Rhodamines with a Chloronicotinic Acid Fragment for Live Cell Superresolution STED Microscopy**

Article abstract of DOI:10.1002/chem.202005134

Formylation of 2,6-dichloro-5-R-nicotinic acids at C-4 followed by condensation with 3-hydroxy-N,N-dimethylaniline gave analogs of the popular TAMRA fluorescent dye with a 2,6-dichloro-5-R-nicotinic acid residues (R=H, F). The following reaction with thioglycolic acid is selective, involves only one chlorine atom at the carbon between pyridine nitrogen and the carboxylic acid group and affords new rhodamine dyes absorbing at 564/ 573 nm and emitting at 584/ 597 nm (R=H/ F, in aq. PBS). Conjugates of the dyes with “small molecules” provided specific labeling (covalent and non-covalent) of organelles as well as of components of the cytoskeleton in living cells and were combined with fluorescent probes prepared from 610CP and SiR dyes and applied in two-color STED microscopy with a 775 nm STED laser.

Full text of DOI:10.1002/chem.202005134

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&
Fluorescent Dyes
Rhodamines with a Chloronicotinic Acid Fragment for Live Cell
Superresolution STED** Microscopy
Florian Grimm,[a] Jasmin Rehman,[a] Stefan Stoldt,[b] Taukeer A. Khan,[b] Jan Gero Schlçtel,[c]
Shamil Nizamov,[a] Michael John,[d] Vladimir N. Belov,*[b] and Stefan W. Hell[b]
Abstract: Formylation of 2,6-dichloro-5-R-nicotinic acids at
C-4 followed by condensation with 3-hydroxy-N,N-dimethyl-
aniline gave analogs of the popular TAMRA fluorescent dye
with a 2,6-dichloro-5-R-nicotinic acid residues (R=H, F). The
following reaction with thioglycolic acid is selective, involves
only one chlorine atom at the carbon between pyridine ni-
trogen and the carboxylic acid group and affords new rhod-
amine dyes absorbing at 564/ 573 nm and emitting at 584/
597 nm (R=H/ F, in aq. PBS). Conjugates of the dyes with
“small molecules” provided specific labeling (covalent and
non-covalent) of organelles as well as of components of the
cytoskeleton in living cells and were combined with fluores-
cent probes prepared from 610CP and SiR dyes and applied
in two-color STED microscopy with a 775 nm STED laser.
Introduction
of linkers and ligands (small molecules) for binding (covalent
or non-covalent) with biological targets. The dye-ligand conju-
gates were applied for selective in vivo labeling of proteins
and cell organelles in neurons, human fibroblasts, U2OS, COS-7
and HeLa cells using confocal and super resolution microsco-
py.[3,6,7e–g,8b,9]
A high demand on bright, photostable and selective fluores-
cent markers for optical microscopy of living matter stimulated
progress in the design, synthesis and screening of the new cell
permeable fluorophores.[1] The core structures are compact,[2]
and the whole dye molecules have reactive centers and, in
some cases, additional solubilizing groups.[3] These features
and the absence of a net electrical charge, are essential for
target-specific probes4 including fluoresceins,[5] rhodamines,[6]
their carbon,[6b,c,d,f] silicon[7] and germanium analogs, as well as
related structures.[8] The design of the probes is focused not
only on fluorescent dyes, but also includes the proper choice
The structures of “live dyes” are based on a xanthene (pyro-
nine) fluorophore and resemble “good old” 5’(6’)-carboxy-
N,N,N’,N’-tetramethylrhodamine[10] (X=O, R=OH). The meso
carbon atom of a positively charged fluorophore can react
with a carboxyl group in the ortho-position of the pendant
phenyl ring (C-3’). This leads to the equilibrium between the
zwitterionic (fluorescent) and spirolactone (non-fluorescent)
forms (Scheme 1). Tetramethylrhodamine (TMR) represents a
very simple and attractive scaffold for construction of the new
dyes. An additional carboxyl group attached to C-5’ or C-6’ is
used for bioconjugation. Thus, 2’,5’- or 2’,6’-dicarboxyphenyl
group (Scheme 1) remained the most conservative structural
[a] F. Grimm, J. Rehman, Dr. S. Nizamov
Abberior GmbH
Hans Adolf Krebs Weg 1, 37077 Gçttingen (Germany)
[b] Dr. S. Stoldt, Dr. T. A. Khan, Dr. V. N. Belov, Prof. Dr. S. W. Hell
Department of Nanobiophotonics
Max Planck Institute for Biophysical Chemistry (MPIBPC)
Am Fassberg 11, 37077 Gçttingen (Germany)
E-mail: vbelov@gwdg.de
[c] Dr. J. G. Schlçtel
Abberior-Instruments GmbH
Hans Adolf Krebs Weg 1, 37077 Gçttingen (Germany)
[d] Dr. M. John
Institute of Organic and Biomolecular Chemistry
Georg-August University
Tammannstr. 2, 37077 Gçttingen (Germany)
[**] STED: stimulated emission depletion
Supporting information and the ORCID identification number(s) for the au-
thor(s) of this article can be found under:
https://doi.org/10.1002/chem.202005134.
