Live-Cell Localization Microscopy with a Fluorogenic and Self-Blinking Tetrazine Probe

Article abstract of DOI:10.1002/anie.201906806

Recent developments in fluorescence microscopy call for novel small-molecule-based labels with multiple functionalities to satisfy different experimental requirements. A current limitation in the advancement of live-cell single-molecule localization micro

Full text of DOI:10.1002/anie.201906806

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Accepted Article
Title: Live-Cell Localization Microscopy with a Fluorogenic and Self-
Blinking Tetrazine Probe
Authors: Philipp Werther, Klaus Yserentant, Felix Braun, Nicolai
Kaltwasser, Christoph Popp, Mathis Baalmann, Dirk-Peter
Herten, and Richard Wombacher
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To be cited as: Angew. Chem. Int. Ed. 10.1002/anie.201906806
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10.1002/anie.201906806
Angewandte Chemie International Edition
Live-Cell Localization Microscopy with a Fluorogenic
and Self-Blinking Tetrazine Probe
Philipp Werther,†,Δ Klaus Yserentant,‡,§,○,Δ Felix Braun,‡,§ Nicolai Kaltwasser,† Christoph Popp,†
Mathis Baalmann,† Dirk-Peter Herten‡,§,♦,* and Richard Wombacher†,*
† Institut für Pharmazie und Molekulare Biotechnologie, Ruprecht-Karls-Universität Heidelberg, Im
Neuenheimer Feld 364, 69120 Heidelberg, Germany.
‡ Physikalisch-Chemisches Institut, Ruprecht-Karls-Universität Heidelberg, Im Neuenheimer Feld
229, 69120 Heidelberg, Germany.
§ CellNetworks, Single-Molecule Spectroscopy, Ruprecht-Karls-Universität Heidelberg, Im
Neuenheimer Feld 267, 69120 Heidelberg, Germany.
○ Fakultät für Biowissenschaften, Ruprecht-Karls-Universität Heidelberg, Im Neuenheimer Feld 234,
69120 Heidelberg, Germany
♦ College of Medical and Dental Sciences, Medical School, University of Birmingham, Edgbaston,
Birmingham, B15 2TT, UK
Δ These authors contributed equally to this work.
* corresponding authors: wombacher@uni-heidelberg.de, d.herten@bham.ac.uk
Keywords:
bioorthogonal chemistry, click chemistry, fluorescent probes, super-resolution imaging, tetrazines
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10.1002/anie.201906806
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Abstract
Recent developments in fluorescence microscopy call for novel small molecule-based labels with
multiple functionalities to satisfy different experimental requirements. A current limitation in the
advancement of live-cell single-molecule localization microscopy, is the high excitation power
required to induce blinking. This is in marked contrast to the minimal phototoxicity required in live-
cell experiments. At the same time, quality of super-resolution imaging depends on high label
specificity, making removal of excess dye essential. Approaching both hurdles, we present a rational
design and synthetic route for a novel type of small molecule label comprising both fluorogenic and
self-blinking features. Bioorthogonal “click” chemistry ensures fast and highly selective attachment
onto a variety of biomolecular targets. Along with spectroscopic characterization, we demonstrate
that the probe overall improves quality and conditions for regular and single-molecule localization
microscopy on live-cell samples. The fluorescent probe is tailor-made for advanced live-cell
microscopy technologies with molecular features that go far beyond the usual requirements of spectral
and photophysical properties for small-molecule labels.
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Introduction
Small molecule fluorophores are essential tools for biological imaging, which is a key method
in modern life sciences. With the rapid development of novel fluorescence-based imaging
techniques, the design and chemical synthesis of fluorophores with improved photophysical
properties has experienced an enormous revival.[1] In particular, super-resolution imaging
techniques such as single-molecule localization microscopy (SMLM), like (d)STORM or PALM,
are heavily dependent upon individual properties of the applied organic fluorophores.[2] Active
control of emissive and dark states required for the localization of individual emitters in SMLM
typically requires high intensity illumination, addition of redox reagents or UV exposure, rendering
SMLM in live specimen challenging.[3] Spontaneously blinking fluorescent probes[4] based on the
silicon rhodamine (SiR) scaffold[5] have recently been developed to enable live-cell SMLM under
physiological conditions. In this approach, a hydroxymethyl nucleophile causes the reversible
intramolecular formation of a non-chromophoric spiroether. This chemical equilibrium of
hydroxymethyl SiR (HM-SiR) leads to stochastically occurring transitions between the excitable
quinoid and the non-excitable spiroether isomer. Hence, strenuous manipulation of the fluorophore
excited state to populate dark states is not required. This enables SMLM recordings without
additional stimulation of on-off-transitions.
