Fluorescent Rhodamines and Fluorogenic Carbopyronines for Super-Resolution STED Microscopy in Living Cells

Article abstract of DOI:10.1002/anie.201511018

A range of bright and photostable rhodamines and carbopyronines with absorption maxima in the range of λ=500-630 nm were prepared, and enabled the specific labeling of cytoskeletal filaments using HaloTag technology followed by staining with 1 μm solution

Full text of DOI:10.1002/anie.201511018

Angewandte
Communications
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International Edition: DOI: 10.1002/anie.201511018
German Edition: DOI: 10.1002/ange.201511018
STED Microscopy
Fluorescent Rhodamines and Fluorogenic Carbopyronines for
Super-Resolution STED Microscopy in Living Cells
Alexey N. Butkevich,* Gyuzel Yu. Mitronova, Sven C. Sidenstein, Jessica L. Klocke,
Dirk Kamin, Dirk N. H. Meineke, Elisa D’Este, Philip-Tobias Kraemer, Johann G. Danzl,
Vladimir N. Belov,* and Stefan W. Hell*
Abstract: A range of bright and photostable rhodamines and
carbopyronines with absorption maxima in the range of l =
500–630 nm were prepared, and enabled the specific labeling
of cytoskeletal filaments using HaloTag technology followed
by staining with 1 mm solutions of the dye–ligand conjugates.
The synthesis, photophysical parameters, fluorogenic behavior,
and structure–property relationships of the new dyes are
discussed. Light microscopy with stimulated emission deple-
tion (STED) provided one- and two-color images of living
cells with an optical resolution of 40–60 nm.
dehalogenase), which is able to selectively and rapidly form
a covalent bond with the substrate.
The variety of fluorescent probes applicable for the
intracellular labeling of living cells is restricted owing to
cell-permeability requirements. Several rhodamine dyes,[4]
carbopyronines,[5] and silicon rhodamines (SiR)[6] are known
to penetrate the outer plasma membranes of intact cells,[7] and
methods for improving their brightness have recently been
proposed.[1h] Unfortunately, the spectral variety of photo-
stable fluorescent dyes suitable for intracellular targeting and
super-resolution imaging in living cells is quite limited, and
only few of them are commercially available (see the
Supporting Information, Figure S1).
In commercial STED microscopes, three depletion
regions are available: l = 592/595 nm, 660 nm, and 765/
775 nm.[8] Red (ꢀ 620 nm) or near-infrared (NIR; ꢀ 750 nm)
depletion lasers are advantageous as they cause less damage
to biological objects and benefit from reduced autofluores-
cence, photobleaching, and light scattering. Therefore, the
present study was focused on the design and evaluation of
new cell-permeant fluorophores for STED microscopy with
orange-red (618 nm) and NIR (775 nm) depletion lasers.
We learned empirically that cell-permeant probes are
preferably electronically neutral (or zwitterionic with a short
charge separation distance and zero net charge) and possess
a compact structure, limited molecular mass (M < 700 Da),
and several heteroatoms as hydrogen-bond donors and
acceptors. Based on these criteria, we prepared several
green-, yellow-, and red-emitting rhodamines, carbopyro-
nines, and silicon rhodamines (Scheme 1). The spectral
properties of these dyes are given in Table 1 and Figure S2.
Only the 6’-carboxy isomers of the fluorophores have been
shown to provide the successful labeling of proteins by means
of HaloTag and SNAP-tag self-labeling techniques.[9] There-
fore, we fixed the attachment of the HaloTag ligand to the 6’-
position (or a topologically identical position) of the pendant
aromatic ring and employed the original bis(oxyethylene)
linker.[3]
F
luorescent dyes are widely used as indispensable markers
in biology-related optical microscopy.[1] The selective, sensi-
tive, and stable imaging of cellular microstructure depends on
the optimal combination of several chemical, biological, and
physical factors. The availability and proper choice of
fluorescent markers—fluorescent proteins (FPs) or synthetic
fluorescent dyes—are key factors to the success of the entire
labeling and imaging sequence. Owing to their superior
brightness and photostability, synthetic dyes represent an
attractive alternative to fluorescent proteins.
