Simultaneous imaging of protonated and deprotonated carbonylcyanide p-trifluoromethoxyphenylhydrazone in live cells by Raman microscopy

Article abstract of DOI:10.1039/c3cc48587k

We report the simultaneous imaging of protonated and deprotonated forms of carbonylcyanide p-trifluoromethoxy-phenylhydrazone (FCCP) molecules in live cells by Raman microscopy. Nitriles, structure-sensitive Raman tags, are used to detect the two distinct

Full text of DOI:10.1039/c3cc48587k

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Simultaneous imaging of protonated and
deprotonated carbonylcyanide p-trifluoro-
methoxyphenylhydrazone in live cells by
Raman microscopy†
Cite this: Chem. Commun., 2014,
0, 1341
5
Received 11th November 2013,
Accepted 26th November 2013
ab
ac
ab
ac
Hiroyuki Yamakoshi, Almar F. Palonpon, Kosuke Dodo, Jun Ando,
DOI: 10.1039/c3cc48587k
c
ac
ab
Satoshi Kawata, Katsumasa Fujita and Mikiko Sodeoka*
www.rsc.org/chemcomm
We report the simultaneous imaging of protonated and deprotonated from the small molecule of interest with Raman signals from
forms of carbonylcyanide p-trifluoromethoxy-phenylhydrazone (FCCP) numerous biomolecules in the cell often makes analysis complicated.
molecules in live cells by Raman microscopy. Nitriles, structure- To overcome this problem, we recently proposed a method for small
sensitive Raman tags, are used to detect the two distinct molecular molecule imaging in live cells by using an alkyne tag (alkyne-tag
6,7
structures, demonstrating the potential of Raman microscopy for Raman imaging, ATRI).
structure-based imaging of bioactive small molecules.
The concept behind ATRI is to tag molecules with an alkyne
moiety whose vibrational frequency lies in the spectroscopically
À1
Small molecules serve as modulators of a myriad of biological systems silent region of the cell (between 1800 and 2700 cm ), so that the
by interacting with biomolecules to regulate their functional activities signal is free from interference with Raman signals of endogenous
or as sensors of a cellular environment. Consequently, the utilization molecules. The alkyne tag is small enough to minimally perturb the
of biologically active small molecules has resulted in many important properties of the small molecule. Besides alkyne, nitrile is another
advances in our understanding of various cellular processes and has small functional groups that show strong Raman signals in the
1
also contributed significantly to drug discovery and development.
cellular silent region. We studied the relationship between Raman
A method that can image the molecular structures of bioactive small shift/intensity and structure of various alkynes and nitriles and
molecules within live cells should provide new insights into many found a strong dependence of the Raman shift frequency of alkyne
7
biological functions and processes. Infrared (IR) spectroscopy is one and nitrile signals on the surrounding chemical structure. This
2
candidate for this purpose, but it is difficult to apply IR methods for finding suggests that both alkynes and nitriles could be good Raman
live-cell imaging due to interference from the strong absorption band probes for structure-based imaging of small molecules. Here, we
of water and low spatial resolution. Another candidate for structure- examine the feasibility of employing Raman microscopy to image
based imaging of small molecules is Raman spectroscopy. This different structural forms of the same small molecule simulta-
3
technique has been widely used for analysis of molecular structures,
neously by using the widely used uncoupler, FCCP (carbonylcyanide
8
and more recently, early work on the detection of structural informa- p-trifluoromethoxyphenylhydrazone), as a model system in which
tion of specific molecules in live cells has produced promising the nitrile group serves as an intrinsic Raman tag.
4
9
results. Recent developments in Raman instrumentation based on
FCCP was first reported in 1962 by Heytler and Prichard. With a
parallel detection have made it possible to acquire chemical images of slightly acidic proton (pK 6.0), FCCP should exist in cells as an
a
5
a sample with high spatial and temporal resolution. As a result, equilibrium mixture of deprotonated and protonated forms (Fig. 1).
Raman microscopy has emerged as a powerful tool for live cell It is believed that both states of FCCP can cross the membrane lipid
imaging applications. However, overlapping of critical Raman signals bilayer, due to the appropriate hydrophobicity (log P 2.42) and the
delocalized anionic character of the molecule, allowing it to act as an
8
a
uncoupler. Uncouplers are an interesting group of compounds that
dissipate the proton gradient across the mitochondrial inner
membrane and ‘‘uncouple’’ the function of the electron transport
chain from ATP synthesis. Therefore, FCCP has been used for
studying mitochondrial functions. Some uncouplers have been used
as fungicides, and mild uncouplers are also expected to have
therapeutic potential based on the hypothesis that mild uncoupling
Sodeoka Live Cell Chemistry Project, ERATO, Japan Sciences and Technology
Agency, 2-1 Hirosawa, Wako-shi, Saitama 351-0198, Japan.
E-mail: sodeoka@riken.jp
b
c
Synthetic Organic Chemistry Laboratory, RIKEN, 2-1 Hirosawa, Wako, Saitama
3
51-0198, Japan
Department of Applied Physics, Osaka University, 2-1 Yamadaoka, Suita, Osaka
69-0871, Japan
Electronic supplementary information (ESI) available: Experimental procedures
5
†
1
0
and spectral data. See DOI: 10.1039/c3cc48587k
can be beneficial to cells in certain pathological states. To our
This journal is ©The Royal Society of Chemistry 2014
Chem. Commun., 2014, 50, 1341--1343 | 1341

