Azido push-pull fluorogens photoactivate to produce bright fluorescent labels

Article abstract of DOI:10.1021/jp907080r

Dark azido push-pull chromophores have the ability to be photoactivated to produce bright fluorescent labels suitable for single-molecule imaging. Upon illumination, the aryl azide functionality in the fluorogens participates in a photochemical conversion to an aryl amine, thus restoring charge-transfer absorption and fluorescence. Previously, we reported that one compound, DCDHF-V-P-azide, was photoactivatable. Here, we demonstrate that the azide-to-amine photoactivation process is generally applicable to a variety of push-pull chromophores, and we characterize the photophysical parameters including photoconversion quantum yield, photostability, and turn-on ratio. Azido push-pull fluorogens provide a new class of photoactivatable singlemolecule probes for fluorescent labeling and super-resolution microscopy. Lastly, we demonstrate that photoactivated push-pull dyes can insert into bonds of nearby biomolecules, simultaneously forming a covalent bond and becoming fluorescent (fluorogenic photoaffinity labeling).

Full text of DOI:10.1021/jp907080r

J. Phys. Chem. B 2010, 114, 14157–14167
14157
Azido Push-Pull Fluorogens Photoactivate to Produce Bright Fluorescent Labels†
Samuel J. Lord,‡ Hsiao-lu D. Lee,‡ Reichel Samuel,§ Ryan Weber,§ Na Liu,§
Nicholas R. Conley,‡ Michael A. Thompson,‡ Robert J. Twieg,§ and W. E. Moerner*,‡
Department of Chemistry, Stanford UniVersity, Stanford, California 94305-5080, and Department of Chemistry,
Kent State UniVersity, Kent, Ohio 44242
ReceiVed: July 24, 2009; ReVised Manuscript ReceiVed: October 2, 2009
Dark azido push-pull chromophores have the ability to be photoactivated to produce bright fluorescent labels
suitable for single-molecule imaging. Upon illumination, the aryl azide functionality in the fluorogens
participates in a photochemical conversion to an aryl amine, thus restoring charge-transfer absorption and
fluorescence. Previously, we reported that one compound, DCDHF-V-P-azide, was photoactivatable. Here,
we demonstrate that the azide-to-amine photoactivation process is generally applicable to a variety of push-pull
chromophores, and we characterize the photophysical parameters including photoconversion quantum yield,
photostability, and turn-on ratio. Azido push-pull fluorogens provide a new class of photoactivatable single-
molecule probes for fluorescent labeling and super-resolution microscopy. Lastly, we demonstrate that
photoactivated push-pull dyes can insert into bonds of nearby biomolecules, simultaneously forming a covalent
bond and becoming fluorescent (fluorogenic photoaffinity labeling).
Introduction
SCHEME 1: Photoconversion of Dark Azide-Substituted
Fluorogens Producing Fluorescent Amine-Substituted
Advances in super-resolution fluorescence imaging by con-
trollable photoactivation of single-molecule emitters (e.g.,
PALM, FPALM, STORM)1-3 require new and optimized
activatable fluorophores. If one single emitter is switched on at
a time in a diffraction-limited region (∼250 nm), its location
can be determined well below the diffraction limit by fitting
the point-spread function (i.e., image of the single molecule).
A super-resolution image of a labeled complex structure can
then be reconstructed from many successive rounds of weak
photoactivation and fitting.4 Several groups have been develop-
ing photoswitchable fluorescent proteins,5-7 organic fluoro-
phores,8-12 and quantum dots13 to build the toolbox of control-
lable emitters.14 Recently, we reported a photoactivatable azido
version of a push-pull fluorophore that contains a 2-dicya-
nomethylene-3-cyano-2,5-dihydrofuran (DCDHF) moiety as a
very strong electron-accepting group.15 In addition to super-
resolution imaging, the ability to photochemically control the
fraction of emitting molecules has additional applications in
pulse-chase experiments, in single-molecule tracking, or in
situations where the number of emitting molecules at a given
time must be kept low.
Push-pull chromophores containing an electron donor, a
conjugated network (π), and an electron acceptor have been
explored for many years for nonlinear optics,16 photoinduced
electron transfer,17 and photorefractivity;18 some molecules in
this class were even found to be good single-molecule labels.19-23
In our approach, a nonfluorescent, blue-shifted azide-π-acceptor
fluorogen precursor is photoconverted to a fluorescent, red-
shifted amine-π-acceptor fluorophore. In the fluorogen, the donor
is absent, but the product fluorophore contains all three necessary
components of the complete donor-π-acceptor push-pull chro-
mophore (Scheme 1). Because the azido fluorogens do not
exhibit the red-shifted charge-transfer band typical of push-pull
Fluorophores, Which May Involve Insertion into C-H or
C-C Bonds
chromophores,24,25 they are not resonant with the wavelengths
used to excite the amino version of the fluorophore (Figure 1
and Table 1) and are therefore dark. In related work, Bouffard
et al.26 designed a chemically caged DCDHF fluorophore in an
effort to detect cysteine: an electron-withdrawing sulfonyl group
was added to the nitrogen and thus interfered with its donor
capability until it was cleaved off by any cysteine, producing
an amine capable of donating electrons. Budyka et al.27 have
previously reported that an azido hemicyanine dye (similar to
the azido stilbazolium reported below) undergoes conversion
to an amine upon irradiation with visible light; however, the
fluorescence properties of this dye were not reported.
While amines are strong electron-donating substituents, azides
are weakly electron-withdrawing.28 Recovering fluorescence
from aryl azides is possible because they are known to be
photolabile. The photochemistry of aryl azides has been studied
extensively;29 the photoreaction most often reported involves
the loss of dinitrogen and rearrangement to a seven-membered
azepine heterocycle. However, electron-withdrawing substituents
on the aromatic ring can stabilize the nitrene intermediate and
promote formation of the amino functionality.30 Because
push-pull chromophores inherently contain a strong electron-
withdrawing substituent, an azido push-pull molecule should
be more prone to photoconvert to the fluorescent amino version
upon irradiation with activating light that is resonant with the
blue-shifted absorption of the azido fluorogen. Initially, we
demonstrated this photoactivation of fluorescence with only one
† Part of the “Michael R. Wasielewski Festschrift”.
* Corresponding author. E-mail: wmoerner@stanford.edu.
‡ Stanford University.
§ Kent State University.
10.1021/jp907080r  2010 American Chemical Society
Published on Web 10/27/2009

14158 J. Phys. Chem. B, Vol. 114, No. 45, 2010
Lord et al.
Figure 1. (left) Spectra of NBD-azide photoactivation. (See the Supporting Information for spectra of the other fluorogens.) Because the azide
cannot participate as a donor in a charge transfer, the fluorogen exhibits blue-shifted absorption (λabs ) 384 nm) with respect to its amine-donor
sister (λabs ) 456 nm). Upon irradiation with UV light, the azido fluorogen (solid black curve) converts to the amino fluorophore (solid gray
curves). Fluorescence (dotted red lines) excited at 440 nm increases significantly after photoconversion. (right) Photoactivation of DCDHF-P-azide
using a 385 nm flashlight (1.1 mW cm-2). The short-wavelength azido absorption peak (triangles) disappears with time, and the long-wavelength
absorption peak (circles) corresponding to the fluorescent amino fluorophore grows in. The time constant (τP ) 85 s) from the exponential fit of the
disappearance of the azido fluorogen is used to calculate the photoconversion quantum yield (ΦP) in Table 1.
member of the DCDHF class of fluorophores.15 Here, we expand
the concept to a broad range of push-pull dyes: DCM,
stilbazolium, NBD, and other DCDHF fluorophores (for struc-
tures see Table 1). We also demonstrate fluorogenic photoaf-
finity labeling of biomolecules using the photochemical azide-
to-amine process.
Hz, Ar, 2H), 6.97 (d, J ) 16 Hz, vinyl, 1H), 1.80 (s, CH3, 6H).
DCDHF-V-P-amine (photoconverted from DCDHF-V-P-azide,
column separated): H NMR (400 MHz, CDCl3, δ) 7.58 (d, J
1
) 16 Hz, vinyl, 1H), 7.50 (d, J ) 8.4 Hz, Ar, 2H), 6.80 (d, J
) 16 Hz, vinyl, 1H), 6.70 (d, J ) 8.8 Hz, Ar, 2H), 4.39 (s,
NH2, 2H), 1.76 (s, CH3, 6H). DCDHF-V-P-amine (pure
synthesized independently): 1H NMR (500 MHz, CDCl3, δ) 7.58
(d, J ) 16 Hz, vinyl, 1H), 7.50 (d, J ) 8.5 Hz, Ar, 2H), 6.80
(d, J ) 17 Hz, vinyl, 1H), 6.70 (d, J ) 8.5 Hz, Ar, 2H), 4.39
(s, NH2, 2H), 1.76 (s, CH3, 6H). DCDHF-V-P-nitro (photocon-
Methods
Chemical Analysis of Photoproducts. For most of the azido
fluorogens, we relied on UV-vis and fluorescence spectroscopy
to identify the amine products, comparing the spectra of the
photoconverted samples to the spectra of independently syn-
thesized versions of the amino fluorophores. The following full
chemical analysis of the photoconversion of DCDHF-V-P-azide
(bolded to signify fluorogen) was originally reported in the
Supporting Information of ref 15. Samples for bulk chemical
studies were photoconverted in ethanol, both with and without
removing dissolved oxygen by bubbling N2, and analyzed using
NMR and HPLC-MS.
