A caged, localizable rhodamine derivative for superresolution microscopy

Article abstract of DOI:10.1021/cb2002889

A caged rhodamine 110 derivative for the specific labeling of SNAP-tag fusion proteins is introduced. The caged rhodamine 110 derivative permits the labeling of cell surface proteins in living cells and of intracellular proteins in fixed cells. The probe requires only a single caging group to maintain the fluorophore in a non-fluorescent state and becomes highly fluorescent after uncaging. The high contrast ratio is confirmed both in bulk and at the single molecule level. This property, together with its high photon yield makes it an excellent dye for photoactivated localization microscopy (PALM), as we demonstrate here.

Full text of DOI:10.1021/cb2002889

Letters
pubs.acs.org/acschemicalbiology
A Caged, Localizable Rhodamine Derivative for Superresolution
Microscopy
Sambashiva Banala,†,§ Damien Maurel,†,§ Suliana Manley,‡ and Kai Johnsson†,*
†
‡
́
́ ́
Institute of Chemical Sciences and Engineering and Laboratory of Experimental Biophysics, Ecole Polytechnique Federale de
Lausanne (EPFL), Lausanne, Switzerland
S
* Supporting Information
ABSTRACT: A caged rhodamine 110 derivative for the
specific labeling of SNAP-tag fusion proteins is introduced.
The caged rhodamine 110 derivative permits the labeling of
cell surface proteins in living cells and of intracellular proteins
in fixed cells. The probe requires only a single caging group to
maintain the fluorophore in a non-fluorescent state and
becomes highly fluorescent after uncaging. The high contrast ratio is confirmed both in bulk and at the single molecule level. This
property, together with its high photon yield makes it an excellent dye for photoactivated localization microscopy (PALM), as we
demonstrate here.
he caging of a fluorophore is defined as its derivatization
with a photocleavable group that converts the fluorophore
derivatives, the partially uncaged fluorophore displays only
relatively weak fluorescence compared to the fully uncaged
fluorophore, and therefore this approach does not exploit the
full potential of synthetic fluorophores for PALM.6 Caged
versions of the fluorophore Tokyogreen with only one
photocleavable group and of rhodamine with a 2-diazoketone
(COCNN) caging group have been reported,7,8 but the specific
coupling of these probes to individual proteins has not been
described yet. Recently, a caged version of a dicyanomethyle-
nedihydrofuran (DCDHF) fluorophore has been described that
can be selectively coupled to Halo-tag fusion proteins.9
DCDHF was efficiently caged by replacing a crucial amino
group with a photolabile azide; the probe is then uncaged by
photoconversion of the azide back to the amino group. The
potential of this caged DCDHF was demonstrated by
performing PALM of Halo-tag fusion proteins in bacteria and
mammalian cells. In summary, there is a generally acknowl-
edged need for new localizable caged fluorophores.
T
into a non-fluorescent state. As the uncaging and resulting
recovery of fluorescence through a light pulse can be achieved
with high spatial and temporal resolution, caged fluorophores
have become important tools in studying numerous biological
processes, including cell lineage and protein trafficking.1,2
Another attractive application of caged fluorophores is in
photoactivated localization microscopy (PALM), which permits
imaging of cellular components with nanometer resolution.3 In
PALM, sparse subsets of dye molecules are stochastically
activated or uncaged, imaged, and bleached over the course of
thousands of raw images. Since the density of activated
molecules depends on the intensity of the activation light, we
can ensure that molecules in each image are spatially well-
separated and thus can be localized by fitting to the point-
spread function.4 The certainty with which a molecule can be
localized depends inversely on the square-root of the number of
photons collected, so the high photon yields of synthetic
fluorophores offer a significant advantage over fluorescent
proteins (FPs) in this respect. Furthermore, there are few
options for good photoactivatable FPs in the green part of the
spectrum for single-molecule-based imaging, and those that do
exist suffer from high background and low contrast ratios.
Caged fluorophores that could be coupled to selected proteins
in living or fixed cells therefore have the potential to become
important tools for superresolution microscopy. Caged versions
of several fluorophores such as fluorescein and rhodamine have
been reported, but caging of these fluorophores required the
attachment of two photocleavable groups.1,2,5,6 The removal of
two caging groups necessitates a longer irradiation time to
recover the whole fluorescence from the illuminated sample.