ꢀ 2021 The Authors. Chemistry - A European Journal published by Wiley-
VCH GmbH. 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 modifications or adaptations are
made.
Scheme 1. Membrane-permeant fluorophores in equilibrium between non-
fluorescent and fluorescent forms. For structures of 565pR, linkers and li-
gands, see Scheme 2 and Figure 3.
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element in all successful membrane-permeant dyes and was
not varied until very recently.[8,11] Incorporation of 2,6-dichloro-
nicotinic residue into the TMR structure represented a very
simple and attractive option to prepare analogs 4-TAMRA dye
(Scheme 2) which was reported very recently and demonstrat-
ed excellent performance in fluorescent probes.[11b] Due to the
presence of the (reactive) halogen atoms in the precursor com-
pounds 4-R (Scheme 2) we expected easy functionalization,
the red shifted emission and increased D0.5 values (see discus-
sion below). These were the main reasons why we embarked
on the synthesis and characterization of the new dyes 5-R (R=
H, F; Scheme 2) and fluorescent probes derived from them.
thylamino)phenol (3) was performed in the presence of air
oxygen, in order to provide oxidation of the intermediate
leuco-forms (the structures not shown) to dyes 4-R. We tried
various solvents (propionic acid, toluene, trifluoroethanol; with
and without acidic catalysts), but only in dichlorobenzene
rhodamines 4-R were formed in moderate yield. Careful addi-
tion of thioglycolic acid to the solutions of compounds 4-R in
DMF at room temperature resulted in selective substitution of
the chlorine between the carboxylic acid group and the nitro-
gen atom[12] and resulted in the new fluorescent dyes 5-R bear-
ing a short linker and the second carboxylate. The NMR spectra
of rhodamines 4-R and 5-R displayed broad signals at +258C;
probably due to protonation of the pyridine nitrogen. At ele-
vated temperatures (758C) the spectra could be interpreted,
and the signals assigned. For compound 5-H, the position of
the carboxymethylthio group is supported by the absence of
the NOE between the CH2S protons and any other protons in
the molecule. For compound 5-F, the position of the carboxy-
methylthio group is supported by the absence of the NOE be-
tween the CH2S group and the aromatic protons attached to
C-4 and C-5 of the xanthen moiety. For compound 5-F, the po-
sition of the carboxymethylthio group is additionally support-
ed by the relatively small high-field shift of 19F signal:
À129 ppm in 4-F and À125 ppm in 5-F (+4 ppm). If the car-
boxymethylthio group were attached to C-6’, we would expect
much stronger high-field shift of 27–31 ppm.[13] Having dyes 5-
R at hand we studied their photophysical properties (Table 1,
Figure 1 and S2) prepared conjugates with small molecules
(Figure 3) and tested them as fluorescent probes for imaging
of various organelles in fixed and living cells (Figures 2–5 and
Supporting Information). As a reference, we chose the structur-
ally and spectrally similar dye 4-TAMRA (Scheme 2) which per-
formed exceptionally well in STED microscopy.[11b] As a supple-
mentary dye we used carbopyronine 610CP-6’-CO2H6c (8)
whose versatility in bioconjugates[6b,d,9c] and spectral properties
allowed to combine it with 4-TAMRA[11b[ and dyes 5-R in label-
ling and superresolution STED microscopy. The wide applicabil-
ity of 610CP prompted to optimize the synthesis of this dye. In-
spired by the published transformations,[11b[ we proved that
Scheme 2. Synthesis of chloropyridine-containing rhodamine dyes 5-R as an-
alogs of 4-TAMRA (with the uniform numeration of the carbon atoms). Re-
agents and conditions: i) LDA, THF, À788C, 1 h; then DMF, À788C to RT, 1 h;
ii) 1,2-dichlorbenzene, 1208C, 3 h; iii) HSCO2H, Et3N, DMF, RT, 1 h.
