DOI: 10.1002/cphc.200800581
DCDHF Fluorophores for Single-Molecule Imaging in
Cells**
Samuel J. Lord,[a] Nicholas R. Conley,[a] Hsiao-lu D. Lee,[a] Stefanie Y. Nishimura,[a]
Andrea K. Pomerantz,[a] Katherine A. Willets,[a] Zhikuan Lu,[b] Hui Wang,[b] Na Liu,[b]
Reichel Samuel,[b] Ryan Weber,[b] Alexander Semyonov,[b] Meng He,[b] Robert J. Twieg,[b] and
W. E. Moerner*[a]
1. Introduction
1.1. Nonlinear Optical Uses for DCDHFs
In the late 1980s and 1990s, the nonlinear optical properties of
push-pull chromophores—structures containing electron
donor and electron acceptor units separated by a p-electron-
rich conjugated linker—were well documented.[1–3] The 2-di-
cyanomethylene-3-cyano-2,5-dihydrofuran (DCDHF) unit is a
useful p-accepting unit, and continues to be used in nonlinear
applications, especially in electro-optic media.[4] Chromophores
containing a DCDHF acceptor and an amine donor, separated
by a p-conjugated system (Figure 1), were also optimized for
photorefractive polymer applications.[5–7] In these materials, the
linear polarizability anisotropy is very important and the push-
pull character of the chromophore increases both the hyperpo-
larizability b and the ground state dipole moment mG via the
charge-transfer absorption and the asymmetric charge distribu-
tion, respectively. Increases in donor and acceptor strength
also red-shift the absorption, lowering the energy required to
produce an intramolecular charge-transfer upon photoexcita-
tion.[8]
1.2. Discovery of DCDHF Fluorescence Suitable For
Single-Molecule Imaging
Most nonlinear optical chromophores are not known to be effi-
cient fluorescent emitters, especially because very strong
charge transfer can produce large excited-state structure dis-
tortions. In the case of photorefractive polymer materials, the
dominant figure of merit is not fluorescence but m2(Da),[9–11]
where Da is the polarizability anisotropy (the tensor that re-
ports how easily the electron distribution of the molecule dis-
There is a persistent need for small-molecule fluorescent labels
optimized for single-molecule imaging in the cellular environ-
ment. Application of these labels comes with a set of strict re-
quirements: strong absorption, efficient and stable emission,
water solubility and membrane permeability, low background
emission, and red-shifted absorption to avoid cell autofluores-
cence. We have designed and characterized several fluorophores,
termed “DCDHF” fluorophores, for use in live-cell imaging based
on the push–pull design: an amine donor group and a 2-dicya-
nomethylene-3-cyano-2,5-dihydrofuran (DCDHF) acceptor group,
separated by a p-rich conjugated network. In general, the
DCDHF fluorophores are comparatively photostable, sensitive to
local environment, and their chemistries and photophysics are
tunable to optimize absorption wavelength, membrane affinity,
and solubility. Especially valuable are fluorophores with sophisti-
cated photophysics for applications requiring additional facets of
control, such as photoactivation. For example, we have reengi-
neered a red-emitting DCDHF fluorophore so that it is dark until
photoactivated with a short burst of low-intensity violet light.
This molecule and its relatives provide a new class of bright pho-
toactivatable small-molecule fluorophores, which are needed for
super-resolution imaging schemes that require active control
(here turning-on) of single-molecule emission.
Figure 1. Schematic structure of the DCDHF fluorophores. The amine donor
and DCDHF acceptor are connected by a p-conjugated linker. The R1R4
groups can be modified (usually without affecting the photophysics) in
order to add reactive functionality or increased solubility. The naming
scheme used in this paper specifies the p system: “DCDHF-(p unit closest to
acceptor)-…-(p unit closest to donor)” with the p units denoted P=phenyl-
ene, V=vinyl, T= thiophene, N=naphthalene, A=anthracene; the amine
donor is not specified because it is present in all structures (see Table 1 for
structure drawings.)
[a] S. J. Lord, N. R. Conley, H.-l. D. Lee, Dr. S. Y. Nishimura, Dr. A. K. Pomerantz,
Prof. K. A. Willets, Prof. W. E. Moerner
Department of Chemistry
Stanford University, Stanford, California 94305 (USA)
Fax: (+1)650-725-0259
E-mail : wmoerner@stanford.edu
[b] Dr. Z. Lu, Dr. H. Wang, Dr. N. Liu, Dr. R. Samuel, R. Weber,
Prof. A. Semyonov, Dr. M. He, Prof. R. J. Twieg
Department of Chemistry
Kent State University, Kent, Ohio 44240 (USA)
[**] DCDHF: 2-dicyanomethylene-3-cyano-2,5-dihydrofuran
ChemPhysChem 2009, 10, 55 – 65  2009 Wiley-VCH Verlag GmbH&Co. KGaA, Weinheim 55

torts in an applied electric field). Chromophores containing a
carbocyanine structure were found to be optimal for such non-
linear optical applications.[12]
In 2003, we discovered[13] that some chromophores designed
for photorefractive polymers,[14] the DCDHFs, have high fluores-
cence quantum yields in glassy polymer films and that they
emit millions of photons before photobleaching. This means
that when the local environment inhibits certain intramolecular
twists, the excited-state distortion is not so large as to prevent
fluorescence. Since then, we have designed, synthesized, and
characterized hundreds of members of the DCDHF class for ap-
plications as fluorophores for single-molecule imaging. This
paper concentrates on molecules specifically optimized for po-
tential use as cellular labels[13,15–24] and the DCDHF class should
be regarded as equally useful for single-molecule experiments
as other dyes in the well-known rhodamine, cyanine, rylene,
etc. classes.