Moreover, attempts to selectively uncage only a subset of a
population of caged fluorophores will mostly generate
fluorophores where only one of the two caging groups has
been removed. In the case of caged fluorescein and rhodamine
Here we present a caged version of rhodamine 110 (Rh110
)
that can be coupled to SNAP-tag fusion proteins and that is
suitable for PALM (Figure 1). The large fluorescence increase
upon uncaging and the brightness and stability of the SNAP-
tag-bound fluorophore make it an attractive probe for
superresolution imaging and the study of dynamic cellular
processes.
The design of our caged rhodamine probe 1 (BG-cRhod)
was based on the observation that ureated derivatives of Rh110
retained much of the fluorescence of the parental compound.10
Particularly, the relative fluorescence intensity (a product of
extinction coefficient and quantum yield) of the ureated
derivative is 35% that of Rh110. The relative ease with which
Received: August 12, 2011
Accepted: October 25, 2011
Published: October 25, 2011
© 2011 American Chemical Society
289
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Letters
Figure 1. Design of the caged rhodamine BG-cRhod and its use for the
labeling of SNAP-tag fusion proteins.
ureated Rh110 derivatives can be synthesized and their good
spectroscopic properties have triggered the generation of a
number of Rh110-based probes for various applications.11−13 On
the basis of these reports, we envisioned the synthesis of a
caged Rh110 derivate for the reaction with SNAP-tag fusion
proteins. SNAP-tag is a protein tag that reacts with O6-
benzylguanine (BG) derivatives carrying different probes in
vitro and in vivo.14,15 In BG-cRhod, a BG is attached via a urea
linkage to Rh110, while the attachment of a photocleavable 4,5-
dimethoxy-2-nitrobenzyl group (DMNB) to the other amino
group of Rh110 would force the probe in its non-fluorescent
lactone configuration (Figure 1). We anticipated that SNAP-tag
fusion proteins could be labeled with BG-cRhod and
subsequently uncaged through irradiation with UV light.
Starting from commercially available Rh110, BG-cRhod was
synthesized in five steps in a total yield of 4.3% (Figure 2, panel
A). The fluorescence properties of BG-cRhod were studied in
vitro before and after reaction with SNAP-tag. First, we
confirmed by HPLC and mass spectroscopy that the photolysis
of BG-cRhod generated the corresponding BG Rh110 derivative
BG-Rhod (Supplementary Figure 1). As expected, BG-cRhod is
non-fluorescent (Φfl < 0.001) and became fluorescent upon
irradiation with 365 nm light, although the fluorescence
quantum yield of BG-cRhod was relatively low (Φ fl = 0.074)
(Figure 2, panel B). We then demonstrated that BG-cRhod
permits the labeling of SNAP-tag with cRhod (Supplementary
Figures 3 and 4). As observed on BG-cRhod, SNAP-tag-bound
cRhod is virtually non-fluorescent (Φfl < 0.002). However, the
fluorescence quantum yield of uncaged SNAP-tag-bound
cRhod increased to 0.36, which is about 5 times higher than
that of the uncaged free substrate (Figure 2, panel B). The
observed increase of fluorescence upon uncaging of SNAP-tag-
bound cRhod is at least 200-fold. We assume that the
background fluorescence observed prior to uncaging is most
likely due to the presence of small amounts of already uncaged
BG-cRhod. The low fluorescence quantum yield of BG-cRhod
is most likely due to quenching through benzylguanine.16,17
Similar behavior has already been observed for other
fluorescent BG derivatives used for SNAP-tag labeling.18−20
The observed quenching in uncaged BG-cRhod relative to that
Figure 2. (A) Synthesis of BG-cRhod. (B) In vitro characterization of
BG-cRhod. Fluorescence increase of free BG-cRhod (triangles) and its
SNAP-tag conjugate (circles) before (open) and after (filled)
uncaging. An enlargement of the fluorescence spectrum of caged
BG-cRhod and caged SNAP-tag-bound cRhod is shown in Supporting
Information. Boc2O = di-tert-butyldicarbonate, DIEA = N,N-
diisopropylethylamine, DPPA = diphenylphosphoryl azide, iPr2NH =
diisopropylamine, TFA = trifluoroacetic acid, DMF = dimethylforma-
mide, THF = tetrahydrofuran.
of SNAP-tag-bound uncaged cRhod is an advantageous feature
of our probe because it reduces potential background
fluorescence from unreacted, uncaged probe.
