Superresolution Imaging of Targeted Proteins in Fixed and Living Cells Using
Photoactivatable Organic Fluorophores
Hsiao-lu D. Lee,† Samuel J. Lord,† Shigeki Iwanaga,† Ke Zhan,‡ Hexin Xie,‡ Jarrod C. Williams,|
Hui Wang,| Grant R. Bowman,§ Erin D. Goley,§ Lucy Shapiro,§ Robert J. Twieg,| Jianghong Rao,‡
and W. E. Moerner*,†
Departments of Chemistry, Radiology, DeVelopmental Biology, Stanford UniVersity, Stanford, California 94305, and
Department of Chemistry, Kent State UniVersity, Kent, Ohio 44244
Received May 21, 2010; E-mail: wmoerner@stanford.edu
Abstract: Superresolution imaging techniques based on sequen-
tial imaging of sparse subsets of single molecules require
fluorophores whose emission can be photoactivated or photo-
switched. Because typical organic fluorophores can emit signifi-
cantly more photons than average fluorescent proteins, organic
fluorophores have a potential advantage in super-resolution
imaging schemes, but targeting to specific cellular proteins must
be provided. We report the design and application of HaloTag-
based target-specific azido DCDHFs, a class of photoactivatable
push-pull fluorogens which produce bright fluorescent labels
suitable for single-molecule superresolution imaging in live bacte-
rial and fixed mammalian cells.
Recently, sequential imaging of sparse subsets of photoactivat-
able/photoswitchable single-molecule fluorophores has enabled
optical imaging beyond the diffraction limit (DL), providing insight
into the subdiffraction world (e.g., PALM, FPALM, STORM).1-3
These single-molecule superresolution (SR) techniques have pro-
vided the impetus for development of new controllable fluorophores
with large numbers of emitted photons N, because the achievable
resolution4 scales as 1/
N. Most previous SR experiments in living
cells5 have used photocontrollable fluorescent proteins.6-9 However,
despite having the advantage of being target-specific, fluorescent
proteins on average provide 10-fold fewer photons before photo-
bleaching than good organic fluorophores.10,11 Small organic
fluorophores have the additional benefit of synthetic design flex-
ibility for tuning target specificity, spectral wavelength, solubility,
and other desired properties. Therefore, targeted bright organic
fluorophores which are compatible with the live-cell environment
and which can be turned on and/or off would be advantageous.
While targeted fluorogens which activate via esterase-mediated
cleavage have been previously demonstrated,12 optical control of
the emitting concentration is necessary for SR.13
Many biological processes can be interrupted by fixing (i.e., killing)
cells, which is required for the immunostaining used in most SR
experiments that rely on organic fluorophores.16,17 While fixation can
be useful, there is a need for techniques and probes that can be used
for SR imaging in liVing cells. Single-molecule imaging in living cells
using exogenous fluorophores faces the dual hurdles of cell perme-
ability and targeted labeling.5 There are exceptions in the literature
that did not use fixation and immunostaining. For instance, Heilemann
et al.17 imaged mRNA in living cells using oligomers labeled with
small organic fluorophores and Conley et al.18 labeled the external
lysines of bacterial cells using a Cy3-Cy5 heterodimer. Very recently,
fluorophores were targeted with trimethoprim and intrinsic cellular
reductants enabled photoinduced blinking.19 These examples demon-
strated some possibilities for live-cell labeling and SR, but there is
still a need for photoactivatable organic labels for SR imaging because
each molecule is localized only once, while, with blinking or
photoswitching, each molecule can be localized a variable number of
times.
Here we present a target-specific photoactivatable organic
fluorophore for use inside living and fixed cells, 3, based on the
commercial HaloTag targeting approach.20-22 This method requires
a genetic fusion to the HaloEnzyme (HaloEnz), which forms a
covalent linkage to the HaloTag substrate, thus labeling the protein
of interest (i.e., a protein-HaloEnz-HaloTag-fluorophore covalent
unit). Specifically, we present the following: (i) the basic photo-
physical properties of a new targeted photoactivatable probe; (ii)
proof-of-principle labeling of known structures in fixed and living
mammalian cells validated by costaining with antibodies or
cotransfection with fluorescent proteins; (iii) specific SR imaging
of microtubules in a mammalian cell with quantification of
resolution enhancement; (iv) demonstration of targeted labeling in
living bacteria with diffraction-limited imaging; and finally, (v) SR
imaging of poorly understood structures inside living bacteria.
As molecules with bright emission for single-molecule imaging,
dicyanomethylenedihydrofuran (DCDHF) push-pull fluorophores emit
millions of photons before photobleaching and can enter living
cells.15,23 Recently, we reported a photoactivatable DCDHF fluorogen
† Department of Chemistry, Stanford University.
‡ Department of Radiology, Stanford University.
§ Department of Developmental Biology, Stanford University.
| Department of Chemistry, Kent State University.