2. General Characteristics and Photophysics
2.1. Environment-Sensing
DCDHF fluorophores exhibit two types of sensitivity to the
local environment: solvatochromism (as a result of the charge-
transfer character of the excitation) and viscosity dependence
(due to suppression of bond twists that permit nonradiative
pathways).[15] A simple consequence of the strong solvato-
chromism that DCDHFs demonstrate is that the fluorescence is
substantially red-shifted with respect to the excitation in bio-
logical media, making it easy to filter out background due to
scatter and autofluorescence, which typically has a smaller
Stokes shift.[25] Solvatochromism in other fluorophores has
been harnessed to report local hydrophobicity, because
changes in polarity cause changes in the color or intensity of
the emission signal.[26] Moreover, the viscosity-dependent
brightness that DCDHFs exhibit has a more direct advantage in
background suppression: copies of DCDHF or DCDHF-labeled
biomolecules not in a rigid region of interest are dim, and con-
tribute little to the fluorescence background. Microscopy in
total-internal-reflection (TIR) mode is not always a viable
option (e.g. if one needs to image away from the glass inter-
face such as at the upper membrane of a cell or if the optical
polarization of TIR cannot be tolerated), and fluorescence read-
out only in certain regions of a cell permits low-background
epifluorescence imaging of those regions.[18,27]
Solvatochromism is an expected outcome of the push–pull
character of donor–p–acceptor chromophores. Conventionally,
the Lippert–Mataga equation [Eq. (1)] may be used to probe
the influence of the solvent polarity on the Stokes shift.[28] In
most push–pull chromophores, the charge separation is great-
er in the excited-state manifold. When such a charge-transfer
fluorophore is excited from the ground to excited state, the
nearby solvent dipoles can reorient around the larger excited-
state dipole moment, thus stabilizing the system and lowering
the energy of the excited state (and simultaneously destabiliz-
ing the ground state). This effect becomes more pronounced
as the solvent polarity is increased, and the Stokes shift in-
creases in more polar solvents. In the Lippert–Mataga approxi-
mation, the orientation polarizability Df is used as a parameter
to represent the degree of molecular rearrangement around a
dipole in a continuous medium, thus leading to the observed
dependence of the Stokes shift on the square of the dipole
moment change:
nA  nF ¼
2
hc
Df mE  mGð Þ
2
a3
þ constant ð1Þ
where Df ¼ er12erþ1 
n21
2n2þ1 and nA and nF are the wavenumbers of
the absorption and emission, mG and mE are the ground- and
excited-state dipole moments, a is the Onsager cavity radius
(assumed to be 5 ), n is the refractive index of the solvent, er
is the relative dielectric constant of the solvent, h is Planck’s
constant, and c is the speed of light. The solvent parameter Df
takes into account only the low-frequency polarizability (i.e.
the slow rearrangement of nuclear coordinates), and removes
the high-frequency polarizability component (from electron re-
distribution, which occurs on the same timescale as photoin-
duced charge transfer in the fluorophore) from the total polar-
izability.[28,29]
Figure 2 demonstrates the influence that the p structure has
on solvatochromism for a series of DCDHF fluorophores with
different acene p units. Increasing the conjugation length
(from a single phenyl group P to larger naphthalene N and an-
thracene A) between the longitudinally situated donor and ac-
ceptor increases the sensitivity of Stokes shift to solvent polari-
ty. This trend can be explained by the fact that the charge re-
distribution in the excited state occurs over a greater distance
in the cases of naphthalene and anthracene, thus resulting in a
larger change in the dipole moment between the ground and
excited state. (This of course assumes that the magnitude of
charge displaced does not change. In push–pull chromo-
phores, the dipole moment eventually plateaus as the p-
system is made very large.)
Figure 2. Solvatochromism exhibited in a Lippert–Mataga plot (see text for
details). The slopes of the fits for DCDHF-A, DCDHF-N, and DCDHF-P (struc-
tures in Figure 3) are 7757, 6921, and 1588 cm1, respectively; these slopes
correspond to the change in dipole (mEmG) values of 9.7, 9.4, and 4.4 D. As
expected, increasing the conjugation length between the donor and accept-
or increases the sensitivity to solvent polarity, because photoinduced charge
transfer across a greater distance results in a larger change in the dipole
moment. (Data from refs. [15] and [20].)
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W. E. Moerner et al.

The fluorescence quantum yield (FF) of DCDHFs increases in
rigid environments as demonstrated in Figure 3 for the three
acene-based fluorophores. A strong dependence of fluores-
cence quantum yield on viscosity of the solvent was also ob-
served.[15] The working model for this dependence is a twisted
intramolecular charge transfer (TICT) state that opens a nonra-
diative relaxation channel.[30] Quantum-mechanical calculations
have indicated that the excited-stated structure with the
lowest energy involves an approximately 908 rotation of the di-
cyano group around the methylene bond (labeled d in
Figure 4).[15] In viscous media, such structural isomerizations
are hindered, decreasing the probability of entering the TICT
state (the pathway on the right in Figure 4). Because the radia-
tive decay rate remains relatively unchanged, the nonradiative
pathway becomes kinetically less accessible and the fluores-
cence quantum yield increases. It is worth noting that this
mechanism relies on local effects, and the strong connection
between fluorescence and viscosity observed for methanol-
glycerol mixtures may arise from networks of hydrogen bonds
than can form around the acceptor portion of the molecule.