We then investigated the use of BG-cRhod for the labeling of
cell surface receptors and subsequent uncaging of labeled
protein. We chose the β-adrenergic receptor β2AR as a model
protein as its SNAP-tag fusion has previously been shown to be
functional.21 HEK293T cells were transiently transfected with a
plasmid for expression of SNAP-β2AR and consecutively
labeled with BG-cRhod (0.3 μM, 1 h) and BG-Cy5 (2 μM,
10 min). Simultaneous labeling with BG-Cy5 was performed to
identify transfected cells expressing SNAP-β2AR prior to
uncaging. Using a 488 nm laser for excitation, no significant
fluorescence of BG-cRhod-labeled SNAP-β2AR could be
detected prior to uncaging (Figure 3, panel A). However,
irradiation with a 405 nm laser resulted in uncaging of labeled
SNAP-β2AR, as demonstrated by the strong fluorescence signal
observed at the plasma membrane of transfected cells (Figure 3,
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Figure 3. (A) Confocal fluorescence images of living HEK293T cells expressing SNAP-β2 adrenergic receptor. Transiently expressed SNAP-β2AR
was labeled with BG-cRhod (green) and then with BG-Cy5 (red). Nuclear DNA staining with Hoechst 33342 was used as a reference (blue). Images
were taken before and after UV irradiation. (B, C) Confocal fluorescence images of fixed U2OS cells expressing SNAP-NLS and SNAP-MEK1. (B)
Transiently expressed SNAP-NLS (nucleus) and (C) SNAP-MEK1 (cytoplasm) were labeled with the SNAP-tag substrates BG-cRhod (green) and
then with BG-Cy5 (red). Nuclear DNA staining with Hoechst 33342 was used as a reference (blue). Images were taken before and after uncaging at
405 nm. Cells were fixed with paraformaldehyde prior to staining.
panel A). The possibility to uncage BG-cRhod-labeled proteins
with a 405 nm laser is important for practical applications as
this laser line is found on most confocal microscopes and
illumination at 405 nm is less phototoxic than illumination with
UV light.
therefore investigated if the selective labeling of SNAP-tag
fusion proteins with BG-cRhod can be achieved after fixation.
To this end, we performed labeling experiments with BG-
cRhod on fixed U2OS cells expressing SNAP-tag fusion
proteins localized either in the nucleus (SNAP-NLS) or in
the cytoplasm (SNAP-MEK1) (Figure 3, panels B and C). Cells
were fixed with paraformaldehyde and consecutively labeled
with BG-cRhod (0.3 μM, 1 h) and then BG-Cy5 (2 μM, 10
min) to facilitate the identification of transiently transfected
cells. After labeling with BG-cRhod and uncaging with a 405
nm laser, cells showed the expected localized fluorescence
signals. In contrast, no significant fluorescence was detected
prior to photoactivation. These experiments demonstrate that
BG-cRhod is suitable for the labeling of SNAP-tag fusion
proteins in fixed cells and suggest that the probe is suitable for
PALM.
For PALM imaging, we used BG-cRhod to label SNAP-tag
fusion proteins localized to mitochondria (Mito-SNAP). U2OS
cells expressing Mito-SNAP were fixed with paraformaldehyde
and labeled with BG-cRhod (0.3 μM, 2 h). Before PALM
imaging, cells were exposed to a high level of 488 nm light to
photobleach any previously uncaged molecules. The sum of all
raw images corresponds to a time-integrated epifluorescence
image (Figure 4, panel A), which is shown for comparison with
the rendered super-resolution PALM image (Figure 4, panel
We then tested if BG-cRhod permits the labeling of
intracellular SNAP-tag fusion proteins. For these experiments,
a fusion protein of SNAP-tag with the kinase MEK1 (SNAP-
MEK1) was chosen as an example of a cytosolic protein and
transiently expressed in U2OS cells. Although labeling of
SNAP-MEK1 with the cell-permeable fluorescent SNAP-tag
substrate TMR-star was achieved, no significant labeling with
BG-cRhod could be observed (Supplementary Figure 5). The
failure to label intracellular SNAP-tag fusion proteins with BG-
cRhod in living cells limits the application range of this
substrate. However, the labeling of intracellular SNAP-tag
fusion proteins with BG-cRhod could be achieved with invasive
approaches such as bead-loading, microinjection or electro-
poration; all of these approaches have been previously been
shown to permit the labeling of SNAP-tag fusion proteins with
cell-impermeable substrates.22−24
Superresolution imaging using point-based localization
(PALM, STORM, etc.) for the elucidation of biological
structures is generally performed on fixed cells.25,26 We
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Letters
microscopy.28−30 These methods rely on synthetic fluoro-
phores subjected to reduction−oxidation or triplet-state cycling
to induce blinking and therefore temporally separated signals as
required by point-localization superresolution microscopy.
