Cell-Permeant Large Stokes Shift Dyes for Transfection-Free Multicolor Nanoscopy

Article abstract of DOI:10.1021/jacs.7b06412

We designed cell-permeant red-emitting fluorescent dye labels with >140 nm Stokes shifts based on 9-iminoanthrone, 9-imino-10-silaxanthone, and 9-imino-10-germaxanthone fluorophores. The corresponding probes selectively targeting mitochondria, lysosomes, and F-actin demonstrate low toxicity and enable stimulated emission depletion (STED) nanoscopy in neurons, human fibroblasts, U2OS, and HeLa cells. In combination with known small Stokes shift dyes, our probes allow live-cell three-color STED nanoscopy of endogenous targets on popular setups with 775 nm STED wavelength.

Full text of DOI:10.1021/jacs.7b06412

Communication
pubs.acs.org/JACS
Cell-Permeant Large Stokes Shift Dyes for Transfection-Free
Multicolor Nanoscopy
Alexey N. Butkevich,*,# Grazvydas Lukinavicius,*,# Elisa D’Este, and Stefan W. Hell
̌
̌
Department of NanoBiophotonics, Max Planck Institute for Biophysical Chemistry, Am Fassberg 11, 37077 Gottingen, Germany
̈
S
* Supporting Information
of living cells. Besides, they should be compatible with the
ABSTRACT: We designed cell-permeant red-emitting
fluorescent dye labels with >140 nm Stokes shifts based
on 9-iminoanthrone, 9-imino-10-silaxanthone, and 9-
imino-10-germaxanthone fluorophores. The corresponding
probes selectively targeting mitochondria, lysosomes, and
F-actin demonstrate low toxicity and enable stimulated
emission depletion (STED) nanoscopy in neurons, human
fibroblasts, U2OS, and HeLa cells. In combination with
known small Stokes shift dyes, our probes allow live-cell
three-color STED nanoscopy of endogenous targets on
popular setups with 775 nm STED wavelength.
popular 775 nm STED wavelength. Following the recent report
by the Klan
́
group12 and an earlier work of Wu and Burgess,13
we had identified 9-aminopyronin scaffold as a promising LSS
analog of rhodamine. Substitution of the bridging oxygen with a
14 group element14 allowed us to develop several selective and
cell-permeant LSS probes based on these fluorophores and
establish a three-color live-cell imaging scheme for standard
STED microscopes.
To this end, we synthesized four 9-iminoanthrone dyes with
a group in the 10-position varying between CMe2, GeMe2,
SiMe2, and SO2 (Figure 1, i) and bearing a protected primary
ulticolor super-resolution fluorescence microscopy or
Mnanoscopy is a valuable method for observing
interactions between intracellular structures and biomolecules,
as well as tracking dynamic processes in cells.1,2 Initially,
fluorescence nanoscopy of living cells relied mainly on
fluorescent proteins, necessitating genetic modifications of
organisms or cells. Small-molecule ligands with nanoscopy-
compatible fluorophores, targeting specific proteins or organ-
elles, avoid these difficulties.3
In recent reports, a number of bright green- to far-red-
emitting fluorophores suitable for nanoscopy have been
designed and employed as live-cell fluorescent markers.4−6
Several dye combinations permitting two-color imaging with
<60 nm resolution were identified. For example, simultaneous
two-color stimulated emission depletion (STED) microscopy
with a single pulsed ∼770 nm de-excitation laser was achieved
by combining fluorophores excitable at ∼580 and ∼640 nm.7 In
living cells, this imaging scheme was realized with markers such
as 580R or 580CP together with SiR, 640SiRH, GeR, or
630GeRH dyes.2b,4,6a,8 The implementation of additional
imaging channels with known live-cell dyes requires sophisti-
cated techniques such as hyperspectral detection1,9 or
fluorescence lifetime recording.10 A straightforward solution
for introducing a third channel on the widely available
nanoscopes with 775 nm STED wavelength relied on using
large Stokes shift (LSS) labels, as demonstrated on fixed
samples.11 A LSS dye can be spectrally separated from small
Stokes shift dyes on the basis of the excitation and/or emission
wavelengths, and this selectivity allows an easy implementation
of an additional color channel into an established two-color
detection scheme employing two small Stokes shift labels.
However, LSS dyes for live-cell labeling have still been missing.