Herein, we introduce a set of cell-permeant fluorescent
markers for living cells and apply them in one- and two-color
super-resolution optical microscopy with stimulated emission
depletion (STED).[2] For the specific labeling of intracellular
targets in living cells, we used a well-established and robust
procedure based on HaloTag fusion proteins.[3] In this
technology, the protein of interest is genetically fused with
an engineered enzyme (modified Rhodococcus rhodochrous
[*] Dr. A. N. Butkevich, Dr. G. Y. Mitronova, M. Sc. S. C. Sidenstein,
B. Sc. J. L. Klocke, Dr. D. Kamin, M. Sc. D. N. H. Meineke,
Dr. E. D’Este, Dr. P.-T. Kraemer, Dr. J. G. Danzl, Dr. V. N. Belov,
Prof. Dr. S. W. Hell
Department of NanoBiophotonics
Max Planck Institute for Biophysical Chemistry
Am Fassberg 11, 37077 Gçttingen (Germany)
E-mail: abutkev@gwdg.de
vbelov@gwdg.de
shell@gwdg.de
Homepage: http://www.mpibpc.gwdg.de/abteilungen/200/
The carbopyronines and Si-rhodamines 1 f–k were pre-
pared from substituted anthrones or 10-silaanthrones
(dibenzo[b,e]silin-10(5H)-ones) 2 as shown in Scheme 2.
This modular approach allowed us to choose the tricyclic
fragment of the fluorophore and the dicarboxylated aromatic
ring independently. Carbopyronine 1e was prepared from the
corresponding carbofluorescein ditriflate[5f] by Buchwald–
Hartwig amidation with BocNHMe[4d] (Boc = tert-butoxycar-
bonyl) followed by deprotection (see the Supporting Infor-
mation for the synthetic method and analytical data). While
Supporting information and ORCID(s) from the author(s) for this
article are available on the WWW under http://dx.doi.org/10.1002/
anie.201511018.
ꢀ 2016 The Authors. Published by Wiley-VCH Verlag GmbH & Co.
KGaA. This is an open access article under the terms of the Creative
Commons Attribution Non-Commercial NoDerivs License, which
permits use and distribution in any medium, provided the original
work is properly cited, the use is non-commercial and no modifica-
tions or adaptations are made.
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Scheme 2. General approach to the red-emitting dyes 1 f–k with a free
carboxyl group for conjugation. PG=protecting group.
conjugates substantially increased upon binding to their
target protein, whereas they emitted poorly in their free
state (or bound to off-target proteins or hydrophobic
structures) because the equilibrium was shifted to the non-
fluorescent spirolactone form (Scheme 1). It was therefore of
interest to correlate the fluorogenic behavior of the new dyes
with their performance in live-cell imaging.
Scheme 1. Membrane-permeant fluorescent dyes for STED microscopy
of living cells that are in equilibrium between non-fluorescent and
fluorescent forms (for the spectral properties, see Table 1).
To evaluate the response of our dyes to the
polarity of the medium, a series of absorption spectra
were recorded in aqueous dioxane solutions with
water contents from 10 to 100% and dielectric
constants (D) from 5.6 to 78.3 (Figure 1).[6h] As
expected, upon increasing the water content, the
colorless spirolactone forms of all dyes underwent
ring opening to the colored and fluorescent zwitter-
ionic forms. Rhodamine dyes 1b–d favor the zwit-
terionic form in all but the most non-polar solvent
systems, whereas the carbopyronines 1e–h and SiR 1i
exist predominantly in the lactone form in solutions
with > 50% dioxane content (D < 34.3). For the SiR
thiophene analogue (650SiR, 1j),[11] cyclization into
the colorless lactone form is impeded by the increase
in angle strain upon formation of a five-membered
lactone fused to a five-membered thiophene ring.
As a measure of the response of the dye to
changes in solvent polarity, we defined D0.5, the
dielectric constant of a dioxane–water mixture at
which the normalized absorption A/Amax (or extinc-
tion e/emax) of the dye is equal to one half of the
Table 1: Spectral properties of cell-permeant dyes in aqueous PBS buffer (pH 7.4) at
room temperature; STED at l=587 (1a), 618 (1b,c), and 775 nm (1d–k).
[c]
Dye
Absorption
Emission
lmax [nm]
Brightness
D0.5
Fluorescence
lifetime
t [ns]
lmax [nm]
rel. to SiR[b]
(e [mÀ1 cmÀ1]) (Ffl)[a]
500R (1a)
515R (1b)
520R (1c)
580R (1d)
580CP (1e)
610CP (1 f)
620CP (1g)
630CP (1h) 628 (6700)
SiR (1i)
650SiR (1j)
670SiR (1k) 670 (150)
501 (88000)
515 (56000)
521 (52000)
581 (58000)
582 (90000)
609 (100000)
617 (73000)
523 (0.93) 2.15
543 (0.86) 1.26
32.5 4.2
19.6 4.1
7.5 4.0
546 (0.79) 1.08
607 (0.95) 1.45
607 (0.69) 1.63
634 (0.59) 1.55
647 (0.17) 0.33
660 (0.06) 0.01
661 (0.41) 1 (ref.)
