7.61 (1H, dd, J 7.0 Hz, 8.4 Hz, H8), 7.52 (1H, d, J
8.4 Hz, H9), 7.49 (1H, dd, J 7.0 Hz, 8.4 Hz, H7), 7.25 (1H, s, H1),
2.62 (3H, s, CH3); dC (CDCl3) 179.2 (C5), 160.3 (C2), 151.8 (C4),
1,7-Bis(tert-butoxycarbonyl)-4,10-bis[2-methyl-3-azathiaxan-
thone]-1,4,7,10-tetraazacyclododecane. 1,7-Bis(tert-butoxycar-
bonyl)-1,4,7,10-tetraazacyclododecane (49 mg, 0.12 mmol),
2-bromomethyl-3-azathiaxanthone,22 (106 mg, 0.35 mmol) and
K2CO3 (35 mg, 0.34 mmol) were stirred in dry CH3CN (4 mL) at
78 ꢃC for 18 h under argon. The reaction was monitored by TLC
to conrm that the brominated starting material had been
consumed. The solvent was removed under reduced pressure
and the residue dissolved in CH2Cl2 (25 mL) and extracted with
water (3 ꢀ 25 mL). The organic layer was dried over MgSO4,
ltered and solvent removed under reduced pressure. The
yellow residue was puried by column chromatography on
silica (CH2Cl2 : 5% MeOH) to give the title compound as a glassy
yellow solid (70 mg, 68%); dH (CDCl3) 9.42 (2H, s, H4), 8.55 (2H,
d, J 8.5 Hz, H6), 7.67 (2H, dd, J 7.0 Hz, 8.5 Hz, H8), 7.60 (2H, dd, J
7.0 Hz, 8.4 Hz, H7), 7.51 (2H, d, J 8.5 Hz, H9), 7.48 (2H, s, H1),
3.51 (4H, br s, Ha), 3.25 (4H, s, Hb), 3.00–2.40 (16H, br m, cyclen
Hs), 1.38 (18H, s, He), dC (CDCl3) 178.8 (C5), 172.5 (Cc) 160.1 (C2),
0
0
0
146.8 (C4 ), 136.0 (C9 ), 132.4 (C8), 130.5 (C6 ), 129.7 (C6), 127.2
0
(C7), 126.2 (C9), 121.9 (C1 ), 118.5 (C1), 24.6 (CH3), m/z (HRMS+)
228.0402 [M + H]+ (C13H10ONS requires 228.0405); Rf ¼ 0.46
(silica, CH2Cl2 : 2.5% MeOH).
0
0
0
151.8 (C4), 147.4 (C4 ), 135.6 (C9 ), 133.2 (C8), 130.2 (C6 ), 129.7
0
(C6), 127.3 (C7), 126.4 (C9), 123.1 (C1 ), 119.3 (C1), 82.7 (Cd), 58.6
(Cb), 57.2 (Ca), 51.0–49.0 (cyclen Cs), 28.1 (Ce), m/z (HRMS+)
851.3629 [M + H]+ (C46H55O6N6S2 requires 851.3624); Rf ¼ 0.58
(silica, CH2Cl2 : MeOH 2%).
2-Bromomethyl-3-azathiaxanthone. To a solution of 2-
methyl-3-azathiaxanthone (500 mg, 2.20 mmol) in carbon
tetrachloride (40 mL) was added N-bromo-succinimde (376
mg, 2.20 mmol) and dibenzoyl peroxide (20 mg, 0.08 mmol).