Therefore, we focused our efforts on the development of LSS
fluorophores capable of penetrating intact plasma membranes
Figure 1. Chemical structures of LSS probes. (i) Protonation−
deprotonation behavior responsible for the environment sensitivity of
the dyes 1−4. (ii) Ligands a−d for labeling of intracellular structures.
amino group or carboxylic acid for conjugation to a suitable
ligand (Figure S1). Three of them, based on 9-iminoanthrone
(CX, 1), 9-imino-10-germaxanthone (GeX, 2), and 9-imino-10-
silaxanthone (SiX, 3) fluorophores, demonstrated similar
absorption and emission bands, with Stokes shifts of 140−
165 nm (Table 1 and Figure S2), good photostability (Figure
S3), high emission quantum yields in aqueous buffers, and
Received: June 20, 2017
Published: August 28, 2017
© 2017 American Chemical Society
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DOI: 10.1021/jacs.7b06412
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Communication
a
Table 1. Photophysical Properties of Dyes 1−4, Measured in 10% Methanol/PBS 7.4 (unless Otherwise Indicated)
λmax, nm
dye
absorption (ε, M−1 cm−1
)
emission (Φfl)
Stokes shift, nm
fluorescence lifetime, ns
CX (1)
459 (28000)
454 (24000)
458 (17000)
599 (0.55)
618 (0.17)
623 (0.28)
140
164
165
3.2
1.2
1.9
GeX (2)
SiX (3)
b
b
c
SO2X (4)
509 (13000)
647 (0.15)
138
1.2
a
b
c
ε, extinction coefficient; Φfl, absolute fluorescence quantum yield. In 10% acetonitrile/H2O, In 30% acetonitrile/H2O.
fluorescence lifetimes varying by ∼1 ns. Their fluorescence
emission was found to strongly decrease in nonpolar solvents
and at pH > 8 (Figures S4 and S5). In pure water, loss of
emission (but not absorption) was observed due to aggregation
of all dyes except CX (1). On the contrary, the 9-imino-10,10-
dioxido-9H-thioxanthone based dye (SO2X, 4) demonstrated
fluorescence and intense absorption in the range >350 nm only
in acidic media (pH < 5). This behavior suggested a
protonation/deprotonation-based mechanism for environment
sensitivity of iminoanthrone dyes (Figure 1, i). Indeed, the
calculated values for the excitation maxima of the correspond-
ing model 9-(methylimino)anthrones and their protonated
forms (at the APFD/6-311++G(2d,p) level of theory) matched
their experimental absorption maxima within 25 nm, with
consistent overestimation of λmax for imines and under-
estimation for iminium forms (Figure S6 and Table S1).
For intracellular targeting of the proposed fluorescent dyes,
we selected two different strategies: membrane potential-based
targeting for directing to mitochondria and protein−ligand
interaction for selective labeling of lysosomes and cytoskeleton
(Figure 1, ii). Accumulation of lipophilic cations, in particular of
triphenylphosphonium (TPP) salts,15 in mitochondria is driven
by the negative potential of the inner mitochondrial membrane.
Weakly basic pH of the mitochondrial matrix (about 7.9)16
limited the LSS fluorophore choice to dyes 1−3 (Figures S5
and S7). All three TPP conjugates 1a−3a provided high-
contrast images of mitochondria (Figure S8) verified by perfect
colocalization with MitoTracker Orange (Figure S9).
Prior to the live-cell nanoscopy studies, we identified
threshold concentrations below which our probes were not
affecting the population distribution over cell cycle, a critical
parameter reflecting cell viability and proliferation (Figure 2a).
Figure 2. Performance of SiX probes targeted to different cellular
organelles. (a) Percentage of cells with different measured DNA
content after 24 h treatment with indicated probes vs DMSO vehicle,
corresponding to cell cycle phases: G1, S, G2, and M. Cells containing
less DNA than haploid (SubG1) are considered non-viable. Data are
presented as mean with standard deviation, N = 4. For the results of t
test, see Table S3. (b) Confocal and STED images of fixed human
fibroblasts stained with primary mouse anti-α-tubulin and SiX-labeled
secondary sheep anti-mouse antibodies. (c) Dependence of micro-
tubule apparent thickness FWHM on the de-excitation power applied.
(d−f) STED images of living human fibroblasts stained with (d) 0.25
μM SiX-actin (3c), (e) 2 μM SiX-TPP (3a), and (f) 3 μM SiX-lyso
(3b). Dashed squares indicate zoom-in areas shown in Figure S15.
Scale bars, 1 μm.
Pepstatin A is a known potent non-covalent inhibitor of the
lysosomal protease cathepsin D (IC50 = 10 nM).17 All
pepstatin-derived probes (1b−4b) showed fluorescence in-
crease upon pepsin binding (Figure S10a), were functionally
active and inhibited BSA hydrolysis by pepsin (Figure S10b).
The apparent Kd values of the probes 1b−3b lay in the range of
40−50 nM (Table S2 and Figure S10c,d) and compared
TPP probes showed little influence on cell cycle below 2.5 μM
over 24 h, comparable to previous results on fluorescent TPP
conjugates.19 The lysosomal probes displayed no detectable
toxic effects within the tested range; nevertheless, high
concentrations (>5 μM) of these probes might influence the
morphology of lysosomes and should be avoided.20 SiX-actin
3c altered the cell viability in sub-micromolar concentrations
(above 0.5 μM), similarly to SiR-actin probe (Figures 2a and
S13).6b Note that the following imaging experiments were
carried out below the established cytotoxicity threshold.
