Carboxylated Photoswitchable Diarylethenes for Biolabeling and Super-Resolution RESOLFT Microscopy

Article abstract of DOI:10.1002/anie.201607940

Reversibly photoswitchable 1,2-bis(2-ethyl-6-phenyl-1-benzothiophene-1,1-dioxide-3-yl)perfluorocyclopentenes (EBT) having fluorescent “closed” forms were decorated with four or eight carboxylic groups and attached to antibodies. Low aggregation, efficient

Full text of DOI:10.1002/anie.201607940

Angewandte
Communications
Chemie
International Edition: DOI: 10.1002/anie.201607940
German Edition: DOI: 10.1002/ange.201607940
Optical Microscopy Very Important Paper
Carboxylated Photoswitchable Diarylethenes for Biolabeling and
Super-Resolution RESOLFT Microscopy
Benoꢀt Roubinet+, Mariano L. Bossi+, Philipp Alt+, Marcel Leutenegger, Heydar Shojaei,
Sebastian Schnorrenberg, Shamil Nizamov, Masahiro Irie,* Vladimir N. Belov,* and
Stefan W. Hell*
Abstract: Reversibly photoswitchable 1,2-bis(2-ethyl-6-
phenyl-1-benzothiophene-1,1-dioxide-3-yl)perfluorocyclopen-
tenes (EBT) having fluorescent “closed” forms were decorated
with four or eight carboxylic groups and attached to antibodies.
Low aggregation, efficient photoswitching in aqueous buffers,
specific staining of cellular structures, and good photophysical
properties were demonstrated. Alternating light pulses of UV
and blue light induce numerous reversible photochemical
transformations between two stables states with distinct struc-
tures. Using relatively low light intensities, EBTs were applied
in biology-related super-resolution microscopy based on the
reversible saturable (switchable) optical linear fluorescence
transitions (RESOLFT) and demonstrated optical resolution
of 75 nm.
achieved with reversibly photoswitchable fluorescent proteins
(Dreiklang, rsEGFP) undergoing thousands of E/Z isomer-
izations.[2] Owing to the thermal stabilities of the non-
fluorescent and fluorescent states and adequate photoisome-
rization quantum yields, the switching between these states
can be performed reversibly, with relatively low light inten-
sities and comparatively low photobleaching. While fluores-
cent proteins demonstrate good performance in RESOLFT
microscopy, synthetic dyes are far inferior in this respect.
Man-made photochromic fluorescent dyes[3] incorporated
into silica gel nano-beads undergo tens of switching cycles
and improve optical resolution.[4] Recently, thiol-assisted
photoswitching of a Cy3-Alexa647 conjugate has been
implemented in the presence of potassium iodide (KI)
under RESOLFT conditions and allowed, for example, to
discern tubulin filaments approximately 130 nm apart.[5] Still,
RESOLFT microscopy of noteworthy subdiffraction resolu-
tion on biological samples has not yet been realized for
synthetic organic fluorophores undergoing reversible photo-
chemical transformation and switching between two stable
states with distinct and stable structures.
The required properties of RESOLFT markers—suffi-
cient photostability, high fluorescent modulation (> 90%),
high brightness (product of the fluorescence quantum yield
(Ffl) and extinction coefficient (e)), thermal stability of the
fluorescent and non-fluorescent forms, satisfactory switching
kinetics, large separation between the absorption bands of the
two stable states—have been achieved for synthetic photo-
chromic dyes in organic solvents,[3,4] but their performance in
purely aqueous solutions remained poor (even in the presence
of solubilizing residues and polar ionic groups).[6] In most
cases, these disappointing results were obtained for bi-
chromophoric compounds with separate photochromic and
fluorescent units, in which the fluorescence quenching was
effected by the resonant energy transfer (RET) from the
fluorescent unit (RET donor) to the colored form of the
photochromic unit (RET acceptor).[7] Alternatively, photo-
switching of the emission signal without destructive readout
caused by the excitation light was realized by using the
electron-transfer process (from the excited state of fluoro-
phore to the “closed form” of the photochromic unit).[8] In
any case, it is difficult to compensate the inherent hydro-
phobicity of the relatively large and complex molecular switch
by adding polar or charged groups.
