Two-photon fluorescence probes for imaging of mitochondria and lysosomes

Article abstract of DOI:10.1039/c3cc41240g

Novel biocompatible cyanines show not only a very large two-photon cross-section of up to 5130 GM at 910 nm in aqueous medium for high-contrast and -brightness two-photon fluorescence live cell imaging but also highly selective subcellular localization properties including localization of mitochondria and lysosomes.

Full text of DOI:10.1039/c3cc41240g

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Two-photon fluorescence probes for imaging of
mitochondria and lysosomes†
Cite this: DOI: 10.1039/c3cc41240g
Wanggui Yang,a Pui Shan Chan,b Miu Shan Chan,ac King Fai Li,d Pik Kwan Lo,c
Nai Ki Mak,*b Kok Wai Cheah*d and Man Shing Wong*a
Received 16th February 2013,
Accepted 6th March 2013
DOI: 10.1039/c3cc41240g
www.rsc.org/chemcomm
Novel biocompatible cyanines show not only a very large two-photon would play a crucial role in the realization of practical applications.4
cross-section of up to 5130 GM at 910 nm in aqueous medium for high- As a result, developing useful biological TPA fluorophores with a
contrast and -brightness two-photon fluorescence live cell imaging large two-photon excited fluorescence (FFLs2) and good solubility in
but also highly selective subcellular localization properties including aqueous medium without aggregation as well as low cytotoxicity still
localization of mitochondria and lysosomes.
remains a great challenge to the scientific community.5
Fluorescence probes that can selectively target specific sub-
Among various conventional bioimaging techniques, two-photon cellular compartments and organelles are particularly useful tools
excited fluorescence microscopy is a very attractive and non-invasive for the study of localization, movements and intracellular concen-
tool for living cell and tissue imaging.1 Since the two-photon tration of a species of interest in living cells as well as for the study
absorption (TPA) process is nonlinearly intensity-dependent, it can of cellular activities and processes. For instance, lysosomes are
provide higher spatial resolution in imaging when compared with membrane-bound acidic organelles responsible for the breakdown
that of the one-photon counterparts. In addition, the use of the low- of unwanted macromolecules. Two-photon probes for such
energy near infrared excitation would enable deeper tissue penetra- lysosomes are still limited.6 Mitochondria produce ATP, the
tion and reduce the photobleaching and photodamage of the tissue energy currency of the cell, through a chain of electron transport
during imaging which is particularly important for prolonged and oxidative phosphorylation pathways and mediate apoptosis
studies.2 However, most of the currently used biological fluoro- which are of great interest to study and monitor. As a result, the
phores by one-photon excitation show only low TPA cross-sections. development of novel TPA biological fluorescence probes that
Furthermore, TPA fluorophores often show substantially lower TPA show a specific targeting property is of practical significance.
cross-sections and fluorescence quantum yield in aqueous medium
It was previously demonstrated that methylpyridinium
than those measured in organic solvents.3 On the other hand, most derived cyanines showed no specific subcellular localization
of the highly active TPA molecules are not designed and suitable for properties although these dyes could afford high brightness for
biological applications as they often are either water insoluble or two photon excited fluorescence (TPEF) images of the HeLa
incompatible with the biological systems or form non-fluorescence cells.7 However, by modifying the functional substituent(s)
aggregates in aqueous medium. To be useful for two-photon excited attached onto the heteroaromatic ring(s), we have successfully
fluorescence microscopic applications, fluorophores need to possess developed novel carbazole-based cyanine fluorophores that
large TPA cross-sections (s2). Besides, other desirable physical and exhibit highly selective subcellular localization properties. In
biological properties including a high fluorescence quantum yield this contribution, we report herein the synthesis and investiga-
(FFL), good water-solubility, high photostability and low cytotoxicity tion of biocompatible, mitochondrial and lysosomal probes
with high two-photon cross-sections in aqueous medium based
on the carbazole-based cyanine framework for TPEF imaging of
live cells. The molecular structures of TPA carbazole-based
cyanines are shown in Fig. 1. It is worth mentioning that the
a Department of Chemistry and Institute of Advanced Materials, Hong Kong Baptist
University, Kowloon Tong, Hong Kong SAR, China. E-mail: mswong@hkbu.edu.hk;
Fax: +852 34117348; Tel: +852 34117069
b Department of Biology, Hong Kong Baptist University, Kowloon Tong, Hong Kong SAR,
China. E-mail: nkmak@hkbu.edu.hk; Fax: +852 34115995; Tel: +852 34117059
c Department of Biology and Chemistry, City University of Hong Kong, Tat Chee Avenue,
Kowloon, Hong Kong SAR, China
d Department of Physics and Institute of Advanced Materials, Hong Kong Baptist
University, Kowloon Tong, Hong Kong SAR, China. E-mail: kwcheah@hkbu.edu.hk;
Fax: +852 34115813; Tel: +852 34117029
R substituent not only acts as a guiding moiety to selectively
localize at a specific subcellular compartment but also affects
the ultimate physical and biological properties of the probe.