The efficiency of SiX de-excitation was estimated in fixed
human fibroblasts treated with combination of primary mouse
anti-α-tubulin and SiX-tagged secondary sheep anti-mouse
antibodies. We were able to reach an average apparent
microtubule full-width at half-maximum (FWHM) of 88
15 nm at full de-excitation laser power (250 mW entering
objective, Figure 2b,c). The fluorescent primary antibody,
tagged with SiX-NHS (3d), was used for labeling acetylated
favorably to the 174
27 nM value obtained for SiR-
lysosome.6a In living human fibroblasts treated with 2 μM
pepstatin A conjugates (1b−4b), the acidic compartments of
lysosomes were stained brightly and selectively with GeX-lyso
(2b) and SiX-lyso (3b) probes (Figure S11). Both LSS probes
colocalized with CellLight Lysosomes-RFP (Figure S12). CX-
lyso probe 1b demonstrated some off-target staining of
mitochondria, while no specific signal was observed for
SO2X-derived probe 4b, and SO2X fluorophore was excluded
from further tests.
Based on these experiments, we identified GeX and SiX
fluorophores as the most suitable for the development of
further selective ligands, and SiX was preferred on the grounds
of its higher emission quantum yield (Table 1). Jasplakinolide-
derived SiX-actin probe 3c for cytoskeletal F-actin17,18 and
amino-reactive ester SiX-NHS (3d) for antibody labeling were
then prepared.
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Communication
tubulin in fixed human fibroblasts. The structure of primary
cilium was visualized with SiX anti-acetylated tubulin, and the
inner diameter of the centriole cylinder of cilium-forming
centrosomes was resolved to 143
5 nm (Figure S14),
comparable to the previously reported diameter of 176 10
nm measured in living human fibroblasts stained with SiR-
tubulin.6b
For single-color live-cell STED nanoscopy, human fibroblasts
were stained with 0.25 μM SiX-actin (3c). We were able to
resolve F-actin stress fibers which are not discernible using
confocal microscopy (Figures 2d and S15a). With 2.5 μM SiX-
TPP (3a), mitochondrial substructures could be resolved
(Figures 2e and S15b). Similarly, lysosomes stained with 3 μM
SiX-lyso (3b) exhibit resolution improvement in STED images
(Figures 2f and S15c). Additionally, actin and lysosomes could
be specifically stained in living HeLa and U2OS cells with SiX-
actin (3c) and SiX-lyso probes (3b), respectively (Figure S16).
We initiated live-cell multicolor confocal and STED imaging
using our LSS probes by examining their concurrent use with
GFP-tagged proteins. Human fibroblasts were transduced with
CellLight Talin-GFP, stained with 0.25 μM SiX-actin (3c) for 1
h and imaged after washing. It was possible to read both
channels with two detectors simultaneously while exciting with
a 485 nm laser, and correct localization of talin in the focal
adhesions at the end of actin stress fibers21 was clearly visible
(Figure S17a,c). The imaging scheme was further expanded to
four colors (3× STED and 1× confocal) with the introduction
of two additional markers: MitoTracker Orange CM-
H2TMRos and GeR-tubulin4 (Figure S17b,d). Four structures
were localized, demonstrating the expected positioning of
cytoskeleton and mitochondria. We also succeeded in imaging
genetically unmodified human fibroblasts by three-color
staining with SiX-lyso, 580CP-actin, and SiR-tubulin;6b
lysosome movement along the microtubules could also be
recorded (supplementary video). Furthermore, all three
mitochondrial LSS probes (1a−3a) supported four-color
imaging when applied in combination with 580CP-actin,
GeR-tubulin,4 and a plasma membrane and endosomal marker
wheat germ agglutinin (WGA) labeled with Alexa Fluor 488
(Figures 3a,b and S18). Interestingly, we found that some
endosomes and mitochondria share the same microtubule
(Figure 3c).
Finally, we demonstrated the application of the new LSS
probes to transfection-free four color staining of living rat
hippocampal neurons using the combination of CX-TPP (1a),
Alexa Fluor 488-labeled secondary antibody against a primary
anti-neurofascin antibody, 580CP-actin, and GeR-tubulin.4
Neurofascin, a marker for the axon initial segment, conven-
iently allowed an easy identification of axons.22 The ubiquitous
periodic organization of the actin cytoskeleton23 perpendicular
to microtubule cables was clearly visible, as well as
mitochondria localized along the microtubule network (Figure
3d).