T
he optical super-resolution techniques, as well as the
fluorescent proteins and synthetic dyes required for them,
revived fluorescence microscopy as one of the most powerful
methods in the life sciences. The diffraction limit no longer
restricts the spatial resolution in optical microscopy; it was
overcome by using photoinduced switching of the fluorescent
markers between dark and emitting states.[1] For example, in
biology-related optical microscopy based on the RESOLFT
concept,[1a] an optical resolution of 30–40 nm has been
[*] Dr. B. Roubinet,[+] Dr. M. L. Bossi,[+] Dipl.-Biophys. P. Alt,[+]
Dr. M. Leutenegger, Dr. H. Shojaei, M. Sc. S. Schnorrenberg,
Dr. S. Nizamov, Dr. V. N. Belov, Prof. Dr. S. W. Hell
Abteilung Nanobiophotonik
Max-Planck-Institut fꢀr biophysikalische Chemie
Am Fassberg 11, 37077 Gçttingen (Germany)
E-mail: vbelov@gwdg.de
shell@gwdg.de
Homepage: http://www.mpibpc.gwdg.de/abteilungen/200/
Prof. Dr. M. Irie
Research Center for Smart Molecules
Department of Chemistry
Rikkyo University
Nishi-Ikebukuro 3-34-1, Toshimaku, Tokyo (Japan)
E-mail: iriem@rikkyo.ac.jp
[+] These authors contributed equally to this work.
Supporting information and the ORCID identification number(s) for
the author(s) of this article can be found under http://dx.doi.org/10.
1002/anie.201607940.
ꢁ 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
modifications or adaptations are made.
Great expectations in the field were associated with the
new photochromic sulfones based on 1,2-bis(2-alkyl-6-aryl-1-
benzothiophen-3-yl)perfluorocyclopentenes
(see
Scheme 1).[9] By irradiation with UV light, the “open” form
Angew. Chem. Int. Ed. 2016, 55, 1 – 6
ꢀ 2016 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
1
These are not the final page numbers!

Angewandte
Communications
Chemie
might promote photodegradation of the closed form. Ethyl
groups at C2(2’) (not methyl groups) are essential for
increasing the fluorescence quantum yields in highly polar
solvents.[11b] Photophysical properties of newly synthesized
dyes 1 and 2 in aqueous buffers are summarized in Table 1
(the absorption and emission spectra are presented in Fig-
ure S6). The differences in properties measured in aqueous
solutions and in methanol (Table S2) are not large. For
instance, the emission efficiencies and fluorescence lifetimes
are reduced by only 20–30% in PBS at pH 7.4, with slightly
larger variations observed for compound 1.
A difficult task was to find and attach to the dye core the
most appropriate hydrophilic residues, to prevent aggregation
of the free dyes and their bioconjugates in aqueous solutions.
Numerous approaches to hydrophilic, water soluble diary-
lethenes with sulfonic,[13] carboxylic acid,[11,14] hydroxy groups,
or inositol residues,[14] as well as poly(ethyleneglycol)
chains,[14a,15] have been already reported. These polar groups
are necessary because the cores of the best synthetic switchers
are inherently hydrophobic, and these compounds are poorly
soluble in water,[14a] form nanoaggregates,[13b] or need cyclo-
dextrins as additives for solubilization.[13a] Moreover, they
often lack a reactive group for bioconjugation. We have found
that the presence of several carboxylic acid groups conferred
the required properties to dyes 1 and 2, despite the common
notion that the carboxylic acid is inferior to the sulfonic or
phosphoric (phosphonic) residue in terms of solubilization.
Indeed, the presence of four or eight carboxylates provided
a fairly good solubility at pH > 5 and inhibited aggregation (in
the micromolar range) even after photocyclization (the closed
isomer aggregates more readily).[15a] Unlike most of the other
solubilizing groups, one of the carboxylates can be conven-
iently transformed into any reactive group. Using mono N-
hydroxysuccinimidyl esters and standard labeling methods,
we have successfully conjugated these two new hydrophilic
diarylethenes with secondary antibodies. Remarkably, all the
bioconjugates retain the photochromic and fluorescent prop-
erties of free dyes 1 and 2. They were used in conventional
(confocal) microscopy and in super-resolution RESOLFT
microscopy.
