Dinuclear ruthenium(ii) polypyridyl complexes as single and two-photon luminescence cellular imaging probes

Article abstract of DOI:10.1039/c3cc48916g

A new series of dinuclear ruthenium(ii) polypyridyl complexes, which possess larger π-conjugated systems, good water solubility and pH resistance, and high photostability, were developed to act as single and two-photon luminescence cellular imaging probes. The Royal Society of Chemistry.

Full text of DOI:10.1039/c3cc48916g

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Dinuclear ruthenium(II) polypyridyl complexes
as single and two-photon luminescence
cellular imaging probes†
Cite this: Chem. Commun., 2014,
50, 2123
Received 22nd November 2013,
Accepted 23rd December 2013
Wenchao Xu, Jiarui Zuo, Lili Wang, Liangnian Ji and Hui Chao*
DOI: 10.1039/c3cc48916g
www.rsc.org/chemcomm
A new series of dinuclear ruthenium(II) polypyridyl complexes, which such as their exceptional nonlinear optical properties, high
possess larger p-conjugated systems, good water solubility and pH luminescence, large Stokes shifts, high photostability and relatively
resistance, and high photostability, were developed to act as single long lifetimes, but also because of their small molecular weight,
and two-photon luminescence cellular imaging probes.
enabling easy penetration of cell membranes for live cell staining
processes.10 Recently, some Ru(II) polypyridyl complexes have been
Live cell imaging has been the focus of increasing interest in recent investigated for applications in live cell imaging;11 however, only a few
years.1 As visualisation of cellular events by confocal fluorescence were successfully applied as two-photon luminescent cellular imaging
microscopy plays an important role in biochemistry and biomedical probes.12 The vast potential of Ru(II) complexes as two-photon
research, a wide variety of fluorescent cellular dyes have been imaging probes remains largely untapped. In the present work,
developed and utilised, such as the commercial organic dyes we synthesised a series of dinuclear Ru(II) polypyridyl complexes,
40,6-diamidino-2-phenylindole (DAPI) and MitoTracker Green. [(phen)2Ru(L1–7)Ru(phen)2]4+ (RuL1–7, phen = 1,10-phenanthroline,
However, most of the commercially available dyes have notable L = 1,3-bis(1-substituted-1H-imidazo[4,5-f][1,10]phenanthroline-2-yl)-
shortcomings including poor water solubility, high toxicity to living benzene), which possess larger p-conjugated systems, good
cells and poor photostability. These commercial organic dyes may water solubility and pH resistance, high photostability, and
also cause extensive cellular damage and unwanted background proved to be useful as single and two-photon luminescent
signals due to the ultraviolet (UV) radiation required for their cellular imaging probes.
excitation and small Stokes shifts.2 These short excitation wave-
The syntheses of RuL1–7 (Scheme 1) were accomplished as
lengths (o650 nm) also inhibit the application of these materials in described in Scheme S1 (ESI†). The bridging ligands were synthe-
thick tissues or live animals due to the resultant short penetration sised through the condensation of 1,10-phenanthroline-5,6-dione,
depth.3 The use of two-photon fluorescence probes is an attractive isophthalic aldehyde, 4-substituted aniline and ammonium acetate
solution to these problems because these molecules exhibit near in refluxing glacial acetic acid over 24 h. The Ru(II) complexes were
infrared (NIR) or longer excitation wavelengths, less phototoxicity, obtained in yields ranging from 56% to 68% by the direct reaction of
deeper penetration depth and reduction of photobleaching.4 L1–7 with appropriate molar ratios of cis-Ru(phen)2Cl2 in ethylene
As such, many laboratories are focused on the development of new glycol. The synthetic details and characterisation data for these
two-photon imaging probes, such as organic dyes,5 luminescent complexes are provided in Fig. S1–S21, ESI.†
d6-metal complexes (e.g., complexes of Ir and Re),6 d8-metal Pt
We studied the electronic absorption and emission spectra
complexes,7 d10-metal Zn(II) salen complexes8 and quantum dots.9 of RuL1–7 in aqueous media (DMSO–H2O, v/v = 1 : 99) at 298 K
However, rationally designed molecules providing efficient two-photon
absorption (TPA) medium compatibility, photostability, membrane-
crossing capabilities and low cytotoxicity are still highly sought.