Compared to PALM, these approaches have been able to
exploit the higher photon counts available from synthetic dyes,
resulting in higher molecular localization precision. However,
for high densities of molecules, it can be difficult to fully
initialize the system into the dark state. Furthermore, it is not
straightforward to control the rate of molecules cycling, to
maintain their temporal separation. Caged dyes such as cRhod
provide an advantage in that their initial state is largely the
caged one, with the possibility of easily photobleaching any
small uncaged population. Moreover, their uncaging is well-
controlled as a function of the intensity of the UV uncaging
light. Another interesting aspect of cRhod for future practical
applications is the possibility to use it in combination with red
FPs or synthetic photoswitchable probes for multicolor
imaging. In light of these general considerations and the
favorable spectroscopic properties of cRhod, we expect that this
dye will become a useful addition to the already available dyes
for superresolution microscopy.
ASSOCIATED CONTENT
■
S
* Supporting Information
Detailed experimental procedures and characterization of
compounds. This material is available free of charge via the
Internet at http://pubs.acs.org.
Figure 4. (A) Summed raw images of fixed U2OS cells expressing
SNAP-Mito (mitochondria). Quasi-stable cells expressing a mitochon-
drial targeting sequence were labeled with BG-cRhod, and a stack of
5000 images was collected with sparse activation using a 405 nm laser
and then summed to create the equivalent of an epi-fluorescence
image. (B) PALM images. The same stack of images was processed for
PALM. Color bar corresponds to the probability density of finding a
molecule in units of molecules per nm2. Number of molecules:
142,433 (left), 16,960 (right). (C) Intensity profiles across a single
mitochondria were analyzed to extract the full width half max from a
Gaussian fit for summed raw (solid line, fwhm = 517 nm) and PALM
(dashed line, fwhm = 135 nm) images. (D) Histograms of the number
of photons and molecular localization uncertainty. The molecules
shown in panel B were analyzed for their photon yield (mean = 3488;
median = 2027) and localization uncertainties, σ (mean = 16 nm,
median = 11.7 nm). Scale bar (left) is 5 μm, scale bar (right, zoom) is
2 μm.
AUTHOR INFORMATION
■
Corresponding Author
*E-mail: kai.johnsson@epfl.ch.
Author Contributions
§These authors contributed equally to this work.
ACKNOWLEDGMENTS
■
This work was supported by the Swiss National Science
Foundation and partially supported by funding from the
European Research Council under the EC FP7/ERC grant
agreement n° 243016-PALMassembly. The authors thank
Grazvydas Lukinavicius and Carine Ben-Adiba for technical
assistance.
B). As expected, features that are obscured in wide-field
imaging are sharply resolved in the PALM image. We used the
information from the raw data to obtain statistics on molecular
photon yields and localization precision. In comparison with
the best red FPs or PA-GFP, which give on average 300−800
photons per molecule (ref 27 and Supplementary Figure S7),
we found a mean value of 3488 photons/molecule, an increase
in photon yields of a factor of ∼5−10. This increased molecular
brightness is translated into an improvement in localization;
because there is a rather long tail in the distribution this is
demonstrated by the median value of uncertainty in position,
which is 11.7 nm, a factor of ∼2 better than for the red FPs.
The mode of the distribution is even lower, 6.1 nm, a factor of
∼3 better than for the red FPs. Thus, cRhod is an excellent
label for PALM imaging in general.
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