A Highly Photostable Near-Infrared Labeling Agent Based on a Phospha-rhodamine for Long-Term and Deep Imaging

Article abstract of DOI:10.1002/anie.201804731

Various fluorescence microscopy techniques require bright NIR-emitting fluorophores with high chemical and photostability. Now, the significant performance improvement of phosphorus-substituted rhodamine dyes (PORs) upon substitution at the 9-position with a 2,6-dimethoxyphenyl group is reported. The thus obtained dye PREX 710 was used to stain mitochondria in living cells, which allowed long-term and three-color imaging in the vis-NIR range. Moreover, the high fluorescence longevity of PREX 710 allows tracking a dye-labeled biomolecule by single-molecule microscopy under physiological conditions. Deep imaging of blood vessels in mice brain has also been achieved using the bright NIR-emitting PREX 710-dextran conjugate.

Full text of DOI:10.1002/anie.201804731

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Accepted Article
Title: A Highly Photostable Near-infrared Labeling Agent Based on a
Phospha-rhodamine for Long-term and Deep Imaging
Authors: Marek Grzybowski, Masayasu Taki, Kieko Senda, Yoshikatsu
Sato, Tetsuro Ariyoshi, Yasushi Okada, Ryosuke Kawakami,
Takeshi Imamura, and Shigehiro Yamaguchi
This manuscript has been accepted after peer review and appears as an
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To be cited as: Angew. Chem. Int. Ed. 10.1002/anie.201804731
Angew. Chem. 10.1002/ange.201804731
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10.1002/anie.201804731
Angewandte Chemie International Edition
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A Highly Photostable Near-infrared Labeling Agent Based on a
Phospha-rhodamine for Long-term and Deep Imaging
Marek Grzybowski,[a] Masayasu Taki,*[a,b] Kieko Senda,[c] Yoshikatsu Sato,[a] Tetsuro Ariyoshi,[d]
Yasushi Okada,[d,e] Ryosuke Kawakami,[f] Takeshi Imamura,[f] and Shigehiro Yamaguchi*[a,c]
Dedication ((optional))
Abstract: Various fluorescence microscopy techniques require
bright NIR-emitting fluorophores with high chemical and
photostability. Herein, the significant performance improvement of
phosphorus-substituted rhodamine dyes (PORs) upon substitution at
the 9-position with a 2,6-dimethoxyphenyl group is reported. The
thus obtained dye PREX 710 was used to stain mitochondria in living
cells, which allowed long-term and three-color imaging in the vis-NIR
range. Moreover, the high fluorescence longevity of PREX 710
long-term fluorescence imaging, which requires exposure to light
in multimolecular cellular environment.
To improve the photostability of organic fluorophores,
various strategies, such as the introduction of a triplet quencher
significantly[4] or the incorporation of a four-membered azetidinyl
group into rhodamine fluorophores[5] have been proposed. As
another approach, we have recently proposed structurally
reinforced p-conjugated skeletons that contain an electron-
withdrawing phosphine oxide (P=O) moiety.[6] Such P=O-
substituted fluorophores enable the acquisition of repeated
images in stimulated emission depletion (STED) microscopy,
where the fluorophores are exposed to an extremely intense
depletion laser field.[7]
allows tracking
a dye-labeled biomolecule by single-molecule
microscopy under physiological conditions. Deep imaging of blood
vessels in mice brain has also been achieved using the bright NIR-
emitting PREX 710-dextran conjugate.
The incorporation of
bathochromic shifts of the absorption and emission maxima.
Indeed, the replacement of the endocyclic oxygen atom in
a
P=O group also leads to
Recent advances in optical microscopy techniques, e.g. multi-
photon, super-resolution, and single-molecule microscopy, have
revolutionized our understanding of the structures and functions
of biomolecules at the sub-cellular and molecular level.[1] In
parallel, various fluorescence-labeling methods as well as
valuable fluorescent chemical probes and proteins have been
developed.[2] In particular, dyes that absorb and emit light in the
near-infrared (NIR) region are ideal to minimize interference
from autofluorescence, which is a major contributor to the
background noise in cells. However, most NIR-emitting dyes
currently used in bioimaging, especially cyanine-based
fluorophores, suffer from inherently low chemical and
photostability.[3] This significantly limits the utility of these dyes in
xanthene scaffolds with
a
P=O moiety generates the
corresponding fluorescein (POF),[8] rhodol (PORL),[9] and
rhodamine (POR)[10] derivatives (Figure 1a), which emit
fluorescence in the far-red to NIR region in PBS (pH = 7.4).
