R1
N/
R2
ON


Photoactivatable DCDHF fluorophores for single-molecule imaging

Samuel J. Lord,a Nicholas R. Conley,a Hsiao-lu D. Lee,a Na Liu,b
Reichel Samuel,b Robert J. Twieg,b W. E. Moernera*

a Department of Chemistry, Stanford University, Stanford CA, 94305-5080
b Department of Chemistry, Kent State University, Kent OH, 44240
ABSTRACT
We have designed and studied the photophysics of a class of organic fluorophores termed “DCDHFs,” which were
originally used as push–pull chromophores for nonlinear optical applications. In this paper, we describe the general
photophysics of many realizations of the DCDHF class of single-molecule emitters. Moreover, we have reengineered a
red-emitting DCDHF fluorophore so that it is dark until photoactivated with a short burst of low-intensity violet light.
Photoactivation of the dark fluorogen leads to conversion of an azide to an amine, which shifts the absorption to long
wavelengths. After photoactivation, the fluorophore is bright and photostable enough to be imaged on the single-
molecule level in living cells. This molecule and its relatives will provide a new class of bright photoactivatable
fluorophores, as are needed for super-resolution imaging schemes that require active control of single-molecule
emission.
Keywords: fluorophores, single-molecule detection, optical imaging, photostability, photophysics, photoactivatable

1. INTRODUCTION
With recent advances in super-resolution cellular imaging techniques using single-molecule localization (e.g. PALM, F-
PALM, STORM)1-3 comes the need for new photoactivatable fluorophores that emit many photons, absorb beyond 500
nm, and are targetable to specific biomolecules. For the past several years, we have been designing and characterizing
several fluorophores, termed “DCDHF” fluorophores, for use in live-cell imaging based on the push–pull design: an
amine donor group and a 2-dicyanomethylene-3-cyano-2,5-dihydrofuran (DCDHF) acceptor group, separated by a π-rich
conjugated network. 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.4-14 We
have now reengineered the DCDHFs to make them photoactivatable: by replacing the amine with an azide, the
fluorescence is quenched; upon photocleaving the azide, the amino fluorophore is regenerated, and the fluorescence
returns.13 In this paper, we review the properties of the DCDHF class in general, and then present the scheme that has
been utilized to make them photoswitchable.

Figure 1. Schematic structure of the DCDHF fluorophores. The amine donor and DCDHF acceptor are connected by a π-
conjugated linker. The R1–R4 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 π system: “DCDHF-
(π unit closest to acceptor)-…-(π unit closest to donor)” with the π units denoted P = phenylene, V = vinyl, T =
thiophene, N = naphthalene, A = anthracene; the amine donor is not specified because it is present in all structures.

* wmoerner@stanford.edu; phone 1 650 723 1727; http://www.stanford.edu/group/moerner/
Reporters, Markers, Dyes, Nanoparticles, and Molecular Probes for Biomedical Applications
edited by Samuel Achilefu, Ramesh Raghavachari, Proc. of SPIE Vol. 7190, 719013
© 2009 SPIE · CCC code: 1605-7422/09/$18 · doi: 10.1117/12.809257
Proc. of SPIE Vol. 7190 719013-1

5000
4000
v DCDHF-A
0 DCDHF-N
E0
3000
C)
DCDHF-P
b 0
St
ok
e.
M Q o
0
w
1000 J.
0.0 0.1 0.2 0.3
z\f