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Fig. 1 (a) Protonated and (b) deprotonated forms of FCCP.
knowledge, FCCP has never been previously visualized in live
cells. It is anticipated that protonation-state-specific imaging of
FCCP could provide invaluable information for medicinal and
biological research.
First, the Raman spectra of deprotonated and protonated FCCP in
solution were compared to determine the difference of nitrile peak
frequencies. The spectra were obtained in aqueous DMSO adjusted to
8
various pH values. Since FCCP has an acidic pK
a
of 6.0, it is almost
completely protonated at pH 4.6 and almost completely deprotonated
11
at physiological pH (pH 7.4). As shown in Fig. 2, the Raman spectra
of deprotonated FCCP showed nitrile Raman signals at 2176 and
À1
2
195 cm (Fig. 2, red). In contrast, protonated FCCP showed a single
À1
strong nitrile signal at 2226 cm (Fig. 2, green). Much to our delight,
the nitrile bands of the protonated and deprotonated forms were
well separated and readily distinguishable in the Raman spectra.
In pH 6.0 buffer, all three peaks were observed, indicating that FCCP
molecules exist as a mixture of protonated and deprotonated forms at
this pH, as expected (ESI,† Fig. S1). In contrast, only the peak of the
protonated form was observed in aprotic DMSO without buffer (ESI,†
Fig. S2). In addition to the difference of nitrile signals, the two forms
showed different signals in the fingerprint region. The Raman spectra
of deprotonated FCCP showed distinct Raman bands at 1154, 1319,
À1
1
360, and 1494 cm . These peaks are very weak for protonated
À1
FCCP; instead, a characteristic signal at 1511 cm was observed
Fig. 2 and ESI,† Fig. S3). Furthermore, two rather similar Raman
signals appearing at the same Raman shift frequency, 1298 cm
(
À1
,
were observed in both spectra (Fig. 2). Since these signals could
Fig. 3 Raman imaging of protonated and deprotonated forms of FCCP in
overlap with strong cellular signals that usually appear in this region, live HeLa cells. Raman images were obtained from HeLa cells treated with
À1
the distinct nitrile signals in the cellular silent region were considered 100 mM FCCP. The signals at 2197 and 2230 cm were assigned to the red
and green channels, respectively, and a merged image was generated. The
more suitable for separately imaging the deprotonated and proto-
nated forms of FCCP molecules in live cells.
laser wavelength was 532 nm. The light intensity at the sample plane was
À2
4
.2 mW mm . The exposure time for each line was 10 s. The total number
of lines was 140.
Next, we performed Raman imaging of deprotonated and
protonated FCCP molecules in live cells. HeLa cells were incubated
with 100 mM FCCP for 30 min, and Raman images were obtained
by laser excitation at 532 nm (Fig. 3). Images were constructed
from the distribution of Raman peaks at 753, 2176, 2197, 2230
À1
À1
1
and 2851 cm . The peaks at 753 and 2851 cm are assigned to
2
cytochrome c and lipid molecules, respectively. The peaks at
176 and 2197 cm correspond to deprotonated FCCP, while
À1
2
À1
the peak at 2230 cm corresponds to protonated FCCP. At a
concentration of 100 mM FCCP, the distribution of cytochrome
c shows granulated structures similar to the reported shapes of
mitochondria treated with the uncoupler. Ionic deprotonated
FCCP exhibited a diffuse distribution in the cytoplasm, whereas
protonated FCCP colocalized with lipid droplets. In lipids,
FCCP is held in a hydrophobic environment and hence should
1
3
Fig. 2 Raman spectra of protonated and deprotonated FCCP. The sample
concentration was 10 mM in DMSO with buffer. The laser wavelength was
À2
5
32 nm. The light intensity at the sample plane was 3 mW mm . The exposure
time for each line was 10 s.
1
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the principle described here should be generally applicable to
the structure-based imaging of various small molecules in live
cells as a means of investigating their functional interactions.
This work was partly supported by a Grant-in-Aid for Young
Scientists (B) (23710276 to H.Y.) from the Ministry of Education,
Culture, Science, and Technology, Japan.
Notes and references
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À1
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À1
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region
is in the manner of utilizing the tag. Instead of using
Raman tags to image the amount or concentration of a small
molecule in live cells regardless of its structural state, in the
present work, we used an intrinsic tag to simultaneously image
different structural states of a given small molecule. Although
FCCP was used as the model compound for this demonstration,
1
1
This journal is ©The Royal Society of Chemistry 2014
Chem. Commun., 2014, 50, 1341--1343 | 1343