The mixture was stirred and irradiated by a 100 W lamp under
1
argon. The reaction was monitored by H-NMR and stopped
aer 14 h. The solvent was removed under reduced pressure
and the crude product was dissolved in CH2Cl2 (30 mL) and
washed with dilute K2CO3 solution (30 mL) to remove excess
succinimide. The organic layer was dried over MgSO4, ltered
and the solvent removed under reduced pressure. Purica-
tion by column chromatography on silica (petroleum ether
(60–80 ꢃC bp): 0–10% EtOAc in 1% increments) yielded a pale
yellow solid (160 mg, 24%), m.p. 189–192 ꢃC, dH (CDCl3) 9.59
(1H, s, H4), 8.58 (1H, d, J 8.5 Hz, H6), 7.66 (1H, dd, J 7.0 Hz, 8.5
Hz, H8), 7.57 (1H, s, H1), 7.55 (1H, d, J 8.5 Hz, H9), 7.54 (1H,
dd, J 7.0 Hz, 8.4 Hz, H7), 4.60 (2H, s, CH2Br); dC (CDCl3) 178.8
Europium(III) chloride complex of 1,7-bis(acetic acid)-4,10-
bis[2-methyl-3-azathiaxanthone]-1,4,7,10-tetraazacyclodode-
cane, [Eu.L2b]Cl. 1,7-Bis(tert-butoxycarbonyl)-4,10-bis[2-methyl-
3-azathiaxanthone]-1,4,7,10-tetraazacyclododecane (41 mg,
0.048 mmol) was dissolved in CH2Cl2 (2 mL) and CF3CO2H
(2 mL) was added. The mixture was stirred at 22 ꢃC under argon
for 16 h. Excess CF3CO2H and CH2Cl2 were removed under
reduced pressure and the yellow residue was redissolved in
CH2Cl2. The solvent was again removed under reduced pressure
0
0
(C5), 157.8 (C2), 152.1 (C4), 147.7 (C4 ), 135.7 (C9 ), 133.2 (C8),
0
0
129.7 (C6), 128.4 (C6 ), 127.4 (C7), 126.3 (C9), 123.1 (C1 ), 119.2
(C1), 32.6 (CH2Br), m/z (HRMS+) 305.9513 [M
+
H]+
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to ensure the removal of all CF3CO2H. Deprotection of the OtBu imaging. Control experiments were undertaken where water
1
groups was conrmed by H-NMR before the residue was dis- was used to inltrate the leaves, to rule out autouorescence or
solved in H2O–CH3OH (4 : 1 v/v, 4 mL). Addition of Eu(OAc)3 emission caused by the inltration procedure.
(23 mg, 0.058 mmol) was followed by adjustment of the pH to
For the root studies, Columbia wild type Arabidopsis thaliana
5.8, by the addition of aq. ammonia solution, and the mixture seeds were sterilized in 1% bleach for 10 min., washed twice in
was stirred at 60 ꢃC for 18 h. Aer allowing the solution to cool sterile water, and planted on either (a)“1/2MS10” half-strength
to room temperature, the pH was raised to 10 by the addition of Murashige and Skoog basal salt medium supplemented with
aq. ammonia solution. The solution was stirred for 1 h causing 1% sucrose and 0.8% agar or (b) “1/2MS” half-strength Mura-
excess Eu3+ to precipitate as Eu(OH)3, which was removed by shige and Skoog basal salt medium supplemented with 0.8%
syringe ltration. Adjustment of the pH to 5.8 by the addition of agar, both at pH 5.7. The plates were vernalized for 2 days at
CH3CO2H, followed by lyophilisation of the solvent, gave the
4
ꢃC, then grown in a growth cabinet at 21 ꢃC. At 5–14 days,
product as the acetate salt. The solid was converted to the seedlings were incubated with 100 mM of the relevant
chloride salt by stirring in H2O for 1 h with Dowex 1 ꢀ 8200– compounds and used for imaging. It was found that the seeds
400 mesh Clꢁ, which had been washed with 1 M HCl and on I1/2MS10O plates grew faster but were otherwise indistin-
´
¨
´
¨
neutralised with water. The Dowex resin was removed by guishable from those grown on I1/2MSO. Control experiments
ltration and the solvent lyophilised to yield the title compound were undertaken using roots incubated with H2O only to
as a yellow solid (20 mg, 47%), m/z (HRMS+) 887.1329 [M ꢁ Cl]+ understand the level of autouorescence and uorescence
(C38H36O6N6S2151Eu requires 887.1336); s(D2O) ¼ 0.56 ms, caused by the experimental procedure. For roots grown in
s(D2O) ¼ 1.57 ms, fem (water) 8.3%; tR ¼ 6.4 min.
liquid medium the following medium was used: 0.5 g Lꢁ1 MES,
0.1 g Lꢁ1 myo-inositol, 10.0 g Lꢁ1 sucrose, 0.303 g Lꢁ1 KNO3,
0.472 g Lꢁ1 Ca(NO3)2, 0.123 g Lꢁ1 MgSO4$7H2O, 0.115 g Lꢁ1
(NH4)2 HPO4, 1 mg Lꢁ1 thiamine, 0.5 mg Lꢁ1 pyridoxine–HCl,
0.5 mg Lꢁ1 nicotinic acid, with a 0.1% micronutrient mix. The
seeds were placed upon a microscope slide with an attached
cover slip lled with medium, held at an angle and their roots
allowed to grow down into the gap between the slide and the
cover slip. The dyes were added under the cover slip before
imaging.