Table 1. Photophysical properties of dyes measured in aqueous PBS at
pH 7.5
Results and Discussion
Synthesis (Scheme 2) and photophysical properties of the
new dyes
[c]
Dye
Absorption
lmax [nm]
(e, [MÀ1 cmÀ1])[a]
Emission
lmax [nm]
(Ffl)[b]
D0.5
Lifetime[d]
[ns]
The commercial availability of 2,6-dichloro-5-R-nicotinic acids
(1-R) allows to replace the benzoic acid residue in the classical
TMR molecule by 2,6-dichloro-5-R-pyridine fragments (R=H, F)
and synthesize compounds 4-R. The reactivity of thiols in nu-
cleophilic substitution of the activated chlorine atoms in pyri-
dines indicated the possibility to prepare 2-chloropyridine-con-
taining rhodamine dyes 5-R (R=H, F). Acids 1-R were lithiated
(at O and C-4 atoms) with an excess of lithium diisopropyla-
mide (THF, À788C) and then transformed into aldehydes 2-R
(existing in a cyclic form; see Supporting Information for de-
tails). Heating of compounds 2-R (R=H, F) with 3-(N,N-dime-
5-H (565pR)
5-F
564 (90 000)
double: 529/
573 (57 000)
551 (78 000)
609 (100 000)
589 (0.31)
597 (0.29)
24
15
2.2
2.0
4-TAMRA[e]
610CP-6’-COOH[f]
573 (0.41)
634 (0.59)
14
36
2.0
3.1
[a] e— extinction coefficient. [b] Ffl— fluorescence quantum yield (abso-
lute value). [c] Dielectrical constant of dioxane (D=2)— water (D=80)
mixture, in which the fluorescent form (Scheme 1) is present to 50% of
its maximal possible concentration (in pure water). [d] Lifetime of the ex-
cited state. [e] Scheme 2; data from ref. [11b]. [f] Figure 3; data from
ref. [6c].
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Figure 1. a) Normalized absorption (solid line) and emission (dashed line)
spectra of pyridine-containing rhodamine 5-H (565pR) in PBS at pH 7.5.
Green bar shows excitation at 561 nm and red bar represent 775 nm STED
laser. b) Normalized absorption (A/Amax) at lmax of 5-H (565pR) plotted vs. die-
lectric constant (D) of dioxane-water mixtures. D0.5 values (Table 1) corre-
spond to the intersection of interpolated graphs with A/Amax =0.5 line.
Figure 3. Chemical structures of fluorescent probes based on dye 5-H (5-F
for actin) connected via amide bounds and linkers with ligands (recognition
units) for tubulin (cabazitaxel), lysomes (pepstatin A), mitochondria [(4-car-
boxybutyl)triphenylphosphonium] and HaloTag fusion protein (HaloTag
amine O2). The values of the fluorescence quantum yields (in aq. PBS) are
given. Blue: structure of 610CP-6’-COOH (carbopyronine) dye incorporated
into tubulin probe (see Figure S6) and used in two-color STED imaging.
