Hydroxylated Fluorescent Dyes for Live-Cell Labeling: Synthesis, Spectra and Super-Resolution STED

Article abstract of DOI:10.1002/chem.201701216

Hydroxylated rhodamines, carbopyronines, silico- and germanorhodamines with absorption maxima in the range of 530–640 nm were prepared and applied in specific labeling of living cells. The direct and high-yielding entry to germa- and silaxanthones tolerates the presence of protected heteroatoms and may be considered for the syntheses of various sila- and germafluoresceins, as well as -rhodols. Application in stimulated emission depletion (STED) fluorescence microscopy revealed a resolution of 50–75 nm in one- and two-color imaging of vimentin-HaloTag fused protein and native tubulin. The established structure–property relationships allow for prediction of the spectral properties and the positions of spirolactone/zwitterion equilibria for the new analogues of rhodamines, carbo-, silico-, and germanorhodamines using simple additive schemes.

Full text of DOI:10.1002/chem.201701216

A Journal of
Accepted Article
Title: Hydroxylated fluorescent dyes for live cell labeling: synthesis,
spectra and superresolution STED** microscopy
Authors: Vladimir N Belov, Alexey N Butkevich, Kirill Kolmakov, Viktor
V Sokolov, Heydar Shojaei, Sven C Sidenstein, Dirk Kamin,
Jessica Matthias, Rifka Vlijm, Johann Engelhardt, and Stefan
W Hell
This manuscript has been accepted after peer review and appears as an
Accepted Article online prior to editing, proofing, and formal publication
of the final Version of Record (VoR). This work is currently citable by
using the Digital Object Identifier (DOI) given below. The VoR will be
published online in Early View as soon as possible and may be different
to this Accepted Article as a result of editing. Readers should obtain
the VoR from the journal website shown below when it is published
to ensure accuracy of information. The authors are responsible for the
content of this Accepted Article.
To be cited as: Chem. Eur. J. 10.1002/chem.201701216
Link to VoR: http://dx.doi.org/10.1002/chem.201701216
Supported by

10.1002/chem.201701216
Chemistry - A European Journal
FULL PAPER
Hydroxylated fluorescent dyes for live cell labeling: synthesis,
spectra and superresolution STED** microscopy
Alexey N. Butkevich,* Vladimir N. Belov,* Kirill Kolmakov, Viktor V. Sokolov, Heydar Shojaei, Sven C.
Sidenstein, Dirk Kamin, Jessica Matthias, Rifka Vlijm, Johann Engelhardt and Stefan W. Hell*
Abstract: Hydroxylated rhodamines, carbopyronines, silico- and
germanorhodamines with absorption maxima in the range of 530–
640 nm were prepared and applied in specific labeling of living
cells. The direct and high-yielding entry to germa- and
silaxanthones tolerates the presence of protected
heteroatoms and may be considered for the syntheses of
various sila- and germafluoresceins, as well as -rhodols.
Application in stimulated emission depletion (STED) fluorescence
microscopy revealed a resolution of 50–75 nm in one- and two-
color imaging of vimentin-HaloTag fused protein and native
tubulin. The established structure-property relationships allow
prediction of the spectral properties and the positions of
spirolactone/zwitterion equilibria for the new analogs of
rhodamines, carbo-, silico- and germanorhodamines using simple
additive schemes.
(covalent ligands of the SNAP-tag,[7] CLIP-tag,[8] and HaloTag
proteins[9]). Several non-covalent ligands (such as docetaxel,
jasplakinolide, or pepstatin A binding to native β-tubulin,[2b] F-
actin[2b] or aspartyl proteases in lysosomes,[2c] respectively)
have been successfully used as conjugates with fluorescent
dyes (see Figure S1, Supporting Information). However, the
affinity and staining specificity of a given marker depends on
the cell line, the nature of the ligand and the dye, the length of
the linker between them and the cell staining buffer
composition.[2b] Moreover, the spectral variety of photostable
dyes suitable for live cell superresolution microscopy still
remains limited.
To address some of the above-stated limitations, we have
recently proposed the introduction of hydroxyl groups into the
fluorescent dye molecules as a method for increasing polarity,
improving solubility in water and preventing unspecific binding.