1
verted from DCDHF-V-P-azide, crude, column enriched): H
NMR (300 MHz, CDCl3, δ) 8.34 (d, J ) 8.7 Hz, Ar), 7.80 (d,
J ) 8.4 Hz, Ar), 7.69 (d, J ) 11 Hz, vinyl), 7.12 (d, J ) 14
Hz, vinyl), 1.83 (s, CH3). DCDHF-V-P-nitro (pure synthesized
1
independently): H NMR (400 MHz, CDCl3, δ) 8.34 (d, J )
8.8 Hz, Ar, 2H), 7.80 (d, J ) 8.8 Hz, Ar, 2H), 7.68 (d, J )
16.8 Hz, vinyl, 1H), 7.12 (d, J ) 16.4 Hz, vinyl, 1H), 1.83 (s,
CH3, 6H).
Purification of DCDHF-V-P-amine and DCDHF-V-P-nitro
by Semiprep HPLC. An ethanolic solution containing ∼1 mg
mL-1 of DCDHF-V-P-azide was photoconverted using a
150-W Xe lamp for 5 min under air. Photoproducts DCDHF-
V-P-amine and DCDHF-V-P-nitro were separated by HPLC on
a Hypersil Hyper Prep 100 BDS-C18 column (10.0 × 250 mm)
with linear gradient elution (5-100% acetonitrile over 25 min,
5 min hold at 100% acetonitrile; balance by volume, 0.1 M
tetraethylammonium acetate buffer, pH 7.5; total flow rate, 4
mL min-1). The UV-vis absorption spectrum of the column
eluent was continuously monitored using a Shimadzu diode
array detector (SPD-M10A). Under these conditions, compounds
DCDHF-V-P-amine and DCDHF-V-P-nitro exhibited retention
times of 20.9 and 22.5 min, respectively. No detectable
DCDHF-V-P-azide(RT)23.6min)remainedafterphotoactivation.
HPLC-MS Characterization of Photoproducts. Ethanolic
solutions of DCDHF-V-P-azide were photoconverted using
diffuse 407 nm laser light under nitrogen or air. The photoac-
tivation products were analyzed by HPLC–MS (Waters 2795
Separations module with 2487 Dual λ Absorbance Detector;
Waters Micromass ZQ mass spectrometer). Gradient elution
(2-95% acetonitrile with 0.1% formic acid over 20 min, 10
min hold at 95% acetonitrile/formic acid; balance by volume,
Column Chromatography and NMR. A solution of photo-
converted DCDHF-V-P-azide in ethanol was separated on a
TLC plate (1:3 acetone/dichloromethane) into two bands: a red
band with lower Rf that was fluorescent under UV light (365
nm) and a yellow band with higher Rf that was nonemissive;
the yellow band was not present when the solution of DCDHF-
V-P-azide was deoxygenated by bubbling N2 before and during
photoconversion. (Adequate separation was not achievable using
dichloromethane and hexanes or dichloromethane alone; there-
fore, we resorted to acetone in the mobile-phase solvent
mixture.)
For chromatography, the photoproducts were separated on a
column using silica gel as the stationary phase and 2:1 hexanes/
acetone as the mobile-phase solvent. Two bands were well
separated: a yellow band of DCDHF-V-P-nitro eluted first, then
a red band of DCDHF-V-P-amine eluted later (see Figure S1,
Supporting Information, for structures). NMR spectra of column-
separated photoproducts confirm these assignments, as compared
to pure, synthesized samples (although the yellow band was
contaminated with some other minor photoproducts).30,31 DCDHF-
1
V-P-azide: H NMR (400 MHz, CDCl3, δ) 7.65 (d, J ) 8.4
Hz, Ar, 2H), 7.61 (d, J ) 16 Hz, vinyl, 1H), 7.13 (d, J ) 8.4

Azido Push-Pull Fluorogens Photoactivation
J. Phys. Chem. B, Vol. 114, No. 45, 2010 14159
TABLE 1: Photophysical and Photochemical Parameters of Various Azido Fluorogens (In Ethanol unless Otherwise Stated)
a Peak absorbance and molar absorption for azido fluorogen. b Peak absorbance and molar absorption for amino fluorophore. c Fluorescence
peak of amino fluorophore. d Overall chemical reaction yield to the fluorescent amino product (see Methods). e Fluorescence quantum yield of
amino fluorophore. DCDHFs become much brighter in rigid environments.20 f Photoconversion quantum yield of azido fluorogens to any
product. g Wavelength used to photoactivate azido fluorogens. h Photobleaching quantum yield of amino fluorophore. Fluorescein in gelatin is
64 × 10-6
.
i In dichloromethane. j In acetonitrile. k In gelatin. l In PMMA.
water with 0.1% formic acid) through a C18 column (2.1 × 40
mm) was employed for the separation. The column eluent was
subjected to electrospray ionization, and positive and negative
ions with m/z from 100-1000 amu were detected.
In the absence of oxygen, photoconversion of DCDHF-V-
P-azide produced DCDHF-V-P-amine (RT ) 11.36 min; ESI-:
m/z ) 301.7, [M - H]-; ESI+: m/z ) 303.5, [M + H]+) as the
only major photoproduct. A putative azo dimer (RT ) 16.97
min; ESI-: m/z ) 599.7, [M - H]-) was observed as a minor
photoproduct.
301.5, [M - H]-; ESI+: m/z ) 303.4, [M + H]+) and DCDHF-
V-P-nitro (RT ) 12.99 min; ESI-: m/z ) 331.5, [M - H]-,
315.5 [M - O - H]-, 301.5 [M - 2O - H]-) as major
products. After several days in air and room lights, an unidenti-
fied species believed to be generated from DCDHF-V-P-nitro
formed in the solution (RT ) 19.15 min; ESI-: m/z ) 367.6).
Bulk Solution Spectroscopy. Bulk solution absorption and
emission spectra were acquired on a Perkin-Elmer Lambda 19
UV-vis spectrometer and a Horiba Fluoromax-2 fluorimeter
using standard 1 cm path length, quartz cuvettes. Fluorescence
quantum yields were referenced against standards with known
quantum yields, corrected for differences in optical density and
In air, photoactivation of DCDHF-V-P-azide produced a
mixture of DCDHF-V-P-amine (RT ) 11.43 min; ESI-: m/z )

14160 J. Phys. Chem. B, Vol. 114, No. 45, 2010
Lord et al.
solvent refractive index.32 All quantitative measurements were
performed at low concentrations (absorbance values less than
0.2) to avoid any complications with dimer or aggregate
formation. Molar absorption coefficients were measured from
dilutions of solutions with known concentrations.
of the population but separated enough to avoid self-quenching
or excimer behavior. From our previous experience with bulk
and single-molecule samples of fluorophores in polymer films,
we are confident that we are safely in this regime.
Because in some measurements the on and off signals fell
outside the dynamic range of the CCD detector, it was more
necessary to use different intensities or gain levels for the dark
and background measurements than for the fluorescent signal.
This required that the intensities and gain levels were accurately
measured and accounted for in the calculation of the turn-on
ratio.
Intermediate Lifetime. The lifetime of the nonfluorescent
intermediate was measured immediately after the photoconver-
sion. Imaging in the widefield epifluorescence microscope at
594 nm (∼1 kW cm-2), a sample in aqueous agarose gel was
repeatedly photoactivated with 0.5 s of high-intensity 405 nm
light (∼1 kW cm-2). These bright bursts of violet light both
photobleached any existing fluorescent species and initiated the
photoconversion of the azido species to fluorescent amino
products, via a nonfluorescent intermediate (putatively a nitrene,
but there may be additional intermediates requiring rearrange-
ment before fluorescence appears). By fitting with a single
exponential the increase of fluorescence over time after the
photobleaching/photoactivating burst, the average lifetime of
the intermediate was determined to be 1.85 s. Presumably, this
lifetime is dependent on the environment, solvent, and temper-
ature; however, this was measured in an aqueous environment
that is not dissimilar to the cell.
Live-Cell Microscopy. For details of Chinese Hamster Ovary
(CHO) cell culture, see ref 34. CHO cells were plated on
fibronectin-coated borosilicate chambered coverslips overnight
prior to imaging. CHO cells were treated with 1 µM dye solution
(1 mM dye stock in ethanol into growth medium) at 37 °C for
1 h, followed by extensive PBS buffer rinses to remove excess
dye. Briefly, cells were imaged at 22 °C in supplemented PBS
buffer. That is, imaging was performed within 45 min after
removing the cell tray from the 37 °C incubator to ensure cell
viability. No changes in cell morphology were observed after
photoactivation. Moreover, previous studies using DCDHFs in
living cells did not encounter complications with toxicity.
Photoaffinity Labeling and Gel Electrophoresis. Photoaf-
finity labeling (PAL) of azido fluorogens was performed on
Chinese Hamster Ovary (CHO) cells. Under red lights, 20 µL
each of ∼1 mM NBD-azide and DCDHF-P-azide stock
solutions in ethanol (kept in the dark to prevent preactivation)
were mixed with separate 200 µL aliquots of CHO cells
suspended in PBS buffer. The mixed solution was photoactivated
using a 365 nm handheld Hg UV lamp (0.4 mW cm-2) for 30
min. The nitrene intermediate inserted into bonds of accessible
biomolecules. For a control, aliquots of the fluorogen stock
solutions were preactivated before mixing with cells; the
preactivated dye is unable to participate in the covalent PAL
bioconjugating photoreaction because the nitrene does not
survive for more than a few seconds (see above).
Photoconversion in ethanol was performed using one of the
following light sources: a 365 nm hand-held UV lamp (0.62
mW cm-2); a 385 nm diode flashlight (1.1 mW cm-2, see Figure
S5, Supporting Information, for spectrum); and the 407 nm line
from a Kr-ion laser (Coherent Innova-301, 3.1 mW cm-2).
The overall chemical reaction yields to fluorescent product
listed in Table 1 were measured from the absorbance values in
the photoactivation spectra. Yield was defined by [amino]f/
[azido]i ) (Aamino/εamino)f/(Aazido/εazido)i, where i and f refer to
initial and final values, respectively. In cases where other
photoproducts contributed significant absorbance at the amino
peak wavelength, the final absorbance value for the amino peak
Aamino,f was corrected for this additional absorbance.