Figure 2. Confocal and STED microscopy (indirect immunofluorescence) of a
nuclear pore complex (NPC) protein NUP98 in fixed Vero cell labeled with
dye 5-H (565pR): a) STED and confocal (upper left corner) images of NPC
with a confocal overview of the whole nucleus (upper right corner), scale
bar 1 mm; b) example of an line profile of one NPC under confocal (black
line) and STED (red line) conditions; the Gaussian fits gave full-width-at-half-
maximum (FWHM) of 220 nm for confocal and 86 nm for STED image; c) dis-
tribution of FWHM values (N=309) under STED conditions. Confocal and
STED images were acquired using a pulsed 561 nm excitation laser (average
power at focal plane: 2.8 mW, 40 MHz). For STED images a pulsed 775 nm
STED laser (average power at focal plane: 60 mW, 40 MHz; pulse length:
1.1 ns).
tion microscopy is possible with the most widely used 775 nm
laser (see below).
The presence of organic solvents shifts the absorption and
emission spectra of compounds 5-R to the blue spectral
region, but only slightly. For example, in methanol and aque-
ous acetonitrile the absorption/ emission maxima of 5-H are
found at 556/ 580 and 559/ 586 nm, respectively; the e-values
are lower than in aqueous solutions (70 000 and 73 000 in
methanol and aq. acetonitrile, respectively). For compound 5-F,
the absorption/ emission maxima in methanol are found at
569 nm and 591 nm, respectively, while the extinction coeffi-
cient (75 000mÀ1 cmÀ1; one band with a shoulder typical for
xanthene dyes) is higher than the value measured in aqueous
PBS solution (see Table 1). Lower extinction coefficients in or-
ganic solvents are due to the presence of the colorless and
non-emissive “closed” forms in the equilibrium with the
“open”, zwitterionic, colored and fluorescent isomers
(Scheme 1). As a measure of the equilibrium between “open”
and “closed” forms (Scheme 1), we use the parameter D0.5: the
dielectric constant of the dioxane-water mixture at which the
normalized absorption A/Amax (or e/emax) of the dye is equal to
50% of the maximal value observed within the entire dioxane-
water gradient.[3,6b,d,e,f,9c,11b] The absorption spectra in dioxane-
water solutions were recorded, with water content varying
from 0 to 100% and dielectric constant (D) changing from 2.0
bis-triflate 6 can be used as a universal precursor to carbopyro-
nine dyes with 6’-COOH group, including 580CP and 610CP
(see Scheme 2 and Supporting Information).
The photophysical properties of these dyes (Table 1) are
given in aqueous buffer at pH 7.5; under conditions relevant
to living cells. The main absorption and emission bands of dye
5-H (565pR) are shown in Figure 1. Small bathochromic and
bathofluoric shifts (13 nm and 16 nm) observed for 565pR in
comparison with 4-TAMRA are explained by the presence of
the electron-acceptor pyridine ring with the chlorine substitu-
ent. Further red shifts were observed for compound 5-F (Fig-
ure S2), and are explained by the presence of the fluorine
atom.[14] All dyes in Table 1 (except 5-F) feature very similar
band shapes and Stokes shifts of 20–30 nm.
Dye 5-F has an absorption band in aqueous PBS with two
maxima at 529 and 573 nm, so that the extinction coefficient
measured at the maximum is lower, but the oscillator strength
(proportional to the band area) is roughly the same, as for
compound 5-H. Importantly, the emission bands of the new
dyes 5-R extend to the far-red region, and STED superresolu-
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Figure 5. Multicolor confocal volume-image (z-projection, 45 frames) of
sperm bundles inside Drosophila melanogaster adult testis (ex vivo). a) DNA
stained with Hoechst (“grey”), b) tubulin stained with 565pR-Tubu (“green”),
c) actin cones stained with SiR-Actin (“magenta”). d) Merge of three channels
shown in a–c. Images were recorded with excitation lasers 405 nm, 561 nm
and 640 nm (for Hoechst, 565pR and SiR dyes, respectively) on a Facility Line
microscope (Abberior Instruments GmbH) equipped with rainbow detection
module. Scale bar 10 mm.