The quality of image reconstruction in SMLM is governed by blinking kinetics, labeling density
and signal specificity.[6] In STORM and PALM the first is controlled by adapting excitation power
and redox buffer properties. In contrast, blinking kinetics of spontaneously blinking fluorophores
is an intrinsic property which can be influenced by a change in the local microenvironment;
Localizing HM-SiR to lipid membranes, for instance, has resulted in improved performance in
SMLM.[7] Regarding labeling density and signal specificity, fast, quantitative and highly selective
labeling reactions with a minimum of unspecific binding are crucial. Among bioconjugation
methods, bioorthogonal reactions such as the inverse electron demand Diels Alder reaction between
1,2,4,5-tetrazines and ring-strained alkenes and alkynes (DAinv) have lately received significant
attention. DAinv is particularly popular due to its fast kinetics, chemoselectivity and
biocompatibility, making it well-suited for live-cell applications.[8] Therefore, tetrazine-modified
small-molecule fluorophores have a broad applicability as they can be installed at virtually any
dienophile-tagged biomolecule. They allow specific labeling in combination with protein tags[9],
peptide tags[10] and unnatural amino acids[11] pushing the label size to a minimum. Moreover, DAinv
enables labeling various biomolecules other than proteins like nucleotides[12], sugars[13], lipids[14]
as well as to exploit small molecule-mediated targeting[15]. Additionally, tetrazine-based labeling
can be used to reduce unspecific signal; Carefully designed tetrazine probes have been shown to
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10.1002/anie.201906806
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undergo an increase in fluorescence when the tetrazine is consumed in DAinv, i.e. when the dye
label is covalently linked to its target structure.[15a, 16] This is a valuable additional feature for live-
cell fluorescence microscopy because it obviates the need for extensive excess dye wash-out and
dramatically reduces background signal.[17] This is particularly advantageous for SMLM as the
localization precision is affected by background signal.[18] Overall, we reason that a broadly
applicable probe for SMLM would comprise all of the above-mentioned features. It should be as
small as possible, self-blinking, fluorogenic, readily suited for bioconjugation and equipped with
generally favorable photophysical properties such as high brightness and photostability.
Here, we report the first merger of all these properties in the fluorogenic, far red-emitting, self-
blinking silicon rhodamine f-HM-SiR. This tetrazine-derivatized HM-SiR is initially strongly
quenched and shows fluorescence enhancement upon bioconjugation in DAinv. We report the
synthesis and photophysical properties of the novel fluorogenic dye and demonstrate its application
in live-cell super-resolution microscopy. The tetrazine functionalization was utilized to attach
f-HM-SiR to intracellular targets by fast and selective “click” chemistry in living cells and to
visualize intracellular dynamics by SMLM. Importantly, the fluorogenicity of f-HM-SiR allows
for minimal- or no-wash procedures in live-cell imaging while its self-blinking feature minimizes
phototoxicity in SMLM experiments.