In our previous study, we reported the dye 580R[1a, 10] (a live-
cell two-color STED imaging marker), a rhodamine with two
hydroxyl groups in allylic positions. In 580R and its
predecessor Atto 590,[1b] the conjugated alkene bonds in
dihydroquinoline fragments provide the bathochromic and
bathofluoric shifts.[1a, 10] However, these fragments are prone
to photooxidation and may negatively affect the
photostablity.[11] Aimed at developing methods for preventing
unspecific binding and improving water solubility of
fluorescent dyes for living cells, we have designed
fluorophores with additional hydroxyl groups (530RH and
575RH, Figure 1) in non-allylic and non-benzylic positions.
Among the multitude of fluorophores reported so far, only
rhodamines,[1] carbopyronines,[1a] and silicon-rhodamines
(SiR)[2] bearing a carboxyl in the ortho-position of the pendant
aromatic ring provide specific vital labeling and perform well
in superresolution fluorescence microscopy. These dyes exist
in equilibrium between zwitterionic (fluorescent) and
spirolactone (non-fluorescent) forms. Many cationic lipophilic
triarylmethanes bind non-specifically and stain membrane
structures.[3]
Numerous
anionic
fluorescent
labels,
commercially available as sulfonates or phosphates, are
hydrophilic and highly water-soluble but do not penetrate the
plasma membrane and are therefore used nearly exclusively
in immunostaining of fixed cells; alternative strategies of
membrane-impermeant label delivery employ cell-penetrating
peptide conjugates[4] and reversible permeabilization.[5]
On the contrary, several rhodamine-type dyes specifically
stain intact cells when applied as conjugates with small
molecule recognition units.[1a, 2a, 2c, 6] The most widely used
recognition units are BG-NH2, BC-NH2 and HaloTag amine
[*]
Dr. A. N. Butkevich, Dr. V. N. Belov, Dr. K. Kolmakov,
Dr. H. Shojaei, Dr. S. C. Sidenstein, Dr. D. Kamin,
Prof. Dr. Dr. h. c. S. W. Hell, Department of
Nanobiophotonics, Max Planck Institute for Biophysical
Chemistry (MPIBPC), Am Fassberg 11, 37077 Göttingen,
Germany
E-mail: abutkev@gwdg.de, vbelov@gwdg.de, shell@gwdg.de
Website: http://www.mpibpc.gwdg.de/abteilungen/200/
Dr. V. V. Sokolov, Department of Chemistry, St. Petersburg
State University, Universitetskiy Pr. 26, 198504 St.
Petersburg, Russia
MSc J. Matthias, Dr. R. Vlijm, Dr. J. Engelhardt, German
Cancer Research Center (DKFZ), Im Neuenheimer Feld 280,
69120 Heidelberg, Germany
Figure 1. Membrane-permeant and fluorescent rhodamines (R),
carbopyronines (CP), silicon- (SiR) or germano-rhodamines (GeR)
designed for STED microscopy of living cells (in free dyes, R = OH; in dye-
ligand conjugates, R = NH-ligand or NH-linker-ligand). H in names stands
for hydroxylated fluorophores.
[**]
STED: stimulated emission depletion
Supporting information for this article is given via a link at the
end of the article.
This article is protected by copyright. All rights reserved.

10.1002/chem.201701216
Chemistry - A European Journal
FULL PAPER
conjugation between the σ* orbitals of exocyclic X–R bonds
and the π*-system of the X-containing tricyclic fragment.[12]
The extent of the red shift falls in the order Si > Ge > Sn (>>
C), as the efficiency of σ*–π* overlap decreases with the
increasing atomic radius of X and C–X bond length. The
corresponding Sn-pyronine dyes are unstable; however, GeR
dye, the direct Ge analog of the widely used fluorogenic dye
SiR,[2a] and its bis-azetidinyl analog (Ge analog of the dye
JF646[6]) remain unknown[13] (Figure S2). To investigate the
properties of the new fluorophores and the influence of
hydroxylation on staining specificity, we prepared dyes listed
in Table 1 (see also Figure S3).