Synthesis of carbazole-based cyanines is shown in Scheme 1.
By adapting the convergent approach established previously,1,2
the Knoevenagel reaction of the corresponding carbazolyl-3-aldehyde
† Electronic supplementary information (ESI) available. See DOI: 10.1039/c3cc41240g or carbazolyl-3,6-dialdehyde and functionalized 4-methyl-pyridinium
c
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Table 1 Summary of photophysical measurements of carbazole-based fluoro-
phores measured in pH 7 phosphate buffer solution and DMSO
abs
a
c
abs d
l
/
lem a
nm
/
smax
GM
/
l
/
lem d
/
nm
max
max
a,b
b,d
nm (e)
FFL
nm (e)
FFL
SPBN 424 (1.99) 600
SPBP 423 (1.20) 595
VPBN 443 (4.26) 582
0.02
0.07
0.003 5130
243
109
435 (1.80) 603
435 (1.53) 600
453 (3.92) 618
0.37
0.45
0.03
a
b
Measured in buffer, e  104 MÀ1 cmÀ1
.
Using rhodamine 6G (F488 =
Fig. 1 Molecular structure of carbazole-based cyanines.
0.95) as a standard and average of two independent measurements.
c
d
Maximum TPA cross-section. Measured in DMSO, e  104 MÀ1 cmÀ1
.
aqueous medium (Table 1) due to the strong intermolecular
interaction between the cyanines and water molecules.8 Never-
theless, upon binding to a host such as ct-DNA and protein, these
cyanines would exhibit a strong fluorescence enhancement. In
addition, these cyanines are found to be stable to the change in
the pH value from 4.0 to 10.6 in the buffer medium as reflected in
the absorption and emission spectra of these cyanines in various
pH buffered solutions (Fig. S2, ESI†). This property is especially
beneficial to the application of imaging in a cell environment, in
particular for acidic lysosomal probes.
Upon excitation at 800 nm by femtosecond laser pulses in
phosphate buffer, moderate fluorescence at around 572–
590 nm is observed. The power-squared dependence of two-
photon excited fluorescence of these cyanines measured at
800 nm fs laser pulses was followed with the slope of 1.8–1.9
indicating that these cyanines are two-photon excitation active
(Fig. S3, ESI†). The TPEF spectra as shown in Fig. 2a are very similar
to those of the one-photon excited counterparts suggesting that
these cyanines have the same emissive states for the two-photon
excitation process. The standard two-photon induced fluorescence
method was used to determine the TPA cross-sections and two-
photon excited spectra of the cyanines in the range of 620–1100 nm
using a femtosecond pulsed laser in buffered solution.9 Fig. 2b
shows the two-photon excited spectra of the three newly synthesized
cyanines in which the bis-cyanine, VPBN, shows a maximum TPA
cross-section (smax) of 5130 GM at 910 nm which is among the
highest smax reported in aqueous medium. Such a large value
highlights the merit of using multi-dimensional structure motifs
to enhance s2.10 Furthermore, the mono-cyanines SPBN and SPBP
afford a smax of 243 and 109 GM at 890 nm in buffered solution,
respectively. Despite the identical donor–acceptor p-conjugated
backbone for SPBN and SPBP, the discrepancy in the TPA
cross-sections could arise from the difference in the solute–
solvent interaction. Nevertheless, these cyanines with such high
TPA cross-sections in aqueous medium have potential for TPA
live cell imaging.
Scheme 1 Synthesis of carbazole-based cyanines.
halide was used as the key step to synthesize functionalized
carbazole-based cyanines. 9-(2-(2-Methoxy-ethoxy)ethyl)-9H-carba-
zole-3-carbaldehyde and 9-(2-(2-methoxyethoxy)ethyl)-9H-carbazole-
3,6-dicarbaldehyde were synthesized according to the published
literature.1,2 1,4-Dibromobutane was reacted with triethylamine to
afford ammonium salt 1 followed by alkylation with 4-methylpyr-
idine to afford functionalized 4-methylpyridinium halide 2. On the
other hand, 1,6-dibromohexane was refluxed with triphenyl-
phosphine to afford phosphonium salt 3 followed by heating with
4-methylpyridine to afford functionalized 4-methylpyridinium
halide 4. The Knoevenagel reaction of 9-(2-(2-methoxyethoxy)-
ethyl)-9H-carbazole-3-carbaldehyde with methylpyridinium halide
2 and 4 afforded the desired mono-cyanines SPBN and SPHP,
respectively. Furthermore, the Knoevenagel reaction of 9-(2-(2-
methoxyethoxy)ethyl)-9H-carbazole-3,6-dicarbaldehyde and halide 2
gave the bis-cyanine VPBN. All of the new cyanines were fully
characterized using 1H and 13C NMR, and HRMS. The data
obtained are in good agreement with the proposed structures.