Other such “molecular rotors” have been reported in the lit-
erature.[31–33] Figure 5 demonstrates the utility of obtaining
strong fluorescence only from viscous local environments:
when studying the plasma membrane of living cells, the ob-
served emission signal is brightest from fluorophores located
in the lipid bilayer.[18,27] Because molecules that are not in the
membrane are dim, they do not contribute to the fluorescence
background. Therefore, there is less need for washing away un-
bound fluorophores or for background-reducing microscopy
configurations (e.g. TIR or confocal[34]). This study, discussed in
more detail below, required a DCDHF derivative with reactive
maleimide functionality ;[35] N-hydroxysuccinimide variants have
also been prepared (see the supporting material of ref. [21]). In
most cases, adding reactive functionality to DCDHFs via single-
bonded alkyl attachments had no affect on the photophysics,
because the added groups do not significantly interact elec-
tronically with the fluorophore’s charge-transfer absorption.
2.2. Photostablility
Inherent photostability is one of the most important parame-
ters of a single-molecule fluorophore: the longer each mole-
cule survives before it photobleaches, the more it reports on
its location, environment, orientation, diffusion kinetics, or
whatever parameter the experiment aims to measure. Singlet
molecular oxygen, produced when triplet oxygen interacts
Figure 3. A) Three acene DCDHF structures with increasing conjugation, and
thus increasing absorption wavelength. B) Demonstrating the quantum-yield
dependence on viscosity : in rigidified frozen samples, the fluorescence emis-
sion increases drastically. The “6” in the names refer to the six carbon tails
on the amine. (Adapted with permission from J. Phys. Chem. A 2007, 111,
8934–8941. Copyright 2007 American Chemical Society.)
Figure 4. Proposed mechanism accounting for the interplay between radia-
tive and nonradiative pathways of DCDHF fluorophores. DCDHF-P in its ex-
cited state S1 can relax either via channel I to S1f or via channel III to S1d. In
channel III, a twist around the methylene bond d produces a stable TICT
state (S1d), from which the molecule can relax back nonradiatively to ground
state G0. In viscous media, this twist is hindered and relaxation to the rela-
tively planar S1f state via channel I becomes more competitive and fluores-
cence emission can compete with the nonradiative pathway; thus, the fluo-
rescence quantum yield increases. (Adapted with permission from J. Phys.
Chem. B 2004, 108, 10465–10473. Copyright 2004 American Chemical Soci-
ety.)
Figure 5. Octaarginine cell-penetrating peptides labeled with DCDHF-V-P in
the plasma membrane of living CHO cells. At low enough concentrations of
labeled peptides (right), single molecules can be observed and tracked as
they interact with the membrane. (Reproduced with permission from J. Am.
Chem. Soc. 2008, 130, 9364–9370. Copyright 2008 American Chemical Soci-
ety.)
ChemPhysChem 2009, 10, 55 – 65  2009 Wiley-VCH Verlag GmbH&Co. KGaA, Weinheim www.chemphyschem.org 57
Single-Molecule Imaging in Cells

with a fluorophore in its triplet state, is often blamed as the
culprit in many photodestruction reactions.[36–38] Therefore, var-
ious oxygen-scavenging systems are often used in single-mole-
cule biophysics experiments to extend the survival time of the
fluorophore.[39,40] However, molecular oxygen is a potent triplet
quencher,[41] because the molecular oxygen triplet ground
state helps couple the fluorophore triplet state back to the
ground singlet state (thus increasing the intersystem-crossing
rate). Therefore, removing oxygen from solution can result in
dim, intermittent fluorescence because emitters become trap-
ped in long-lived dark states. Careful experiments have dem-
onstrated that removing oxygen can even reduce the total
number of cycles to the excited state while simultaneously im-
peding the emission rate.[41–44] Because oxygen removal can
generate inconsistent behavior from structure to structure, flu-
orophores with inherently robust photostability are always de-
sirable.
Two fundamental measures of fluorophore photostability are
the total number of photons emitted for a single molecule
before it permanently photobleaches (Ntot,e) and the photo-
bleaching quantum yield (FB) which is the probability of pho-
tobleaching for each photon absorbed. These two parameters
scale inversely to each other (Ntot,e=FF/FB), but can also be re-
garded as independent useful tests, measured using different
experiments and assumptions. For the Ntot,e measurement,
single-molecule time traces from movies are used to extract
the total number of detected photons before photobleaching
molecule-by-molecule, where all the photons (minus back-
ground) contributing to a single-molecule spot are spatially
and temporally integrated. Results from hundreds of single
molecules are histogrammed[20] or plotted versus a probability
distribution,[23] and an average number of photons detected is
extracted from an exponential fit. To convert to the photons
emitted, the electron-multiplying and conversion gains of the
camera as well as collection efficiency D of the microscopy
setup must be measured: D=hQFcollFoptFfilter, which is the prod-
uct of the camera quantum efficiency hQ, the angular collection
factor Fcoll determined by the objective NA, the transmission
factor through the objective and microscope optics Fopt, and
the transmission factor through the various filters Ffilter, respec-
tively.[34] Typical values of D are approximately 10% for epi-
fluorescence. True photon-counting detectors (e.g. avalanche
photodiodes) provide an alternative detection scheme which
reduces error introduced by inaccuracies in camera gain
values, but imaging with such detectors requires confocal
scanning and measuring molecules one at a time. Note that
the Ntot,e calculation does not depend on irradiance, wave-
length, or extinction coefficient (unless the molecule is excited
so strongly as to experience excitation from the first excited
state to higher excited or triplet states that lead to bleaching,
and this regime is to be avoided).