Bis-hydroxylated dye 530RH with 6'-carboxy-Q-rhodamine
(RhQ-CO2H)[14] core was prepared according to Scheme 1,a.
While the parent RhQ-CO2H is cell-permeant and its conjugate
with HaloTag amine provided specific staining in living
cells,[1d] the dye is poorly soluble in aqueous media and its
secondary amino groups are prone to acylation, rendering its
NHS ester unstable. To get rid of these drawbacks, a new
dye 530RH, having two 2,2,2-trifluoroethyl groups to block its
nitrogen atoms from acylation and two hydroxyl groups to
offset the hydrophobic properties of N-trifluoroethyl
substituents, was designed. These substituents impart only
slight bathochromic and bathofluoric shifts but shift the
equilibrium between zwitterionic and spirolactone forms
significantly towards the latter,[1a] facilitating cell membrane
penetration. Following the same logic, the dye 560CP (Figure
S4) and its hydroxylated analog 570CPH were designed as
useful derivatives of the N,N'-unsubstituted carbopyronine,
which itself has not yet found any application in fluorescent
labeling.
Table 1. Spectral properties of cell-permeant dyes in aqueous PBS buffer (pH
7.4) at room temperature (STED at 775 nm, unless noted otherwise).
Absorption
λmax, nm
Emission
λmax, nm
(Φfl)[a]
Fluor.
lifetime
τ, ns
Brightness
[c]
Dye
D0.5
rel. to SiR[b]
(ε, M-1 cm-1)
RhQCO2H[1d]
530RH
540 (70000)
532 (56000)
561 (0.79)
1.45
1.31
<5.6
29.6
4.0
4.0
553
(0.89)[d,e]
560CP
570CPH
6-ROX[f]
575RH
GeR
561 (61000)
571 (79000)
575 (82000)
574 (55000)
634 (97000)
631 (61000)
645 (93000)
641 (51000)
588 (0.76)[e]
600 (0.71)
602 (0.76)
597 (0.74)
655 (0.43)
651 (0.60)
661 (0.41)
662 (0.42)
1.22
1.47
1.63
1.07
1.09
0.96
1 (ref.)
0.56
71.0
58.5
<5.6
<5.6
65.2
72.2
64.5
72.4
4.2
4.0
4.3
4.3
2.7
3.2
2.7
3.2
630GeRH
SiR[2]
640SiRH
H denotes hydroxylated fluorophores. [a] absolute values; [b] relative brightness
expressed as (ε×Φfl)dye / (ε×Φfl)SiR; [c] see ref.[1a] for the definition of D0.5; [d]
pulsed STED at 631 nm; [e] CW gated STED at 660 nm (LEICA microsystems);
[f] 6'-COOH-X-Rhodamine.
Substitution of an oxygen atom in pyronines with a 14 group
element atom (X
= Si, Ge, Sn) leads to significant
bathochromic and bathofluoric shifts.[2,12] The effect is due to
lower LUMO energy of these fluorophores explained by
Scheme 1. a) Synthesis of the bis-hydroxylated rhodamine dye 530RH (an analog of RhQ-CO2H). b) Synthesis of hydroxylated ROX dyes (6-COOH-X-
Rhodamines) 575RH (13a) and 13b. Alternative schemes indicate better yielding sequences. PPSE = trimethylsilyl polyphosphate.
This article is protected by copyright. All rights reserved.

10.1002/chem.201701216
Chemistry - A European Journal
FULL PAPER
The synthesis of hydroxylated ROX dyes – 6'-carboxy-
derivatives of X-rhodamine (Rhodamine 101), known to have
high fluorescence quantum yields both in organic and
aqueous solutions – is shown in Scheme 1,b. The scrambling
condensation between acetate 9 and benzophenone 12a
afforded two other dyes (13b,c) besides the expected 575RH
(13a) due to two consecutive acid-catalyzed reactions: retro-
Friedel-Crafts dissociation of 12a to compounds 10 and 11 (or
trimellitic acid), followed by Friedel-Crafts acylation of 9 with
11. As a result, a new benzophenone with 2-hydroxy- or 2-
acetoxyjulolidine fragment was formed, leading to
dihydroxylated dye 13b. 6-ROX (13c) arose similarly from the
retro-Friedel-Crafts byproduct 10.