Scheme 1. Photochemical Activation Produces 2 from 1a
a A mixture of photoproducts is produced,14,15 but the primary amine
with R1dR2dH is the significant product (see Table 1 for reaction yield of
primary amine). HaloTag versions of 1 and a separate fluorophore are also
shown (3 and 4).
10.1021/ja1044192  XXXX American Chemical Society J. AM. CHEM. SOC. XXXX, xxx, 000 9 A

based on photocaging the fluorescence by replacing the amine donor
with a poorly donating but photolabile azide, which can then be
converted back to an amine using low-intensity blue light.14 Azido
DCDHF fluorogens exhibit high turn-on ratios (the increase in emission
from the fluorescent form compared to the dark fluorogen) of
325-1270-fold, an attractive property for SR imaging.15 (For a detailed
discussion of the photophysical properties of the fluorogens, as well
as the methods used to characterize them, see refs 11 and 15.)
Compared with the original DCDHF-V-P-azide,14 3 exhibits similar
spectral changes upon optical pumping of the aryl azide (Figure 1)
but a higher photoconversion quantum yield (ΦP, see Table 1).
Presumably the oxygen on the aryl azide stabilizes the intermediate
nitrene, making photoconversion more favorable.14,15,24 The photo-
conversion of 3 was so sensitive such that an additional activating
blue laser was not necessary; instead, the diffuse ambient light (e.g.,
the blue light emitted from a nearby computer monitor in an otherwise
dark room) was sufficient to activate sparse sets of the fluorogen. The
thermal activation rate and activation by the 594 nm imaging laser
were significantly lower, as measured in complete darkness in a
covered sample (see Supporting Information (SI)). Because the
fluorogen’s sensitivity to photoconversion is so high, it was possible
to set the level of the ambient light such that the bleaching rate from
594 nm pumping was similar to the activation rate. For instance, with
room lights off and a nearby computer monitor on, we maintained a
steady-state concentration of isolated emitters. This reduced the
complexity of the experiment by allowing the use of only one laser.17
Moreover, because no blue or UV laser was necessary for activation,
photodamage to the imaging sample was greatly reduced. (The
drawback to this high sensitivity is that it increases the difficulty of
preventing photoactivation before imaging. We successfully minimized
preactivation by performing all preparations in complete darkness or
under dim red lights only.)
We verified the specificity of 3 in mammalian culture. Wild-
type HeLa cells and HeLa cells transfected to express HaloEnz-
R-tubulin were stained live with 3, fixed, and immunostained with
Alexa488-mAb to R-tubulin. Fluorescence images after photo-
activation clearly demonstrate that 3 is only retained in the cells
that expressed-HaloEnz-R-tubulin (Figure 2A-G). Next, live CHO
cells were cotransfected with HaloEnz-R-tubulin and R-tubulin-
eGFP, and the labeling by 3 was shown to colocalize well with the
eGFP labeling (Figure 2H-I).
Most importantly, BS-C-1 cells were transfected with HaloEnz-
R-tubulin, fixed, stained with 3, and washed before SR imaging by
PALM. Comparing DL and SR fluorescence images (Figure 2J,K),
the microtubule structure is clearly imaged with a resolution beyond
the optical diffraction limit. After corroborating the utility of the
DCDHF fluorophores for labeling known structures in mammalian
cells, we then moved our attention to cells with protein organization
that is not fully understood.
Bacteria are tiny (about a thousand bacteria could fit within the
volume of one HeLa cell), and the details of protein localization in
prokaryotes are poorly understood, yet essential for function and
phenotype.25 DL imaging of labeled proteins in bacteria only
Figure 1. Absorption of 1 in ethanol decreases during irradiation with 1.1
mW/cm2 at 385 nm shown at 15, 30, 90, and 150 s (left down arrow).
Concurrently, the absorption of amine photoproduct 2 grows proportionally
(center up arrow). The bright fluorescence from 2 when pumped at 550 nm
is the dotted curve; the heavy dashed curve is dim fluorescence from the
original sample of 1 (the small emission signal is most likely from the
preactivated amine contaminants in the azide sample). Preactivation can
be minimized by keeping the samples in complete darkness. Compound 3
has the same photophysical properties as 1.
Table 1. Photophysical/Photochemical Parameters
λabs,azide
(nm)b
λabs,amine/λfl,amine
(nm)c Yieldd ΦPe
DCDHF-V-P-azidea 424 570/613 65% 0.0059
1 (and 3) 443 572/627 ∼50% 0.095
4 - 598/629 - -
a DCDHF-V-P-azide is the earlier underivatized azido-DCDHF from ref
9 for comparison. b Peak absorbance for azido fluorogen. c Absorbance and
fluorescence peak wavelengths of the amino fluorophore. d Overall chemical
reaction yield to the primary fluorescent amine. e Photoconversion quantum
yield of azido fluorogens to any product (i.e., the probability of
photoconverting after absorbing one photon). For reference, the value of ΦP
for mEos is on the order of 10-5 (see ref 11 for more detailed
comparisons). For all DCDHFs, the fluorescence quantum yield of the
photoactivated form varies greatly depending on the precise
nanoenvironment, typically <0.1% in buffer to >30% when rigidized (e.g.,
in the membrane or when bound to proteins).23 For measurement details,
see SI. Compound 3 has the same photophysical properties as 1.