Figure 4. Imaging of cellular structures and organelles in living cells stained
with 565pR probes (for structures, see Figure 3): a) confocal image of mito-
chondria network in a living human fibroblast labeled with 565pR-Mito with
a close-up (white frame; scale bar 2 mm) of a selected region; scale bar:
10 mm; b) STED and confocal (upper left corner) microscopy images of tubu-
lin network in a living U2OS cell stained with 565pR-Tubu; scale bar: 2 mm;
c) two-color image of lysosomes (confocal) stained with 565pR-Lyso (green)
and tubulin network (STED) stained with 610CP-Tubu in a living human fi-
broblast; scale bar: 2 mm; d) two-color STED and confocal (upper left corner)
images of a monoclonal vimentin-Halo expressing U2OS cell; vimentin and
tubulin were stained with a 565pR-Halo (green) and 610CP-Tubu (magenta)
probes, respectively; scale bar 2 mm. Confocal and STED images were record-
ed with Facility Line (a–c) or Expert Line microscopes (d) equipped with a
pulsed 561 nm (for 565pR-probes) and 640 nm (for 610CP-Tubu) excitation
lasers, respectively, and a pulsed 775 nm STED laser (repetition rate: 40 MHz;
pulse duration: 1.1 ns). The color separation in c was enabled by rainbow de-
tection.
SiR as a bench-mark dye[7a-g] or carbopyronines[6b,c]) suggest
that the dye and its conjugates may be fluorogenic. The rela-
tively low D0.5 values observed for the reported dyes are typical
for all rhodamines without halogen atoms (F, Cl) in the N-alkyl
groups (2,2,2-trifluorethyl) and/or xanthene fluorophore (C-3,
C-6).[3,6c] This may be a limitation typical for rhodamines, if only
fluorogenic probes are required (e.g., for “no wash” experi-
ments). Thus, low D0.5 values may help to exclude the dyes
from screening and save work in their synthesis. However, if
the fluorogenic effect is caused by the ligand (due to pi-pi
stacking of two aromatic systems),[6b] then the dyes with low
D0.5 values can be considered and screened.
to 80.4, respectively (Figure 1). This series of spectra allow to
evaluate the response of the dyes to the solvent polarity, cal-
culate and directly compare the D0.5 parameters of the dyes,
even if their absorption and emission spectra are different. 4-
TAMRA and compound 5-F (surprisingly!) have the lowest D0.5
values (14–15).[11b] Introduction of the chlorosubstituted pyri-
dine ring (an acceptor group) favors the closed-ring lactone
form (due to destabilization of the positive charge on the
meso carbon atom opposite to oxygen: C-1’) and increases D0.5
value of 565pR to 24 (Table 1). It is not clear, why the presence
of an additional electron-acceptor (fluorine) in compound 5-F
does not increase the D0.5 value even further. The D0.5 value of
610CP is higher (36), because the >C(CH3)2 center disrupts the
conjugation and does not stabilize the positive charge on a
fluorophore so efficient, as the oxygen atom does. Notably,
high D0.5 values are required but not sufficient for fluorogenici-
ty (fluorogenic response) of the dyes and their conjugates (in-
crease in fluorescence intensity upon selective binding with
the target). Thus, all non-fluorogenic dyes (4-TAMRA, 565pR)
have low values of D0.5, while higher values of D0.5 (e.g., 64 for
Fluorescent conjugates and imaging performance of the
dyes
Indirect immunofluorescence is a standard approach for evalu-
ation of the imaging performance under confocal and STED
conditions. The STED wavelengths in the near IR region
À775 nm for Abberior Instruments and Leica Microsystems,
765 nm for PicoQuant— are present in all commercial STED mi-
croscopes, because in this spectral region the absorbance of
cells and tissues is low. Therefore, as in the previous studies,
most of red-emitting dyes are designed for the use with
775 nm or 765 nm STED lasers. To prepare bioconjugates, com-
pounds 5-H and 5-F (Scheme 2) were converted into NHS
esters (for experimental details, see Supporting Information)
and conjugated with proteins or small molecules (ligands)
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(Figure 3), as recognition units for subcellular structures in
living specimen. The fluorescence quantum yields of com-
pounds in Figure 3 were measured in aqueous PBS and varied
in the range 28–34% for all conjugates of 565pR and was
lower (4.6%) for 5-F-Actin probe (probably due to stronger ag-
gregation of the fluorinated dye). The better performing dye
5-H (565pR) was conjugated to goat anti-rabbit IgG secondary
antibodies and tested in fixed cells for staining of nuclear
porin 98 (NUP98) protein (Figure 2). In a fluorescence micro-
scope, the NUP98 protein stained with fluorescent dyes looks
like bright dots of similar sizes. Under confocal and STED con-
ditions 565pR provided bright images with low background.