The hydroxylated analogs of the unknown bis-(N-azetidinyl)-
GeR and JF646[6] – dyes 630GeRH and 640SiRH, respectively
– were prepared following the general route on Scheme 2.
The method involves a regioselective bromination of di-O-
TIPS-protected bis(3-hydroxyphenyl)silanes or -germanes
16b,c (TIPS group is required for selectivity) and a double
lithium-halogen exchange on dibromides 16 followed by a
reaction with dimethylcarbamoyl chloride to yield germa- and
silaxanthones 17. The intermediates 17 are general
precursors to the variety of sila- and germafluoresceins and -
rhodols, and the proposed approach offers a significant
improvement with regard to the number of steps,[6, 15] yield[16]
and functional group tolerance as compared to earlier
preparations.
Figure 2. Normalized extinction ε/εmax at λmax of the dyes from Table 1
versus dielectric constant D of dioxane-water mixtures (575RH and 6-ROX
are unresponsive and are not included). The D0.5 values correspond to the
intersection of interpolated graphs with ε/εmax = 0.5 line.[1a]
As expected, the colorless spirolactones of GeR, fluorinated
rhodamine 530RH and carbopyronine 560CP undergo ring
opening to the colored and fluorescent zwitterionic forms in
systems with high water content, while electron-rich ROX
derivatives remain unresponsive, existing predominantly in
the zwitterionic form (Figure S5). Hydroxylation of the N-alkyl
substituents shifts the position of the equilibrium towards the
spirolactone form as can be expected from the weak –I effect
of β-hydroxy substituents (the increase in D0.5 observed upon
transition from SiR to 640SiRH and from GeR to 630GeRH is
7-8 units). The hydroxylated dyes appear photophysically
very similar to the parent fluorophores, demonstrating up to
0.5 ns longer fluorescence lifetimes and, in the case of
630GeRH, an improved fluorescence quantum yield.
In agreement with our earlier observations,[1a] HaloTag(O2)
amine ligands derived from silico- and germanorhodamines,
as well as from fluorinated carbopyronines 560CP and
570CPH, demonstrated significant fluorogenic response
(increase in fluorescence intensity upon covalent binding to
HaloTag protein) in the presence of serum proteins
background (Figure S6). The magnitude of the response was
consistently smaller for hydroxylated dyes, suggesting a
decreased binding affinity of hydroxylated ligands. We can
therefore conclude that moderate or high values of D0.5 seem
to be required, but not sufficient for the desirable fluorogenic
Scheme 2. Synthesis of GeR and SiR dyes. a) n-BuLi, -78 °C, then
Me2GeCl2 or Me2SiCl2; b) NBS; c) n-BuLi or tert-BuLi, -78 °C, then
Me2NCOCl; d) 20, tert-BuLi (2 eq), -78 °C to rt, then 6 M HCl, 80 °C; e) 21,
n-BuLi, THF-pentane, -100 °C to rt; f) TBAF, then Tf2O, pyridine; g) 3-(tert-
butylsilyloxy)azetidine, Pd2(dba)3, XPhos, K2CO3 (3 eq), dioxane, 100 °C,
then TBAF, then TFA, CH2Cl2.
behavior of triarylmethane fluorescent labels[1a, 2c]
.
For the evaluation of performance of our new dyes in
superresolution microscopy, living HeLa and U2OS cells
expressing a vimentin-HaloTag fusion protein were incubated
for 20 min with 1 µM solutions of 530RH, 570CPH, 575RH,
630GeRH, as well as non-hydroxylated GeR and 6-ROX
(13c), conjugated to HaloTag(O2) amine ligand. For labeling
of tubulin filaments, HeLa cells were incubated with
noncovalent β-tubulin ligands, prepared from GeR and
630GeRH and N-Boc-deprotected docetaxel, connected via
8-aminooctanoic acid linker[2b] (Figure S1). All dye conjugates
mentioned above provided specific staining and good imaging
performance in confocal and STED microscopy (Figures S7–
S17), with hydroxylated dyes generally requiring higher
To evaluate the response of our dyes to the polarity of the
media, a series of absorption spectra were recorded in
aqueous dioxane solutions with varying water content. The
spirolactone-zwitterion equilibrium is shifted in favor of the
colored and fluorescent zwitterionic form as the water content
increases (Figure 2). For each dye, the D0.5 parameter,[1a] an
interpolated dielectric constant of the dioxane-water mixture
at which the normalized absorption A/Amax (or extinction
ε/εmax) of this dye equals one half of the maximal value
observed across the entire dioxane-water gradient, was
determined (Table 1).