We have previously reported that POFs and PORLs exhibit
remarkable photostability, surpassing that of their oxygen- and
silicon-substituted counterparts.[8a,11] As for POR dyes, two
groups have independently reported dyes that contain
P(=O)Me[10a] and P(=O)OH or P(=O)OR moieties,[10b] and their
utility as NIR-emissive probes in fluorescence imaging. In the
present study, we synthesized a series of tetraethyl rhodamine
PORs that bear different aryl groups at the 9-position (Figure 1b).
We evaluated the substituent effects on the chemical and
photophysical properties and discovered that POR dye 1c
exhibited outstanding performances in multi-facet applications,
including not only multicolor imaging in the vis-NIR region, but
also long-term time-lapse data acquisition, single-molecule
analysis, and deep imaging in vivo.
[a]
Dr. M. Grzybowski, Prof. Dr. M. Taki, Prof. Dr. Y. Sato, Prof. Dr. S.
Yamaguchi
Institute of Transformative Bio-Molecules (WPI-ITbM)
Nagoya University, Furo, Chikusa, Nagoya 464-8601 (Japan)
E-mail: taki@itbm.nagoya-u.ac.jp, yamaguchi@chem.nagoya-
u.ac.jp
[b]
[c]
Prof. Dr. M. Taki
POR dyes 1a–c were synthesized by the treatment of the
common xanthone precursor
PRESTO, Japan Science and Technology Agency (JST)
4-1-8 Honcho, Kawaguchi, Saitama 332-0012 (Japan)
Ms. K. Senda, Prof. Dr. S. Yamaguchi
4
with the corresponding
aryllithium (1a,c) or Grignard (1b) reagents (Scheme S1). The
structure of 1b was determined by single- crystal X-ray
diffraction analysis (Figure S1). Unlike the oxygen-bridged
parent rhodamine, the xanthene skeleton of 1b is slightly bent
due to the larger diameter of the phosphorus atom (Figure S1c).
The dihedral angle between the mean planes of the two
benzene rings is 16.1(1)°. The analysis of the bond lengths in
the xanthene skeleton revealed a typical quinoidal character,
which is indicative of an efficient delocalization of the positive
charge over the entire p-conjugated skeleton (Figure S1b).
The photophysical properties of 1a–c were measured in 10
mM PBS at pH = 7.4 (Table S1, Figures S3–S5). These
compounds show absorption (labs) and emission (lem) maxima in
the NIR region with large molar absorption coefficients
Department of Chemistry, Graduate School of Science, and
Integrated Research Consortium on Chemical Sciences (IRCCS),
Nagoya University, Furo, Chikusa, Nagoya 464-8602 (Japan)
Dr. T. Ariyoshi, Prof. Dr. Y. Okada
Laboratory for Cell Polarity Regulation, Center for Biosystems
Dynamics Research (BDR), RIKEN, Suita, Osaka 565-0874 (Japan)
Prof. Dr. Y. Okada
[d]
[e]
Department of Physics and Universal Biology Institute (UBI),
Graduate School of Science, and International Research Center for
Neurointelligence (WPI-IRCN), The University of Tokyo, 7-3-1
Hongo, Bunkyo-ku, Tokyo 113-0033 (Japan)
Prof. Dr. R. Kawakami, Prof. Dr. T. Imamura
[f]
Department of Molecular Medicine for Pathogenesis, Graduate
School of Medicine
Ehime University, Shitsukawa, Toon-city, Ehime, 791-0295 (Japan)
Supporting information for this article is given via a link at the end of
the document.
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10.1002/anie.201804731
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and in a solution containing 10 mM GSH. Moreover, 1b and 1c
are stable at pH = 4–10, i.e., in the biologically relevant range.