Diarylethenes 1 and 2 were obtained via three different
routes: using a Suzuki–Miyaura cross coupling of C6(6’)-
diiodide derivative[11a] (Scheme S2/S3) with either 3,5-difor-
mylphenyl boronic (route A; 23% yield over 2 steps), or 5-
boronoisophthalic acid (route B; 46% yield), or a pinacol
ester of 3,5-di(tert-butoxycarbonyl)phenylboronic acid
(route C; 13% over 2 steps).[16] An additional Jones oxidation
step in route A or an acidic treatment (with CF3COOH in
CH2Cl2) in route C was applied. To improve the hydrophilic
properties and suppress the aggregation even further, we
prepared diarylethene 2 with eight carboxylic groups. This
was achieved in 41% yield by a similar strategy using
a Suzuki–Miyaura cross-coupling followed by cleavage of
tert-butyl esters under acidic conditions.[16] The amino-reac-
tive mono NHS esters of these dyes were prepared by the
gradual and controlled addition of the measured quantities of
N,N’-dicyclohexylcarbodiimide (DCC) or 1-ethyl-3-(3-dime-
thylaminopropyl)carbodiimide (EDC) to solutions of 1 or 2
and N-hydroxysuccinimide in DMF with catalytic amounts of
Scheme 1. Photoswitchable EBTs 1 and 2 decorated with four or eight
carboxylic acid groups; see Table 1 for spectral properties of the
“open” (OF, non-fluorescent) and the “closed” (CF, fluorescent)
forms.[12] For compounds with R=H and R=C6H5 see Refs. [9a,b,c]
and [9d,e], respectively.
transforms to the fluorescent yellow “closed” form.[10] Illumi-
nation with visible (blue) light restores the “open” isomer.
Remarkably, the fluorescent “closed” forms emit green light
with quantum efficiencies of up to 0.92,[11] and the photo-
chromic (switching) and fluorescent (signal) functions are
combined in one chemical entity. Using these sulfone
derivatives as leads, we planned to prepare photochemically
switchable fluorophores applicable in purely aqueous buffers
that possess a reactive group and perform well (as bioconju-
gates) in commercially related RESOLFT microscopes using
moderate light intensities of 1–10 kWcmꢀ2 and switching
times in the ms-ms range.
After screening many substituents attached to positions
C2(2’) and C6(6’) in 1,2-bis(1-benzothiophene-1,1-dioxide-3-
yl)perfluorocyclopentene, we have chosen a symmetrical core
structure with 2(2’)-ethyl and 6(6’)-phenyl groups (Scheme 1,
dyes 1 and 2).[11] These groups, and the absence of strong
electron-donor residues at C6,6’ provide a relatively low (but
not too low) quantum efficiency of the ring-opening reaction
(Table 1). It was sufficient for a high number of excitation–
emission cycles of the closed isomer in a read-out process,
before its back-isomerization to the open form takes place
(erase or refresh step). In contrast, strong electron donors at
C6,6’ slow down the ring-opening reaction drastically[11a,c] and
Table 1: Photophysical properties of free dyes 1 and 2 in PBS (pH 7.4).
Parameter
State
1
2
lmax abs [nm]/
OF[a] 340/12400
337/16500
e [mꢀ1 cmꢀ1
]
lmax abs [nm]/
CF
450/35000
448/45000
e [mꢀ1 cmꢀ1
]
lmax em [nm]
F
fl
CF
CF
CF
534, 558
0.48
522, 550
0.57
[b]
t [ns]
FOF!CF
FCF!OF
1.72ꢁ0.05
2.04ꢁ0.05
[c]
0.19ꢁ0.04
0.23ꢁ0.04
1.2ꢂ10ꢀ3 ꢁ3ꢂ10ꢀ4 2.0ꢂ10ꢀ3 ꢁ3ꢂ10ꢀ4
[d]
[a] lowest energy absorption peak. [b] fluorescence quantum yield;
fluorescein in 0.1m NaOH was used as standard. [c] At 365 nm. [d] At
470 nm (see Supporting Information for details).
2
www.angewandte.org
ꢀ 2016 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Angew. Chem. Int. Ed. 2016, 55, 1 – 6
These are not the final page numbers!

Angewandte
Communications
Chemie
4-(N,N-dimethyl)aminopyridine (DMAP).[16] The stable
100 and 200 cycles are shown, respectively). These measure-
mono-NHS ester of dye 1 was isolated by preparative RP-
HPLC, while mono-NHS ester of dye 2 could be only
generated in solution without isolation. Then, the secondary
antibodies were labeled with these mono-NHS esters and
purified by gel-filtration.[16] MALDI-TOF mass spectrometry
and UV/Vis spectroscopy were used to evaluate the degrees
of labeling (DOL; see Table S1 in the Supporting Informa-
tion). We varied the DOL values to achieve the best
immunofluorescence staining and acquire the best-possible
images under confocal and RESOLFT conditions.