2. GENERAL PHOTOPHYSICS
2.1 Solvatochromism
Because of the push–pull character of DCDHF chromophores, their photophysics are dominated by photoinduced
intramolecular charge transfer. For instance, adding electron-donating and -accepting groups to a conjugated unit tends
to red-shift the absorption by lowering the energy required to produce a charge-transfer upon photoexcitation.15, 16 For
this reason, DCDHF fluorophores—with their strong amine donor and DCDHF acceptor groups—exhibit relatively red-
shifted absorption (out to beyond 700 nm14).
The static Stokes shift of DCDHF fluorophores is dependent on solvent polarity, because the excited-state electronic
dipole can be better stabilized by polar solvent molecules. The Lippert-Mataga equation uses solvent orientation
polarizability Δf to approximate the effect of solvent polarity and dipole moment change on the Stokes shift:
( )
constant
2
3
2
+−Δ=− afhc
GE
FA
μμνν (1)
where
12
1
12
1
2
2
+
−−+
−=Δ n
nf
r
r
ε
ε

and Aν and Fν are the wavenumbers of the absorption and emission, μG and μE are the ground- and excited-state dipole
moments, a is the Onsager cavity radius (assumed to be 5 Å), n is the refractive index of the solvent, εr is the relative
dielectric constant of the solvent, h is Planck’s constant, and c is the speed of light. The solvent parameter Δf 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 redistribution, which occurs on the same time scale as
photoinduced charge transfer in the fluorophore) from the total polarizability.17, 18

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 (nomenclature described in Figure 1) are 7757, 6921, and 1588 cm–1, respectively; these
slopes correspond to the change in dipole (μE – μG) values of 9.7, 9.4, and 4.4 D. As expected, increasing the
conjugation length between the donor and acceptor 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 references 6 and
10.)

By correlating the Stokes shift with solvent polarity (Figure 2), we were able to calculate changes in the dipole moment
upon photoexcitation (μE – μG) for various DCDHF fluorophores. Increasing the conjugation between the amine donor
and DCDHF acceptor from a benzene to a naphthalene and to an anthracene increases the Δμ.6, 10
Proc. of SPIE Vol. 7190 719013-2

2.2 Viscosity sensitivity
The charge-transfer in DCDHF fluorophores has a strong effect on their intensity of fluorescence, not only their
emission color (i.e. Stokes shift). In solution at room temperature, the fluorescence quantum yields (ΦF) of DCDHFs are
typically very low; in polar solvents, the values of ΦF are normally less than 1%. However, in rigidized environments
(e.g. ice, glasses, polymer films), the fluorescence increases dramatically. This can be explained by a twisted
intramolecular charge-transfer (TICT) state,19 which opens a nonradiative deexcitation pathway: In solution, the TICT
state is easily accessible by bond rotations; in a rigid environment, those bond twists are hindered, and the fluorescence
pathway becomes dominant.6 This photophysical property can be harnessed by targeting the DCDHFs to locations in
which their bond twists are hindered (e.g. the cell membrane20, 21 or embedded in an enzyme binding pocket). Because
the fluorescence is brighter from the targeted position than in solution, background from unbound copies is suppressed.
2.3 Photostability
One of the most important properties of a probe for single-molecule studies it the photostability. The more photons a
reporter emits before photobleaching (Ntot,e), the better the target can be localized, tracked, or imaged above the
background noise. The value for Ntot,e can be measured from a single-molecule sample either molecule-by-molecule with
a point detector with single-molecule sensitivity (i.e. avalanche photodiode) or in widefield using a CCD camera. In
either case, in order to convert from the photons detected to the number emitted, the detection efficiency of the imaging
system must be measured.22 For an independent measure of photostability, the photobleaching quantum yield (ΦB) can
be measured from a bulk sample if the laser irradiance (Iλ) and wavelength (λ), and absorption cross-section (σλ =
2303ελ/NA) are known, from which the ratio of the bleaching rate (RB) to the absorption rate (Rabs) can be calculated:
⎟⎠
⎞⎜⎝
⎛===Φ
hcI
RR
R
λσττ λλBabsBabs
B
B
11
(2)
where τB 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. The photobleaching quantum yield—the probability of bleaching with each photon absorbed—must
be very small to ensure that the emitters last long enough for adequate imaging.
DCDHFs typically emit millions of photons before photobleaching (and thus have very low ΦB values), making them as
photostable as some of the most popular single-molecule fluorophores, such as rhodamines (see Table 1).10
3. STRUCTURE-PROPERTY DESIGN
3.1 Long-wavelength absorption and emission
The most obvious structure-property relationship for organic fluorophores is that increasing the π-conjugation length
increases the size of the “box” and thus redshifts the absorption wavelength. We have synthesized and characterized
several long-wavelength DCDHFs, with extended conjugation using rigid acene groups or multiple rings.10, 14 Because
autofluorescence from endogenous fluorophores is strongly pumped by wavelengths shorter than 500 nm,23 red
fluorophores are important for low-background cell imaging.
3.2 Water solubility and membrane permeability
Although complete hydrophilicity is not desirable (e.g. entering the lipid membrane requires some degree of
hydrophobicity), biological imaging requires fluorophores that are water soluble. Because many of the DCDHF
structures are best soluble in organic solvents, there was a need to add functional groups that would impart greater water
solubility while maintaining the beneficial characteristics of the parent molecule. Thus, we added hydroxyl, carboxylic
acid, and sulfonate groups to two parent DCDHF fluorophores.12 These groups imparted increasing water solubility,
respectively; moreover, the photophysical and environment-sensing properties of the fluorophores remained. We have
also measured photostability parameters (Ntot,e and ΦB) of DCDHFs in aqueous environments, such as gelatin films and
cell membranes.10 Conversely, to enhance membrane permeability and residence time, we appended long alkyl chains
from the DCDHF fluorophores, making them more hydrophobic and enabling them to embed into the lipid bilayer.20
Proc. of SPIE Vol. 7190 719013-3

