A ratiometric fluorescent molecular probe for visualization of mitochondrial temperature in living cells

Article abstract of DOI:10.1039/c4cc10349a

Mitochondrial thermodynamics is the key to understand cellular activities related to homeostasis and energy balance. Here, we report the first ratiometric fluorescent molecular probe (Mito-RTP) that is selectively localized in the mitochondria and visualize the temperature. We confirmed that Mito-RTP could work as a ratiometric thermometer in a cuvette and living cells.

Full text of DOI:10.1039/c4cc10349a

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A ratiometric fluorescent molecular probe for
visualization of mitochondrial temperature in living
cell
Cite this: DOI: 10.1039/x0xx00000x
Received 00th January 2012,
Accepted 00th January 2012
Mitsumasa Homma, Yoshiaki Takei, Atsushi Murata, Takafumi Inoue and
Shinji Takeoka*
DOI: 10.1039/x0xx00000x
www.rsc.org/
By contrast, low molecular weight chemical probes are easy
to handle for staining either cultured or primary cells. In
addition, membrane permeability and intracellular localization
of chemical probes can be controlled by their physicochemical
properties13. However, the accuracy of singleꢀmolecule
fluorescence imaging is limited to using moving cells and
transported organelles because the fluorescence intensity is also
Mitochondrial thermodynamics is the key to understand cellular
activities related to homeostasis and energy balance. Here, we
report the first ratiometric fluorescent molecular probe (Mito-
RTP) that is selectively localized in the mitochondria and
visualize the temperature. We confirmed that Mito-RTP could
work as a ratiometric thermometer in cuvette and living cell.
Cellular events and functions are performed by a series of influenced by the focus point. The ratiometric property is a
chemical reactions that can be described in terms of powerful means of measuring activities within a living cell
thermochemistry. Living cells efficiently utilize these chemical because this method can negate fluorescence changes related to
reactions to produce the optimum temperature for biomolecular the concentration of fluorophore or focus drift.
dynamics and enzymatic reactions. Furthermore, it is presumed
As far as we know, this is the first study to report a small
that temperature variations must exist when metabolic activities molecular ratiometric thermosensor for organelles. In this
vary e.g., at each stage of the cell cycle, between different kinds paper, we selected mitochondria as the first targeted small
of cells, during environmental changes or when abnormal cells organelle because it plays a key role in maintaining thermal
appear. Therefore, intracellular or subcellular thermometry is a homeostasis in the cell16)
.
potent analytical method for understanding molecular
A
B
D
biological functions related to temperature. Recently, various
types of fluorescent thermometers have been developed1,2
.
Some thermometers, including polymer nanogels3, polymer
dots4,5
,
quantum
dots7,
nanodiamonds8,
polymer
nanoparticles9,10, have high spatial resolution in cultured cells
but their intracellular localization is difficult to control.
Uchiyama et al11 recently constructed
a polymerꢀbased
fluorescent thermometer and revealed that each organelle has a
different temperature within the cell. Although this method can
determine the temperature of each organelle, it could not avoid
the effects of heat diffusion. Therefore, the importance of
C
developing
a direct organelle thermometry method was
recognized. Kiyonaka et al12 has also developed a genetically
encoded fluorescent thermometer. The methodology is based on
a temperature sensitive coiledꢀcoil GFP and is capable of
measuring the temperature of a targeted organelle directly.
However, the technique is time consuming in terms of setting
up the gene expression.
Fig. 1 In vitro evaluation of MitoꢀRTP in various parameters. (A)
Chemical structure of MitoꢀRTP. Thermosensitive rhodamine B and
thermoinsensitive CS NIR dye were conjugated to each end of the
linker. (B) Fluorescence intensity ratio (Rhodamine B/CS NIR dye,
calculated from peak intensities of rhodamine B and CS NIR dye) was
determined at various temperatures (normalized at 34oC). Fluorescence
Department of Life Science & Medical Bioscience, Graduate School
of Advanced Science & Engineering, Waseda University, TWins 2ꢀ2
Wakamatsuꢀcho, Shinjukuꢀku, Tokyo 162ꢀ8480 (Japan). Eꢀmail:
takeoka@waseda.jp
spectra of (C) rhodamine B (λex = 563 nm) and (D) CS NIR dye (λex
=
722 nm) measured with a spectrophotometer every 3oC from 25oC to
43oC.