HPLC analyses
Reverse phase HPLC analysis was performed at 298 K on a
Perkin Elmer system, comprising a Perkin Elmer Series 200
pump, Perkin Elmer Series 200 autosampler, Perkin Elmer
Series 200 UV/Vis detector and Perkin Elmer Series 200 uo-
rescence detector. Either a 150 ꢀ 4.66 mm 4 micron Phenom-
enex Synergi 4m Fusion-RP 80i column or an XBridge C-18
10 cm ꢀ 3.5 mm column was used with a ow rate of 1 mL minꢁ1
and run times varying from 15–25 min. A solvent system of H2O
(0.1% HCOOH)/CH3CN (0.1% HCOOH) (gradient elution) was
used. The UV/Vis and uorescence detectors were set at 340 nm
and 615 nm respectively.
Microscopy
Cells from liquid cultures were pipetted into 32 mm diameter
Petri dishes with 22 mm diameter glass cover-slips in the
bottom. Roots and leaves were placed upon glass microscope
slides and covered with a glass cover-slip. Confocal microscopy
Plant materials
The following tobacco BY-2 cell lines were grown in the dark at images were acquired using a pair of Leica SP5 II laser scanning
25 ꢃC with shaking in BY-2 medium: wild type lines BY2-1, BY2-2 confocal microscopy systems, equipped with
variable
a
and BY2-JI, along with cell lines expressing GFP-TDP6, RFP- temperature and atmosphere control unit to maintain the
TDP6, GFP-Lifeact. The lanthanide complexes were added to the constant environment required for the relevant experiment. The
culture medium immediately before imaging at
a nal lanthanide complexes were excited using either a 405 nm diode
concentration of 100 mM. Control experiments were undertaken laser or a 355 nm NdYAG 3rd harmonic laser at 30% power
on cells with no dyes added to rule out autouorescence. The (12 mW). Emitted light was collected between 605 and 720 nm
BY-2 growth medium contains 4.4 g Lꢁ1 Murashige and Skoog utilising the HyD detector on the Leica SP5, or between 600 and
medium with 10 mL Lꢁ1 Kao & Michayluk vitamins, 250 mg Lꢁ1 720 nm on one of the PMTs (photomultiplier tubes) to look at
sucrose, 68.4 g Lꢁ1 of glucose, 0.2 mg Lꢁ1 2,4- dichlor- the lanthanide emission. Images were also recorded between
ophenoxyacetic acid, 0.5 mg Lꢁ1 zeatine, 1 mg Lꢁ1 NAA at pH 410 and 500 nm to look at the organic emission when required.
5.6. In order to isolate protoplasts from BY-2 cells, 500 mg GFP and green probes were excited using the 488 nm line of an
cellulase, 100 mg pectinase, 0.037 g CaCl2.2H2O, 75 mg KCl, argon laser between 15 and 20% power (6–10 mW). For these
200 mg and 12 g sucrose were dissolved in 50 mL H2O and the experiments on the Leica system emitted light was collected
pH was adjusted to 5.7 before lter sterilisation. The cells were between 500 and 550 nm and on the Zeiss system emitted light
le in this solution at room temperature overnight, before being was collected using a long pass 505 nm lter. MitoTracker
centrifuged and then re-suspended in growth medium before Orange was excited using the 488 nm line of an argon laser at
dye incubation and imaging.
30% power and emitted light was collected between 550 and
For the leaf experiments, Nicotiana benthamiana plants were 700 nm on the Leica system, whereas on the Zeiss system a long
grown in soil. Once the leaves were grown to approximately 3 cm pass 560 nm lter was used.
in diameter leaf inltration was undertaken using a 1 mL
For all images either a 40x/1.3 plan Apo UV oil immersion or
syringe lled with a 100 mM solution of the complex, followed by a lBlue 63x/1.4 plan UV oil immersion objective lens was used.
9364 | RSC Adv., 2014, 4, 9356–9366
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Gain, digital offset line average and speed values were opti-
mised in each experiment. The control experiments were
undertaken with the same laser power and gain settings to
ensure that no autouorescence was visible. For multi-colour
imaging, the channels were acquired sequentially. The images
were exported in TIFF format, and gures were assembled using
ImageJ 1.47 and Inkscape 0.48 soware.
T. Gunnlaugsson, Angew. Chem., Int. Ed., 2012, 51, 9624; (c)
M. Liu, Z. Ye, C. Xin and J. Yuan, Anal. Chim. Acta, 2013,
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and O. S. Woleis, Chem. Commun., 2009, 5000.