The conjugates obtained with secondary antibodies were
highly fluorescent and photochromic; we used them for
immunostaining of tubulin filaments and nucleopore com-
plexes in Vero cells. Confocal measurements (Figure S1)
showed their selective binding with primary antibodies and
tubular network in the whole fixed cells. Cells immunolabeled
with dyes 1 and 2 were used for pump–probe fluorescence
measurements in a modified confocal microscope using
a wide-field illumination (Figure S2). For that, the stained
cells were mounted onto an object slide with a cavity filled
with PBS (pH 7.4). Switching between the open and closed
forms was performed by laser light of 375 nm and 491 nm
wavelength, respectively. The measurement was initialized by
a 10 ms pulse of 491 nm light (17 kWcmꢀ2) to switch all the
fluorophores in the observation area to the “dark” state. Then
a pulse sequence was applied consisting of a 20 ms pulse of
375 nm light (0.8 kWcmꢀ2), a 500 ms delay, a 10 ms pulse of
491 nm light (17 kWcmꢀ2) and another 2 ms delay. This pulse
sequence was repeated 200 times (in Figure 1 and Figure S3,
ments showed that half of the initial fluorescence is bleached
after performing about 11 switching cycles for dye 1 and
21 switching cycles for dye 2, respectively. A bi-exponential fit
of the obtained decay curve of the ring-opening (“off”-
switching) reaction gave the time constants of t1 = 241 ms and
t2 = 1.31 ms for dye 1 (79% of the amplitude belongs to t1 and
21% to t2). Under these irradiation conditions, the corre-
sponding decay times for dye 2 are t1 = 126 ms (77%) and t2 =
674 ms (23%). The residual fluorescence per cycle was
evaluated by comparison of the maximal fluorescence in
a cycle and the average value of the residual fluorescence
recorded during the last 100 ms illumination with 491 nm light
(as a mean value of 100 cycles). The residual fluorescence was
found to be 4.3% for dye 1 and 1.7% for dye 2, respectively.
To demonstrate the applicability of dyes 1 and 2 for
RESOLFT microscopy, fixed immunolabeled Vero cells were
imaged using a modified 1C RESOLFT QUAD Scanning
microscope (Abberior Instruments, Gçttingen). The imaging
was performed by applying the following pulse sequence:
switching the diarylethenes to their fluorescent “closed”
isomers with 355 nm light (130 Wcmꢀ2) for 50 ms, 200 ms delay,
application of the doughnut-shaped 488 nm beam
(36 kWcmꢀ2) for 1.2 ms to switch the diarylethenes in the
periphery of the focal spot to their non-fluorescent “open”
isomers. Finally, the remaining area with the fluorescent
isomers was probed for 80 ms with a Gaussian shaped 488 nm
light beam (9.7 kWcmꢀ2). The same pulse sequence was
applied to record the confocal images except that the off-
switching was omitted. Using this imaging scheme, it was
possible to acquire RESOLFT images with an optical
resolution of 74 nm FWHM[17] and confocal images with
a
resolution of 175 nm FWHM.[17] The confocal and
RESOLFT images are given in Figure 2, Figure S4 (tubulin
filaments), and Figure S5 (nuclear pore complexes). The
relatively high confocal resolution (Figure 2 and Figure S4)
results from the 355 nm activation spot that is significantly
smaller than the excitation spot at 488 nm wavelength. This
two-step process of activation and excitation yields an
effective confocal point spread function (PSF) which can be
described by the product of the activation, excitation, and
detection PSFs.[18]
Both diarylethenes show a high switching contrast in the
pump–probe measurements. Although the switching-off rate
of the free dye 2 is only about twice the rate of dye 1 (Table 1),
it turned out that during RESOLFT measurements, dye 2
(attached to a protein) switches off much faster than dye
1 (1.5 ms for dye 2 versus 7 ms for dye 1); this is probably the
reason why dye 1 bleached faster and provided poorer
resolution (85 nm). The resolution achieved in the RESOLFT
measurements was limited mainly by photobleaching and not
by the residual fluorescence after off-switching.