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Based on our previous technique9, we designed
a
completely merged (Fig. 2A right). These observations
mitochondriaꢀtargeting ratiometric temperature probe (Mitoꢀ indicated that MitoꢀRTP was easily able to permeate the cell
RTP). MitoꢀRTP is composed of two kinds of rhodamine dyes; membrane owing to its hydrophobicity and that 30 min
namely rhodamine B and CS NIR dye (Fig. 1A). The incubation was sufficient time to stain the mitochondria.
fluorescence intensity of rhodamine B decreased linearly with
We also evaluated the effect of defocusing on the
elevated temperature14. This property is due to the changes in fluorescence intensity ratio because mitochondria are
ratio (photons emitted/photon absorbed), which depends on the transported in any direction by motor proteins18. Fluorescence
rotation of diethylamino groups on the xanthene ring, resulting intensities of both rhodamine B and CS NIR dye showed a
in the decrease in the quantum yield with increasing maximal value at z = 0, where cells were in focus, and
temperature. While, the fluorescence intensity of CS NIR dye15 decreased by defocusing as the objective lens moved up and
was expected to be stable in various temperatures because the down from z = 0. The fluorescence intensity ratio, however,
molecular rotation in this structure is restricted. These two remained constant, demonstrating the advantage of
fluorophores were coupled at each end of the ratiometric measurement (Fig. 2 B).
a
hexamethylenediamine linker to provide a ratiometric property.
We further evaluated the temperature sensitivity of Mitoꢀ
Furthermore, due to their hydrophobicity, rhodamine RTP localized in mitochondria by observing the temperatureꢀ
fluorophores are known to readily pass through the cell dependent fluorescence intensities. The temperature of the
membrane and specifically localize to the mitochondria16. In extracellular buffer was controlled from 34oC to 41oC (0.5oC/5
order to maintain the stability of MitoꢀRTP, amide bonds of the min) using a stage incubator and measured with a digital
hexamethylenediamine linker were methylated to avoid thermocouple having an accuracy of ±0.1oC. The fluorescence
forming the deprotonation inducing interꢀmolecular quenching intensities of rhodamine B (Fig. 2 C) and CS NIR dye (Fig. 2D)
state15, 17. Thus, MitoꢀRTP stains the intracellular mitochondria during gradual heating were scattered due to the focus drift
within a short period of time (<30 min) after simply adding the along the zꢀaxis. However, a linear decline in the fluorescence
dye to the extracellular solution. The cells are then visualized in intensity ratio (rhodamine B/CS NIR dye) was clearly observed
order to monitor the mitochondrial thermodynamics. As such, as the extracellular temperature increased (r = ꢀ0.808, P =
the technique is unaffected by changes to the focal point, 1.10×10ꢀ88 by a Pearson’s correlation coefficient test) (Fig. 2
concentration of the fluorophore, pH or ionic strength.
E). The slope of the linear fit for the average values was ꢀ
MitoꢀRTP was synthesized by 10 steps of organic chemical 2.72%/oC. The accuracy of the temperature measurement was
reactions. A full description of each reaction step is described determined to be 0.6 oC.
in the Supporting Information (Fig. S1). Each compound was
characterized by NMR and ESIꢀMS (Fig. S2 – S12). The
A
fluorescence spectrum of MitoꢀRTP (Fig. S14) showed both
absorption and emission wavelengths of the thermally sensitive
rhodamine B and thermally insensitive CS NIR dye, which
were well separated for dual fluorescence imaging.
Temperature sensitivity of MitoꢀRTP from 25oC to 43oC
was evaluated in a cuvette with a spectrophotometer. As
B
anticipated, the fluorescence intensity of rhodamine
B
reversibly responded to changes in temperature (Fig. 1C),
whereas that of CS NIR dye was constant (Fig. 1D). The
fluorescence intensity ratio of these two fluorophores
(Rhodamine B/CS NIR dye, calculated from peak intensities of
rhodamine B and CS NIR dye) showed a linear decline with
increasing temperature (Fig. 1B, S15). From the slope of the
linear fit, the thermal sensitivity of MitoꢀRTP was ꢀ2.65%/oC.
Within living cells, conditions such as pH and ionic strength
vary between locations and with time. Fluorescent
thermometers should be insensitive to such environmental
parameters. In our MitoꢀRTP, the fluorescence intensity ratio
did not change when the pH was varied between 4 and 10 (Fig.
S16A) and ionic strength between 0 and 500 mM KCl (Fig.
S16B). These results indicate that MitoꢀRTP has a ratiometric
D
C
E
Fig. 2 Evaluation of MitoꢀRTP properties with a conventional
fluorescence microscope. (A) Localization of MitoꢀRTP in living HeLa
property with temperature. In addition, methylation of the cells. Fluorescence images of rhodamine B in MitoꢀRTP (Left), mitoꢀ
tracker green (center) and their merged images (right). Scale bar
indicate 5 ꢁm. (B) Focus independent fluorescence ratio of MitoꢀRTP.