8 (a) H. Sigel, The Lanthanides and Their Interrelations with
Biosystems, Metal Ions in Biological Systems, CRC Press, 1st
edn, 2003, vol. 40; (b) S. Mohandessi, M. Rajendran,
D. Magda and L. W. Miller, Chem.–Eur. J., 2012, 18, 10825;
(c) E. J. New, D. Parker, D. G. Smith and J. W. Walton,
Curr. Opin. Chem. Biol., 2010, 14, 238.
Spectral imaging
Spectral imaging in cells was achieved using a custom built
microscope (modied Zeiss Axiovert 200M), using a Zeiss
APOCHROMAT 63x/1.40 NA oil objective combined with a low
voltage 365 nm pulsed UV LED focused, collimated excitation
source (1.2 W, ca. 1200 nJ mmꢁ2 s). For rapid spectral acquisition
the microscope was equipped at the external port with a Peltier
cooled 2D-CCD detector (Ocean Optics) used in an inverse
100 Hz time gated sequence. The spectrum was recorded from
400–800 nm with a resolution of 0.24 nm and the nal spectrum
was acquired using an averaged 10 000 scan duty cycle. Probe
lifetimes were measured on the same microscope platform
using a novel emission decay proling sequence (time corre-
lated counting with time-gated tail tting) cooled PMT detector
(Hamamatsu H7155) interchangeable on the external port, with
the application of preselected interference lters. Both the
control and detection algorithm were written in LabView2011.
The probe lifetime was determined by using a single exponen-
tial tting algorithm to the monitored signal intensity decay.
Time gated imaging was achieved using the same 10 Hz
sequence (50–200 summed duty cycle)as for spectral acquisi-
tion, using a gated and cooled high resolution ThorLabs EOS
Monochrome mCCD camera.
9 M. Elbanowski and B. Makowska, J. Photochem. Photobiol., A,
1996, 99, 85.
10 (a) S. Aime, M. Botta, M. Fasano, E. Terreno, P. Kinchesh,
L. Calabi and L. Paleari, Magn. Reson. Med., 1996, 35, 648;
(b) P. Harvey, A. M. Blamire, J. I. Wilson, A. M. Funk,
K.-L. N. A. Finney, P. K. Senanayake and D. Parker, Chem.
Sci., 2013, 4, 4251.
11 (a) K. Hanaoka, K. Kikuchi, S. Kobayashi and T. Nagano, J.
Am. Chem. Soc., 2007, 129, 13502; (b) H. E. Rajapakse,
N. Gahlaut, S. Mohandessi, D. Yu, J. R. Turner and
L. W. Miller, Proc. Natl. Acad. Sci. U. S. A., 2010, 107,
13582; (c) D. G. Smith, B. K. McMahon, R. Pal and
D. Parker, Chem. Commun., 2012, 48, 8520.
12 J. Yuan and G. Wang, TrAC, Trends Anal. Chem., 2006, 25,
490.
13 (a) R. Pal and D. Parker, Org. Biomol. Chem., 2008, 6, 1020; (b)
B. K. McMahon, R. Pal and D. Parker, Chem. Commun., 2013,
49, 5363; (c) D. G. Smith, R. Pal and D. Parker, Chem.–Eur. J.,
2012, 18, 11604.
14 J. Yu, D. Parker, R. Pal, R. A. Poole and M. J. Cann, J. Am.
Chem. Soc., 2006, 128, 2294.
15 R. A. Poole, G. Bobba, M. J. Cann, J.-C. Frias, D. Parker and
R. D. Peacock, Org. Biomol. Chem., 2005, 3, 1013.
16 J. C. Frias, G. Bobba, M. J. Cann, C. J. Hutchison and
D. Parker, Org. Biomol. Chem., 2003, 1, 905.
17 Y. Bretonniere, M. J. Cann, D. Parker and R. Slater, Org.
Biomol. Chem., 2004, 2, 1624.
Acknowledgements
We thank the ERC (FCC 266804: SJB, DP, RP), CISbio (JWW) and
Durham University for support, and Dr Chris Ottley (Geology)
for the ICP-MS measurements.
18 E. J. New and D. Parker, Org. Biomol. Chem., 2009, 7, 851.
19 E. J. New, A. Congreve and D. Parker, Chem. Sci., 2010, 1, 111.
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21 L.-O. Palsson, R. Pal, B. S. Murray, D. Parker and A. Beeby,
Dalton Trans., 2007, 5726.
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