All of the cyanines are highly soluble in common organic
solvents and water. Both of the mono-cyanines show very
similar absorption characteristics with a broad and structureless
charge-transfer band at B424 nm in phosphate buffer. On the other
hand, the bis-cyanine shows a distinct overlapping of two absorp-
tions at the long wavelength region which is relatively red-shifted
(i.e. lmax = 443 nm) as compared to that of the mono-cyanines
(Fig. S1, ESI†). This suggests that the two conjugated arms of the
bis-cyanine are structurally and spectroscopically non-equivalent.
Upon excitation at the absorption maximum, these cyanines exhibit
relatively strong fluorescence at B600 nm in organic solvents such
as CH3CN and DMSO but very weak and blue-shifted emission in
Fig. 2 (a) The two-photon fluorescence spectra excited at 800 nm fs laser pulses
and (b) two-photon excited spectra of the cyanines in phosphate buffer solution.
c
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Fig. 3 Cytotoxicity of SPBN, SPHP and VPBN on HK-1 cells. Cells were treated
with various concentrations of the compounds (0.5–8 mM) for 24 hours. MTT
reduction assay was carried out to determine the cell viability. Data are expressed
as mean value Æ standard derivation of three separate trials.
To ascertain the potential of these cyanines for cellular
imaging, the cytotoxicity of the cyanines towards HK-1 cells
was assessed by MTT cell viability assay. Fig. 3 shows the
cytotoxicity data of HK-1 cells after treatment with various
concentrations (0.5–8 mM) of the cyanines for 24 h. The
cytotoxicities of these cyanines are well below 20% at most of
the dye concentrations indicating no significant cytotoxicity of
these cyanines. Such low cytotoxicity is beneficial to prolonged
cellular imaging.
To assess the ability of these newly developed cyanines as
probes for live cell imaging and determine the location of the
probes in the cells, the colocalization of these cyanines with
various one-photon fluorescence organelle trackers in HK-1
cells was carried out. The one-photon fluorescence images
showed that all the cyanines were efficiently taken up by the
HK-1 cells at a concentration of 5 mM affording bright images.
The one-photon fluorescence images of HK-1 cells co-stained
with the cyanines and Mito Tracker or Lyso Tracker are shown
in Fig. S4 and S5 (ESI†). Interestingly, the fluorescence image
of SPBN greatly overlapped with that of the Mito Tracker
indicating that SPBN is localized to the mitochondria; in
contrast, the fluorescence image of VPBN largely colocalized
with that of Lyso Tracker suggesting that the intracellular
localization of VPBN is at the lysosomes (Fig. S5, ESI†) despite
the same targeting triethylamino moiety being incorporated. In
addition, the fluorescence images of SPHP and Mito Tracker
are almost identical indicating that SPHP can exclusively stain
mitochondria (Fig. S5, ESI†).
Fig. 5 (a) TPEF and (b) transmission images of HK-1 cells upon incubation with
VPBN for 24 h at a concentration of 2 mM. (c) One-photon confocal microscopic
images of the same sample after being co-stained with Lyso Tracker (100 nM) for
10–20 minutes in the dark, (d) overlapped images of (a)–(c).
brightness for TPEF images with high resolution. It is also
clearly shown that the SPHP probe shows better contrast and
brightness than that of SPBN (Fig. S6, ESI†), which is attributed
to a larger two-photon excited fluorescence (FFLs2) of SPHP.
In summary, three new carbazole-based cyanine fluoro-
phores have been developed as highly active two-photon
fluorescence probes for live cell imaging. These probes not
only show no significant cytotoxicity but also exhibit highly
selective subcellular localization properties including localiza-
tion of mitochondria and lysosomes. These cyanines have been
found to show large two-photon cross-sections in aqueous
medium in which a record high two-photon cross-section of
5130 GM at 910 nm has been obtained for VPBN. The TPEF
images of HK-1 cells with these cyanines show good to excellent
contrast and brightness. This study demonstrates that
subtle structure modifications provide a means to optimize
the functional properties of carbazole-based fluorophores for
bio-imaging applications.
This work was supported by GRF (HKBU 203212), Hong
Kong Research Grant Council, and Faculty Research Grant
(FRG2/11-12/123), Hong Kong Baptist University.
Fig. 4 and 5 depict the two-photon excited fluorescence
(TPEF) images of HK-1 cells with SPHP and VPBN excited at
850 nm and 910 nm, respectively, under the same incubation
conditions (2 mM, 24 h). The selective staining properties of
SPHP to mitochondria and VPBN to lysosomes were clear and
further confirmed with the one-photon tracker dyes. Consistent
with the very large 2PA cross-section, VPBN affords excellent
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Fig. 4 (a) TPEF and (b) transmission images of HK-1 cells upon incubation with
SPHP for 24 h at a concentration of 2 mM. (c) One-photon confocal microscopic
images of the same sample after being co-stained with Mito Tracker (100 nM) for
10–20 minutes in the dark, (d) overlapped images of (a)–(c).
c
This journal is The Royal Society of Chemistry 2013
Chem. Commun.