672 (0.36) 0.40
696 (0.03) 0.0001
<5.6 3.9
34.6 3.6
36.4 3.1
62.8 1.0
72.7 0.4
64.5 2.7
<5.6 2.5
645 (93000)
650 (42000)
–
1.9[d]
[a] See the Supporting Information for details. [b] Brightness relative to SiR:
(e”Ffl)dye/(e”Ffl)SiR. [c] See Figure 1 and the main text. [d] In methanol with 1%
(v/v) of trifluoroacetic acid.
fluorination of the rhodamine tricyclic core led to small blue
shifts of the absorption and emission bands (compare 515R
and 520R), fluorination of the carbopyronine (1g,h) and Si-
rhodamine (1k) cores resulted in dyes with red-shifted
absorption and emission maxima, so that the new analogues
are excitable at l = 600–670 nm (Table 1). However, the
emission quantum yields and fluorescence lifetimes of the
fluorinated carbopyronines (1g,h) and Si-rhodamine dye 1k
were significantly reduced.
Fluorogenic behavior (a marked fluorescence intensity
increase in response to an intracellular event of interest)[10]
has recently been observed for the SiR dye 1i bearing
a HaloTag, SNAP-tag, or CLIP-tag ligand,[6h] and for SiR-
based actin and tubulin probes.[6k] The emission of these
maximal value observed across the entire dioxane–water
gradient (Figure 1). On this scale, low values of D0.5 corre-
spond to non-fluorogenic dyes (rhodamines 520R and 580R
(1c,d) and the SiR thiophene analogue 1j), whereas inter-
mediate (20–40: fluorinated rhodamines and carbopyronines
1a,b,e, and f) and high values of D0.5 (60–70: 1g, SiR)[13]
indicate fluorogenic behavior. Dye 1k (D0.5 > 70) exists
predominantly in the colorless lactone form and will likely
be too dark for imaging. As evident from Figure 1, fluorina-
tion of the fluorophore core (rhodamine 1b vs. 1c and
carbopyronines 1g,h vs. 1 f) or the introduction of N-
trifluoroethyl groups (1a) favored lactone ring closure in
more polar solvent systems. This effect can be explained by
the destabilization of the positive charge at the C9 position of
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Figure 1. Normalized extinction, e/emax, at lmax of the dyes 1a–j versus
the dielectric constant, D, of dioxane–water mixtures. The D0.5 values
Figure 2. Confocal and STED images (raw data) of vimentin filaments
in a living HeLa cell that expresses vimentin–HaloTag fusion protein
after incubation with 520R (1c-Halo; 1 mm for 20 min). a) STED image
with the corresponding confocal data in the bottom left corner.
b,c) Magnified views of the region marked in (a) of the confocal and
STED images, respectively. Optical resolution ca. 40 nm (see Fig-
ure S9). Scale bars: 500 nm; excitation power: 1.6 mW; STED power:
14 mW; pixel dwell time: 20 ms; pixel size: 18 nm for the STED, 20 nm
for the confocal image. Each line was scanned twice, and the counts
were accumulated.
correspond to the intersection of interpolated graphs with a e/emax
=
0.5 line.[12] The absorbance of 580CP (1e), 650SiR (1j), and rhodamine
dyes 1a–d is non-monotonic at high D values owing to fluorophore–
solvent interactions or dye aggregation.[6h]
the xanthene, dihydroanthracene, or dihydrosilaanthracene.
On the contrary, the replacement of N,N-dimethylamino (1 f)
with N-methylamino groups (1e) did not significantly affect
D0.5, which may reflect the comparatively small difference
between the Hammett sp parameters for these substituents
(À0.83 vs. À0.70).[14]
resolutions of approximately 40 nm for 500R and 520R and
approximately 50 nm for 515R in living samples (Figures S7–
S9). The dyes 515R and 520R were found to be more
photostable than Citrine under STED conditions (Fig-
ure S10), and the photostabilities increase in the order
Citrine < 515R < 520R.