In this work, we focus on Ru(II) polypyridyl complexes, not
only because of their outstanding photochemical properties,
MOE Key Laboratory of Bioinorganic and Synthetic Chemistry, State Key Laboratory
of Optoelectronic Materials and Technologies, School of Chemistry and Chemical
Engineering, Sun Yat-Sen University, Guangzhou 510275, P. R. China.
E-mail: ceschh@mail.sysu.edu.cn; Fax: +86-20-84112245; Tel: +86-20-84110613
† Electronic supplementary information (ESI) available: Procedures and addi-
Scheme 1 The chemical structure of complexes RuL1–7
.
tional experiments. See DOI: 10.1039/c3cc48916g
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(Table S1, ESI†). All of the complexes showed good solubility.
RuL1–7 each show intense absorption features (e on the order
of 104 Mꢀ1 cmꢀ1) between 230 nm and 600 nm (Fig. S22, ESI†).
The emission spectra of RuL1–7 exhibit lem values at approxi-
mately 603 nm with quantum yields (f) of 0.041–0.054, using
[Ru(bpy)3]2+ as a standard13 (Fig. S23, ESI†). A luminescence decay
experiment performed at room temperature determined the life-
times of RuL1–7 to be B987–1114 ns through fitting the data to a
single exponential decay function. The Stokes shifts of RuL1–7 were
measured to be B146–153 nm. These large Stokes shifts offer
advantages over popular commercial cellular dyes such as DAPI
(Stokes shift = 46 nm) or SYTO-17 (Stokes shift = 18 nm).14
Fig. 2 OPM images of living HeLa cells incubated with 10 mM RuL4 for 2 h
at 37 1C and then further incubated with DAPI (a) and MitoTracker Green (b).
The two-photon absorption (TPA) properties of RuL1–7
were also studied. With reference to rhodamine B,15 the largest
two-photon absorption cross-sections (d) of RuL1–7 were esti-
conducted two-photon fluorescence microscopy (TPM) imaging
experiments (Fig. S27b–32b, ESI†). The TPM images of HeLa cells
stained with RuL4 (Fig. 1b) showed similarities with OPM (one-
photon fluorescence microscopy) images, confirming penetration of
the dye into the cell cytoplasm. Due to the two-photon absorption,
the background signal was strongly suppressed, resulting in a TPM
image of higher resolution and better signal to noise contrast.
To determine the location of the probes within the cells,
co-localisation studies of RuL4 with several well-known one-photon
fluorescence probes were conducted in HeLa cells. The OPM
images of HeLa cells co-stained with RuL4 and DAPI or MitoTracker
Green (Fig. 2) individually demonstrated that the localisation of
RuL4 in the cells was similar to that of MitoTracker Green, but not
identical. To better understand the location of the dye in the cell, a
z-stack experiment (three-dimensional visualisation) was processed.
HeLa cells were co-stained with MitoTracker Green and RuL4 and
imaged by serially scanning at increasing depths along the z-axis.
Fig. 3 shows that in xy images of the cells obtained at a depth z,
most of the cell cytoplasm was stained green with MitoTracker
Green and red with RuL4 (the overlap was yellow), but the nuclear
regions were blank. Images involving co-localisation with
MitoTracker Green clearly showed that the observed luminescence
was evenly distributed throughout the cytoplasm, but excluded
from the nucleus. Inductively coupled plasma mass spectrometry
(ICP-MS) experiments were carried out to quantify the amounts of
ruthenium in the nucleus and the cytoplasm of a RuL-stained cell.