On the other hand, the photobleaching quantum yield can
be measured from a bulk sample if the laser irradiance (Il) and
wavelength (l), and absorption cross-section (sl=2303el/NA)
are known, from which the ratio of the bleaching rate (RB) to
the absorption rate (Rabs) can be calculated using Equation
(2):
FB ¼
RB
Rabs
¼ 1tBRabs
¼ 1
tBslIl
l
hc
  , ð2Þ
where tB is the time constant in an exponential fit of the decay
of bleaching fluorescence, h is Planck’s constant, and c is the
speed of light. If measured under similar conditions to a
single-molecule imaging experiment but at approximately an
order of magnitude higher concentration, FB is an independ-
ent check of the photostability. Because of experimental varia-
bility (standard errors of the mean for Ntot,e or FB can be as
high as 30%) and differences in fluorescence quantum yields
among fluorophores, the two photostability measures may not
be exactly inverse; however, trends in each measure should be
similar among fluorophores.
In Table 1, values for Ntot,e and FB are reported for many
DCDHFs, as well as for the standard fluorophores rhodami-
ne 6G and fluorescein. Many DCDHFs exhibit comparable or
superior photostability to rhodamine 6G, which is a demonstra-
bly good single-molecule emitter; moreover, all DCDHFs mea-
sured are at least an order of magnitude more photostable
than fluorescein. Table 1 also contains extinction coefficients,
absorption and fluorescence emission maxima, and FF values
in solution and in polymer films.
3. Structure-Property Design
3.1. Long-Wavelength Absorption
One of the more straightforward structure-property relation-
ships is the red-shift that accompanies an increase in the
length of the conjugated linker between the donor and ac-
ceptor. Assuming a particle-in-a-box model, increasing the
length of the fluorophore should lower the energy and spac-
ing between the ground and excited state, and thus produce
red-shifted absorption. This can be seen, for example, in going
from DCDHF-P to DCDHF-V-P : there is a 75 nm absorption
red-shift (in toluene) with the addition of a vinyl group.
Because cis–trans isomerism and bond twists involving the
vinyl group can reduce the fluorescence quantum yield, it may
be preferable to extend the conjugation using more rigid
groups. For instance, replacing the phenyl group with a naph-
thalene or anthracene increases the absorption wavelength in
toluene 40 nm for DCDHF-N and nearly 100 nm for DCDHF-A
(see Figure 3). Table 1 includes various other red DCDHF fluoro-
phores with combinations of vinyl, phenyl, and thiophene link-
ers (see Figure 1 for nomeclature.)
3.2. Constraining the Amine Twist
Although quantum-mechanical modeling revealed methylene-
bond twists as one major source of viscosity sensing in
DCDHFs (see Figure 4), the various other twists in the molecule
may also significantly contribute to lowering the fluorescence
quantum yield in solution. Moreover, the methylene bond
does not have synthetic “handles” with which to constrain ro-
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W. E. Moerner et al.

Table 1. Names, structures, and photophysical parameters of various DCDHFs and standard fluorophores. The structures are generalized: the methyl
groups on the amine and dihydofuran ring may vary in length and composition; the data presented is representative of chromophores with varying R1R4
groups, because varying the length of the alkyl chains has little effect on the photophysics. See text for details on photophysical parameters. (Data from
refs. [19, 20] , and [24] .)
Compound Structure emax
[m1 cm1][a]
labs
[nm][a]
lem
[nm][a]
FF in toluene
{PMMA}
FB in gelatin {PMMA}
(106)
Ntot,e in PMMA
(106)
DCDHF-P 71000 486 505 0.044 {0.92} 6.6 2.4
DCDHF-P with 1 constrain-
ing ring
95900[b] 495 515 0.10
DCDHF-P with 2 constrain-
ing rings
90400[b] 503 527 0.21 1.1
DCDHF-V-P 45500 562 603 0.02 {0.39} 2.8 ~1.9
DCDHF-T 100000 514 528 0.11 ~0.91
DCDHF-V-T 114000 614 646 0.02 ~0.23
DCDHF-N 42000 526 579 0.85 {0.98} 3.4 1.1
DCDHF-V-N 58100[b] 574 671 0.01
DCDHF-V-(1,4)N 22200[b] 538 661 0.01
DCDHF-A 35000 585 689 0.54 1.7 2.2
DCDHF-P-P 31000 506 623 0.82 4.5
DCDHF-T-T 71800 634 679 0.50 ACHTUNGTRENNUNG{2.1}
DCDHF-V-T-T 49800 708 779 0.13 ACHTUNGTRENNUNG{3.4}
DCDHF-P-T 44000 591 663 0.21 6.4
DCDHF-T-P 22000 575 631 0.74 2.9
DCDHF-V-T-P 47300 611 723 0.07 ACHTUNGTRENNUNG{0.13}
DCDHF-P-T-P 28000 541 709 0.34 0.91
Rhodamine 6G 105000[c] 530[c] 556[c] 0.95[c] 3.5 1.4
ChemPhysChem 2009, 10, 55 – 65  2009 Wiley-VCH Verlag GmbH&Co. KGaA, Weinheim www.chemphyschem.org 59
Single-Molecule Imaging in Cells

tations, because modifying the cyano groups would also dras-
tically alter the photophysics.