This article is protected by copyright. All rights reserved.

10.1002/chem.201701216
Chemistry - A European Journal
FULL PAPER
concentrations (e.g., 4-5 µM for 630GeRH instead of 1 µM or
below for GeR).
Isomerically pure 6-ROX dye is one of the “big four” dyes
(FAM, JOE, TAMRA and ROX) dominating in the dye-
terminator DNA sequencing, but has not yet been applied to
live-cell imaging. We have demonstrated that the
fluorescence of all 6-ROX dyes (13a-c) may be efficiently
switched off by de-excitation at 775 nm, making them useful
complementary partners in two-color STED nanoscopy with
SiR or GeR labels. Figures 3, S16 and S17 demonstrate that
HaloTag(O2) amine conjugate of 575RH in combination with
GeR-tubulin or 630GeRH-tubulin ligand provide high quality
two-color images in the most blue-shifted dye pair still
applicable for the widely used 775 nm STED laser line.[17]
Comparison of the Figures S9 and S10 confirms that
hydroxylation of the rhodamine core in the dye 575RH
improves image quality as related to the commercially
available 6-ROX (Table S1).
Figure 4. An example of normal cell division (2 min between images) in the
presence of 640SiRH bound to CID-SNAP and 580R bound to CAL1-Halo
after incubation with 0.4 µM 580R-Halo[1a] (4 h) followed by addition of
0.4 µM 640SiRH-BG and imaging for 12 h. Cell viability in the presence of
dyes in the centromeres was verified by measuring cell morphology of
Drosophila S2 cells using a holographic microscope (Holomonitor M4). For
the corresponding time-lapse movie, see supplementary Movie.
In view of an expanding palette of live-cell compatible dyes
and increasing fluorophore substitution diversity, we propose
a general method relying on simple additive schemes to
estimate the positions of absorption/emission maxima and
D0.5 values of the substitution pattern analogs of
triarylmethane dyes. For example, using the data of our
previous study,[1a] the properties of dye 560CP in the same
solvent (PBS, pH 7.4) have been accurately predicted before
its synthesis (Scheme 3).
Figure 3. Two-color STED image (raw data) of vimentin-HaloTag fusion
protein (green; labeled with 1 µM 575RH-Halo) and endogeneous tubulin
Scheme 3. Three reference dyes (A, M, N) are chosen in such a way that
M and N differ from the unknown dye X in one structural element. Dye A
has one common structural element with dye M, and another common
element with dye N. The known changes in properties between A and M
(or N) allow estimation of λ and D0.5 for X. Assuming Δλ(AN) ≈ Δλ(MX) and
Δλ(AM) ≈ Δλ(NX) (where Δλ(AN) = λ(N) – λ(A), valid for both absorption
and emission maxima; similarly for ΔD0.5), the estimated values for 560CP
are: λabs(560CP) = λabs(580CP) + Δλabs(520R,500R) = 582+(500–521) nm =
561 nm; D0.5(560CP) = D0.5(580CP) + ΔD0.5(520R,500R) = 34.6+(32.5–7.5)
= 59.6. For details, see Figures S20, S21 and Table S2 in the Supporting
Information.
(magenta; labeled with
5
µM GeR-tubulin) in living HeLa cells,
simultaneous incubation time 20 min, followed by 10 min washing. (a)
STED image with confocal part in bottom left corner. (b) and (c): zoomed
(confocal and STED) views of the region marked in (a) in separate colors.
Scale bars 2 µm; pixel dwell time: 12 µs for both color channels; pixel size:
28 nm for STED and confocal image.