Results and Discussion
First, we examined synthetic strategies on how to access fluorophore probes that combine both
tetrazine and the desired structural hydroxymethyl motif in the SiR scaffold. Synthesis with
tetrazines requires mild reaction conditions as they are highly base sensitive and react with strong
nucleophiles. Therefore, a mild Lewis acid-mediated Friedel-Crafts reaction[16e] was selected as the
key step for the assembly of the HM-SiR derivative. We set out to test the feasibility of this strategy
for the synthesis of the simple unfunctionalized HM-SiR without tetrazine (Fig. 1) and found that
this four step route using methoxymethyl (MOM) protection group chemistry enabled synthesis of
HM-SiR at 22 % overall yield (Scheme S2). This approach complements previous strategies
relying on the addition of aryl lithium reagents to Si-anthrones[4a] and could offer a short and mild
synthesis route for similar fluorophores in the future. Our attention then turned to the synthesis of
the tetrazine-modified HM-SiR derivative, in the following termed fluorogenic HM-SiR
(f-HM-SiR). For this purpose, we synthesized the appropriate tetrazinyl benzaldehyde (5,
Scheme S1) as electrophile for the Friedel-Crafts reaction. Conversion with the diarylsilane (6,
Scheme S1) gave access to the desired f-HM-SiR, which carries both a 3-hydroxyl and a 6-tetrazine
substituent (Fig. 1). Both substituents are crucial for the intramolecular modulation of the probe’s
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spectral properties: While fluorescence of f-HM-SiR is strongly quenched by the tetrazine moiety
(Fig 1a), it is restored by conversion with dienophile-equipped biomolecular targets in DAinv
(Fig. 1a, 1b). The resulting Diels-Alder product of f-HM-SiR still exhibits the pH-dependent
equilibrium between absorbing (open) and non-absorbing form (closed spiroether) and thereby
spontaneously switches between fluorescent and non-fluorescent states. Consequently, external
control of the emitter density which is otherwise essential in SMLM becomes obsolete.
Figure 1. f-HM-SiR, a fluorogenic and spontaneously blinking fluorophore for bioorthogonal DAinv a) Spiro-cyclization
equilibrium of spontaneously blinking and fluorogenic f-HM-SiR and its Diels-Alder cycloadduct (DAinv-Product). b)
Fluorescence enhancement of f-HM-SiR upon conversion in DAinv at pH 4 in aqueous buffer; c) pH-dependent fluorescence
of f-HM-SiR (blue) and DAinv-Product (red).
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In order to evaluate the fluorogenicity of f-HM-SiR in DAinv, it was treated with
bicyclo[6.1.0]non-4-yne (BCN) dienophile, which resulted in a 10-fold fluorescence enhancement
upon reaction (Fig. 1b). In addition, we determined a substantial increase in brightness of the
respective isolated cycloadduct DAinv-Product (SI, Table S1). To assess the self-blinking
properties of f-HM-SiR, we studied the reversible spirocyclization reaction. Both, the quinoid and
1
the spiroether isomer of f-HM-SiR, could be observed in H NMR (Fig. S3). The pH dependent
equilibrium between the two isomers was investigated by absorbance (Fig. S2) and fluorescence
(Fig. 1c) spectroscopy. Here, we determined an equilibrium constant of pKcycl = 4.0 (±0.1)
indicating that the non-fluorescent spiroether form of f-HM-SiR significantly prevails at
physiological conditions (99.9 %).[4a] The isolated DAinv-Product showed a significant shift to
pKcycl = 5.2 (±0.1), indicating that the proportion of the fluorescent quinoid form of the target-
bound f-HM-SiR is increased but remains below 1 % at physiological pH (99.4 % spiroether). The
higher abundance of the quinoid isomer of target-bound f-HM-SiR infers that the emissive state
will be more populated compared to unreacted dye leading to an overall reduction of unspecific
signals. Consequently, two mechanisms of fluorogenicity could be observed: one corresponds to
the loss of the quenching tetrazine, the other to the changed tendency of the product to form the
spiroether. Following up on those results, we set out to evaluate the general suitability of f-HM-SiR
for bioconjugation. A purified HaloTag-EGFP was modified with HaloTag ligand-BCN
(HTL-BCN) and subsequently reacted with f-HM-SiR. In-gel fluorescence showed specific
labeling, while the pH-dependent emission of the EGFP-bound dye was retained (Fig. S7).
Based on these promising photophysical characteristics, we moved on to apply the probe in live-
cell wide-field fluorescence imaging to further evaluate the fluorogenicity of f-HM-SiR in the
context of cellular labeling. Given the flexibility of tetrazine-based “click” chemistry, we tested a
variety of procedures (Fig. 2a). Protein labeling was demonstrated using the enzymatic self-labeling
HaloTag as a fusion with histone H2A or mitochondrial import receptor subunit TOM20 in
combination with HaloTag ligand dienophiles. Further, we used triphenylphosphonium (TPP), a
small molecule organelle marker, to localize the respective dienophile conjugate TPP-BCN to the
mitochondrial matrix.[19] Staining and fluorescence imaging of nuclear and mitochondrial targets
were carried out in HeLa cells. Cells were transiently transfected with H2A-HaloTag and treated
with HTL-BCN (10 µM) for 30 min. Additional staining of unmodified HaloTag proteins with
HTL-TMR subsequent to HTL-BCN incubation served as a transfection and co-localization control.