Therefore, in order to experimentally test the influence of
other bond twists on the fluorescence quantum yield, a series
of fluorophores were synthesized with increasing constraint on
the amine rotation (angle a in Figure 4) produced by tetrahy-
droquinoline rings.[19] Table 2 demonstrates that restraining the
amine not only increases the fluorescence quantum yield but
also slightly red-shifts the absorption and emission. However,
the quantum yields of molecules in solution do not approach
the values measured in the rigid polymer film, which indicates
that the amine bond alone is not responsible for the viscosity
sensing.
These experimental results do not entirely match the calcu-
lations,[15] and suggest that the TICT mechanism in DCDHFs is
more complex than just one bond rotation. Further computa-
tional studies or ultrafast measurements might help clarify the
exact mechanism of viscosity sensing of DCDHF fluorophores.
3.3. Water Solubility
Biophysics and cell-imaging experiments require fluorophores
that can be used in aqueous environments, and most of the
molecules described so far require relatively nonpolar solvents.
Full water-solubility is not always necessary or desirable (e.g.
fluorescent lipid analogs must have some hydrophobicity) ;
however, many applications do require fluorophores that are
soluble and photostable in water.
The original work on molecules in the DCDHF class was car-
ried out in organic solvents, in which the fluorophores are very
soluble. For single-molecule cell experiments, stock fluoro-
phore solutions in DMSO or ethanol were diluted into the
aqueous buffer;[18] because of the very low dye concentrations,
aggregation in water was not a problem. Nevertheless, in
order to introduce true water solubility, synthetic efforts were
undertaken to add alcohol, carboxylic acid, and sulfonic acid
groups to two DCDHFs.[22] As expected, sulfonic acid groups
imparted the most solubility (up to 104 ppm). Significant water
solubility was achieved without compromising desirable pho-
tophysical properties of the DCDHF class of fluorophores.
3.4. Photoactivation
Sophisticated photophysics (e.g. photoswitching, fluorogenic
reactions, multicolor emission) are required for more complex
imaging modalities now becoming popular. For instance,
super-resolution imaging schemes (PALM, F-PALM, STORM)[45–47]
require active control over fluorescence in order to “turn on”
only a few emitters in each
imaging cycle.[48,49] The DCDHFs
are synthetically tailorable, and
designing in such sophisticated
qualities are possible. Disrupting
the push–pull character of the
fluorophore corresponds to sig-
nificant changes in the absorp-
tion and emission wavelengths
and fluorescence quantum yield,
rendering such modified
DCDHFs promising candidates
for fluorogenic probes.[23,50]
Because amines are strong
electron-donating substituents
and azides are weakly electron-
withdrawing,[51] replacing the
amine on the DCDHF fluoro-
phore with an azide should dis-
rupt the donor–p–acceptor char-
acter of the molecule and blue-
Table 1. (Continued)
Compound Structure emax
[m1 cm1][a]
labs
[nm][a]
lem
[nm][a]
FF in toluene
{PMMA}
FB in gelatin {PMMA}
(106)
Ntot,e in PMMA
(106)
Fluorescein 92300[c] 483[c] 515[c] 0.79[c] 64
[a] In toluene. [b] In dichloromethane. [c] In ethanol.
Table 2. Influence of constraining the amine from twisting on photophysical properties. (Data from ref. [19] .)
Parent
Compound
Number of
constraining rings
emax [m1 cm1][a] labs [nm][b] lem [nm][b] FF in toluene {PMMA}
DCDHF-P
0 486 505 0.044 {0.92}
1 95900 495 515 0.10
2 90400 503 527 0.21
DCDHF-V-P
0 562 603 0.02 {0.39}
1 17400 581 618 0.028
2 80900 594 628 0.053
DCDHF-N
0 40900 527 576 0.85 {0.98}
1 545 607 0.64
DCDHF-V-N
0 58100 574 671 0.0049
1 44200 600 699 0.30
DCDHF-V-(1,4)N
0 22200 538 661 0.0067
1 37500 571 677 0.0096
[a] In dichloromethane. [b] In toluene.
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W. E. Moerner et al.

shift the absorption and fluorescence. Moreover, aryl azides are
known to be photolabile, often losing dinitrogen and rearrang-
ing to seven-membered azepine heterocycles.[52] However,
Platz et al.[53] demonstrated that electron-withdrawing substitu-
ents on the benzene can stabilize the nitrene intermediate and
promote the formation of amines and azo groups versus rear-
rangement to the azepine. Because the DCDHF is a very strong
electron-withdrawing substituent, an azido DCDHF should
photoconvert to a fluorescent amino DCDHF upon irradiation
with resonant light.[23]
Scheme 1 displays several possible photoconversion path-
ways. With the loss of N2, the azide on 1a can be photocon-
verted to a reactive nitrene. The nitrene can then convert into
an amine (1b) or a nitro group (1c) ; both these structures
were actually isolated and characterized.[23] Structure 1d is hy-
pothetical, but has literature precedent: nitrenes are reactive
enough to insert into carbon-carbon bonds of nearby mole-
cules. This approach, called photoaffinity labeling, can be used
to bioconjugate aryl azides to biological targets of interest.[54]
The azido DCDHF could act as a fluorogenic photoaffinity
label : photoactivation should both induce fluorescence and
form a covalent bond between the fluorophore and a neigh-
boring biomolecule.