To date, reversibly photoswitchable fluorescent proteins
still offer a significantly lower switching fatigue (about 100 ꢀ
more cycles),[19] yet we have shown the possibility to prepare
reversibly photoswitchable organic dyes that perform in
purely aqueous solutions and are suitable for biology-related
RESOLFT microscopy. Importantly, the simple EBT scaffold
presented a viable core structure and was decorated with four
Figure 1. Fluorescence emission of cells immunolabeled with dye
1 (top) and 2 (bottom) showing the on/off switching cycles upon
irradiation with the light pulse sequence mentioned in the text (gray).
The red line indicates the maximum fluorescence signal detected
within a switching cycle, while the black line shows the overall
fluorescence gained per cycle. Both plots were normalized to their
maximum value. The insets show one switching cycle as the mean
value of 100 cycles (blue). All measurements were taken with 20 ms
bins. Displayed intensities were corrected according to the dead-time
of the sensors.
Angew. Chem. Int. Ed. 2016, 55, 1 – 6
ꢀ 2016 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.angewandte.org
3
These are not the final page numbers!

Angewandte
Communications
Chemie
Acknowledgements
We thank Jꢂrgen Bienert (MPIBPC), Dr. Holm Frauendorf,
Dr. Michael John and co-workers (Institut fꢂr organische und
biomolekulare Chemie, Georg-August-Universitꢃt, Gçttin-
gen, Germany) for recording spectra. We are indebted to Jens
Schimpfhauser and Jan Seikowski (MPIBPC) for the syn-
thesis of 1,2-bis(2-ethyl-6-iodo-1-benzothiophen-1,1-dioxide-
3-yl)perfluorocyclopentene and 3,5-di(tert-butycarboxyphe-
nyl)boronic acid, pinacol ester. We are grateful to Dr. Ellen
Rothermel (MPIBPC) for the preparation and immunolabel-
ing of cells. We thank Prof. Stefan Jakobs (MPIBPC) for
lending us the Abberior RESOLFT microscope. We are
indebted to the Bundesministerium fꢂr Bildung und For-
schung (BMBF, Germany) for funding in the program KMU-
innovativ: Photonik/ Optische Technologien (FKZ
13N12995; to SWH).
Keywords: diarylethenes · fluorescence · optical microscopy ·
photochromic dyes · water solubility
[1] For the idea of RESOLFT, see: a) S. W. Hell, S. Jakobs, L.
Kastrup, Appl. Phys. A 2003, 77, 859 – 860; for examples, see:
b) M. Hofmann, C. Eggeling, S. Jakobs, S. W. Hell, Proc. Natl.
Acad. Sci. USA 2005, 102, 17565 – 17569; c) E. Betzig, G. H.
Patterson, R. Sougrat, O. Wolf Lindwasser, S. Olenych, J. S.
Bonifacino, M. W. Davidson, J. Lippincott-Schwartz, H. F. Hess,
Science 2006, 313, 1642 – 1645; for reviews, see: d) S. W. Hell,
Nat. Methods 2009, 6, 24 – 32; e) S. W. Hell, Science 2007, 316,
1153 – 1158.
[2] a) T. Brakemann, A. C. Stiel, G. Weber, M. Andresen, I. Testa, T.
Grotjohann, M. Leutenegger, U. Plessmann, H. Urlaub, C.
Eggeling, M. C. Wahl, S. W. Hell, S. Jakobs, Nat. Biotechnol.
2011, 29, 942 – 947; b) T. Grotjohann, I. Testa, M. Leutenegger,
H. Bock, N. T. Urban, F. Lavoie-Cardinal, K. I. Willig, C.
Eggeling, S. Jakobs, S. W. Hell, Nature 2011, 478, 204 – 208;
c) J. G. Danzl, S. C. Sidenstein, C. Gregor, N. T. Urban, P. Ilgen,
S. Jakobs, S. W. Hell, Nat. Photonics 2016, 10, 122 – 128.
[3] M. Bossi, V. N. Belov, S. Polyakova, S. W. Hell, Angew. Chem.
Int. Ed. 2006, 45, 7462 – 7465; Angew. Chem. 2006, 118, 7623 –
7627.
Figure 2. RESOLFT images of the whole fixed Vero cells immunos-
tained with primary antibodies against a-Tubulin and with the diary-
lethene 2 attached to the secondary antibodies. All images show raw
data. The RESOLFT images were recorded before confocal images.