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Figure 2. Cell-permeable f-HM-SiR can be specifically conjugated in live cells by DAinv. a) Prior to reaction with dienophile,
f-HM-SiR resides in quenched form. Target-bound f-HM-SiR spontaneously switches between fluorescent on- and off-form.
b) HeLa cells transiently expressing H2A-HaloTag were incubated with (upper row) or without (lower row) HTL-BCN
(10 µM) for 30 min., washed and labeled with f-HM-SiR (2 µM) (magenta) prior to imaging. Reference images (green) were
generated by labeling with HTL-TMR after incubation HTL-BCN. c) HeLa cells transiently expressing TOM20-HaloTag
were incubated with TPP-BCN (10 µM) and HTL-TMR (green), washed and labeled with f-HM-SiR (2 µM) (magenta) prior
to imaging. d) Background intensity of HeLa cells incubated with various SiR derivatives (2 µM), n=20. Medium was
replaced once after incubation with dyes. Note the broken y-axis and change of scale. e) Representative images for
diagram in d). Settings for image acquisition, processing and display were identical for all shown images. Outlines of cells
indicated by white line. Scale bars in b, c, e): 10 µm.
After wash-out of excess HTL-BCN, cells were incubated with f-HM-SiR and imaged without
dye removal. Averaging 2.000 frames of the recorded blinking signal resulted in an image with
good co-localization to the HTL-TMR control (Fig. 2b). In the same fashion, mitochondrial labeling
with trans-cyclooctene (TCO) as dienophile was equally successful (Fig. S8). Similarly, HeLa cells
treated with TPP-BCN and f-HM-SiR show expected mitochondrial structures (Fig. 2c). Co-
staining with TOM20-HaloTag and HTL-TMR verifies the specificity of the mitochondrial staining
at good contrast. This demonstrates the efficient background suppression when using f-HM-SiR in
live-cell fluorescence microscopy. In control experiments without HTL-BCN we noted slight
unspecific signal upon addition of f-HM-SiR (Fig. 2b). We attribute this to the presence of
lysosomes with luminal pH~4. In this context, we validated the previously observed fluorogenicity
of f-HM-SiR in comparison to other silicon rhodamine dyes like HM-SiR (lacking a tetrazine
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moiety), tetrazine-substituted SiR-Tz, and bare SiR-COOH (Fig. S6). To this end, we incubated
wild type HeLa cells with the respective probes and quantified cellular fluorescence intensities
(Fig. 2d,e). The average fluorescence from HeLa cells incubated with f-HM-SiR was comparable
to untreated cells, while the other three SiR dyes caused significantly higher signal. The observed
signal intensities directly correlate to the residual background resulting from nonreacted dye excess
in labeling experiments. These tendencies were even more pronounced under no-wash conditions
(Fig. S9). This highlights the improved background suppression of f-HM-SiR compared to existing
SiR derivatives owing to the close proximity of tetrazine to the chromophoric center.
Unspecific signal reduces contrast and structure representation quality in fluorescence imaging.