Figure 6 demonstrates the utility of the azido DCDHF as a
fluorogen; before photoactivation, cells incubated with 1a are
dark; after photoactivation, the cells light up as 1a converts to
1b. The intensity of activating light required to generate fluo-
rescent DCDHF molecules is very low, at least three orders of
magnitude lower than the intensity of the imaging light. This
helps ensure that the high-energy blue or UV activating light
does not kill the cells of interest or alter their morphology. Fur-
thermore, photobleaching is three orders of magnitude less
likely than photoconverting, so the activated fluorophore
emits millions of photons before photobleaching (see Table 3).
The photophysics of this azido DCDHF fluorogen are favora-
ble compared to other popular photoswitchable or photoacti-
vatable molecules: it is red-shifted, emits many photons, and
requires only low doses of blue light (see Table 3). However,
the disadvantages of the azido DCDHF system are: it is not ge-
netically targeted, as fluorescent proteins are; DCDHFs with a
primary amine exhibit more blinking than those with secon-
dary or tertiary amines (see Figure 7 and reference [13]) ; and
the photoactivation is irreversible (once the fluorophore pho-
tobleaches, it cannot be reactivated, which is possible in some
photoswitches[55–60]).
In some imaging scenarios, irreversibility is acceptable; be-
cause the millions of photons emitted are not spread over
Scheme 1. Photoactivation reactions of the azido DCDHF fluorogen. Aryl
azides are known to be photolabile ; the loss of dinitrogen leaves a reactive
nitrene intermediate, which can rearrange to form a seven-membered aze-
pine heterocycle (not shown), an amine (1b), or a nitro (1c) group. Com-
pounds 1a and 1c are not fluorescent when pumped at long wavelengths,
but photoproducts 1b and 1d are fluorescent. Compound 1d is hypotheti-
cal and the result of nitrene inserting into CC bonds of a nearby biomole-
cule. (Adapted with permission from J. Am. Chem. Soc. 2008, 130, 9204–
9205. Copyright 2008 American Chemical Society.)
Figure 6. Azido DCDHFs are photoactivatable. In contrast to the amine
group, the azide group is not electron donating; therefore, charge-transfer
band of the chromophore is disrupted and the absorption is significantly
blue-shifted, so it is no longer resonant with the imaging laser. Irradiance
with low-intensity 407 nm light converts the azide to an amine, which re-
pairs the donor-acceptor character of the fluorophore and returns the ab-
sorption and emission to longer wavelengths. See Scheme 1. A) Three CHO
cells incubated with azido fluorogen are dark before activation. B) The fluo-
rophore lights up in the cells after activation with a 10 s flash of diffuse,
low-irradiance (0.4 Wcm1) 407 nm light. The white-light transmission image
is merged with the fluorescence images (white), excited at 594 nm
(~1 kWcm1). Scalebar: 20 mm. C) Single molecules of the activated fluoro-
phore in a cell under higher magnification. Scalebar: 800 nm. D) Absorption
curves in ethanol showing photoactivation of fluorogen 1a (labs=424 nm)
over time to fluorescent product 1b (labs=570 nm). Different curves repre-
sent time points between 0 and 1320 s of illumination by 3.1 mWcm1 of
diffuse 407 nm light (trends denoted by arrows). Dashed line is the absorb-
ance of pure, synthesized 1b. (Inset) Dotted line is weak pre-activation fluo-
rescence of 1a excited at 550 nm; solid line is strong post-activation fluores-
cence resulting from exciting 1b at 550 nm, showing an increase in the fluo-
rescence emission. (Adapted with permission from J. Am. Chem. Soc. 2008,
130, 9204–9205. Copyright 2008 American Chemical Society.)
ChemPhysChem 2009, 10, 55 – 65  2009 Wiley-VCH Verlag GmbH&Co. KGaA, Weinheim www.chemphyschem.org 61
Single-Molecule Imaging in Cells

many activating cycles, the local-
ization precision can be high for
each molecule. Blinking, howev-
er, is not particularly desirable,
but can sometimes be used to
reduce the emitter concentra-
tions in super-resolution micros-
copy methods.[49] This issue may
be addressed in future work by
finding a DCDHF version that
does not blink as a primary
amine, or via fluorogenic photo-
affinity labeling that produces
the secondary amine (because
DCDHFs with a secondary amine
are less prone to blink). When
combined with a targeting
moiety that brings the molecule
close to the location of interest,
fluorogenic photoaffinity makes
such targeting permanent,[54,61, 62]
and thus could resolve two disadvantages of the azido DCDHF
system simultaneously.
4. Cell and Biological Imaging
4.1. Screening DCDHFs in Cell-like Media
Imaging single molecules in living cells has strict criteria ; not
only must the emitter be bright and photostable, but also it
must be red-shifted enough to avoid background from cellular
autofluorescence from flavins and other endogenous fluoro-
phores pumped at wavelengths shorter than approximately
500 nm,[25] be water-soluble and membrane-permeable, be
washable after incubation, and be specifically targetable.
Quantifying photophysical parameters of single molecules in
living cells is not always possible. A more practical approach is
to screen fluorophores in cell-like media such as protein gels,
agarose gels, or poly(vinyl alcohol) aqueous films. For instance,
Figure 8 (left) shows single copies of DCDHF-N-6 fluorophores
embedded in gelatin, an aqueous protein gel. Photostability
parameters (Ntot,e or FB) measured in gelatin offer a better
measure of how well the fluorophore will perform in cells than
testing only in organic solvents and polymer films; Table 1 in-
cludes several values for FB in gelatin.