¨
mated to be 322–386 Goppert-Mayer (GM) units (1 GM = 1 ꢁ
10ꢀ50 cm4 sꢀ1 photonꢀ1) (Table S1, Fig. S24 and S25, ESI†),
hundreds of times larger than those of commercially available
dyes used in two-photon excited (TPE) microscopy (0.16 GM for
DAPI and 1 GM for Cascade Blue fluorescent dyes15a) and
significantly larger than some recently reported two-photon
bio-available organometallic molecular probes.1c,6–8
High cell viability is essential for biological applications.
Cell viability assays using HeLa cells were conducted via the
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)
assay. Fig. S26 (ESI†) shows that RuL1–7 all demonstrated high cell
viability over a 24 hour incubation period at several concentrations
(from 5 mM to 20 mM). This indicates that RuL1–7 each presented
low toxicity during luminescence cell imaging under the applied
conditions (incubation times of 2 h, ruthenium complex concentra-
tions of 10 mM). To investigate the application of RuL1–7 in living cell
imaging, the uptake of RuL1–7 by HeLa cells was evaluated. Confocal
microscopy images showed efficient uptake of RuL1–7 (initial
concentration = 10 mM) by HeLa cells within 2 h (Fig. 1a and
Fig. S27a–S32a, ESI†). The Ru(II) complexes were functionalised with
different substituents with the expectation that different dyes may
reside in different locations within the live cell. Unexpectedly, there
were only small differences between the RuL1–7 complexes in this
respect, so RuL4 was chosen as the model complex in the next
several experiments. To demonstrate the advantage of using RuL1–7
as two-photon luminescence cellular dyes in living cells, we
Fig. 3 Three-dimensional luminescence images of live HeLa cells
incubated with 10 mM RuL4 for 2 h at 37 1C. The cells were co-stained
green with MitoTracker Green. Panel (a) shows an xy image obtained at
z = 8.4 mm, while panels (b) and (c) display the xz and yz cross sections
(z = 1.3–18.9 mm) taken at the lines shown in panel (a), respectively.
(d) The distribution analysis of RuL1–7 in HeLa cells by ICP-MS.
Fig. 1 OPM (a) and TPM (b) images of HeLa cells incubated with RuL4
(10 mM) for 2 h at 37 1C. The wavelengths for one- and two-photon
excitation were 458 and 830 nm, respectively.
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proved to be effective as single and two-photon luminescence
cellular imaging dyes. Fluorescence microscopy imaging and
ICP-MS revealed that most of the novel ruthenium complexes
stained the cell cytoplasm, rather than the nucleus. Due to their
low cytotoxicities to living cells, photostabilities, pH resistance,
membrane permeabilities, and especially their large two-photon
absorption cross sections, RuL1–7 are believed to have great potential
as biocompatible dyes for living cells both in OPM and TPM
imaging. These novel dinuclear ruthenium complexes provide a
platform for the design of new TPM imaging dyes.
Fig. 4 Photostability comparison of RuL4 and MitoTracker Green in HeLa
cells. (a) and (b) OPM images of HeLa cells stained with RuL4 and
MitoTracker Green, respectively. The images were taken under successive
irradiation. The wavelengths for RuL4 and MitoTracker Green irradiation
were 458 and 488 nm, respectively.
This project was financially supported by NSFC (21071155,
21172273, 21171177, 91122010, J1103305), the 973 Program
(2014CB845604), and the Program for Changjiang Scholars and
Innovative Research Team at the University of China (No. IRT1298).
Notes and references
As expected, the results showed a substantial difference between
the nucleus (B0.03 pg Ru per cell) and the cytoplasm (B0.3 pg Ru
per cell), suggesting that more than 90% of the ruthenium was
distributed in the cell cytoplasm (Fig. 3d).
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dyes, the photostabilities of RuL1–7 were examined in comparison to
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