The fluorescent images of 309 NUP98 complexes were ana-
lyzed, and the optical resolution assessed as full-width-at-half-
maximum (FWHM) of the emission profiles of individual ob-
jects and gave an average value of 96 nm. For that we used a
pulsed 775 nm STED laser with an average power of 60 mW,
40 MHz measured at the focal plane and a pulse length of
1.1 ns. Although, the dye 565pR has a particularly weak absorb-
ance at 775 nm (Figure 1), a relatively strong improvement in
optical resolution was observed.
havior in many bioconjugates.[6b,c,d,f,9c] Different excitation
lasers and the optimized light distribution between two ac-
quisition channels enabled very efficient color separation and
negligible crosstalk, though the emission maxima of 565pR and
610CP were separated only by 40–45 nm and band shapes
were very similar (for optical spectra and excitation wave-
length, see Supporting Information). Since the dye 610CP can
be also exited with 561 nm laser light, we collected the emis-
sion of 565pR-Lyso in the range from 571 nm to 600 nm, cut-
ting off the possible emission of 610CP. The detection channel
for the probe 610CP-Tubu was set from 650 nm to 725 nm.
Therefore, the two channels in Figure 4c and S6 has been
nicely separated using the rainbow detection giving the op-
portunity to see movement of the lysosomes along the micro-
tubule filaments (Figure 4c and S6). The binding to mitochon-
dria (565pR-Mito), tubulin (565pR-Tubu), and lysosomes (565pR-
Lyso) was non-covalent, while the vimentin probe (565pR-Halo)
was applied in living U2OS cells expressing a vimentin-HaloTag
fusion protein[9c] and provided covalent binding (Figure 4d and
S4, S5). We also used 610CP-Tubu for co-staining. For imaging
we used the Abberior Instruments expert line equipped with
the detection windows of 580 nm À630 nm and 650 nm to
720 nm. The color separation of both probes was still possible,
although the weak emission from 610CP-Tubu was observed in
the detection window from 580 nm À630 nm.
The 565pR dye conjugates (Figure 3) were prepared and
tested in living human fibroblast and U2OS cells (Figures 4–6
and S3–S6).
We used microscopes manufactured by Abberior Instru-
ments GmbH (Expert and Facility Line), in order to get confocal
and STED images. The new model (Facility Line) has a pair of
gradient-coated light filters coupled with the so-called “rain-
bow detection module”, in order to freely define the edges of
the detection windows in the range between 400 and 800 nm.