In addition to the labeling of cytoskeleton proteins, the new
dyes allow for equally specific staining of nuclear components.
Living U2OS and Drosophila S2 cells expressing SNAP- and
Halo-fusion constructs of different nuclear proteins (TRF2,
PML, CID, CAL1) were incubated for 10-30 minutes with 0.5-
In conclusion, the proposed cell- and nucleus-permeant
fluorescent dyes allow flexible single- and dual-color labeling
in living cells. In STED imaging with de-excitation at 775 nm,
several dye pairs have been validated (575RH & 635GeRH,
610CP & 640SiRH, and 580R & 640SiRH). The improved
synthetic approach to silico- and germanorhodamines has
been developed, and the new dyes 640SiRH, GeR and
635GeRH as conjugates with docetaxel offer direct and
specific visualization of native tubulin filaments in non-
transfected cells. Hydroxylation of fluorophores, especially of
lipophilic rhodamines, improves the staining quality at the
cost of the necessity to use higher dye loadings. In
submicromolar concentrations used for imaging, the dyes of
the present study show no evidence of cytotoxicity. The
design of future dye analogs can be streamlined with
accumulation of photophysical data and estimation of
properties of the new candidates using simple additive
schemes. The degree of predictive precision achieved by
using the measured values for the known structural analogs
is higher than the accuracy provided by the present-day
computational methods (DFT, TD-DFT), especially for the
red-emitting fluorophores.[18] Further increasing of the spectral
and structural variety of cell-permeant fluorophores will
1.0 µM of the chosen dye combination (610CP-BG
&
640SiRH-Halo or 580R-Halo[1a] & 640SiRH-BG; BG = SNAP-
tag ligand) resulting in bright staining free from non-specific
background (Figure S18). The excellent spectral separation of
these combinations allowed for colocalization experiments
without spectral unmixing, mapping the nuclear protein
interaction with sub-diffraction resolution (Figure S19).
Using the so-called holographic microscopy, we have
monitored cell morphology and proliferation of living S2 cells
with the constant presence of 580R and 640SiRH in the
media over 12-16 hours and observed normal cell division
(Figure 4) with proper staining of the kinetochore proteins,
verified with fluorescence microscopy. Therefore, we
conclude that the presence of the protein tags as well as the
staining with our dye conjugates does not negatively affect
cell viability.
This article is protected by copyright. All rights reserved.

10.1002/chem.201701216
Chemistry - A European Journal
FULL PAPER
[3]
[4]
L. C. Zanetti-Domingues, C. J. Tynan, D. J. Rolfe, D. T. Clarke, M.
Martin-Fernandez, PLoS One 2013, 8, e74200.
contribute to the design of new experiments in life sciences,
including those with more sophisticated multiple color channel
separation techniques, such as fluorescence lifetime imaging
and hyperspectral detection.
a) Z. Qian, A. Martyna, R. L. Hard, J. Wang, G. Appiah-Kubi, C.
Coss, M. A. Phelps, J. S. Rossman, D. Pei, Biochemistry 2016, 55,
2601–2612; b) J. P. Richard, K. Melikov, E. Vives, C. Ramos, B.
Verbeure, M. J. Gait, L. V. Chernomordik, B. Lebleu, J. Biol. Chem.
2003, 278, 585–590; c) M. Silhol, M. Tyagi, M. Giacca, B. Lebleu, E.
Vives, Eur. J. Biochem. 2002, 269, 494–501.
Acknowledgements
[5]
[6]
K. W. Teng, Y. Ishitsuka, P. Ren, Y. Youn, X. Deng, P. Ge, A. S.
Belmont, P. R. Selvin, eLife 2016, 5, e20378.