Sample Preparation for Microscopy. Samples for aqueous
bulk photostability measurements and quantitative single-
molecule measurements were prepared using 5-10% (by mass)
gelatin (type A, Bloom 200, MP Biomedicals) or 1% poly(vinyl
alcohol) (PVA, 72000 g mol-1, Carl Roth Chemicals) in purified
water. The gelatin solution was liquefied at 37 °C; then a small
volume (<0.5 µL) of dye stock solution in ethanol was mixed
with 10 µL of gelatin, sandwiched between two Ar-plasma-
etched glass coverslips, and allowed to gel at room temperature.
Single-molecule samples for the movie were made in 1%
poly(methyl methacrylate) (PMMA, Tg ) 105 °C, MW ) 75000
g mol-1, atactic, polydispersity ∼2.8, PolySciences Inc.) in
toluene by mass. For polymer samples, a small volume of stock
dye solution was mixed into an aliquot of PVA in water or
PMMA in toluene, then the mixture was spin-coated onto an
Ar-plasma-etched glass coverslip at 3000 rpm for 30 s. Thick
samples of PMMA were prepared by drop casting 200-400
µL of 10% (by mass) PMMA in toluene and allowing the film
to dry for several hours.
Microscopy. Samples were studied using an Olympus IX71
inverted microscope in an epifluorescence configuration33 using
488 or 514 nm illumination from an Ar-ion laser (Coherent
Innova) or the 594 nm line from a HeNe laser (Meredith
Instruments, 5 µW output); the irradiance at the sample was
typically 0.5-1.0 kW cm-2. The emission was collected through
a 100×, 1.4 N.A. oil-immersion objective, filtered using
appropriate dichroic and long-pass filters to remove scattered
excitation light, and imaged onto an electron-multiplying Si
EMCCD camera (Andor iXon+) with integration times of
20-100 ms.
Turn-On Ratio. To measure the effective turn-on ratio, the
signal on the camera from many activated fluorophores in a
PMMA film was divided by the signal from the same location
in the sample before activation; the background intensity level
measured in an undoped film was subtracted from both signals
(see Figure S6, Supporting Information). This measurement is
experimentally relevant because it not only uses illumination
intensity levels comparable to those used in actual imaging but
also considers the decrease in contrast due to a chemical reaction
yield that is less than unity (see Results and Discussion).
The CHO cells were then lysed using RIPA buffer and passed
through a 200 µL pipet tip 50 times. The insoluble portion of
the lysate was spun down at 4 °C, 14 000 rpm, and discarded.
The soluble supernatant was mixed with SDS and heated to 95
°C. The lysate was then separated on a 12% polyacrylamide
SDS gel to separate the PAL fluorescence from unbound
fluorophores. After the electrophoresis completed, the gel was
destained using a 10% acetic acid solution in water and
methanol. To image the gels, a GE Healthcare Typhoon 8600
scanner was used with 488 nm excitation, a 526 short-pass filter,
For the bulk experiments, the fluorogens were doped into a
thick PMMA polymer film at approximately 1-2 orders of
magnitude higher concentration than single-molecule experi-
ments but otherwise imaged under similar conditions. This
approach assumes that we are working in a concentration regime
where emitters are dense enough to get a statistical sampling

Azido Push-Pull Fluorogens Photoactivation
J. Phys. Chem. B, Vol. 114, No. 45, 2010 14161
and 600 PMT sensitivity setting. The protein in the gel was
then stained with Coomassie Brilliant Blue and destained
overnight. The Coomassie bands were imaged using white light
(BioRad Gel Doc scanner with Universal Hood II).
cold solution was filtered through a Celite pad, and finally the
solid was rinsed several times with ethyl acetate. The organic
phase was separated, washed with saturated brine solution, and
dried over anhydrous MgSO4, and and solvent was removed
under reduced pressure. The crude product obtained was flash
chromatographed on a silica gel column eluted with hexane/
ethyl acetate (3:1). The eluent was concentrated and the product
separated as pale yellow solid. Yield: 1.2 g (65.4%). Mp: 85
Synthesis
DCDHF-P-azide, 2-(4-(4-Azidophenyl)-3-cyano-5,5-dimeth-
ylfuran-2(5H)-ylidene)malononitrile. To a 200 mL round-bottom
flask with stirbar was added 2-[3-cyano-4-(4-fluorophenyl)-5,5-
dimethyl-5H-furan-2-ylidene]-malononitrile (0.20 g, 0.716 mmol)
and DMSO (8 mL). The mixture was stirred at room temper-
ature, then sodium azide (0.08 g, 1.23 mmol) was added and
the reaction continued to react at room temperature. After 2 h,
the reaction mixture was completely homogeneous. The product
mixture was poured into ice-water (300 mL) and stirred for
0.5 h. The yellow precipitate was filtered off by suction filtration.
The solid was recrystallized from 1-propanol to give the final
product as a yellow solid (0.19 g, 88% yield). Mp 184 °C. IR
(neat): 2924, 2228, 2113, 1567, 1533, 1366, 1279, 1188, 1111,
841 cm-1. 1H NMR (400 MHz, DMSO): δ 7.94 (ddd, J ) 9.2,
2.8, 2.0 Hz, 2H), 7.40 (ddd, J ) 9.2, 2.8, 2.0 Hz, 2H), 1.78 (s,
6H). 13C NMR (100 MHz, DMSO): δ 177.1, 176.6, 145.0,
130.6, 123.4, 120.3, 112.3, 111.4, 111.3, 101.6, 100.3, 55.2,
24.9. Anal. Calcd for C16H10N6O: C, 63.57; H, 3.33; N, 27.80%.
Found: C, 63.51; H, 3.44; N, 27.40%. UV-vis (CH2Cl2): λmax
1
°C (lit. mp: 85.5-87.5 °C). H NMR (400 MHz, CDCl3): δ
9.97 (s, 1H), 8.11-8.17 (m, 2H), 7.40 (t, 8.4 Hz, 1H).
2-Azido-5-formyl-benzonitrile. In a 100 mL round-bottomed
flask, sodium azide (600 mg, 9 mmol) was dissolved in DMSO
(20 mL), and 3-cyano-4-fluorobenzaldehyde (1.0 g, 6.7 mmol)
was added and stirred at 50 °C for 6 h. The reaction mixture
was cooled and poured into water (200 mL) and stirred at rt for
1 h. The precipitate formed was filtered, washed with water,
1
and air-dried. TLC and H NMR spectra showed the material
was pure and used as such for reaction. Yield: 1.0 g (86.5%).
Mp: 115 °C. IR (neat) 2869, 2141, 1696, 1680, 1579 cm-1. 1H
NMR (CDCl3, 400 MHz): 9.97 (s, 1H), 8.13-8.15 (m, 2H),
7.28 (d, J ) 8.8 Hz, 1H). 13CNMR (CDCl3): 188.4, 148.65,
135.73, 134.20, 132.92, 119.38, 114.34, 104.88.
2-{4-2-(4-Azido-3-cyano-phenyl)-Winyl-3-cyano-5,5-dimethyl-
5H-furan-2-ylidene}malononitrile. A mixture of 2-azido-5-
formyl-benzonitrile (0.78 g, 4.5 mmol) and 2-dicyanomethylene-
3-cyano-4,5,5-trimethyl-2,5-dihydrofuran (924 mg, 4.6 mmol)
was dissolved in ethanol/THF mixture (30 mL, 2:1). Then
ammonium acetate (359 mg, 4.65 mmol) was added and stirred
at 0-25 °C for 16 h. TLC showed the starting material was
completely consumed. The solvent was removed by rotary
evaporation, and the crude product was purified by silica gel
column chromatography using hexane/EAC (30%) as eluent.
The reddish product obtained was recrystallized from dichlo-
romethane/1-propanol. Yield: 350 mg (22%). Mp: 178 °C. IR
(neat) 2920, 2228, 2134, 1622, 1586, 1553, 1381 cm-1. 1H NMR
(400 MHz, CDCl3): δ 7.85 (m, 2H), 7.54 (d, J ) 16.4 Hz, 1H),
7.34 (d, J ) 8.4 Hz, 1H), 6.91 (d, J ) 16.4 Hz, 1H), 1.78 (s,
6H). 13CNMR (100 MHz, DMSO): δ 177.00, 174.48, 145.57,
143.98, 135.29, 134.54, 131.37, 120.80, 116.53, 115.22, 112.59,
111.73, 110.56, 103.24, 100.40, 99.44, 55.50, 25.04. Anal. Calcd
for C19H11N7O: C, 64.59; H, 3.14; N, 27.75. Found: C, 64.25;
H, 3.44; N, 27.50. UV-vis (EtOH): λmax ) 415 nm.
) 384 nm, ε ) 2.9 × 104 L mol-1 cm-1
.
DCDHF-V-P-azide, (E)-2-(4-(4-Azidostyryl)-3-cyano-5,5-
dimethylfuran-2(5H)-ylidene)malononitrile. The synthesis was
previously reported in the Supporting Information of ref 15.
Literature procedures were followed for the synthesis of the
precursors 4-azidobenzaldehyde35 and 3-cyano-2-dicyanometh-
ylene-4,5,5-trimethyl-2,5-dihydrofuran.36 The 4-azidobenzalde-
hyde was isolated in 78% yield. Other reagents were commer-
cially available and were used as received.
To a 100 mL round-bottom flask with stirbar was added
4-azidobenzaldehyde (0.30 g, 2 mmol), 2-(3-cyano-4,5,5-
trimethyl-5H-furan-2-ylidene)-malononitrile (0.40 g, 2 mmol),
pyridine (5 mL), and acetic acid (several drops). The mixture
was stirred at room temperature for 2.5 days. TLC showed the
desired azido product had been formed as the main product.