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 R1–R4 groups, because varying the length of the alkyl chains
has little effect on the photophysics. See text for details on photophysical parameters. (Data from references 9, 10, and
14.)
Compound

Structure

εmax
(M–1 cm–1)a
λabs
(nm)a
λem
(nm)a
ΦF in toluene
{in PMMA}
ΦB in gelatin
{in PMMA}
Ntot,e
in PMMA
DCDHF-P 71,000 486 505 0.044 {0.92} 6.6×10
–6 2.4×106
DCDHF-V-P 45,500 562 603 0.02 {0.39} 2.8×10
–6 ~1.9×106
DCDHF-N 42,000 526 579 0.85 {0.98} 3.4×10
–6 1.1×106
DCDHF-A 35,000 585 689 0.54 1.7×10–6 2.2×106
DCDHF-P-P 31,000 506 623 0.82 4.5×106
DCDHF-V-T-T 49,800 708 779 0.13 {3.4×10–6}
DCDHF-P-T 44,000 591 663 0.21 6.4×106
Rhodamine 6G 105,000c 530c 556c 0.95c 3.5×10–6 1.4×106
fluorescein 92,300c 483c 515c 0.79c 64×10–6
aIn toluene. bIn dichloromethane. cIn ethanol.

Proc. of SPIE Vol. 7190 719013-4

4. PHOTOACTIVATION
Given the current interest in super-resolution optical imaging in cells, several groups have been developing fluorescent
proteins,24-26 organic fluorophores,27-31 and quantum dots32 that can be optically controlled to ensure that only one
molecule is active at a time in a diffraction-limited region.33

Table 2. Photophysical properties of the photoactivatable DCDHF in Scheme 1 and other photoswitchable fluorophores.
Values reported in ethanol unless otherwise stated. The columns correspond to, respectively, the maximum absorption
and emission wavelengths; the extinction coefficient; the fluorescence, photoconversion, and photobleaching quantum
yields; and the average total number of photons emitted before photobleaching.
λabs (nm)
λfl
(nm)
εmax
(M–1cm–1)
ФF
ФP
ФB
in gelatin
Ntot,e
in gelatin
DCDHF-V-P-azide 424 552 29,100 n/a good (0.0059) n/a n/a
DCDHF-V-P-amine 570 613 54,100 0.025–0.39 n/a 4.1×10–6 2.3×106