Only under strongly basic conditions (pH > 11), 1b and 1c
undergo hydrolysis to the corresponding phospha-rhodols and -
fluoresceins, which we have already reported.[9a] Furthermore,
1b and 1c exhibited high stability in fetal bovine serum (FBS)
(Figure S17), suggesting that sterically protected PORs should
be suitable for practical applications in various biological
systems including in cells and blood.
a)
b)
R1
Ar
Y
N
R2
O
O
P
P
R
X
N
POF: X = OH, Y = O
PORL: X = NR’2, Y = O
POR: X = NR’2, Y = N+R’2
1a: R1 = H, R2 = Me
1b: R1 = R2 = Me
1c: R1 = R2 = OMe (PREX 710)
R = Ph, Me, OH, OEt
The steric bulk of the aryl group at the 9-position also plays a
crucial role for the improvement of the photostability. Among 1a-
c, compound 1c exhibited the highest photostability, i.e.,
negligible photobleaching (99.5% of the dye remained intact)
was observed even after a solution of 1c was exposed for 2 h
under a Xe lamp irradiation (435 mW/cm2), whereas a more
significant decrease was observed for 1a (17%) and 1b (13%)
under the same conditions (Figure S19). Although the exact role
of the methoxy groups in 1c for the suppression of the
photobleaching is not yet fully understood, these may affect the
kinetics of the dark states (triplet and radical anion) in the
photobleaching.[15] Overall, the examination of the photophysical
properties, chemical stability, and photoresistance revealed an
excellent potential for 1c as an NIR-emissive fluorophore. We
therefore named this dye PREX 710, where PREX has a
meaning of Photo-REsistant Xanthene dye.
We next prepared a PREX 710 derivative that bears an NHS
ester (PREX 710 NHS, Scheme S1) for labeling amino groups of
biomolecules, and then evaluated the performance of PREX 710
in fluorescence imaging. The high photostability of PREX 710 is
retained even in conjugation with an antibody. The PREX 710-
conjugated IgG displayed much higher resistance to
photobleaching in PBS buffer than fluorescent antibodies
labeled with commercially available NIR dyes such as Cy5.5,
Alexa Fluor 680, Alexa Fluor 700, and STELLA Fluor 700
(Figure S21). The photostability in the immunocytochemistry
application was further examined by repetitive imaging of HeLa
cells after staining microtubules with these dyes. Almost 80% of
the initial fluorescence intensity of PREX 710 was retained even
after continuous irradiation with an excitation laser at 670 nm for
10 min (174 images in total), whereas the emission faded
completely when using Cy5.5 under the same imaging
conditions (Figure 2a). Superior photostability of PREX 710 was
also observed in the comparison with STELLA Fluor 700
(Figures S31 and S32).
The photostability of PREX 710 in single-molecule imaging
was then assessed in comparison with Alexa Fluor 647, a widely
used cyanine dye.[16] Both dyes were conjugated to NeutrAvidin
to immobilize on a biotinylated coverslip. Fluorescence signals
were recorded by total internal reflection fluorescence
microscopy (Movie S1). After 2 min of observation (1200 frames
in total) at 60 W/cm2 irradiance, more than 80% of the initial
fluorescence intensity of PREX 710 remained (Figure 2b),
whereas the signal rapidly decayed within 30 s for Alexa Fluor
647 (Figure 2c). Under these imaging conditions, repetitive
blinking events was often observed with PREX 710 (Figure S23).
The single-step blinking behavior would reflect the transition to
and from the triplet state, which is a good indicator to guarantee
that signals from each single molecule are actually recorded.
The signals from single PREX 710 molecules were strong
Figure 1. Molecular structures of P=O-containing xanthene dyes. a) POF:
phospha-fluorescein; PORL: phospha-rhodol; POR: phospha-rhodamine. b)
PORs used in this study (1a-c).
(e ~1 ´ 105 M–1 cm–1) and sufficient fluorescence quantum yields
(FF ~0.13). Their longer labs/lem compared to those of PORs
involving P(=O)Me[10a] and P(=O)OEt[10b] moieties were ascribed
to the electron-withdrawing inductive effect of the phenyl group
on the phosphorus atom. Compound 1c, which bears a 2,6-
dimethoxyphenyl group, exhibited labs and lem values of 712 nm
and 740 nm, respectively (Figure S5), which are slightly longer
wavelengths than those of 1a and 1b, and hence indicative of an
electronic perturbation by the aryl group at the 9-position. The
brightness (FF ´ e) is comparable to the existing NIR-fluorescent
silicon-rhodamine dye SiR700.[12,13]
To assess the potential utility of 1a-c as labeling reagents,
we initially evaluated the chemical stability of these dyes against
nucleophiles. The absorption changes of their aqueous solutions
were monitored at pH = 4.2, 7.4, and 10.3. Compound 1a, which
contains a relatively unhindered o-tolyl group at the 9-position,
exhibited a gradual decrease to 40% of the initial value after 8 h
at pH = 10.3 (Figure S8). This degradation is probably due to the
nucleophilic addition of a hydroxide at the 9 position to form the
+
+
corresponding carbinol.[9] The pKR value of 1a (pKR = 8.8,
Figure S11), which is an index for the thermodynamic stability of
carbocations, is much lower than that of silicon-rhodamine dye
+
SiR650 (pKR = 10.8, Figure S12). This difference is due to the
strong electron-withdrawing effect of the phosphine-oxide moiety,
resulting in an enhanced electrophilic character of the positively
charged xanthene skeleton.