Nevertheless, the ultimate test of a fluorophore is whether it
is visible on the single-molecule level in a living cell. Figure 8
shows two DCDHF-A-6 molecules in the plasma membrane of
a living Chinese hamster ovary (CHO) cell. Figure 6 demon-
strates that photoactivating the azido DCDHF fluorogen is pos-
sible in living cells ; moreover, single copies of the photoacti-
vated fluorophore were visible and trackable as they diffused
in the cell (Figure 7). Although these are only qualitative meas-
ures, they are evidence for the utility of these fluorophores as
cellular probes.
Table 3. Photophysical properties of the photoactivatable DCDHF in Scheme 1 and other photoswitchable
fluorophores.[a]
labs [nm] lfl [nm] emax [m1 cm1] FF FP[b] FB in gelatin Ntot,e in gelatin
1a 424 552 29100 n/a good (0.0059) n/a n/a
1b 570 613 54100 0.025–0.39 n/a 4.1106 2.3106
Dronpa[c] 503 518 95000 0.85 very good ~3.2105 –
PA-GFP[d] 504 517 17400 0.79 moderate ~6.9105 ~140,000
EYFP[e] 514 527 84000 0.61 moderate 5.5105 ~140,000
Cy3/Cy5[f] 647 662 200000 0.18 very good – ~670,000
PC-RhB[g] 552 580 110000 0.65 moderate – ~600,000
[a] Values for 1a and 1b reported in ethanol unless otherwise stated. In the other systems, we calculated esti-
mations from available information (see discussion in the supporting material of reference [23]). [b] Quantum
yield of photoconversion; for the DCDHF system, from azide with 407 nm illumination. Quantitative values for
other systems have not been reported, so we include qualitative comparisons. [c] Aqueous photophysical
values for the reversibly photoswitchable GFP called Dronpa from refs. [57] and [67] . [d] Aqueous and in-cell
photophysical values for the irreversibly photoswitchable GFP called PA-GFP as reported in refs. [25, 56], and
[68]. [e] Photophysical values, as reported in refs. [25, 55, 68], and [69] . [f] Aqueous photophysical values of a
Cy3/Cy5 dimer on hybridized DNA, as reported in refs. [58] and [68] . [g] Photophysical values of a photoswitch-
able rhodamine B embedded in a poly(vinyl alcohol) film, as reported in ref. [59] ; the range of Ntot,e values is for
rhodamine 6G in water from ref. [70] and tetramethyl rhodamine in lipid membranes from ref. [68] .
Figure 7. Top: The trajectory of a single copy of the amino DCDHF fluoro-
phore diffusing in a CHO cell after photoactivation. Gray lines indicate
frames when the fluorophore was dark and not tracked (i.e. blinking or out
of focus). Bottom: A background-subtracted intensity time-trace of the mol-
ecule in the trajectory on the left. Gray lines indicate when the fluorophore
was dark (i.e. initially blinking events, then finally bleaching). (Adapted with
permission from J. Am. Chem. Soc. 2008, 130, 9204–9205. Copyright 2008
American Chemical Society.)
62 www.chemphyschem.org  2009 Wiley-VCH Verlag GmbH&Co. KGaA, Weinheim ChemPhysChem 2009, 10, 55 – 65
W. E. Moerner et al.

4.2. DCDHFs as Lipid Analogs
Quantifying the brightness and photostability of emitters in
cells, while difficult, is ultimately necessary to confirm their effi-
cacy. For instance, we measured the diffusion behavior of
single emitters of seven DCDHF structures in the cell mem-
brane,[18] and found by measuring signal-to-background levels
and total photons detected that DCDHF-N-6 performed com-
parably to TRITC, a rhodamine derivative. For this experiment,
DCDHF versions with long alkyl chains on the amine donor
were synthesized in order to mimic the polar head and nonpo-
lar tail of membrane lipids. These DCDHF lipid analogs insert
well into the lipid bilayer, where higher viscosity brightens the
fluorophore to the point that it is visible and trackable, even
with electron-multiplying camera integration times as short as
30 ms. Charged DCDHF versions exhibited slower diffusion ki-
netics than the neutral versions, possibly indicating that they
partitioned into different lipid environments, or possibly result-
ing from stronger association of neighboring lipid molecules
around the additional charges.
4.3. Self-Quenched DCDHF Molecular Beacons
DCDHF emitters can be used for other types of photophysical
readouts of molecular associations. We attached the amine-re-
active NHS ester of DCDHF-V-P to the two ends of a DNA hair-
pin,[21] thereby generating a self-quenched dimer molecular
beacon which fluoresces upon hybridization.[63] In this study,
identical DCDHFs were covalently attached to the ends of a
single-stranded DNA molecular beacon in an H-dimer configu-
ration (i.e. transition dipoles oriented parallel).[64] This interac-
tion results in splitting of the electronic excited state into a
higher and a lower energy level. Excitation to the lower energy
level is dipole-forbidden, as evidenced by the resulting blue-
shifted absorption peak. Following photoexcitation from the
ground state to the higher energy level of the electronic excit-
ed state, rapid internal conversion to the lower energy level
occurs, from which fluorescence is a forbidden transition;
therefore, fluorescence from the DCDHFs is quenched in the
closed molecular beacon. Upon addition of target DNA, which
binds to the loop region of the beacon and causes it to open,
the H-dimer configuration is broken and the DCDHFs spatially
separate and become bright.