The reaction was stopped and poured into ice-water (1 L). After
stirring for 2 h, the precipitate was filtered off by suction
filtration. The solid was recrystallized from 1-propanol to give
the final product as a dark red solid (0.55 g, 84% yield). This
compound decomposes at around 180 °C before melting
(observed under microscope and DSC). IR (neat): 2227, 2119,
1600, 1530, 1281, 1184, 823 cm-1. 1H NMR (400 MHz, CDCl3):
δ 7.67 (d, J ) 8.8 Hz, 2H), 7.60 (d, J ) 16.4 Hz, 1H), 7.15 (d,
J ) 8.4 Hz, 2H), 7.0 (d, J ) 16.4 Hz), 1.83 (s, 6H). 13C NMR
(100 MHz, CDCl3): δ 175.2, 173.5, 145.9, 144.8, 130.8, 130.5,
120.1, 114.3, 111.6, 110.8, 110.2, 99.9, 97.6, 51.1, 26.5. Anal.
Calcd for C18H12N6O: C, 65.85; H, 3.68; N, 25.60%. Found: C,
65.58; H, 3.74; N, 25.94%. UV-vis (CH2Cl2): λmax ) 433 nm,
DCDHF-V-PF-azide, 2-{4-2-(4-Azido-3-fluoro-phenyl)-vinyl-
3-cyano-5,5-dimethyl-5H-furan-2-ylidene}malononitrile. 4-Azido-
3-fluorobenzaldehyde. In a 100 mL round-bottomed flask,
sodium azide (2.60 g, 40 mmol) was dissolved in DMSO (80
mL). To the mixture, 3,4-difluorobenzaldehyde (3.30 mL, 30
mmol) was added and stirred at 40 °C for 5 h. The reaction
mixture was poured into water (200 mL) and stirred at rt for
2 h. The precipitate formed was filtered, washed with water,
and dried under vacuum. Finally, the product was recrystallized
from dichloromethane/1-propanol. Yield: 2.5 g (50%). Mp: 43
1
°C. IR 2959, 2135, 1697, 1682, 1608, 1579, 1502 cm-1. H
ε ) 2.7 × 104 L mol-1 cm-1
.
NMR (400 MHz, CDCl3): δ 9.96 (s, 1H), 7.61-7.75 (m, 2H),
7.10-7.22 (m, 1H). 19F NMR (470 MHz, CDCl3): -124.85 (m,
1F).
DCDHF-V-PCN-azide, 2-{4-2-(4-Azido-3-cyano-phenyl)-vi-
nyl-3-cyano-5,5-dimethyl-5H-furan-2-ylidene}malononitrile. 3-Cy-
ano-4-fluorobenzaldehyde. In a 50 mL single-neck flask, 3-bromo-
4-fluorobenzaldehyde (2.5 g, 12.3 mmol) and CuCN (1.26 g,
14.3 mmol) were mixed with NMP (5 mL). The mixture was
slowly warmed until the temperature reached 170 °C and stirred
at that temperature for 40 h. TLC showed complete consumption
of starting material; the temperature was gradually reduced to
80 °C; ethyl acetate (7 mL) was added dropwise followed by
water (3 mL); and stirring was continued at rt for 20 min. The
2-{4-2-(4-Azido-3-fluoro-phenyl)-Winyl-3-cyano-5,5-dimethyl-
5H-furan-2-ylidene}malononitrile. A mixture of 4-azido-3-fluo-
robenzaldehyde (1.0 g, 6.0 mmol) and 2-dicyanomethylene-3-
cyano-4,5,5-trimethyl-2,5-dihydrofuran (1.20 g, 6.0 mmol) was
dissolved in 25 mL of pyridine, and a few drops of acetic acid
were added. The mixture was stirred at room temperature for
48 h, poured into water, stirred for 2 h at room temperature,
and kept in the refrigerator overnight, and the precipitate

14162 J. Phys. Chem. B, Vol. 114, No. 45, 2010
Lord et al.
obtained was filtered and air-dried. The crude product was
purified by silica gel column chromatography using hexane/
EAC (30%) as eluent, and reddish brown product obtained was
then recrystallized from dichloromethane/1-propanol. Yield: 250
mg (15%). Mp: 193 °C. IR (neat): 3092, 2330, 2130, 1602,
DCM-azide, 2-{2-[2-(4-Azidophenyl)-vinyl]-6-methylpyran-
4-ylidene}-malononitrile. 2-{2-[2-(4-Aminophenyl)-Winyl]-6-me-
thylpyran-4-ylidene}-malononitrile. A mixture of 2,6-dimeth-
ylpyran-4-ylidene-malononitrile(1.2g,6.8mmol),4-aminobenzaldehyde
(1.0 g, 8.2 mmol), and piperidine (0.68 mL, 6.9 mmol) was
dissolved in 1-propanol (150 mL) and refluxed for 48 h. The
reaction mixture was cooled, poured into water (500 mL), and
stirred for 5 h, and the precipitate formed was filtered and air-
dried. The crude product was purified by silica gel column
chromatography using hexane/EAC 30-50% as eluent. The
product was finally recrystallized from dichloromethane/1-
propanol. Yield: 1.50 g (80%). Mp: 249 °C. IR (neat): 3478,
1
1513 cm-1. H NMR (400 MHz, CDCl3): δ 7.53 (d, J ) 16.4
Hz, 1H), 7.37 (m, 2H), 7.14 (t, 1H), 6.88 (d, J ) 16.4 Hz, 1H),
1.78 (s, 6H). 13C NMR (CDCl3): 174.36, 172.67, 156.37, 153.86,
144.40, 144.37, 132.53, 131.55, 131.48, 126.10, 126.07, 121.89,
121.87, 116.17, 115.98, 115.26, 111.21, 110.46, 109.93, 100.50,
97.49, 26.28. 19F NMR (470 MHz, CDCl3): -124.35 (s, 1F).
HRMS (EI): Calcd for C18H11FN6O (M+) 369.08, found 369.08.
UV-vis (CH2Cl2): λmax ) 427 nm.
1
2957, 2200, 1647 cm-1. H NMR (400 MHz, DMSO): δ 7.36
(d, J ) 8.4 Hz, 2H), 7.31 (s, 1H), 6.91 (d, J ) 16 Hz, 1H),
6.69 (bs, 1H), 6.57 (d, J ) 16 Hz, 1H), 6.55 (d, J ) 8.4 Hz,
2H), 5.86 (s, 2H), 2.40 (s, 3H). 13C NMR (DMSO): δ 163.12,
160.71, 156.05, 151.12, 138.47, 129.61, 121.57, 115.31, 113.18,
111.53, 104.88, 104.22, 18.80. Anal. Calcd for C17H13N3O: C,
74.17; H, 4.76; N, 15.26. Found: C, 73.91; H, 4.54; N, 15.38.
UV-vis (CH2Cl2): λmax ) 436 nm, ε ) 2.73 × 104 L mol-1
DCDHF-V-PF4-azide, (E)-2-(4-(4-Azido-2,3,5,6-tetrafluo-
rostyryl)-3-cyano-5,5-dimethylfuran-2(5H)-ylidene)malononi-
trile. Previously reported in the Supporting Information of
Pavani et al.37
4-Azido-2,3,5,6-tetrafluorobenzaldehyde.38- 41
To a 100 mL
.
round-bottom flask with stirbar was added pentafluoroben-
zaldehyde (1.96 g, 0.01 mol), sodium azide (0.72 g, 0.011
mol), acetone (15 mL), and water (15 mL). The mixture was
warmed to reflux under nitrogen for 10 h. TLC showed all
the pentafluorobenzaldehyde was consumed, and so the
reaction was stopped and cooled to room temperature. The
product mixture was diluted with 20 mL of water. The crude
product was extracted with ether (30 mL × 5). The combined
organic layer was dried over anhydrous MgSO4. The solvent
was removed at room temperature under vacuum. Sublimation
of the residue (50 °C/0.2 mm) gave the final product as a
white solid (1.20 g, 55% yield). Mp 44 °C (lit. 44-45 °C,
ref 38). IR (neat): 3377, 2121, 1704, 1644, 1480, 1398, 1237,
1066, 1000, 615 cm-1. 1H NMR (400 MHz, CDCl3): δ 10.26
(m, 1H). 19F NMR (470 MHz, CDCl3): δ -149.6 (m, 2F),
-155.6 (m, 2F).
cm-1
.
2-{2-[2-(4-Azidophenyl)-Winyl]-6-methylpyran-4-ylidene}-mal-
ononitrile. A solution of NaNO2 (552 mg, 8.0 mmol) in 8 mL
of water was added dropwise to a solution of the 2-{2-[2-(4-
aminophenyl)-vinyl]-6-methylpyran-4-ylidene}-malononitrile (1.10
g, 4.0 mmol) in 4 M HCl (46 mL) at 0-5 °C. After stirring the
mixture at this temperature for 45 min, a solution of NaN3 (520
mg, 8.0 mmol) in water (8 mL) was slowly added to the mixture
at the same temperature. Stirring was continued for 1 h below
5 °C and then at room temperature for overnight. The precipitate
obtained was filtered and air-dried. The crude product was
purified by silica gel column chromatography using hexane/
EAC (7:3) as eluent and finally recrystallized from dichlo-
romethane/1-propanol. Yield: 550 mg (45%). Mp: 195 °C. IR
1
(neat): 3078, 2208, 2118, 1655 cm-1. H NMR (400 MHz,
(E)-2-(4-(4-Azido-2,3,5,6-tetrafluorostyryl)-3-cyano-5,5-dimeth-
ylfuran-2(5H)-ylidene)malononitrile. To a 100 mL round-bottom
flask with stirbar was added 4-azido-2,3,5,6-tetrafluoroben-
zaldehyde (0.22 g, 1 mmol) and 2-(3-cyano-4,5,5-trimethyl-
5H-furan-2-ylidene)-malononitrile (0.22 g, 1.1 mmol), 5 mL
pyridine, and several drops of acetic acid. The mixture was
stirred at room temperature for 2.5 days. TLC showed the
desired azido product had been formed as the main product.