The high electrophilicity of 1a also manifests in its reactivity
toward a reduced form of glutathione (GSH), which is the most
abundant low-molecular-weight thiol in living cells. The
absorption and emission bands of 1a decreased to 9% of the
initial value when 10 mM GSH was added to a solution of 1a in
PBS (pH = 7.4) (Figure S13). The dissociation constant toward
GSH (Kd = 1.34 ± 0.05 mM, Figure S16) is much lower than that
of SiR650 (Kd > 100 mM), which carries the same o-tolyl
group.[14] Although, in general, it is considered that
decolorization of 1a caused by GSH is a fatal drawback as a
labeling reagent in fluorescence live-cell imaging, 1a with Kd
=
1.34 mM for GSH could serve as a promising NIR probe to
understand GSH homeostasis (0.5 – 10 mM) in living cells.
In contrast to the high reactivity of 1a, sterically protected 1b
and 1c with a 2,6-disubstituted phenyl group showed high
resistance toward nucleophiles. Negligible spectral changes
were observed for 1b and 1c both under weakly basic conditions
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enough and were traced for more than a few minutes. It should
be noted that the single-molecule fluorescence of conventional
dyes can be traced only for a few seconds under the same
conditions (Figure S24), and can only be extended to 10-30 s
even in a special buffer with additives and/or oxygen removal to
prevent bleaching.[4] In addition to the comparison in practical
conditions, we made a quantitative comparison by compensating
for the difference in the absorption coefficients of these dyes at
640 nm. Namely, five times stronger laser power was used for
the excitation of PREX 710 than for Alexa Fluor 647 (Figure
S25; Movies S2 and S3). The duration of the signals from single
molecules was statistically analyzed for PREX 710 (N = 278)
and Alexa Fluor 647 (N = 274). The average time constant for
photobleaching was estimated by fitting the frequency
distribution of the longevity to a single exponential distribution
(Figures S26 and S27). The time constant for PREX 710 was
34.6 s, more than 10 times longer than Alexa Fluor 647 (3.0 s).
The data were better fitted with a mixture of two exponential
distributions [PREX 710: t1 = 12.4 s (49.2%) and t2 = 52.9 s
(50.8%); Alexa Fluor 647: t1 = 1.7 s (85.7%) and t2 = 8.8 s
(14.3%)]. The longer time constant component would reflect the
process of bleaching after multiple rounds of blinking that was
not detected with the current imaging conditions (200-1000 ms
exposure). The higher mixing proportion (50.8%) for PREX 710
is consistent with the suppression of photobleaching from the
dark states as discussed previously. More practically, three
times more photons were detected from a single PREX 710
molecule than Alexa Fluor 647 before bleaching (photon budget),
even with our system adjusted to the spectrum of Alexa Fluor
647. These results collectively indicate that PREX 710 is far
more superior to conventional fluorescent dyes used as the
probe for the single fluorescent molecule imaging. Each single
molecule can be imaged and traced for much longer time or
much more frames without bleaching even in an environment
like in the living cells, where oxygen depletion or
supplementation with anti-fading reagents is difficult to apply.
PREX 710 itself is membrane-permeable and predominantly
localized in the mitochondria of living cells (Figure S34). Taking
advantage of this feature and the other characteristics of PREX
710, we wanted to demonstrate its high utility in practical
fluorescence imaging. Firstly, the NIR excitation and emission
properties of PREX 710 effectively reduce spectral cross-talk
with other dyes, which allows conducting multi-color imaging
beyond 500 nm. In fact, fluorescence signals observed from the
plasma membrane, nucleus, and mitochondria, which were
stained with DiI, SiR-DNA, and PREX 710, respectively, could
be clearly separated using a simple wide-field microscope
equipped with filter sets for commercially available dyes (Figure
3a). Moreover, we demonstrated the utility of PREX 710 as a
labeling reagent to track cell movement. For that purpose, HeLa
cells were stained with PREX 710 and monitored at intervals of
30 s (exposure time: 500 ms). It should be noted that the
movement of the mitochondria and acidic organelles in the cell
cycle including the cell division was successfully observed for
more than 7 h thanks to the low concentration of PREX 710 (0.2
µM) and the reduced phototoxicity of the irradiation conditions
using NIR light (Movie S4).[17] Although further evaluations of the
potential effects of PREX 710 on the cell behavior and
a)
PREX 710
Cy5.5
b) PREX 710
0 s
20 s
40 s
120 s
•••
c) Alexa Fluor 647
0 s
20 s
40 s
120 s
•••
Figure 2.