Figure 3. Azido DCDHFs are photoactivatable: DCDHF-V-P-azide photoconverts to the fluorescent DCDHF-V-P-amine.
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 repairs the donor-acceptor
character of the fluorophore and returns the absorption and emission to longer wavelengths. See Scheme 1. (A) Three
CHO cells incubated with azido fluorogen are dark before activation. (B) The fluorophore lights up in the cells after
activation with a 10-s flash of diffuse, low-irradiance (0.4 W cm–1) 407-nm light. The white-light transmission image is
merged with the fluorescence images (white), excited at 594 nm (~1 kW cm–1). Scalebar: 20 μm. (C) Single molecules
of the activated fluorophore in a cell under higher magnification. Scalebar: 800 nm. (D) Absorption curves in ethanol
showing photoactivation of the azido fluorogen (λabs = 424 nm) over time to fluorescent amino DCDHF product (λabs =
570 nm). Different curves represent time points between 0 and 1320 s of illumination by 3.1 mW cm–1 of diffuse 407-
nm light (trends denoted by arrows). Dashed line is the absorbance of pure, synthesized amino DCDHF. (Inset) Dotted
line is weak pre-activation fluorescence of the azido fluorogen excited at 550 nm; solid line is strong post-activation
fluorescence resulting from exciting the amino fluorophore at 550 nm, showing an increase in the fluorescence
emission. (Adapted with permission from J. Am. Chem. Soc. 2008, 130, 9204–9205. Copyright 2008 American
Chemical Society.)
Proc. of SPIE Vol. 7190 719013-5

Recently, we reported a photoactivatable azido version of a DCDHF fluorophore.13 Because DCDHFs are push–pull
chromophores, they require both an electron donor group and an acceptor group connected by a conjugated π system; the
donor and acceptor provide a charge-transfer band in the absorption that is red-shifted compared to the π system itself.15,
16 Replacing the amine donor with a weakly electron-withdrawing34 azide disrupts the charge-transfer band and blue-
shifts the absorption of the chromophore to the point where it is no longer in resonance with the pumping laser (see
Table 2 and Figure 3); therefore the azido DCDHF fluorogen is “dark.”
The photochemistry of aryl azides has been well studied,35 and they are known to photodegrade to amine species.36 The
azido DCDHF fluorogen can thus be photoactivated by photoconverting the azide to an amine, returning the donor–
acceptor property of the chromophore. Figure 3 and Table 2 demonstrate that the molecule DCDHF-V-P-azide does
indeed photoconvert to a fluorescent product. This photoconversion occurs with relatively high photoconversion
quantum yield (ФP), so the fluorogen need only absorb several photons before converting, on average. After irradiation
with blue light, the azido fluorogen converts to the long-wavelength DCDHF-V-P-amine, which is fluorescent. The
fluorescence quantum yield (ФF), like other DCDHFs studied,6 exhibits strong dependence on viscosity or rigidity:
values range from low (2.5%) in ethanol to much higher in a rigid polymer environment (39%). Finally, one of the most
important properties for single-molecule imaging and localization precision37, 38 is the number of photons a single
molecule emits before photobleaching (or, conversely, the photobleaching quantum yield, ФB). The values for the
DCDHF fluorogen are favorable, emitting millions of photons before photobleaching. It is clear that these ideas can be
applied to all the fluorophores listed in Table 1 in future work.
5. CONCLUSION
Single-molecule imaging and super-resolution imaging have expanded the need for new photostable fluorophores with
complex photochemistries. We have described the DCDHF class of single-molecule fluorophores, including a version
that is photoactivatable. With their solvatochromism, viscosity sensitivity, photostability—and now their ability to be
photoactivated—DCDHF fluorophores should be regarded as useful for single-molecule experiments along with other
dyes in the well-known classes (e.g. rhodamine, cyanine, rylene, etc.).
Proc. of SPIE Vol. 7190 719013-6

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