In summary, we introduced a series of new LSS probes based
on iminoanthrone scaffolds suitable for live-cell imaging of
lysosomes, mitochondria, and actin, as well as for immunostain-
ing, at different physiological pH levels. The probes are cell-
permeant, compatible with STED nanoscopy, and nontoxic at
the recommended concentrations. The iminoanthrone dyes
enable, for the first time, in combination with our previously
introduced live-cell 580CP8, GeR4, and SiR6a,b labels, far-red
three-color live nanoscopy with widely available 775 nm STED
microscopes. Tuning the STED wavelength and power for a
Figure 3. Four-color microscopy with CX-TPP probe. (a) Normalized
absorption and emission spectra of the dyes Alexa Fluor 488 (yellow),
CX (magenta), 580CP (green), and GeR (cyan) utilized for
simultaneous multicolor imaging, related laser lines (solid vertical
lines), and detection windows (transparent rectangles). De-excitation
laser (black line) is set at 775 nm. (b) Excitation/detection scheme
used for four-color imaging. (c) Four-color image of living human
fibroblasts stained with 1 μM 580CP-actin, 2 μM GeR-tubulin, 2 μM
CX-TPP (1a), and 5 μg/mL Alexa Fluor 488-WGA conjugate for 1 h
at 37 °C in DMEM growth media (acquired in DMEM growth media
after washing two times with HBSS). White arrowheads indicate
microtubule sites co-occupied by mitochondria and endosomes. (d)
Four-color image of living rat hippocampal neurons (16 DIV) stained
with 1 μM 580CP-actin, 2 μM GeR-tubulin, 0.5 μM CX-TPP (1a) for
1 h at 37 °C, followed by anti-neurofascin and anti-mouse Alexa Fluor
488 labeling for 5 min and 30 s, respectively. Scale bars 1 μm.
particular dye combination will allow further optimization of
resolution. The new probes are particularly suitable for dual-
color imaging with GFP fusion proteins and a single laser
excitation source, resulting in reduced bleaching and permitting
simultaneous signal readout in two channels. Their wide
applicability opens up further exciting opportunities for live-
and fixed-cell multicolor imaging with standard fluorescence
microscopes as well as other nanoscopy methods.
ASSOCIATED CONTENT
* Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/jacs.7b06412.
■
S
Synthetic procedures and characterizations of the
compounds, including Figures S1−S18 and Tables S1−
S3 (PDF)
Supplementary video: three-color STED movie of living
human fibroblasts stained with 2 μM SiX-lyso, 5 μM
580CP-actin, and 5 μM SiR-tubulin (AVI)
AUTHOR INFORMATION
Corresponding Authors
*alexey.butkevich@mpibpc.mpg.de
■
*grazvydas.lukinavicius@mpibpc.mpg.de
ORCID
Grazvydas Lukinavicius: 0000-0002-7176-1793
̌
̌
Stefan W. Hell: 0000-0002-9638-5077
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DOI: 10.1021/jacs.7b06412
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Communication
Wildanger, D.; Vicidomini, G.; Kastrup, L.; Hell, S. W. Opt. Express
2011, 19, 3130−43.
Author Contributions
#A.N.B. and G.L. contributed equally to this work.
(11) Sidenstein, S. C.; D’Este, E.; Bohm, M. J.; Danzl, J. G.; Belov, V.
N.; Hell, S. W. Sci. Rep. 2016, 6, 26725.
Notes
The authors declare no competing financial interest.
(12) Horvath, P.; Sebej, P.; Solomek, T.; Klan, P. J. Org. Chem. 2015,
80, 1299−311.
ACKNOWLEDGMENTS
The authors acknowledge Jan Seikowski, Jens Schimpfhauser,
(13) Wu, L.; Burgess, K. Org. Lett. 2008, 10, 1779−82.
(14) Koide, Y.; Urano, Y.; Hanaoka, K.; Terai, T.; Nagano, T. ACS
Chem. Biol. 2011, 6, 600−8.
■
and Jurgen Bienert for assistance with synthesis and character-
̈
(15) Ross, M. F.; Kelso, G. F.; Blaikie, F. H.; James, A. M.; Cocheme,
H. M.; Filipovska, A.; Da Ros, T.; Hurd, T. R.; Smith, R. A.; Murphy,
M. P. Biochemistry (Moscow) 2005, 70, 222−30.
ization of the probes, Dr. Shamil Nizamov (Abberior GmbH,
Gottingen) for a sample of Abberior STAR 470SXP dye, and
̈
Dr. Mariano L. Bossi for helpful discussions. G.L. is grateful to
the Max Planck Society for the Nobel Laureate Fellowship
award. The authors thank Dr. Dirk Kamin, Dr. Sebastian
Schnorrenberg, and Tanja Gilat for technical assistance and
acknowledge Dr. Vladimir N. Belov and Jaydev Jethwa for
proofreading the manuscript.
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