A comparison of the photostability. a) Confocal images of
microtubule immunolabeled with PREX 710 (left) and Cy5.5 (right) after the
rectangular area marked by dashed lines was repeatedly scanned for 10 min
with excitation light at 670 nm. Scale bar: 50 µm. Single-molecule images of
(b) PREX 710 and (c) Alexa Fluor 647 immobilized on a glass surface. Images
were acquired continuously at 10 frames per second for 120 s with excitation
light at 640 nm (60 W/cm2) in PBS at pH = 7.4.
metabolism are still required, PREX 710 and its derivatives
should be useful tools for the investigation of the dynamics of
living organisms, tissues, cells, and molecules.
Finally, we used PREX 710 for the in vivo imaging of blood
vessels in the deep region of mouse brain. For that purpose,
Aminodextran (70 kDa, Thermo Fisher) was labeled with PREX
710 NHS to provide the dextran-conjugate with a DOL value of
3.5, which was subsequently injected into the bloodstream via
the tail vein (7.0 mg/mL in PBS; injection volume: 0.1 mL).
Confocal images were recorded at an excitation wavelength of
638 nm and the 3-D image was reconstructed using 41 images
acquired at 10 µm Z-steps (depth: 400 µm, Figure 3b). Intense
fluorescence signals were observed up to a depth of ~0.3 mm
from the surface, despite the fact that the excitation efficiency of
PREX 710 at 638 nm is only approximately one-third of the
maximum, indicating sufficiently high brightness of the conjugate.
As the detection range for PREX 710 extends to much longer
wavelengths relative to those of other commonly used
fluorescent proteins for in vivo imaging such as YFP and
mCherry, PREX 710 should also be useful for multi-color deep
imaging with a two-photon microscope.[18]
In summary, we have developed the photostable, water-
soluble NIR fluorophore PREX 710 for multi-facet and high-
performance bioimaging applications. We have successfully
demonstrated that PREX 710 can be used for single-molecule,
deep-tissue, and long-term imaging, as well as multicolor-
fluorescence imaging in the vis-NIR region. PREX 710
selectively binds to mitochondria, while its NHS ester can be
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Environment and Materials” in “Network Joint Research Center
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a)
DiI
SiR-DNA
Keywords: near-infrared • labeling agent • photoresistance •
phospha-rhodamine • optical imaging
PREX 710
Overlay
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Figure 3. a) Three-color imaging of cell membrane (cyan), nucleus (yellow),
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PREX 710; scale bar: 20 µm. b) 3-D image of blood vessels in mouse brain
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We would like to thank N. Sugimoto (Nagoya University) for
support with cell biology experiments, and for Dr. M. Hirai for
assistance with X-ray diffraction analysis. This work was
supported by JSPS KAKENHI Grant Numbers 17H05839,
16K13097 (MT), JP15H05952, JP15H04962 (TI), 16H05119,
17H19511 (YO) and JP16H06280 (Advanced Bioimaging
Support); JST CREST JPMJCR15G2 (YO). MG thanks the
JSPS for a postdoctoral fellowship for foreign researchers. This
work was performed in part under the Research Program of
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Entry for the Table of Contents (Please choose one layout)
Layout 1:
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Photostable NIR dye: PREX 710 is
a P=O-containing rhodamine dye that
is substituted with a 2,6-
Marek Grzybowski, Masayasu Taki,*
Kieko Senda, Yoshikatsu Sato,Tetsuro
Ariyoshi, Yasushi Okada, Ryosuke
Kawakami, Takeshi Imamura, Shigehiro
Yamaguchi*
dimethoxyphenyl group at the C9-
position. The NIR-emission and high
fluorescence longevity of PREX 710
allow multi-color imaging, cell-
tracking, and long-term single-
molecule tracking under physiological
conditions as well as deep-tissue
imaging in vivo.
Page No. – Page No.
A Highly Photostable Near-
infrared Labeling Agent Based
on a Phospha-rhodamine for
Prolonged and Deep Imaging
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