Fluorescence images of rhodamine B (red) and CS NIR dye (green) at
each focus point (Left) Scale bar indicate 20 ꢁm. Fluorescence
intensities of both rhodamine B (red) and CS NIR dye (green)
approached the maximum at z = 0 and decreased with defocusing,
whereas the fluorescence ratio (black) was always constant (Right).
Fluorescence intensities of (C) rhodamine B (average values shown in
hexamethylenediamine linker prevents formation of the
quenching state of rhodamine fluorophores. This gives a stable
fluorescence property against environmental parameters within
the physiological range.
We then confirmed that MitoꢀRTP at a 1 ꢁM concentration
within a cancer cell line (HeLa cells). 1ꢁM MitoꢀRTP displayed
no significant cytotoxicity (Fig. S18) and this concentration
was used for all the HeLa cell experiments. The localization of red) and (D) CS NIR dye (average values shown in green) under
gradual heating from 34oC to 41oC (E) fluorescence intensity ratio
MitoꢀRTP was observed from the fluorescence intensity of
(average values shown in black). The linear fit of the average values of
rhodamine B and CS NIR dye with mitoꢀtracker green. The
the fluorescence intensity ratio indicate that thermal sensitivity was ꢀ
fluorescence images of both rhodamine B in MitoꢀRTP (Fig.
2.72%/oC (r = ꢀ0.808, n = 26 ROIs, P = 1.1×10ꢀ88 by Pearson’s
2A left) and mitoꢀtracker green (Fig. 2A center) were
correlation coefficient test).
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It was concluded that the ratiometric measurement of Mitoꢀ
In conclusion, we have successfully constructed a
RTP could appropriately compensate the focus drift caused by ratiometric fluorescent chemical thermosensor, which
cellular movement and transportation of mitochondria inside specifically localizes to mitochondria in a relatively short time
the cell. As such, MitoꢀRTP generates a temperatureꢀdependent period (< 30 min). We confirmed that MitoꢀRTP could work as
fluorescence change in mitochondria within living HeLa cells.
a thermosensor both in vitro and in living cells. Ratiometric
The utility of MitoꢀRTP as a mitochondrial thermosensor measurement using MitoꢀRTP was able to negate the effects of
was examined by monitoring mitochondrial temperature defocusing along the zꢀaxis caused by cellular movements and
changes induced by external chemical stimuli using HeLa cells. concentration changes in the fluorophore. This novel system
We
applied
carbonyl
cyanide
4ꢀ(trifluoromethoxy) enabled us to visualize the FCCPꢀcoupled temperature
phenylhydrazone (FCCP), which transports protons across the elevation in the mitochondria within HeLa cells. Mitochondrial
mitochondrial inner membrane, to prevent the coupling of ATP thermodynamics plays a key role in cellular activities related to
synthesis and oxidative phosphorylation, leading to heat homeostasis and energy balance. Therefore, MitoꢀRTP is a
generation12, 19, 20, 21. Fluorescence intensities of rhodamine B powerful tool for analyzing how mitochondria activate in living
(Fig. 3A) and CS NIR dye (Fig. 3 B) in MitoꢀRTP were widely cells. We believe this technique will provide information
distributed because each region of interest (ROI) (N = 5 cells, n concerning the relationship between cellular activities and
= 19 ROIs) detected signals from a different focus point. The changes in temperature.
fluorescence intensity ratio, however, showed a gradual
decrease from around 150 seconds after addition of FCCP (Fig.
3C), but was constant for the DMSO control (Fig. 3D). The
observed time lag (t = 0 to 150 s) is thought to be due to the
diffusion time of FCCP, which was added dropwise to the
extracellular buffer. The fluorescence intensity ratio of FCCP
treated cells (t = 330) significantly decreased compared with
We thank Dr. H. Nakamura (Waseda U.), Prof. E. Latz (Bonn
U.) and Dr. G. Horvath (Bonn U.), Prof. S. Ishiwata (Waseda U.), Dr.
K. Oyama (Waseda U.) and Dr. S. Arai (WABIOS) for microscopic
experiments and technical advices. This work was supported by
Waseda U. Grant for Special Research Project and the JSPS Coreꢀtoꢀ
Core Program.
that of the DMSO control cells (t = 330) (
student’s ꢀtest) (Fig. 3E). Fluorescence ratio images (Fig. 3G)
indicated that the heterogeneous temperature elevation in FCCP
treated cells (t = 330) compared with untreated cells (t = ꢀ90) (
=8.1 × 10ꢀ5 by student’s
ꢀtest) (Fig. 3F). Mitochondrial
P
= 3.7 × 10−5 by
t
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Fig. 3 Visualization of mitochondrial temperature in HeLa cells in the
presence of 10 ꢁM FCCP. Time course fluorescence signals of (A)
rhodamine B (average values shown in red) and (B) CS NIR dye
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(average values shown in black) (N = 5 cells, n = 19 ROIs). (D)
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