The reaction was stopped and poured into 500 mL of ice
water. After stirring for 2 h, the precipitate was filtered off
by suction filtration. The solid was recrystallized from
1-propanol. After recrystallization, part of the azido product
was converted to the corresponding amino compound. The
mixture was adsorbed on silica gel, placed at the top of a
silica column, and eluted (CH2Cl2/EtOAc ) 20:1). Fractions
containing only the first product were combined and con-
centrated to give an orange product (40 mg, 10% yield). This
is the final azido product, (E)-2-(4-(4-azido-2,3,5,6-tetrafluo-
rostyryl)-3-cyano-5,5-dimethylfuran-2(5H)-ylidene)malono-
nitrile. Recrystallization could not be done on this compound
since it has high photoreactivity and it readily converts to
the corresponding amino compound in solvents (like pro-
panol) in daylight. IR (neat): 2933, 2228, 2124, 1586, 1557,
CDCl3): δ 7.50 (d, J ) 8.4 Hz, 2H), 7.35 (d, J ) 16 Hz, 1H),
7.03 (d, J ) 8.4 Hz, 2H), 6.64 (bs, 1H), 6.63 (d, J ) 16 Hz,
1H), 6.50 (bs, 1H), 2.36 (s, 3H). 13C NMR (100 MHz, CDCl3):
δ 161.70, 158.63, 155.94, 141.83, 136.52, 131.02, 128.90,
119.47, 117.40, 114.68, 107.01, 106.22, 19.71. Anal. Calcd for
C17H11N5O: C, 67.77; H, 3.68; N, 23.24. Found: C, 67.51; H,
3.64; N, 22.97. UV-vis (CH2Cl2): λmax ) 405 nm.
Stilbazolium-azide, 4-[2-(4-Azido-phenyl)-vinyl]-1-methyl-
pyridinium Iodide (4′-Azido-4-stilbazolium Methiodide). 4-(2-
Pyridin-4-yl-Winyl)-phenylamine (4′-Amino-4-stilbazole). The pro-
cedure was adapted from a literature method for preparing 4′-
amino-4-stilbazoles as described by Loew et al.42 To an oven-
dried flask charged with nitrogen was added 4-iodoaniline (5.59
g, 25.5 mmol), 4-vinylpyridine (3.68 g, 35 mmol), palladium(II)
acetate (14.4 mg, 0.064 mmol), tri-o-tolylphosphine (39.0 mg,
0.128 mmol), triethylamine (7.08 g, 70 mmol), and acetonitrile
(25 mL). The flask was fitted with a reflux condenser and then
charged again with nitrogen. A bubbler was quickly attached
to the top of the condenser, and then the flask was heated in an
oil bath. The mixture was stirred at reflux for 48 h, after which
the flask was removed from heat and cooled to room temper-
ature. The mixture was poured into cold water, and then the
precipitate was collected via vacuum filtration. The product was
placed in the vacuum oven and dried overnight at 55 °C at
approximately 20 mmHg vacuum until it reached a constant
mass. The product was collected as a yellow powder in the
amount of 4.26 g (85% yield). Mp: 270-274 °C. 1H NMR (400
MHz, DMSO-d6): δ 8.45 (dd, J ) 1.6, 6.1 Hz, 2H), δ 7.44 (dd,
J ) 1.6, 6.1 Hz, 2H), δ 7.37 - 7.32 (m, 3H), δ 6.87 (d, J )
1
1489, 1372, 1253, 998 cm-1. H NMR (400 MHz, CDCl3):
δ 7.63 (d, J ) 16.8 Hz, 1H), 7.31 (d, J ) 16.4 Hz, 1H), 1.82
(s, 6H). 13C NMR (100 MHz, CDCl3): δ 174.5, 172.5, 146.9
(m), 144.4 (m), 142.0 (m), 139.4 (m), 130.7, 121.4 (t, J )
9.8 Hz), 111.1, 110.3, 109.5, 102.6, 97.8, 51.3, 26.3. 19F NMR
(470 MHz, CDCl3): δ -143.5 (2F), -155.2 (2F). UV-vis
(EtOH): λmax ) 407 nm, ε ) 2.7 × 104 L mol-1 cm-1
.

Azido Push-Pull Fluorogens Photoactivation
J. Phys. Chem. B, Vol. 114, No. 45, 2010 14163
16.4 Hz, 1H), δ 6.58 (ddd, J ) 1.9, 2.6, 8.6 Hz, 2H), δ 5.49 (s,
2H). 13C NMR (100 MHz, DMSO-d6): δ 150.3, 145.7, 134.2,
129.0, 124.1, 120.6, 120.3, 114.2. λmax (CH3CN) ) 348 nm (ε
) 2.96 × 104 M-1 cm-1).
the nonfluorescent azido fluorogens photoconvert to longer-
wavelength amino versions that are fluorescent (e.g., see spectra
in Figure 1 for NBD-azide; the other fluorogens follow the same
trend and spectra may be found in the Supporting Information).
Although there are several photoproducts, we confirmed that
the fluorescent amino product of the DCDHF-V-P-azide
photoreaction is dominant (via mass spectrometry and NMR
analyses of the column- and HPLC-separated photoproducts
described in Methods; see chemical reaction yields in Table
1). For the other fluorogens, the presence of an amino fluoro-
phore in the photoreaction mixture is corroborated by recovery
of the long-wavelength push-pull absorption and emission
spectra. (In the case of DCM-azide, the absorption spectra of
the photoreaction mixture include peaks that obscure the amino
species; nevertheless, fluorescence excitation spectra reveal only
one fluorescing species, which overlaps with the absorption
curve of the independently synthesized DCM-amine. See
Supporting Information.)
4-[2-(4-Azido-phenyl)-Winyl]-pyridine (4′-Azido-4-stilbazole). A
solution of sodium nitrite (1.76 g, 25.5 mmol) in 10 mL of water
was added dropwise to a solution of 4-(2-pyridin-4-yl-vinyl)-
phenylamine (2.0 g, 10.2 mmol) in 102 mL of 4 M HCl at 2-3
°C. After stirring the mixture at this temperature for 1 h, a
solution of sodium azide (1.32 g, 20.4 mmol) in 10 mL of water
was slowly added dropwise, maintaining the temperature at 2-3
°C. Stirring was continued for 30 min at this temperature, and
then the ice bath was allowed to come to ambient temperature
overnight while stirring continued. Saturated aqueous sodium
bicarbonate was added carefully to the mixture with stirring
until the evolution of gases subsided. The precipitate was filtered
out and dried overnight in the funnel to yield 1.875 g of the
product as a beige powder (83% yield). Mp: 88.2-89.4 °C. 1H
NMR (400 MHz, CDCl3): δ 8.51 (dd, J ) 1.4, 6.1 Hz, 2H), δ
7.46 (ddd, J ) 1.9, 2.7, 8.5 Hz, 2H), δ 7.29 (dd, J ) 1.4, 6.1
Hz, 2H), δ 7.18 (d, J ) 16.4 Hz, 1H), δ 6.98 (ddd, J ) 2.0,
2.7, 8.5 Hz, 2H), δ 6.90 (d, J ) 16.4 Hz, 1H). 13C NMR (100
MHz, CDCl3): δ 150.3, 144.4, 140.3, 133.1, 132.0, 128.4, 125.7,
120.8, 119.5. λmax (CH3CN) ) 323 nm (ε ) 3.92 × 104 M-1
cm-1). IR νmax (cm-1) ) 2122 (N3).
Taking into account both the localization precision43,44 and
the Nyquist-Shannon sampling,45,46 the best emitters for pho-
toactivation super-resolution imaging maximize the number of
localized unique molecules per area per time.47 In other words,
the probe must be easily photoactivated to avoid cell damage
from short-wavelength illumination; must be bright; must emit
many photons, then disappear; must densely label the sample;
and must have a high contrast between on and off states. (For
static structures, reversible switching is not ideal because
reactivation of an emitter that has already been localized is
superfluous and only adds complexity to any image reconstruc-
tion.) The azido push-pull chromophores we present in this
paper meet many of these critical requirements: several are
photoconverted with high efficiency without a catalyst, emit
millions of photons before irreversibly photobleaching, exhibit
high turn-on ratios, and possess moderate molar absorption
coefficients and quantum yields.
Photostability. One of the most important parameters for
single-molecule fluorophores is photostability, one measure of
which is the photobleaching quantum yield (ΦB) or the
probability of bleaching with each photon absorbed (see eq 1).
A very low value of ΦB corresponds to not only a bright, long-
lived fluorophore but also higher localization precision for
photoactivation imaging.43,44
We have reported previously that the DCDHF molecules emit
millions of photons and therefore have low values for ΦB,20,23
and this is also true of some of the new activated fluorophores
reported here. These photostable emitters enable precise local-
ization and sophisticated imaging schemes: we demonstrated
previously that single molecules of DCDHF-V-PF4-azide in
PMMA can be photoactivated and localized to less than 20 nm
standard deviation in all three dimensions. Two molecules
separated by 36 nm were resolved in three dimensions (see
Figure 2B for an example of activation and imaging of single
molecules).37
4-[2-(4-Azido-phenyl)-Winyl]-1-methyl-pyridinium Iodide (4′-
Azido-4-stilbazolium Methiodide). In a 100 mL round-bottom
flask equipped with a stirbar and covered with foil, 4-[2-(4-
azido-phenyl)-vinyl]-pyridine (0.57 g, 2.56 mmol) was mixed
with iodomethane (0.95 g, 6.7 mmol) in acetonitrile (20 mL)
and stirred at room temperature for 36 h. A precipitate had
formed, and TLC showed a single spot. So, the solution was
placed in the refrigerator for 30 min and then filtered, rinsing
with diethyl ether. The product was collected as a yellow powder
(0.482 g). Diethyl ether was added to the filtrates and then placed
in the refrigerator for 2 h. Orange/brown crystals formed which
were collected via filtration (0.15 g). The total amount of product
1
collected was 0.63 g (68% yield). Mp: 166.2-168.0 °C. H
NMR (400 MHz, DMSO-d6): δ 8.86 (d, J ) 6.9 Hz, 2H), δ
8.20 (d, J ) 6.9 Hz, 2H), δ 8.01 (d, J ) 16.4 Hz, 1H), δ 7.80
(ddd, J ) 1.8, 2.6, 8.5 Hz, 2H), δ 7.49 (d, J ) 16.4 Hz, 1H),
δ 7.26 (ddd, J ) 1.8, 2.6, 8.5 Hz, 2H), δ 4.26 (s, 3H). 13C
NMR (100 MHz, DMSO-d6): δ 152.89, 145.56, 141.80, 140.08,
132.54, 130.31, 123.90, 123.28, 120.37, 47.39. λmax (CH3CN)
) 373 nm (ε ) 4.07 × 104 M-1 cm-1). IR νmax (cm-1) ) 2118
(N3). Anal. Calcd for C14H13IN4: C, 46.17; H, 3.60; N, 15.38.