In the red spectral region (with a STED wavelength of
775 nm), the derivatives of rhodamine 580R (1d-Halo),
carbopyronines (1e–h-Halo), and Si-rhodamines (1i–k-
Halo) were tested as fluorescent markers for the vimentin–
HaloTag fusion protein. For three dyes (580R, 580CP, and
610CP; Figure 3), the confocal and STED images were bright
and free from background fluorescence, and the staining was
uniform. The optical resolution was ꢁ 70 nm for 580CP,
ꢁ 60 nm for 580R, and approximately 50 nm for 610CP
(Figures S11–S13). The dyes 620CP and 650SiR gave more
background (Figures S15, S16), whereas the difluorinated
dyes 630CP and 670SiR produced insufficiently bright
images.
The two dyes 580R and 580CP can be combined with
SiR[6h,k] in two-color STED microscopy as all three can be
depleted at l = 775 nm with a single STED laser, ensuring
perfect alignment of the two color channels.[4e] Whereas SiR is
excited at l ꢂ 640 nm, and its fluorescence is detected at 650–
700 nm, 580R and 580CP can be excited at l ꢂ 560 nm or
590 nm and detected at 605–625 nm, enabling clear color
separation for 580R (Figure S5) with low crosstalk between
the channels. Figures 4 and S17 show two-color STED images
of living HeLa cells (vimentin fibers labeled with 580R or
580CP, respectively; tubulin labeled with a SiR tubulin
probe).[6k] As evident from both Figures, a larger cross section
for stimulated emission of the red-shifted dye at the STED
wavelength results in higher optical resolution for the long-
wavelength (SiR) channel. An independent choice of the
STED power for each channel would improve the resolution
In agreement with this trend, fluorinated carbopyronines
showed a significant fluorogenic response, comparable to that
of SiR. A more than ten-fold increase in fluorescence
intensity was observed upon covalent binding of 1g,h to the
HaloTag protein (Figure S3), whereas the conjugates of the
rhodamine dyes 1b–d-Halo, 610CP (1 f-Halo), and 650SiR
(1j-Halo) were much less responsive.
The magnitude of the response parallels the increase in
fluorescence intensity of the tagged dyes 1a–k-Halo, bound
nonspecifically and reversibly to bovine serum albumin, upon
addition of the anionic surfactant sodium dodecyl sulfate
(SDS, see Figure S4), as shown previously for SiR.[6h,k]
Interestingly, when the cationic surfactant cetyltrimethylam-
monium bromide was added instead of SDS, the fluorescence
intensity decreased (Figure S4), suggesting that the fluoro-
genic response of the dyes 1g, 1h, and SiR may (at least
partially) be due to interactions of the positively charged
tricyclic fluorophore core with the local negative charges on
the HaloTag protein surface in immediate proximity to its
active site.
Three structurally similar green-emitting rhodamines,
500R, 515R,[4b] and 520R (1a–c), were designed for STED
wavelengths of 590 nm[8] and 618 nm (this work) applicable
for the fluorescent proteins YFP and Citrine. For live-cell
nanoscopy, we labeled vimentin filaments in living cells
expressing a vimentin–HaloTag fusion protein (515R: HeLa,
U2Os, Ptk2; 500R, 520R: HeLa; Figure 2). Upon incubation
with 1 mm of the dye–ligand conjugates 500R-, 515R-, and
520R-Halo for 20 min and washing for 10 min in dye-free cell
culture medium, the cells exhibited bright and uniform
staining of the vimentin fibers with virtually no nonspecific
background fluorescence. STED microscopy with a 587 nm
(1a) or 618 nm (1b,c) pulsed depletion laser afforded optical
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and CLIP-tags, as their recognition units are inherently more
hydrophilic than the HaloTag ligand.
Even though fluorogenic properties are advantageous for
fluorescent labels, the rhodamine derivatives 1a–d showed no
distinct correlation between their D0.5 values and image
quality, as all four dyes bound specifically and produced
images with negligible background (Table 1). The excellent
performance of the dye 580R in live-cell imaging despite its
bulky and rigid polycyclic rhodamine core suggests that the
hydroxylation of a fluorophore is a valuable tactic for
rendering the dye hydrophilic, highly polar, and soluble in
aqueous buffers without compromising its cell permeability.