This feature enables more freedom in choosing fluorescent
markers primarily according to their biolabeling performance,
with fewer limitations due to spectral overlap and predefined
detection intervals. All conjugates shown in Figure 3 gave spe-
cific and bright staining of mitochondria (565pR-Mito), tubulin
(565pR-Tubu), lysosomes (565pR-Lyso) and vimentin (565pR-
Halo). Most of the mitochondria-selective fluorescent probes
contain the positively charged and lipophilic triphenylphos-
phonium residue.[15] According to this principle, we used 5-(tri-
phenylphosphonium)pentanoic acid, linked it with 565pR via
1,6-diaminohexane spacer and thus prepared 565pR-Mito (Fig-
ure 4a). For staining of microtubules (Figure 4b and S3), we
prepared a fluorescent conjugate of the anticancer drug caba-
zitaxel. Along with docetaxel and larotaxel, cabazitaxel binds
(non-covalently) to tubulin and facilitates microtubule forma-
tion. It was shown[6f] that cabazitaxel[16] often provides superior
images than docetaxel which was used in most of the earlier
studies. Then we tested the lysosome probe (565pR-Lyso). Ly-
sosomes are cell organelles containing hydrolytic enzymes
which digest unwanted substances in the cytoplasm. As a
ligand we used pepstatin A, which inhibits cathepsin D— a
major lysosomal aspartic endopeptidase— and which has been
previously incorporated into the fluorescent probes.[6f,7f,8b,17]
We used 1,6-diaminohexane as a linker binding the carboxyl
groups of pepstatin A and 565pR dye. For better visualization
we co-stained the microtubulin filaments with a probe contain-
ing dye 610CP providing high specificity and fluorogenic be-
For staining of actin, we used conjugate of 5-F with the es-
tablished actin ligand des-bromo-des-methyljasplakinolide
(bound via l-lysine residue and 6-aminohexanoic acid linkers
with a fluorescent dye).[18,19] Actin protein plays a crucial role in
cellular function and motility.[20] It can be present either as a
monomer (G-actin; globular) or, upon polymerization, it may
form filaments (F-actin): flexible fibers with a diameter of 4–
7 nm and length of up to several micrometers. In living cells,
both forms of actin are present in equilibrium; they are essen-
tial for the proper mobility and contraction of cells during cell
division, cell motility, cytokinesis, vesicle and organelle move-
ment, cell signaling, as well as the establishment and mainte-
nance of cell junctions and cell shape. We tested the actin
probe based on dye 5-F (5-F-Actin in Figure 3) in concentra-
tions of 0.5 mm, 1 mm and 5 mm on living and fixed U2OS cells.
The specific and bright staining was observed only for fixed
cells, with concentration of 1 mm being the optimal one (see
Figure S7). No significant staining was observed with living
cells (also for the structural analog incorporating compound 5-
H). It is possible to explain these results by cell impermeability:
the low D0.5 values (Table 1) indicate that the dyes are zwitter-
ionic, and the zwitterions do not cross the membranes as
good as unpolar spirolactone forms do (Scheme 1). Another
explanation may be based on the presence of the pyridine
ring which might inhibit binding with native F-actin. The STED
effect demonstrated by the dye 5-F in Figure S7 is strong; it is
explained by the red-shifted emission band of 5-F (emission
maximum 597 nm) compared to the dye 5-H (589 nm; Table 1),
which already showed quite remarkable resolution improve-
ment in response to the STED laser (Figure 2).
To extend the scope of labeling, we stained sperm bundles
inside Drosophila melanogaster adult testis (ex vivo) with fluo-
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rescent probes 565pR-Tubu and SiR-Actin.[7e] Figure 6 shows
the specificity of vital tubulin (green) and actin (magenta) la-
beling (confocal mode) referenced by the position of DNA
(grey). It illustrates the highly dynamic nature, uniform direc-
tion, and characteristic morphology of sperm bundles and
actin cones inside Drosophila melanogaster adult testis, re-
vealed even in ex vivo imaging.
above. Remarkably, even in deeper stacks of the tissue stained
with the two probes 565pR-Tubu and 610CP-Actin, STED imag-
ing was possible and increased the optical resolution of the
fluorescent structures. Now, it was clearly seen that the fila-
ments of tubulin and actin did not co-localize.