Found: C, 46.24; H, 3.69; N, 15.39.
NBD-azide, 7-Azido-4-nitrobenzoxadiazole. In a 50 mL
round-bottom flask equipped with a stirbar, 7-chloro-4-nitroben-
zofurazan (1.5 g, 7.52 mmol) and sodium azide (0.54 g, 8.27
mmol) were stirred in ethanol (15 mL) for 6 h at 35 °C. TLC
indicated complete consumption of the starting material. The
solution was poured into ice water, forming a precipitate, which
was filtered and washed with water. The product was collected
as a yellow powder in the amount of 1.45 g (94% yield). Mp:
The photobleaching quantum yield is defined as the prob-
ability of photobleaching after absorbing a photon or the ratio
of the bleaching rate RB to the rate of absorbing photons Rabs
1
101.1-101.9 °C. H NMR (400 MHz, CDCl3): δ 8.53 (d, J )
8.2 Hz, 1H), δ 7.08 (d, J ) 8.2 Hz, 1H). 13C NMR (100 MHz,
CDCl3): δ 145.9, 143.6, 138.1, 132.2, 128.6, 114.9. λmax
(CH2Cl2) ) 388 nm (ε ) 1.44 × 104 M-1 cm-1). IR νmax (cm-1)
) 2114 (N3).
RB(P)
Rabs
1
1
ΦB(P)
)
)
)
(1)
τ
B(P)Rabs
λ
hc
τB(P)σλIλ
(
)
Results and Discussion
The various azido push-pull chromophores in Table 1 are
all photoactivatable: upon pumping the blue-shifted absorption,
where τB(P) is the decay constant in the exponential fit; the
absorption cross-section is related to the molar absorption

14164 J. Phys. Chem. B, Vol. 114, No. 45, 2010
Lord et al.
Photoconversion was measured by monitoring changes over time
in absorbance values of the reactant and photoproduct of interest
in ethanol. The photoconversion quantum yield ΦP is defined in
eq 1 above, with τP as the average decay constant from the expo-
nential fit of the decaying absorption values for the starting material
(see Figure 1). Note that ΦP is the probability that the starting
material will photoconvert for each photon absorbed; a fraction of
those photoconverted molecules become fluorescent because the
photoreaction yield is less than unity.
Figure 2 shows that DCDHF-V-P-azide was readily activated
in living CHO cells without obvious photodamage. By adding
four electron-withdrawing fluorines to the benzene ring, we were
able to further stabilize the nitrene and increase ΦP by more
than a factor of 2 (compare DCDHF-V-P-azide and DCDHF-
V-PF4-azide). Other acceptor groups and structures resulted in
even higher ΦP values; for instance, NBD-azide needs to absorb
only a few photons on average before photoreacting. These high
photoconversion efficiencies enable activation at lower intensi-
ties; thus, super-resolution imaging by photoactivation should
achieve higher cycling rates without causing photodamage to
living cells. A drawback of experiments taking advantage of
these very high ΦP values is that they require near complete
darkness (or red lights) during sample preparation to prevent
preactivation of the fluorogens. Stock fluorogen solutions in
ethanol in the dark were resistant to thermal activation for
several days or weeks (data not shown).
Figure 2. (A) Two living Chinese Hamster Ovary (CHO) cells
incubated with DCDHF-V-P-azide before activation and after a 5 s
flash of diffuse, moderate-irradiance (13 W cm-2), 407 nm light. The
594 nm light (500 W cm-2) for imaging was illuminating the sample
the entire time, except for the brief period of 407 nm activation. False
color: gray is the white-light transmission image and red the thresholded
fluorescence images. Bar, 15 µm. (B) Snapshots from a movie of single
molecules of DCDHF-V-PF4-amine embedded in a PMMA film
immediately after photoactivation of the corresponding azide (see
Supporting Information for the entire movie). The inset shows the same
frame immediately before activation. Preactivated molecules were first
prebleached using high-intensity 514 nm light, and then the sample
was imaged at lower intensities (2 kW cm-2). To activate, a 100 ms
flash of low-intensity 407 nm light (0.2 kW cm-2) was applied.
Pseudocolor scale: the number of counts in this 100 ms frame ranges
from 459 at the minimum pixel to 6644 at the brightest pixel (which
corresponds to approximately 40-578 photons detected per pixel per
100 ms). Bars, 2 µm.
Turn-On Ratio. Photostability is not the only important
parameter determining the ultimate resolution of a photoacti-
vation image. Nyquist-Shannon sampling requires that there
be at least two emitters per final resolution element, adding
further restriction on the emitters: not only must the labeling
be very dense but also the turn-on ratio (i.e., the contrast between
the bright and dark states of the molecule) must be very high,
lest many weakly emitting “off” molecules in a diffraction-
limited spot drown out the signal from the single “on”
molecule.45-49 In other words, the limit of interest occurs when
the background-subtracted intensity from one bright molecule
Ion equals that of noff dark fluorogens (i.e., when Ion ) noffIoff)
coefficient by the equation σλ ) (1000)2.303ελ/NA ) 2.1 × 10-16
cm2 for DCDHF-V-P-amine; Iλ is the irradiance at the sample;
λ is the excitation wavelength; h is Planck’s constant; and c is
the speed of light. (This definition will also be used for the
photoconversion efficiency ΦP, see below.) The average decay
constant for a two-exponential fitting function, ∑ni))12 Rie(-t/τ ), is
i
given by
R1τ21 + R2τ22
R1τ1 + R2τ2
τ¯ ) f1τ1 + f2τ2 )
(2)
Ion
noffIoff
Ioff
R )
)
) noff ) 1270 ( 500
(3)
where fi ) Riτi/Σj Rjτj is the fractional area under the multiex-
ponential curve.32 Photobleaching quantum yield scales with the
inverse of total number of photons emitted, and a lower value
for ΦB indicates better photostability.
Ioff
where data from DCDHF-V-P-azide in PMMA provide the
numerical value. However, the assumption that every dark
molecule becomes fluorescent is not correct; to the contrary,
non < noff is the common situation. One could measure R by
averaging over many single molecules; however, this would
select only the fluorogens that become fluorescent and would
be artificially inflated. In other words, the simple ratio R is not
an experimentally relevant parameter.
Alternatively, we measured an effective turn-on ratio that
better corresponds to how many localizations we can get for a
given region. In a bulk experiment in a PMMA film, we
integrated the background-subtracted intensities over a large
region before Soff and after Son activation (see Figure S6,
Supporting Information). Not all copies of the fluorogen convert
to the fluorescent species, as the simple ratio R assumes above;
the overall chemical reaction yield p can be 90% or lower (see
Table 1). Therefore, the total emitters that will turn on are the
reaction yield times and the number of precursor molecules:
non ) pnoff. The ratio of the background-subtracted signals in a
bulk experiment gives the effective turn-on ratio Reff, which is
the experimentally relevant parameter
As shown in Table 1, adding electron-withdrawing fluorines
to the aromatic group (DCDHF-V-PF-amine and DCDHF-V-
PF4-amine) had little effect on the photostability parameter ΦB.
DCM-amine is also a strong single-molecule emitter, with a
ΦB comparable to most DCDHFs. DCM-amine has a higher
fluorescence quantum yield in solution, and thus is more likely
to be bright throughout a sample (not just in rigid environments,
which is a feature of DCDHFs20,23). While NBD-amine is
brightly fluorescent in all environments, it is many times less
photostable than DCDHFs. The stilbazolium-amine fluorophore
is also not as photostable as the DCDHFs or even DCM-amine
(a precise value was not determined).
Photoconversion Efficiency. The probability of photocon-
verting an azido fluorogen to any product per photon absorbed
is the photoconversion quantum yield (ΦP), listed in Table 1
for the various push-pull fluorogens and demonstrated in the
Figure 1 right panel for DCDHF-P-azide. The higher the value
of ΦP, the more sensitive the fluorogen is to the activating light,
so less potentially cell-damaging blue or UV irradiation is
required to activate fluorescence.

Azido Push-Pull Fluorogens Photoactivation
J. Phys. Chem. B, Vol. 114, No. 45, 2010 14165
Son
Soff
nonIon
pnoffIon
noffIoff
Ion
Reff
)
)
)
) p
) pR ) pnoff )
noffIoff
Ioff
non ) 325 ( 15 (4)
The value Reff corresponds directly to the maximum number
of localizations non in a diffraction-limited spot before the
aggregate signal (noffIoff) of all the dark fluorogens required for
that number of localizations equals the signal from one on
molecule Ion. In the case of DCDHF-V-P-azide, we measured
(multiple times using different samples, concentrations, and
imaging parameters) the Reff to be 325 ( 15, which is over
three times larger than that in ethanol.15 Moreover, the value of
Reff is approximately a quarter of that of the R value measured
on the single-molecule level; this discrepancy could imply that
the reaction yield is p ) 25% or may result from preactivation
(see below) or simply from different experimental conditions
between the two experiments.