The choice of fluorescent markers for live-cell nanoscopy
will often be guided by the available instrumentation. The
green-emitting rhodamines 500R (1a), 515R (1b), and 520R
(1c) performed exceptionally well with depletion lasers at
587 nm (1a) and 618 nm (1b,c). Dye 520R is superior both in
terms of attainable optical resolution and photostability in
repeated imaging, making it potentially suitable for the time-
lapse nanoscopy of dynamic biological processes. Taking into
account that in STED microscopy, green/yellow FPs clearly
outperform their red counterparts, the new synthetic dyes are
advantageous over FPs in terms of their imaging performance
in all spectral regions. With depletion at 775 nm, both
rhodamine 580R (1d) as well as the carbopyronines 580CP
and 610CP (1e,f) performed well in live-cell imaging. As
longer depletion wavelengths offer the advantage of dimin-
ished interactions with biological matter, the combination of
SiR with 580R or 580CP dyes enables high-quality two-color
live-cell nanoscale imaging and will be a natural choice for the
most widely used 775 nm STED laser.
Figure 3. Confocal and STED images (raw data) of vimentin filaments
in a living HeLa cell that expresses the vimentin–HaloTag fusion
protein after incubation with 610CP (1 f-Halo; 1 mm for 20 min).
a) STED image with the corresponding confocal data in the bottom left
corner. b,c) Magnified views of the region marked in (a) of the
confocal and STED images, respectively. Optical resolution ca. 50 nm
(see Figure S13). Scale bars: 500 nm; pixel dwell time: 20 ms; pixel
size: 20 nm for both the STED and the confocal image. Each line was
scanned twice, and the counts were accumulated.
Acknowledgements
Figure 4. Two-color STED image (raw data) of vimentin (green) and
tubulin (red) in a living HeLa cell. Vimentin filaments were labeled
with 580R applied as 1d-Halo (1 mm) via the HaloTag fusion protein,
whereas endogenous tubulin was directly labeled with the SiR tubulin
probe[6k] (0.5 mm). a) STED image with the corresponding confocal
image in the bottom left corner. b,c) Magnified views of the region
marked in (a) of the confocal and STED image, respectively, of the two
colors. Scale bars: 2 mm; pixel dwell time: 40 ms for the SiR tubulin
channel (red), 20 ms for 580R (green); pixel size: 20 nm for the STED
and the confocal image. Color channels were recorded pixel by pixel;
each line was scanned three times, and the counts were accumulated.
We thank Prof. Y. Okada (RIKEN Quantitative Biology
Center, Osaka, Japan) for the gift of b-tubulin-Halo plasmid,
T. Gilat and Dr. E. Rothermel (MPIBPC, Gçttingen,
Germany) for cell culture and transfection, M. Pulst, J.
Bienert (MPIBPC), Dr. M. John, Dr. H. Frauendorf, and co-
workers (Institut für Organische und Biomolekulare Chemie,
Georg-August-Universität, Gçttingen, Germany) for UV/Vis,
NMR, and ESI-MS spectra, Prof. M. L. Bossi (University of
Buenos-Aires, Argentina) for measuring fluorescence life-
times, and Dr. S. Vos and Prof. P. Cramer (MPIBPC) for
access to a Tecan microplate reader. S.W.H. acknowledges
a grant from the Bundesministerium für Bildung und
Forschung (BMBF 513) within the program “Optische
Technologien für Biowissenschaften und Gesundheit” (FKZ
13N11066). J.G.D. was supported by funds from the People
Programme (Marie Curie Actions) of the European Unionꢀs
Seventh Framework Programme (FP7/2007–2013; REA grant
agreement PIEF-GA-2011-299283).
in the green channel and increase the signal intensity in the
red channel.
In conclusion, the position of the equilibrium responsible
for the fluorogenic properties of dyes 1a–k (Scheme 1) can be
represented by a D0.5 value (Table 1), which can be explained
in terms of electronic factors and predicted for future dye
analogues. The fluorogenic behavior of a dye can be tuned by
the introduction of fluorine atoms in the alkyl chains (e.g.,
500R), the fluorophore core (515R, 620CP), or the pendant
aromatic ring.[15] The principles of dye design exemplified in
this study may enable the development of fluorogenic dyes of
other classes (e.g. coumarins, rhodols, acridines, Ge-[6c] and
P-rhodamines).[16] We expect that our fluorescent dyes will
perform just as well, or even better, when used with SNAP-
Keywords: carbopyronines · fluorescence · living cells ·
optical microscopy · rhodamines
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3294 www.angewandte.org
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Angew. Chem. Int. Ed. 2016, 55, 3290 –3294