We thank J. Bienert (MPIBPC), Dr. H. Frauendorf, Dr. M. John and co-
workers (Institut für organische und biomolekulare Chemie, Georg-August-
Universität, Göttingen, Germany) for recording spectra; Prof. Z. Novák
(Eötvös University, Budapest, Hungary) for the gift of trifluoroethyliodonium
salt; J. Shimpfhauser and J. Seikowski (MPIBPC) for assistance with the
synthesis; Dr. E. Rothermel and T. Gilat (MPIBPC) for the preparation and
immunolabeling of cells; Dr. I. Chung, C. Bauer and PD Dr. K. Rippe
(Genome Organization and Function, DKFZ) for the U2OS cells used for
the nuclear staining and for cloning the TRF2-SNAP and PMLIII-Halo
fusion constructs; Dr. A.-L. Pauleau and Prof. S. Erhardt (Zentrum für
Molekulare Biologie der Universität Heidelberg, ZMBH) for the Drosophila
S2 cells stably expressing CID-SNAP and CAL1-Halo fusion proteins; Dr. A.
Giske (LEICA Microsystems, Germany) for testing 560CP dye with 660 nm
STED laser. S.W.H., A.N.B. and H.S. acknowledge a grant from the
Bundesministerium für Bildung und Forschung (FKZ 13N14122). This work
was also supported by the Netherlands Organisation for Scientific
Research (NWO; grant number 680-50-1501 to R.V.) and EMBO (grant
number ALTF 1516-2015 to R.V.).
J. B. Grimm, B. P. English, J. Chen, J. P. Slaughter, Z. Zhang, A.
Revyakin, R. Patel, J. J. Macklin, D. Normanno, R. H. Singer, T.
Lionnet, L. D. Lavis, Nat. Methods 2015, 12, 244–250.
M. J. Hinner, K. Johnsson, Curr. Opin. Biotechnol. 2010, 21, 766–
776.
A. Gautier, A. Juillerat, C. Heinis, I. R. Correa, Jr., M. Kindermann, F.
Beaufils, K. Johnsson, Chem. Biol. 2008, 15, 128–136.
G. V. Los, L. P. Encell, M. G. McDougall, D. D. Hartzell, N.
Karassina, C. Zimprich, M. G. Wood, R. Learish, R. F. Ohana, M.
Urh, D. Simpson, J. Mendez, K. Zimmerman, P. Otto, G. Vidugiris, J.
Zhu, A. Darzins, D. H. Klaubert, R. F. Bulleit, K. V. Wood, ACS
Chem. Biol. 2008, 3, 373–382.
[7]
[8]
[9]
[10] J. Hanne, F. Gottfert, J. Schimer, M. Anders-Osswein, J. Konvalinka,
J. Engelhardt, B. Muller, S. W. Hell, H. G. Krausslich, ACS Nano
2016, 10, 8215–8222.
[11] S. Nizamov, M. V. Sednev, M. L. Bossi, E. Hebisch, H. Frauendorf,
S. E. Lehnart, V. N. Belov, S. W. Hell, Chem. Eur. J. 2016, 22,
11631–11642.
[12] Y. Koide, Y. Urano, K. Hanaoka, T. Terai, T. Nagano, ACS Chem.
Biol. 2011, 6, 600-608.
[13] For structures of the known Ge-rhodamines (GeR) and Ge-
xanthene dyes, see Figure S3 in Supporting Information and
patents: a) T. Nagano, K. Hanaoka, Y. Koide, T. Egawa, US
20140057312A1; b) J. Braddock-Wilking, T. L. Bandrowski, J. B.
Carroll II, US 20150166581; c) T. Nagano, K. Hanaoka, K.
Hirabayashi, Y. Yoki, US 20150212073A1; d) W. Zhou, P.
Mesenheimer, M. Zhou, US 20140272990A1.
Keywords: fluorescent dyes • rhodamines •
germanorhodamines • optical microscopy • living cells
[1]
a) A. N. Butkevich, G. Y. Mitronova, S. C. Sidenstein, J. L. Klocke, D.
Kamin, D. N. Meineke, E. D'Este, P. T. Kraemer, J. G. Danzl, V. N.
Belov, S. W. Hell, Angew. Chem. Int. Ed. 2016, 55, 3290–3294; b) F.
Bottanelli, E. B. Kromann, E. S. Allgeyer, R. S. Erdmann, S. Wood
Baguley, G. Sirinakis, A. Schepartz, D. Baddeley, D. K. Toomre, J.