Approximating the diffraction limit to be 250 nm in diameter,
the area of the diffraction-limited spot is about 50 000 nm2. With
the maximum of 325 localizations in each diffraction-limited
spot, the average distance between each localization is ap-
proximatedby(50 000nm2/325)1/2 )12nm.TheNyquist-Shannon
criterion45,46 requires a sampling of at least twice the desired
resolution, limiting the resolution to about 25 nm (in two
dimensions). Our calculations here are similar to those in the
Supporting Information of Shroff et al.47
As a side note, this measured value of Reff should be
considered a lower limit because any preactivated molecules
contribute to the signal in the frames before activation, thus
reducing the measured value. The fraction q of preactivated
molecules was kept low by protecting the fluorophore stock
solution and samples from ambient room light; regardless, some
preactivation does inevitably occur. The effect preactivation has
on measuring Reff by including a signal from preactivated
molecules in the dark measurement is calculated by including
the fraction q of preactivated molecules
Figure 3. (A) Schematic of nonspecific fluorogenic photoaffinity
labeling (PAL) of whole cells. The nitrene intermediate resulting from
the photoconversion of an azide to an amine is reactive enough to insert
into bonds of nearby biomolecules. The reaction simultaneously turns-
on fluorescence and covalently links the probe to the biomolecule. (B)
Gel electrophoresis of CHO-cell lysate demonstrating fluorogenic PAL
using NBD-azide. (Similar results were observed with DCDHF-P-
azide, data not shown.) The left panel shows the stained protein, imaged
with white light; the right panel is fluorescence from NBD-amine
photochemically cross-linked to proteins, imaged using 488 nm. The
left lane in the gel (+PAL) is protein covalently labeled with NBD-
amine by PAL. The right lane (-PAL) is a control performed by mixing
into the cell solution preactivated NBD-amine, which is fluorescent
but cannot participate in the covalent PAL bioconjugation reaction.
The fluorescence signal in the control lane was significantly lower. The
blurry fluorescence on the bottom of the gel is from the unbound dye
at the front edge; equal brightness in both lanes indicates equal dye
concentration in the PAL and control.
Son
nonIon
Reff,preact
)
)
)
Soff,preact
noffIoff + qnoffIon
nonIon
Reff,true
pR
1 + qR
)
)
(5)
qnoffIon
noffIoff
1 + qR
noffIoff 1 +
(
)
Thus, the multiplicative correction factor to convert from
measured to true effective turn-on ratio is (1 + qR). Even 0.1%
preactivation could artificially deflate the measured value by
more than half (assuming the measured R of one isolated
molecule is 1270). Therefore, minimizing preactivation (or,
alternatively, maximizing prebleach as done for Figure 2B)
before measuring the effective turn-on ratio can increase the
value and bring it closer to the true ratio. However, prebleaching
is not always an optionsif the sample is highly light-
sensitivesso Reff remains a practical measure of the lower limit
for the turn-on ratio.
molecule binding, binding-pocket chemistries, and protein-protein
interactions.50-53 Aryl azides are common PAL tags because
the nitrene intermediate is reactive and long-lived (we measured
a lifetime of nearly 2 s, see Methods). Here, we demonstrate
fluorogenic PAL: a dark precursor that can become both
fluorescent and bioconjugated in one illumination step. Figure
3 shows proteins from CHO cells labeled with an azido
push-pull fluorogen by PAL and purified by denaturing gel
electrophoresis (see Methods). This demonstrates that nonspe-
cific fluorogenic PAL is possible; moreover, it should be possible
to engineer a binding pocket for one of these azido push-pull
fluorogens, increasing the PAL reaction yield and producing a
targeted fluorogenic bioconjugation system. Additional targeting
strategies to place the fluorogen at a position of interest can
also be envisioned.
Photoaffinity Labeling. Besides super-resolution imaging,37
the azido push-pull fluorogens can be applied to other
biological imaging schemes. For instance, the well-known
photoaffinity labeling (PAL) requires a chemically inert binding
molecule that becomes reactive upon illumination; the reactive
photoproduct forms a covalent bond to a biomolecule to which
it is bound or near. This technique has been used to study small-
Some azide-based fluorogenic PAL ligands have been re-
ported previously.54-58 In these earlier studies, the light required

14166 J. Phys. Chem. B, Vol. 114, No. 45, 2010
Lord et al.
(9) Zhu, M.; Zhu, L.; Han, J. J.; Wu, W.; Hurst, J. K.; Li, A. D. Q.
Spiropyran-Based Photochromic Polymer Nanoparticles with Optically
Switchable Luminescence. J. Am. Chem. Soc. 2006, 128, 4303–4309.
(10) Fukaminato, T.; Umemoto, T.; Iwata, Y.; Yokojima, S.; Yoneyama,
M.; Nakamura, S.; Irie, M. Photochromism of Diarylethene Single Molecules
in Polymer Matrices. J. Am. Chem. Soc. 2007, 129, 5932–5938.
(11) Fo¨lling, J.; Belov, V.; Kunetsky, R.; Medda, R.; Scho¨nle, A.; Egner,
A.; Eggeling, C.; Bossi, M.; Hell, S. W. Photochromic Rhodamines Provide
Nanoscopy with Optical Sectioning. Angew. Chem., Int. Ed. 2007, 46, 6266–
6270.
to excite fluorescence is in the high-energy ultraviolet region
(e.g., 280-350 nm), which prohibits ultrasensitive detection in
living cells because these short wavelengths pump cellular
autofluorescence and cause cell damage. Moreover, photocon-
version in these previous cases did not produce fluorophores
photostable enough to be applied to single-molecule imaging.
Our azido push-pull fluorogens can be activated and imaged
using visible light, and several produce fluorophores that can
be easily imaged on the single-molecule level.
(12) Conley, N. R.; Biteen, J. S.; Moerner, W. E. Cy3-Cy5 Covalent
Heterodimers for Single-Molecule Photoswitching. J. Phys. Chem. B 2008,
112, 11878–11880.
(13) Han, G.; Mokari, T.; Ajo-Franklin, C.; Cohen, B. E. Caged Quantum
Dots. J. Am. Chem. Soc. 2008, 130, 15811–15813.
Conclusion
(14) Fernandez-Suarez, M.; Ting, A. Y. Fluorescent Probes for Super-
Resolution Imaging in Living Cells. Nat. ReV. Mol. Cell Biol. 2008, 9, 929–
943.
A wide variety of azido fluorogens are photoactivatable to
produce bright push-pull fluorophores, some of which emit
millions of photons before photobleaching. For instance, the
various DCDHF derivatives as well as DCM should prove to
be high-quality single-molecule emitters given their photosta-
bility and brightness (especially in rigid environments). In
addition, the azido push-pull molecules can participate in
fluorogenic PAL reactions to label biomolecules covalently
in situ. For cell imaging, these fluorogens will ultimately need
to be targeted to biomolecules of interest but in any case do
not require the addition of other chemicals (e.g., catalysts, thiols,
or oxygen scavengers). Azido push-pull fluorogens thus provide
a useful new class of photoactivatable probes for a variety of
applications in biological labeling and super-resolution imaging.
(15) Lord, S. J.; Conley, N. R.; Lee, H. D.; Samuel, R.; Liu, N.; Twieg,
R. J.; Moerner, W. E. A Photoactivatable Push-Pull Fluorophore for Single-
Molecule Imaging in Live Cells. J. Am. Chem. Soc. 2008, 130, 9204–9205.
(16) Nicoud, J. F.; Twieg, R. J. Nonlinear Optical Properties of Organic
Molecules and Crystals. In Design and Synthesis of Organic Molecular
Compounds for Efficient Second Harmonic Generation; Chemla, D. S., Zyss,
J., Eds.; Academic Press: New York, 1987; Vol. 1, pp 227-296.
(17) Debreczeny, M. P.; Svec, W. A.; Wasielewski, M. R. Optical
Control of Photogenerated Ion Pair Lifetimes: An Approach to a Molecular
Switch. Science 1996, 274, 584–587.
(18) Ostroverkhova, O.; Moerner, W. E. Organic Photorefractives:
Mechanisms, Materials, and Applications. Chem. ReV. 2004, 104, 3267–
3314.
(19) Willets, K. A.; Ostroverkhova, O.; He, M.; Twieg, R. J.; Moerner,
W. E. New Fluorophores for Single-Molecule Spectroscopy. J. Am. Chem.
Soc. 2003, 125, 1174–1175.
(20) Willets, K. A.; Nishimura, S. Y.; Schuck, P. J.; Twieg, R. J.;
Moerner, W. E. Nonlinear Optical Chromophores as Nanoscale Emitters
for Single-Molecule Spectroscopy. Acc. Chem. Res. 2005, 38, 549–556.
(21) Lord, S. J.; Lu, Z.; Wang, H.; Willets, K. A.; Schuck, P. J.; Lee,
H. D.; Nishimura, S. Y.; Twieg, R. J.; Moerner, W. E. Photophysical
Properties of Acene DCDHF Fluorophores: Long-Wavelength Single-
Molecule Emitters Designed for Cellular Imaging. J. Phys. Chem. A 2007,
111, 8934–8941.
(22) Lu, Z.; Liu, N.; Lord, S. J.; Bunge, S. D.; Moerner, W. E.; Twieg,
R. J. Bright, Red Single-Molecule Emitters: Synthesis and Properties of
Environmentally Sensitive Dicyanomethylenedihydrofuran (DCDHF) Fluo-
rophores with Bisaromatic Conjugation. Chem. Mater. 2009, 21, 797–810.
(23) Lord, S. J.; Conley, N. R.; Lee, H. D.; Nishimura, S. Y.; Pomerantz,
A. K.; Willets, K. A.; Lu, Z.; Wang, H.; Liu, N.; Samuel, R.; Weber, R.;
Semyonov, A. N.; He, M.; Twieg, R. J.; Moerner, W. E. DCDHF
Fluorophores for Single-Molecule Imaging in Cells. ChemPhysChem 2009,
10, 55–65.
