Fig. 6i. However, the cellular staining result obtained using
NIM-2 was noteworthy. In contrast to the results observed in
normal cells, cancer cells showed bright spots by confocal
microscopy (Fig. 6e–h) when the spectra were recorded at
450–590 nm in PMT 1 and 550–740 nm in PMT 2. Additionally,
the merge gallery revealed bright spots on cancer cells but not
normal cells. Evaluation of the l scanning spectra suggests
a different emission wavelength of NIM-2 between bright spots
(ꢃ595 nm) and others cancer cell lines as well as normal cells
(Fig. 6j). Similar results were also observed in other cancer cell
lines. These results indicate that NIM-2 molecules generally
spread in the cytoplasm of living cells and accumulate in cancer
cells, which results in the generation of bright spots. These
distinct cellular staining patterns indicate that NIM-2 can be
used to differentiate cancer cells from normal cells once suitable
filters (550–620 nm) are constructed—as shown in the inset image
in Fig. 6j, in which only cancer cells presented bright spots in the
cell staining. Unfortunately, observation of NIM-3 under
confocal microscopy was not successful owing to its higher
absorption energy.
showed good permeability and were well dispersed in the cyto-
plasm. First, investigation of NIM-1 revealed that NIM-4 may
serve as a precursor of DNA or protein labelling markers, for
example, conjugate to isothiocyanate and succinimidyl ester
functional groups with suitable spacers.28 Second, the weak
luminescence of NIM-2 and NIM-3 can achieve bright cellular
imaging owing to their AIEE properties. More importantly,
NIM-2 can be used in cancer cell recognition. Finally, we have
developed color fluorescent probes, with varying emission
wavelengths at a fixed excitation wavelength, for application in
cellular imaging. Eventually, the fluorescence-environment
dependence, large Stokes shift, long emission wavelength,
permanent fluorescence quantum yields, and AIEE of these
naphthalimide derivatives fluorophores can be used to develop
ultrasensitive fluorescent molecular probes to study a variety of
biological events and processes.
Acknowledgements
This work was supported financially by the National Science
Council (NSC 99-2113-M-005 -011-MY2) of Taiwan.
Investigation of the brightness of NIM derivatives in
cells
Notes and references
Based on the above optical results, the apparent fluorescence
emission of NIM-1 was observed, while NIM-2 and NIM-3
posses AIEE properties. It is reasonable that NIM-1 presents
cellular brightness because its emission ability is strong in most of
solvents or environments. However, the cellular staining results
of compounds NIM-2 and NIM-3 are interesting. Indeed, the
quantum yields of NIM-2 and NIM-3 in most solvents were very
low while strong fluorescence only occurred when NIM-2 dis-
solved in very low ET30 solvents. Thus, the bright cellular image
of NIM-2 can be explained by two possibilities. First, the cellular
environment in which the NIM-2 molecule was located should
have a lower polarity condition. Second, molecules aggregated
with very high concentration; that is, molecular imaging of NIM-
2 is the representation of AIEE. Both of these possibilities would
result in a strong emission phenomenon of this compound in
cells. Moreover, the bright cellular image of NIM-3 should be
attributed to one more possibility; fluorescence enhancement
arose from FONs phenomenon under acidic condition. It is
known that cellular cytoplasm is acidic and lysosomes maintain
at lower pH.27 Therefore, it is possible that neutral NIM-3 can
permeate the cell membrane and be protonated in the cytoplasm
to form FONs, and consequently show bright cellular images.
From this cursory study, we suggest that both the NIM-2 and
NIM-3 cases of molecular imaging are due to the presentation of
AIEE, but that they function through different mechanisms.
Nevertheless, we support colorful cellular probes with similar
exciting light sources (under fluorescent microscopy, Fig. 5) for
the application of molecular imaging.
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Conclusions
The results of this study indicate that colorful fluorophores were
developed from NIM derivatives and their large Stokes shifts can
prevent self-quenching and measurement errors caused excita-
tion light and scattering. NIM-1, NIM-2, and NIM-3 clearly
€
}
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3176 | J. Mater. Chem., 2011, 21, 3170–3177
This journal is ª The Royal Society of Chemistry 2011