Conclusions
The thickness and dynamic behavior of actin filaments
makes them an attractive object for observation with superre-
solution optical microscopy.[6d,18–20] Figure 5 shows confocal
and STED images of tubulin fibers stained with 565pR-Tubu
(green) and actin stained with 610CP-Actin[6d,18] (magenta) in a
Drosophila melanogaster wild type egg chamber (ex vivo). Ob-
serving the confocal counterpart, we cannot exclude co-locali-
zation of these structures. The STED images were acquired
with an Abberior Instruments (Facility Line) microscope accord-
ing to the optical settings given in Figure 6 and mentioned
Two fluorescent dyes related to the popular X-carboxy-tetra-
methylrhodamine (X-HO2C-TMR; x=4, 5, 6), but with a 6-chlor-
onictonic acid residue and a carboxymethylthio linker, were
prepared and characterized by absorption and emission spec-
tra. The D0.5 value for dye 5-H is somewhat higher than that of
the structurally related 4-TAMRA, and this suggests higher cell-
permeability of the probes based on 5-H dye. The emission
maxima of the new dyes 5-H and 5-F are far from 775 nm
(STED wavelength), but both dyes showed remarkable STED
effect and substantially improved the optical resolution in fluo-
rescence microscopy of fixed and living cells. To demonstrate
that, the dyes were conjugated with secondary antibodies and
small molecules: a taxol, pepstaine A, (w-aminoalkyl)triphenyl-
phosphonium bromide, Halo Tag amine, jasplakinolide deriva-
tive. We showed that introduction of a thiol linker is regiospe-
cific, and only one chlorine atom (between the carboxylic acid
residue and the pyridine-nitrogen) is substituted. The new fluo-
rescent probes were tested in living cells and in a tissue of dro-
sophila melanogaster and showed specific staining of the tar-
gets. The dye 5-H has been combined with “a red-shifted”car-
bopyronine 610CP-6’-COOH (whose synthesis was optimized)
and used in two-color staining experiments. Although the
emission spectra of 610CP and 5-H are very close, color separa-
tion was possible with the two detection windows of 580–
630 nm and 650–720 nm. An even better separation was ach-
ieved by using the so-called “rainbow detection”, when the de-
tection boarders were varied and set individually.
Acknowledgements
The authors are grateful to Dr. Holm Frauendorf and co-work-
ers for measuring HRMS (Institut für organische und biomole-
kulare Chemie der Georg-August-Universität Gçttingen). We
thank Dr. Leonard Drees for providing wild type Drosophila
melanogaster. For the help with STED microscopy and scientific
discussions, we thank Dr. Christian A. Wurm (Abberior Instru-
ments GmbH). We acknowledge the financial support by Bun-
desministerium für Bildung und Foschung: grants nr.
13N14120 to F.G. and J.R. and 13N14122 to S.W.H. Open access
funding enabled and organized by Projekt DEAL.
Figure 6. Confocal and STED images of Drosophila melanogaster wild type
egg chamber (ex vivo). a) Three color image of DNA (cyan) stained with
Hoechst, tubulin (green) stained with 565pR-Tubu and actin (magenta)
stained with 610CP-Actin probes. b) confocal image of a chosen area in a).
c) STED image of a chosen area in a). Images were recorded with a facility
line (Abberior Instruments GmbH) equipped with excitation lasers 405 nm,
561 nm and 640 nm (for Hoechst, 565pR and 610CP dyes, respectively), a
pulsed 775 nm STED laser (repetition rate: 40 MHz; pulse duration: 1.1 ns)
and rainbow detection. Scale bar 10 mm (in a) and 2 mm (in b and c).
Conflict of interest
The authors declare no conflict of interest.
Keywords: dyes/pigments · fluorescence · fluorescent probes ·
scanning probe microscopy
Chem. Eur. J. 2021, 27, 6070 –6076
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6075 ꢀ 2021 The Authors. Chemistry - A European Journal published by Wiley-VCH GmbH

Full Paper
doi.org/10.1002/chem.202005134
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Manuscript received: November 27, 2020
Accepted manuscript online: January 26, 2021
Version of record online: March 5, 2021
Chem. Eur. J. 2021, 27, 6070 –6076
www.chemeurj.org
6076 ꢀ 2021 The Authors. Chemistry - A European Journal published by Wiley-VCH GmbH