Figure 2. Evidence that the HaloTag-targeted fluorogen correctly labels
specific proteins and enables SR imaging in mammalian cells. (A) Phase image
of fixed WT HeLa cells. (B) The cells in A imaged in the DCDHF-V-P channel.
(C) The cells in A imaged in the Alexa488 channel. (D) Phase image of fixed
HeLa expressing HaloEnz-R-tubulin labeled with 3. (E) The cells in D imaged
in the DCDHF-V-P channel. (F) The cells in D imaged in Alexa488 channel.
(G) Overlay of E, F, and additional blue DAPI channel to show nuclei. (H)
Live CHO cells cotransfected to express both HaloEnz-R-tubulin and R-tubulin-
eGFP labeled with 3 and imaged in DCDHF-V-P channel. (I) Cells from H
imaged in the EGFP channel. (The higher background in H may be the result
of nonspecific binding and imperfect washing of untargeted fluorophores.) (J)
Fixed BS-C-1 cells expressing HaloEnz-R-tubulin labeled with 3 imaged using
conventional diffraction-limited imaging. Indicated microtubule measures 450
( 40 nm fwhm. (K) Same cell as J with SR imaging. Indicated microtubule
measures 85 ( 15 nm fwhm. See SI for sample preparation procedures.
B J. AM. CHEM. SOC. 9 VOL. xxx, NO. xx, XXXX
C O M M U N I C A T I O N S

produces diffuse blobs, complicating meaningful interpretation. For
these reasons, living bacterial studies benefit greatly from SR
imaging.11,26 We used HaloTag-DCDHFs to highlight protein
localization patterns in live Caulobacter crescentus bacteria,26-29
an organism which features the biologically interesting ability to
divide asymmetrically. Elucidating the mechanisms of asymmetric
cell division and intracellular organization requires understanding
how cytoskeletal proteins localize through the life cycle of the cell.25
In this Communication, a polar protein PopZ27 and midplane
proteins FtsZ28 and AmiC29 were expressed as HaloEnz fusions.
PopZ, FtsZ, and AmiC have distinct roles: PopZ anchors the
chromosomal origin at the “swarmer” pole; FtsZ and AmiC are
recruited to the midplane and are components of the cell division
machinery.25 DL imaging using HaloTag targeting of the (nonphoto-
activatable) fluorophore 4 shows correct PopZ localization at cell
poles and FtsZ at the cellular division plane as expected (Figure
3), confirming that this HaloTag labeling system does not signifi-
cantly interfere in phenotype.
SR images produced by photoactivation of fluorogen 3 not only
display the expected localization patterns but also reveal additional
detail unseen in the DL images of Figure 3. For PopZ at the cell pole,
the protein forms an asymmetric cap-like structure with a curvature
that hugs the shape of the bacterial membrane (Figure 4A-C). Also,
in the case of AmiC, the protein localizes to the cellular midplane, as
expected (Figure 4D-E). The SR images of AmiC may reveal a tighter
organization than seen in DL microscopy, but further study is necessary
before any definitive statement can be made. In either case, these SR
images provide new detail not available in DL images.
The target-specific DCDHF single-molecule fluorogen presented
here represents the first successful installation of target specificity to
small organic photoactivatable fluorogens for single-molecule SR
imaging. Compared to existing schemes, the photoactivation of the
fluorogen requires neither other additives (e.g., thiols for Cy530 or redox
chemicals17) nor activation by UV light and thus can be used inside
living cells. SR imaging has been directly demonstrated for fixed
mammalian and live bacterial cells; additional effort to improve
washout for live mammalian cells is an important topic for future work.
This and future photoactivatable fluorogens should be helpful tools
for SR imaging in the complex environment within the living cell.
Acknowledgment. This work was supported in part by Grant
No. R01-GM086196 from the National Institute of General Medical
Sciences. We thank S. Pfeiffer for BS-C-1 cells.
Supporting Information Available: Procedures, chemical synthesis,
analysis, additional figures, and complete ref 20. This material is
available free of charge via the Internet at http://pubs.acs.org.
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JA1044192
Figure 3. Diffraction-limited imaging of 4 inside live C. crescentus cells
expressing fusion proteins to FtsZ and PopZ. These proteins localize as
expected,25 indicating that the HaloTag-DCDHF labeling does not disrupt
typical cellular behavior.
Figure 4. SR imaging of protein fusions inside live C. crescentus cells using
3. (A-C) PopZ forms a polymeric network at the poles of the cells. Compared
to the DL images in Figure 3, these SR images reveal distinct shapes of the
PopZ structure, including the cap-like network in C. (D-E) AmiC is recruited
to the division plane early in the cell cycle. These SR images indicate that
AmiC may form a structure that hugs the membrane. For details of imaging
and image processing, see SI. The SR images are extracted from localizations
over 75 s with a mean localization precision of 32 ( 12 nm.
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