E. Rothman, J. Bewersdorf, Nat. Commun. 2016, 7, 10778; c) B.
Hein, K. I. Willig, C. A. Wurm, V. Westphal, S. Jakobs, S. W. Hell,
Biophys. J. 2010, 98, 158–163; d) S. C. Sidenstein, E. D'Este, M. J.
Bohm, J. G. Danzl, V. N. Belov, S. W. Hell, Sci. Rep. 2016, 6, 26725.
a) G. Lukinavicius, K. Umezawa, N. Olivier, A. Honigmann, G. Yang,
T. Plass, V. Mueller, L. Reymond, I. R. Correa, Jr., Z. G. Luo, C.
Schultz, E. A. Lemke, P. Heppenstall, C. Eggeling, S. Manley, K.
Johnsson, Nat. Chem. 2013, 5, 132–139; b) G. Lukinavicius, L.
Reymond, E. D'Este, A. Masharina, F. Gottfert, H. Ta, A. Guther, M.
Fournier, S. Rizzo, H. Waldmann, C. Blaukopf, C. Sommer, D. W.
Gerlich, H. D. Arndt, S. W. Hell, K. Johnsson, Nat. Methods 2014,
11, 731–733; c) G. Lukinavicius, L. Reymond, K. Umezawa, O.
Sallin, E. D'Este, F. Gottfert, H. Ta, S. W. Hell, Y. Urano, K.
Johnsson, J. Am. Chem. Soc. 2016, 138, 9365–9368; d) Y. Kushida,
T. Nagano, K. Hanaoka, Analyst 2015, 140, 685–695.
[14] V. P. Boyarskiy, V. N. Belov, R. Medda, B. Hein, M. Bossi, S. W.
Hell, Chem. Eur. J. 2008, 14, 1784–1792.
[15] a) T. Egawa, K. Hirabayashi, Y. Koide, C. Kobayashi, N. Takahashi,
T. Mineno, T. Terai, T. Ueno, T. Komatsu, Y. Ikegaya, N. Matsuki, T.
Nagano, K. Hanaoka, Angew. Chem. Int. Ed. 2013, 52, 3874–3877;
b) T. Egawa, Y. Koide, K. Hanaoka, T. Komatsu, T. Terai, T.
Nagano, Chem. Commun. 2011, 47, 4162–4164; c) K. Hirabayashi,
K. Hanaoka, T. Takayanagi, Y. Toki, T. Egawa, M. Kamiya, T.
Komatsu, T. Ueno, T. Terai, K. Yoshida, M. Uchiyama, T. Nagano,
Y. Urano, Anal. Chem. 2015, 87, 9061–9069.
[2]
[16] Q. A. Best, N. Sattenapally, D. J. Dyer, C. N. Scott, M. E. McCarroll,
J. Am. Chem. Soc. 2013, 135, 13365–13370.
[17] Abberior Instruments GmbH offers STED microscopes with the
pulsed 595 nm and 775 nm depletion lasers; Leica Microsystems –
with 592 nm (gated; CW), 660 nm (gated; CW) and 775 nm (pulsed)
depletion lasers; PicoQuant GmbH offers a STED microscope with a
765 nm depletion laser.
[18] S. Fleming, A. Mills, T. Tuttle, Beilstein J. Org. Chem. 2011, 7, 432–
441.
This article is protected by copyright. All rights reserved.

10.1002/chem.201701216
Chemistry - A European Journal
FULL PAPER
Entry for the Table of Contents (Please choose one layout)
Layout 1:
FULL PAPER
Modular design for portable dyes:
hydroxylated rhodamines, carbo-
pyronines, Si- and Ge-rhodamines
absorbing at 530–640 nm showed
specific labeling of living cells and
provided optical resolution of 50–75
nm in one- and two-color STED
microscopy. The established
A. N. Butkevich,* V. N. Belov,* K.
Kolmakov, V. V. Sokolov, H. Shojaei, S.
C. Sidenstein, D. Kamin, J. Matthias, R.
Vlijm, J. Engelhardt and S. W. Hell*
Page No. – Page No.
Hydroxylated fluorescent
dyes for live cell labeling:
synthesis, spectra and
superresolution STED
microscopy
structure-property relationships allow
predicting the properties of the new
dye analogs.
This article is protected by copyright. All rights reserved.