Figure 9 displays the beacon scheme and the sensitivity to
the addition of target sequence in single-molecule imaging. In
addition to achieving a high fluorescence turn-on ratio, this
system affords other advantages, including a one-pot synthesis
of the molecular beacons, non-fluorescent colorimetric detec-
tion of DNA, a two-fold “on” signal (i.e. emission from two fluo-
rophores instead of only one, as in a traditional fluorophore-
quencher molecular beacon), and the ability to use two-step
photobleaching (Figure 9c) of the molecular beacon in single-
molecule studies to reduce false positives resulting from stray
fluorescent impurities or singly labeled beacons. Although
Figure 9. Molecular beacons using DCDHF-V-P as a self-quenchable fluoro-
phore. Single-molecule fluorescence images of surface-immobilized beacons
a) before and b) after adding target oligonucleotide (scale bars : 5 mm).
c) Single-molecule time traces of opened beacons display two-step photo-
bleaching, indicating that two DCDHF fluorophores emit from each bright
spot. d) Closed and open conformations of the surface-immobilized molecu-
lar beacon: the two DCDHFs (indicated by arrows) are arranged in an H-
dimer configuration and the beacon is dark (self-quenched) ; upon binding
of the target oligonucleotide sequence, the two fluorophores become sepa-
rated and both become bright (no longer self-quenched). (Adapted with
permission from J. Phys. Chem. B 2007, 111, 7929–7931. Copyright 2007
American Chemical Society.)
Figure 8. Surface plot of emission from (left) four distinct DCDHF-N-6 single
molecules in a gelatin layer and (right) two distinct DCDHF-A-6 single mole-
cules in a CHO cell membrane. The ability to see single emitters in cells is a
strong qualitative test of their utility. In gelatin, photostability and other im-
portant photophysical parameters can be directly quantified. (Adapted with
permission from J. Phys. Chem. A 2007, 111, 8934–8941. Copyright 2007
American Chemical Society.)
ChemPhysChem 2009, 10, 55 – 65  2009 Wiley-VCH Verlag GmbH&Co. KGaA, Weinheim www.chemphyschem.org 63
Single-Molecule Imaging in Cells

these were in vitro tests of the beacon, sensing single oligonu-
cleotide target sequences in living cells may be possible.
4.4. DCDHFs Labeling Cell-Penetrating Peptides
DCDHFs have also been used to label specific biomolecules of
interest to enable studies of the molecules in conjunction with
living systems. For example, to shed light on the debated cell-
entry mechanisms of cell-penetrating peptides, we labeled a
cell-penetrating peptide of the guanidinium-rich oligoarginine
type[65] using a reactive maleimide group on a DCDHF-V-P flu-
orophore.[27] The DCDHF-labeled oligoarginines were then
added to living cells and observed as they interacted with the
plasma membrane. This experiment was performed at normal
oxygen concentration as the use of DCDHF-V-P affords high-
contrast imaging without the use of oxygen-scavenger sys-
tems.
Figure 5 shows the labeled oligoarginines on a cell at differ-
ent concentrations; single copies are easily visible and track-
ACHTUNGTRENNUNGable on the surface of the cell at nanomolar concentration of
labeled oligoarginines, with low background from fluorophores
in the surrounding solution. In this case, since the free upper
membrane of the cell was of interest, a TIR imaging configura-
tion was not a feasible option. The use of viscosity-sensing
DCDHFs maintained a low enough background to enable
single-molecule tracking in epifluorescence mode on the top
surface of the cell, which does not interact with the coverslip.
Quantitative parameters such as diffusion coefficients and
residence times of the DCDHF-labeled oligoarginines in the
plasma membrane were extracted from single-molecule traces.
These values were used to demonstrate that oligoarginines in-
teract with the plasma membrane either via a multiple-entry
mechanism or via a mechanism distinct from passive diffusion
or endocytosis.
5. Conclusions and Outlook
The DCDHF organic fluorophores are attractive as single-mole-
cule emitters, with their high photostability, bright emission,
sensitivity to local environment, solubility, and range of colors
and reactive chemistries. By harnessing their structure-property
relationships, we have designed and optimized DCDHFs for
cell-labeling applications by red-shifting the absorption wave-
length, adding lipid-like tails, increasing water solubility, and
increasing the brightness by constraining bond rotations.
Moreover, the push–pull character of the fluorophores offers
a distinctive handle for new fluorogenic reactions. Because
each component of the donor–p–acceptor structure is re-
quired for effective charge transfer interaction, disrupting any
one part of the fluorophore can render it dark; chemically or
photochemically regenerating that portion of the molecule re-
stores fluorescence. This property of DCDHFs can be used to
design probes that become fluorescent only after a specific
bioconjugation reaction or irradiation with photoactivating
light. Additionally, this approach could be applied to other
push–pull fluorophores. With photoswitching and fluorogenic
probes becoming more important as complex and sophisticat-
ed microscopy techniques[49,66] are applied to cell imaging,
DCDHF fluorophores will be a valuable tool.
Acknowledgements
We warmly thank our additional collaborators for their contribu-
tions: S. Bunge, P. J. Schuck, J. Bertke, P. R. Callis, and P. A.
Wender. We also thank M. A. Thompson for conversations about
relative photoactivation quantum yields. This work was support-
ed in part by the National Institutes of Health through the NIH
Roadmap for Medical Research, Grant No. P20-HG003638-04.
Keywords: biosensors · fluorescent probes · imaging agents ·
photochemistry · single-molecule studies
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Received: September 4, 2008
Published online on November 21, 2008
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Single-Molecule Imaging in Cells