Scale bar: 1 mm. The line profiles A–D (averaged over ten adjacent
lines) display the regions indicated in the RESOLFT image. The
data (dots) was fitted with a Lorentzian function (solid line) for the
RESOLFT (red) and the confocal (blue) image. The FWHM was deter-
mined on the fits A and B as indicated by the small black arrows.
or eight carboxylic acid groups. Compounds 1 and 2 under-
went reversible photochemical transformations and switched
between two stable states with distinct structures in aqueous
PBS buffer and without stabilizing reagents and additives.
The results of the present study allow for the first time man-
made photoswitchable fluorophores to be compared with
photoswitchable fluorescent proteins. Though the perfor-
mance of synthetic dyes in RESOLFT microscopy is still
inferior to that of fluorescent proteins (in respect of bleach-
ing, number of switching cycles, and phototoxic effects of UV
light), the simplicity of EBT structures is encouraging.
Indeed, low aggregation, efficient photoswitching in aqueous
buffers, and chemical reactivity were gained straightfor-
wardly, by introducing numerous freely rotating COOH
groups to a highly symmetrical core structure. All these
results can be very helpful for future work. It will focus on the
design and characterization of reversibly photoswitchable and
fatigue-resistant dyes that isomerize into a stable fluorescent
form by irradiation with light of 405 nm and longer wave-
lengths. Moreover, their ability for labeling living cells (using
SNAP-Tag or HaloTagꢁ technologies) will be evaluated.
[4] J. Fçlling, S. Polyakova, V. Belov, A. van Blaaderen, M. L. Bossi,
S. W. Hell, Small 2008, 4, 134 – 142.
[5] J. Kwon, J. Hwang, J. Park, G. R. Han, K. Y. Han, S. K. Kim, Sci.
Rep. 2015, 5, 17804.
[6] a) N. Soh, K. Yoshida, H. Nakajima, K. Nakano, T. Imato, T.
Fukaminato, M. Irie, Chem. Commun. 2007, 5206 – 5208;
b) S. M. Polyakova, V. N. Belov, M. L. Bossi, S. W. Hell, Eur. J.
Org. Chem. 2011, 3301 – 3312.
[7] a) J. M. Endtner, F. Effenberger, A. Hartschuh, H. Port, J. Am.
Chem. Soc. 2000, 122, 3037 – 3046; b) L. Giordano, T. M. Jovin,
M. Irie, E. A. Jares-Erijman, J. Am. Chem. Soc. 2002, 124, 7481 –
7489; c) T. Fukaminato, T. Sasaki, T. Kawai, N. Tamai, M. Irie, J.
Am. Chem. Soc. 2004, 126, 14843 – 14849; d) M. Frigoli, G. H.
Mehl, Eur. J. Org. Chem. 2004, 636 – 642.
[8] T. Fukaminato, T. Doi, N. Tamaoki, K. Okuno, Y. Ishibashi, H.
Miyasaka, M. Irie, J. Am. Chem. Soc. 2011, 133, 4984 – 4990.
[9] a) Y. C. Jeong, S. I. Yang, K. H. Ahn, E. Kim, Chem. Commun.
2005, 2503 – 2505; b) Y.-C. Jeong, D. G. Park, I. S. Lee, S. I. Yang,
K.-H. Ahn, J. Mater. Chem. 2009, 19, 97 – 103; c) Y.-C. Jeong, S. I.
Yang, E. Kim, K.-H. Ahn, Tetrahedron 2006, 62, 5855 – 5861;
4
www.angewandte.org
ꢀ 2016 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Angew. Chem. Int. Ed. 2016, 55, 1 – 6
These are not the final page numbers!

Angewandte
Communications
Chemie
d) T. Wu, B. Johnsen, Z. Qin, M. Morimoto, D. Baillie, M. Irie, N.
[14] a) Y. Shoji, A. Yagi, M. Horiuchi, M. Morimoto, M. Irie, Isr. J.
R. Branda, Nanoscale 2015, 7, 11263 – 11266; e) M. Bꢃlter, S. Li,
M. Morimoto, S. Tang, J. Hernando, G. Guirado, M. Irie, F. M.
Raymo, J. Andrꢄasson, Chem. Sci. 2016, 7, 5867 – 5871; for
a recent application in PALM/STORM microscopy, see: f) O.
