Fig. 4 (a) Bright-field image of the HeLa cells stained with ZnL7.
(b) Two-photon microscopy image of the same cells with excitation at
840 nm. (c) Merged image of (a) and (b).
single-photon fluorescence microscopy (1PFM) for HeLa cells.
It is also worthy to note that ZnL4 could be photobleached
under two-photon excitation (Fig. S23–24w), which might be
due to the intrinsic higher power 2PFM laser used (ca. 30 times
higher than 1PFM laser).
Fig. 3 One-photon fluorescence colocalization images of HeLa cells
incubated with probes (2 mM, 0.5 h) and commercial dyes or GFP.
(a) Differential interference contrast (DIC) image, (b) image of
commercial marker, (c) image of ZnSalen, (d) merged images of
(b) and (c). Row A: colocalization study of ZnL4 and ER Tracker Green
(Pearson’s coefficient: 0.74). Row B: colocalization study of ZnL7 and
LysoTracker Green DND-26 (Pearson’s coefficient: 0.70). Row C:
colocalization study of ZnL9 and FYVE-EGFP (Pearson’s coefficient:
0.66).
In conclusion, we present a novel class of ZnSalen
complexes ZnL1–10 with lipophilic and cationic properties
conjugates. These luminescent complexes exhibited chemo-
and photostability, low cytotoxicity to living cells, and
subcellular selectivity, which are essential to be ideal optical
probes in living cell imaging. More importantly, the high 2PA
cross section of these compounds makes them potentially
applicable in 2PFM imaging.
The project was supported by the National Key Basic
Research Support Foundation of China (NKBRSFC)
(2010CB912302) and the National Scientific Foundation of
China (grand no.20971007). Y.H. thanks National Funding
for Fostering Talents of Basic Sciences (J0630421).
hydrophobic environments (Fig. S9w). The confocal micro-
scopy images showed that, except ZnL5, the other Zn
complexes could be taken up efficiently by HeLa cells and
the optimum concentrations are 2 mM. Through comparison
with colocalization of subcellular markers, we found that
ZnL3–10 localized in organelles such as lysosome, endosome
and ER whereas no staining of mitochondria, Golgi apparatus
and cell membrane was observed. As shown in Fig. 3, we
selected fluorescent images of HeLa cells incubated with
ZnL4,7,9 (2 mM, 0.5 h) and commercially available lysosome,
endosome and ER markers. The other images for
colocalization study of other ZnSalen complexes were given
in ESIw. In particular, ZnL4 with cyclic alkyl conjugate at
salicyladehyde were specifically colocalized with an endoplasmic
reticulum marker (Fig. 3A) and ZnL3,6 with linear alkyl
conjugate exhibited similar subcelluar labeling (Fig. S12 and
Fig. S14, repectivelyw). ZnL7 tailed by mopholino mainly
exhibited lysosomal specificity while partly colocalized with
early endosome marker (Fig. 3B). ZnL8 tailed by mopholino
derivative showed similar organelle localization (Fig. S16w).
With cationic conjugates such as pyridinum and triphenyl
phosphonium (TPP) ions which were supposed to accumulate
in mitochondria,17 ZnL9–10 displayed punctate luminescence
in the cytoplasm and only colocalizated with FYVE-EGFP
(Fig. 3C and Fig. S19,w respectively), an early endosome
marker and the reason need to be elucidated. These results
demonstrate the efficiency of these conjugates to tailor
luminescent ZnSalen in specific organelles.
Notes and references
1 D. J. Stephens and V. J. Allan, Science, 2003, 300, 82–86.
2 R. Y. Tsien, L. Ernst and A. Waggoner, Handbook of Biological
Confocal Microscopy, Springer, 2006.
3 W. Denk, J. Strickler and W. Webb, Science, 1990, 248, 73–76.
4 C. Xu, W. Zipfel, J. B. Shear, R. M. Williams and W. W. Webb,
Proc. Natl. Acad. Sci. U. S. A., 1996, 93, 10763–10768.
5 V. Fernandez-Moreira, F. L. Thorp-Greenwood and
M. P. Coogan, Chem. Commun., 2010, 46, 186–202.
6 P. Wu, E. M. Wong, D. L. Ma, G. M. Tong, K. M. Ng and
C. M. Che, Chem.–Eur. J., 2009, 15, 3652–3656.
7 C. K. Koo, L. Y. So, K. L. Wong, Y. M. Ho, Y. W. Lam,
M. W. Lam, K. W. Cheah, C. W. Cheng and W. M. Kwok, Chem.
Eur. J., 2010, 16, 3942–3950.
8 C. K. Koo, K. L. Wong, C. W. Y. Man, Y. W. Lam, K. Y. So,
H. L. Tam, S. W. Tsao, K. W. Cheah, K. C. Lau, Y. Y. Yang,
J. C. Chen and M. H. W. Lam, Inorg. Chem., 2009, 48, 872–878.
9 S. W. Botchway, M. Charnley, J. W. Haycock, A. W. Parker,
D. L. Rochester, J. A. Weinstein and J. A. G. Williams, Proc. Natl.
Acad. Sci. U. S. A., 2008, 105, 16071–16076.
10 P. Wang, Z. Hong, Z. Xie, S. Tong, O. Wong, C.-S. Lee, N. Wong,
L. Hung and S. Lee, Chem. Commun., 2003, 1664–1665.
11 C.-C. Kwok, S.-C. Yu, I. H. T. Sham and C.-M. Che, Chem.
Commun., 2004, 2758–2759.
12 Y. Gao, J. Wu, Y. Li, P. Sun, H. Zhou, J. Yang, S. Zhang, B. Jin
and Y. Tian, J. Am. Chem. Soc., 2009, 131, 5208–5213.
13 S. I. Pascu, P. A. Waghorn, T. D. Conry, B. Lin, H. M. Betts,
J. R. Dilworth, R. B. Sim, G. C. Churchill, F. I. Aigbirhio and
J. E. Warren, Dalton Trans., 2008, 2107–2110.
To demonstrate the application of ZnSalen complexes in
two-photon fluorescence microscopy (2PFM) imaging in living
cells, we used ZnL4 and ZnL7 as probes to stain HeLa cells.
The 2PFM image and the merged image confirm that the
molecules of ZnL4 and ZnL7 are presented within the cells
(Fig. 4, Fig. S21w) and showed similar images to that using
14 A. J. Gallant, J. H. Chong and M. J. MacLachlan, Inorg. Chem.,
2006, 45, 5248–5250.
15 C. Xu and W. W. Webb, J. Opt. Soc. Am. B, 1996, 13, 481–491.
16 N. S. Makarov, M. Drobizhev and A. Rebane, Opt. Express, 2008,
16, 4029–4047.
17 L. F. Yousif, K. M. Stewart and S. O. Kelley, ChemBioChem,
2009, 10, 1939–1950.
c
This journal is The Royal Society of Chemistry 2011
Chem. Commun., 2011, 47, 2435–2437 2437