In SMLM it additionally leads to decreased localization precision. Therefore, efficient background
suppression is particularly important here. Thus, small molecule probes bearing bioorthogonal
attachment functionalities that come along with fluorogenic properties possess great potential for
super-resolution microscopy. In the past, super-resolution microscopy with fluorogenic tetrazine
dyes has been achieved in fixed cells.[16g, 16h] Encouraged by the advantageous features of
f-HM-SiR in live-cell labeling, we studied its suitability for SMLM in living cells. HeLa cells
transiently transfected with TOM20-HaloTag were incubated with HTL-TCO*, washed and labeled
with f-HM-SiR (2 μM). To minimize background signal for live-cell SMLM excess fluorophore
was washed out in this experiment. Imaging under highly inclined illumination conditions allowed
localization of individual spontaneously blinking emitters. The image reconstructed from
500 frames shows the expected structure for outer mitochondrial membrane localized TOM20
(Fig. 3a) and enabled us to follow dynamics at sub-diffraction resolution (movie S1). Comparison
with an image averaged over 500 frames in Fig. 3b highlights the achieved resolution improvement
of f-HM-SiR in live-cell SMLM. From the reconstructed image, it is possible to differentiate the
outer membrane of the mitochondrial network (Fig. 3b,d) and we were able to determine a mean
mitochondrial diameter of about 375 nm from 16 cross-sectional profiles (Fig. 3e,f). SMLM image
sequences were recorded at a laser intensity of 1 kW/cm2 at 640 nm, at which we did not observe
any considerable phototoxicity, even in long time experiments (movie S5). As these moderate laser
intensities mitigate photobleaching, we were interested in the capability of f-HM-SiR for long-term
SMLM. In order to genuinely quantify the number of localizations over time unbiased by cellular
movement, we chemically fixed the cells after addition of the probe. The average number of
localizations sampled over 200 s showed no significant decrease (Fig. 3g). At the same time, super-
resolution images reconstructed from frame subsets do not indicate any noticeable loss in
reconstruction quality. Finally, we tested how the fluorogenicity of f-HM-SiR affects localization
of unspecifically bound label in comparison to the non-fluorogenic HM-SiR. Comparison of the
background localizations within a cell per area and time unit shows a more than 2-fold reduction
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for f-HM-SiR (Fig. 3h). Together, these observations suggest a high suitability of f-HM-SiR to
probe cellular dynamics in live-cell SMLM.
Figure 3. Spontaneous blinking of f-HM-SiR enables SMLM with improved resolution and reduced background. a) COS-7
cells transiently expressing TOM20-mCherry-HaloTag were incubated with HTL-TCO (10 µM), washed and labeled with
f-HM-SiR (2 µM) for SMLM imaging. Also see movie S1 b) Average projection of 500 raw data frames used for
reconstruction in a). c) Merged zoom-ins of boxed regions in a) (red) and b) (green). d) Line profile along boxed region in
c) comparing normalized intensities in averaged raw data (green) and SMLM reconstruction (red). e) Averaged cross-
sectional profiles of labeled mitochondria ±1 standard deviation, n=16. Individual profiles were aligned to minimum between
peaks (see methods). See Fig. S11 for all profiles. f) Peak to peak distances in cross-sectional profiles shown in e). g)
Long-term stability of f-HM-SiR labeled TOM20-HaloTag in fixed COS-7 cells. Mean localizations per frame normalized to
number of localizations in first frame (black line) ±1 standard deviation, n=8. h) Background localization rate for HM-SiR
and f-HM-SiR in non-transfected HeLa cells. 20 datasets each. Scale bars a, b): 5 µm, c): 1 µm.
To fully make use of excitation-independent on-off switching of f-HM-SiR, we performed
long-term SMLM experiments, this time omitting unnecessary dye wash-out in order to fully utilize
the probe’s fluorogenicity. First, we turned our attention to the nuclear target protein H2A-HaloTag.
After incubating HeLa cells with HTL-BCN and subsequent linker wash-out, cells were labeled
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with f-HM-SiR. Mere buffer medium change and direct image acquisition was sufficient to afford
high resolution in the reconstructed image (Fig. 4a). In contrast to an average projection (Fig. 4b,c),
only the super-resolved image allows for an identification of individual H2A proteins and variations
in local H2A density. Subsequently, to visualize a more dynamic cellular target we localized the
dienophile to the mitochondrial matrix with TPP. HeLa cells were incubated with TPP-BCN,
washed and stained with f-HM-SiR. Medium was replaced once to minimize extra-cellular
deposition of the probe before imaging the cells. We choose a moderate excitation power of
1 kW/cm2 to facilitate long time imaging. As TPP accumulates in the mitochondrial matrix, the
reconstructed image in Fig. 4d shows the expected pattern of homogeneously filled tubes. The
averaged intersection from eleven cross-sectional profiles (Fig. 4e) has a median FWHM of 346 nm
(Fig. 4f), which is in accordance with the median of the peak-to-peak distance of 375 nm
determined for mitochondria labeled at the outer membrane (Fig. 3e,f and Fig. S11c for a direct
comparison).