Nevskyi, D. Sysoiev, A. Oppermann, T. Huhn, D. Wçll, Angew.
Chem. Int. Ed. 2016, DOI: 10.1002/anie.201606791; Angew.
Chem. 2016, DOI: 10.1002/ange.201606791.
Chem. 2013, 53, 303 – 311; b) “Photochromic molecule”: M. Irie,
C. C. Yamada, JP2012172139, 2012; c) “Water-soluble photo-
chromic molecule”: H. Tokiwa, M. Irie, K. Ikeda, T. Otsubo
(Rikkyo Educational Corporation and Josho Gakuen Educa-
tional Foundation), EP 2902402, 2015.
[15] a) T. Hirose, M. Irie, K. Matsuda, Adv. Mater. 2008, 20, 2137 –
2141; b) T. Hirose, M. Irie, K. Matsuda, Chem. Asian J. 2009, 4,
58 – 66; c) T. Hirose, K. Matsuda, M. Irie, J. Org. Chem. 2006, 71,
7499 – 7508.
[10] The “closed” form is yellow and emits green light.
[11] a) K. Uno, H. Niikura, M. Morimoto, Y. Ishibashi, H. Miyasaka,
M. Irie, J. Am. Chem. Soc. 2011, 133, 13558 – 13564; b) Y. Takagi,
T. Kunishi, T. Katayama, Y. Ishibashi, H. Miyasaka, M.
Morimoto, M. Irie, Photochem. Photobiol. Sci. 2012, 11, 1661 –
1665; c) F. Gillanders, L. Giordano, S. A. Dꢅaz, T. M. Jovin, E. A.
Jares-Erijman, Photochem. Photobiol. Sci. 2014, 13, 603 – 612.
[12] The “open” form is in equilibrium between two isomers:
parallel (p) and antiparallel (ap), and the fluorescent “closed”
form is formed only from an anti-parallel conformer (one of the
Woodward–Hoffmann rules). For reviews on DAEs, see: a) M.
Irie, Chem. Rev. 2000, 100, 1685 – 1716; b) M. Irie, T. Fukami-
nato, K. Matsuda, S. Kobatake, Chem. Rev. 2014, 114, 12174 –
12277.
[16] For synthetic procedures, details and analytical data, see the
Supporting Information. Dyes 1 and 2 are base-sensitive; under
alkaline conditions they react with water giving products with
a -CF2CF2C(O)-fragment instead of initial perfluorocyclopen-
tene ring. The same transformation occurred in wet DMSO.
[17] Full width at half minimum (FWHM)of the Lorentzian fit.
[18] The point spread function (PSF) is the spatial intensity distrib-
uton of a point-like source imaged by an optical system.
[19] T. Grotjohann, I. Testa, M. Reuss, T. Brakemann, C. Eggeling,
S. W. Hell, S. Jakobs, eLife 2012, 1, e00248, DOI: 10.7554/
eLife.00248.
[13] a) M. Takeshita, M. Irie, Chem. Commun. 1997, 2265 – 2266;
b) M. Takeshita, N. Kato, S. Kawauchi, T. Imase, J. Watanabe, M.
Irie, J. Org. Chem. 1998, 63, 9306 – 9313.
Received: August 15, 2016
Published online: && &&, &&&&
Angew. Chem. Int. Ed. 2016, 55, 1 – 6
ꢀ 2016 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.angewandte.org
5
These are not the final page numbers!

Angewandte
Communications
Chemie
Communications
Optical Microscopy
Glow the distance: Diarylethenes with
a fluorescent “closed” form and carbox-
ylic groups reversibly switch between
”dark” and green-emitting states. In
aqueous buffers their antibody conju-
gates specifically stain cellular structures.
RESOLFTmicroscopy based on reversible
saturable (switchable) optically linear
fluorescence transitions (induced by UV
and blue light) demonstrated optical
resolution of 75 nm.
B. Roubinet, M. L. Bossi, P. Alt,
M. Leutenegger, H. Shojaei,
S. Schnorrenberg, S. Nizamov, M. Irie,*
V. N. Belov,* S. W. Hell*
&&&& — &&&&
Carboxylated Photoswitchable
Diarylethenes for Biolabeling and Super-
Resolution RESOLFT Microscopy
6
www.angewandte.org
ꢀ 2016 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Angew. Chem. Int. Ed. 2016, 55, 1 – 6
These are not the final page numbers!