Figure 4. f-HM-SiR reveals cellular dynamics in live HeLa cells with improved resolution. a) HeLa cells transiently
expressing H2A-HaloTag were incubated with HTL-BCN (10 µM), washed and labeled with f-HM-SiR (2 µM.) A
reconstruction from 333 frames corresponding to 6.67 seconds acquisition time is shown. b) Zoom-in of boxed region in a).
c) Corresponding averaged image for boxed region in a). d) HeLa cells were incubated with TPP-BCN (10 µM), washed
and labeled with f-HM-SiR (2 µM.) Reconstruction from 500 frames (10 seconds) is shown. e) Averaged cross-sectional
profiles from mitochondrial tubules after alignment, ±1 standard deviation, n=11. f) Width of individual profiles shown in e).
See Fig. S11 for position off all profiles. g) Zoom in of boxed region in d). Average (left) and reconstructions from
500 frames at different time points (right). Arrow head indicates mitochondrial fusion event. h) Average (top left) and
reconstructions of boxed region in g). Localizations are colored with respect to their relative time of appearance within a
single reconstruction. Scale bars: a) 5 µm, d) 10 µm, g) 2 µm, h) 1 µm.
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We were also able to follow mitochondrial dynamics over 200 s with a temporal resolution of 10 s
as shown in Fig. 4g and movie S2. Considering the inherent trade-off between high excitation
power (resulting in high photon output and thus localization precision) and cell viability, the
achieved spatial resolution is reasonable (for photon count and localization uncertainty see
Fig. S10). The time-resolved super-resolution imaging was not limited by fluorophore bleaching,
emphasizing the performance of f-HM-SiR. Beyond visualizing motion dynamics, it was also
possible to observe mitochondrial fusion events (Fig. 4g) and fission events (Fig. 4h). Temporal
resolution in SMLM depends on the time during which emitter localizations are accumulated for
reconstructing a super-resolved image. Increased time resolution can be achieved by shortening the
accumulation time at the cost of reconstruction quality, i.e. the captured localization density along
the imaged structures.
To capture fast cellular dynamics, we additionally made use of the exact time at which individual
localizations were recorded. We visualized this additional information by color coding the
appearance time of localizations within each reconstruction from red (beginning) over green to blue
(end) of each reconstruction window (Fig. 4h, movie S3). In this representation, structures which
remain quasi stationary during data recording for one reconstruction will appear green while
forming structures will be colored red and collapsing structure will be colored blue (Fig. 4h,
arrows). The fact that most mitochondria in the shown example are colored green further indicates
that the achieved time resolution of 10 s is sufficient to capture the observed mitochondrial
dynamics.
Overall, these results show that the combination of fluorogenicity and self-blinking in f-HM-SiR
greatly improves the potential of SMLM applications for live-cell experiments. While
fluorogenicity significantly reduces artifacts from unspecific probe deposition, self-blinking
enables live-cell SMLM over relatively long time scales due to the low excitation power required.
Conclusion
In summary, we presented the first combination of fluorogenic and self-blinking properties in a
single fluorescent probe and demonstrated its advantage for live-cell localization microscopy. The
tetrazine moiety strongly quenches the fluorescence prior to reaction and enables fluorogenic
bioconjugation to various dienophile-modified cellular targets. f-HM-SiR exhibits excellent cell
permeability, high brightness and photostability, all of which are important criteria for application
in live-cell imaging. Exploiting the self-blinking properties, we demonstrate its application in live-
cell SMLM without the necessity of stabilizing buffers or high excitation power. f-HM-SiR allows
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live-cell localization microscopy after simple media replacement. SMLM imaging of intracellular
dynamics can be performed over long time periods and at a high spatio-temporal resolution. We
expect that our molecular design will stimulate the future development of multifunctional
fluorescent probes as tailor-made tools for bioimaging.
Acknowledgements
R.W. acknowledges funding from the Deutsche Forschungsgemeinschaft DFG (SPP1623, WO
1888/1-2) and D.-P.H. from the Federal Ministry of Education and Research (BMBF/VDI;
MorphiQuant3d and Switch-Click-Microscopy).
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Research Article
A rationally designed small molecule
label comprising fluorogenic and self-
blinking features for bioorthogonal
chemistry and super-resolution
microscopy in living cells.
P. Werther Δ, K. Yserentant Δ, F. Braun,
N. Kaltwasser, C. Popp, M. Baalmann,
D.P. Herten*, R. Wombacher*
Page No. – Page No.
Live-Cell Localization Microscopy
with a Fluorogenic and Self-Blinking
Tetrazine Probe
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