(24) Doub, L.; Vandenbelt, J. M. The Ultraviolet Absorption Spectra
of Simple Unsaturated Compounds. I. Mono- and p-Disubstituted Benzene
Derivatives. J. Am. Chem. Soc. 1947, 69, 2714–2723.
(25) Stevenson, P. E. Effects of Chemical Substitution on the Electronic
Spectra of Aromatic Compounds: Part I. the Effects of Strongly Perturbing
Substituents on Benzene. J. Mol. Spectrosc. 1965, 15, 220–256.
(26) Bouffard, J.; Kim, Y.; Swager, T. M.; Weissleder, R.; Hilderbrand,
S. A. A Highly Selective Fluorescent Probe for Thiol Bioimaging. Org.
Lett. 2008, 10, 37–40.
Acknowledgment. We thank Marissa K. Lee and Nathan R.
Hobbs for assistance. This work was supported in part by Award
Number R01GM086196 from the National Institute of General
Medical Sciences.
Note Added after ASAP Publication. This paper was pub-
lished on the Web on October 27, 2009. A text correction has
been made to the Turn-On Ratio section of the Results and
Discussion. The corrected version was reposted on March 8,
2010.
Supporting Information Available: Spectra of all fluoro-
gens, live-cell images, activation flashlight spectrum, images
from effective turn-on ratio measurements, PAL results with
pure BSA protein, general schemes, and a single-molecule
movie. This material is available free of charge via the Internet
at http://pubs.acs.org.
References and Notes
(1) Betzig, E.; Patterson, G. H.; Sougrat, R.; Lindwasser, O. W.;
Olenych, S.; Bonifacino, J. S.; Davidson, M. W.; Lippincott-Schwartz, J.;
Hess, H. F. Imaging Intracellular Fluorescent Proteins at Nanometer
Resolution. Science 2006, 313, 1642–1645.
(27) Budyka, M. F.; Biktimirova, N. V.; Gavrishova, T. N.; Kozlovskii,
V. I. Azido Derivative of a Hemicyanine Dye with High Sensitivity to
Visible Light. MendeleeV Commun. 2007, 17, 159–160.
(28) Hansch, C.; Leo, A.; Taft, R. W. A Survey of Hammett Substituent
Constants and Resonance and Field Parameters. Chem. ReV. 1991, 91, 165–
195.
(29) Schriven, E. F. V., Ed. Azides and Nitrenes: ReactiVity and Utility;
Academic Press: Orlando, FL, 1984.
(30) Soundararajan, N.; Platz, M. S. Descriptive Photochemistry of
Polyfluorinated Azide Derivatives of Methyl Benzoate. J. Org. Chem. 1990,
55, 2034–2044.
(31) Cline, M. R.; Mandel, S. M.; Platz, M. S. Identification of the
Reactive Intermediates Produced upon Photolysis of p-Azidoacetophenone
and its Tetrafluoro Analogue in Aqueous and Organic Solvents: Implications
for Photoaffinity Labeling. Biochemistry 2007, 46, 1981–1987.
(32) Lakowicz, J. R. Principles of Fluorescence Spectroscopy; Springer
Science: New York, 2006.
(2) Hess, S. T.; Girirajan, T. P. K.; Mason, M. D. Ultra-High Resolution
Imaging by Fluorescence Photoactivation Localization Microscopy. Biophys.
J. 2006, 91, 4258–4272.
(3) Rust, M. J.; Bates, M.; Zhuang, X. Sub-Diffraction-Limit Imaging
by Stochastic Optical Reconstruction Microscopy (STORM). Nat. Methods
2006, 3, 793–795.
(4) Moerner, W. E. New Directions in Single-Molecule Imaging and
Analysis. Proc. Natl. Acad. Sci. U.S.A. 2007, 104, 12596–12602.
(5) Dickson, R. M.; Cubitt, A. B.; Tsien, R. Y.; Moerner, W. E. On/
Off Blinking and Switching Behavior of Single Green Fluorescent Protein
Molecules. Nature 1997, 388, 355–358.
(6) Lukyanov, K. A.; Chudakov, D. M.; Lukyanov, S.; Verkhusha,
V. V. Photoactivatable Fluorescent Proteins. Nat. ReV. Mol. Cell Biol. 2005,
6, 885–891.
(7) Lippincott-Schwartz, J.; Patterson, G. H. Fluorescent Proteins for
Photoactivation Experiments. Methods Cell Biol. 2008, 85, 45–61.
(8) Bates, M.; Blosser, T. R.; Zhuang, X. Short-Range Spectroscopic
Ruler Based on a Single-Molecule Switch. Phys. ReV. Lett. 2005, 94,
108101-1–108101-4.
(33) Moerner, W. E.; Fromm, D. P. Methods of Single-Molecule
Fluorescence Spectroscopy and Microscopy. ReV. Sci. Instrum. 2003, 74,
3597–3619.
(34) Vrljic, M.; Nishimura, S. Y.; Moerner, W. E.; McConnell, H. M.
Cholesterol Depletion Suppresses the Translational Diffusion of Class II

Azido Push-Pull Fluorogens Photoactivation
J. Phys. Chem. B, Vol. 114, No. 45, 2010 14167
Major Histocompatibility Complex Proteins in the Plasma Membrane.
Biophys. J. 2005, 88, 334–347.
(35) Bartoszewska, B.; Szczerek, I. Synthesis and use of 2,6-Bis(p-
Azidobenzylidene)Cyclohexanone. Organika 1979, 31–5.
(36) Moerner, W. E.; Twieg, R. J.; Kline, D. W.; He, M. Fluorophore
Compounds and Their Use in Biological Systems. US Patent Application
2005, Application number 10/604,282.
(37) Pavani, S. R. P.; Thompson, M. A.; Biteen, J. S.; Lord, S. J.; Liu,
N.; Twieg, R. J.; Piestun, R.; Moerner, W. E. Three-Dimensional, Single-
Molecule Fluorescence Imaging Beyond the Diffraction Limit by using a
Double-Helix Point Spread Function. Proc. Nat. Acad. Sci. U.S.A. 2009,
106, 2995–2999.
(38) Keana, J. F. W.; Cai, S. X. New Reagents for Photoaffinity
Labeling: Synthesis and Photolysis of Functionalized Perfluorophenyl
Azides. J. Org. Chem. 1990, 55, 3640–3647.
(48) Shroff, H.; Galbraith, C. G.; Galbraith, J. A.; White, H.; Gillette,
J.; Olenych, S.; Davidson, M. W.; Betzig, E. Dual-Color Superresolution
Imaging of Genetically Expressed Probes within Individual Adhesion
Complexes. Proc. Nat. Acad. Sci. U.S.A. 2007, 104, 20308–20313.
(49) Biteen, J. S.; Thompson, M. A.; Tselentis, N. K.; Bowman, G. R.;
Shapiro, L.; Moerner, W. E. Super-Resolution Imaging in Live Caulobacter
Crescentus Cells using Photoswitchable EYFP. Nat. Methods 2008, 5, 947–
949.
(50) Kotzyba-Hibert, F.; Kapfer, I.; Goeldner, M. Recent Trends in
Photoaffinity Labeling. Angew. Chem., Int. Ed. 1995, 34, 1296–1312.
(51) Vodovozova, E. Photoaffinity Labeling and its Application in
Structural Biology. Biochemistry (Moscow) 2007, 72, 1–20.
(52) Dorma´n, G.; Prestwich, G. D. Using Photolabile Ligands in Drug
Discovery and Development. Trends Biotechnol. 2000, 18, 64–77.
(53) Baruah, H.; Puthenveetil, S.; Choi, Y.; Shah, S.; Ting, A. Y. An
Engineered Aryl Azide Ligase for Site-Specific Mapping of Protein-Protein
Interactions through Photo-Cross-Linking. Angew. Chem., Int. Ed. 2008,
47, 7018–7021.
(39) Keana, J. F. W.; Cai, S. X. Functionalized Perfluorophenyl Azides:
New Reagents for Photoaffinity Labeling. J. Fluorine Chem. 1989, 43, 151–
154.
(54) Dockter, M. E. Fluorescent Photochemical Surface Labeling of
Intact Human Erythrocytes. J. Biol. Chem. 1979, 254, 2161–2164.
(55) Dreyfuss, G.; Schwartz, K.; Blout, E. R.; Barrio, J. R.; Liu, F.;
Leonard, N. J. Fluorescent Photoaffinity Labeling: Adenosine 3′,5′-Cyclic
Monophosphate Receptor Sites. Proc. Natl. Acad. Sci. U.S.A. 1978, 75,
1199–1203.
(40) Stromgaard, K.; Saito, D. R.; Shindou, H.; Ishii, S.; Shimizu, T.;
Nakanishi, K. Ginkgolide Derivatives for Photolabeling Studies: Preparation
and Pharmacological Evaluation. J. Med. Chem. 2002, 45, 4038–4046.
(41) Wiegand, M.; Lindhorst, T. K. Synthesis of Photoactive Alpha-
Mannosides and Mannosyl Peptides and their Evaluation for Lectin Labeling.
Eur. J. Org. Chem. 2006, 2006, 4841–4851.
(56) Thevenin, B.; Shahrokh, Z.; Williard, R.; Fujimoto, E.; Kang, J.;
Ikemoto, N.; Shohet, S. A Novel Photoactivatable Cross-Linker for the
Functionally-Directed Region-Specific Fluorescent Labeling of Proteins. Eur.
J. Biochem. 1992, 206, 471–7.
(57) Moreland, R. B.; Dockter, M. E. Preparation and Characterization
of 3-Azido-2,7-Naphthalene Disulfonate: A Photolabile Fluorescent Precur-
sor Useful as a Hydrophilic Surface Probe. Anal. Biochem. 1980, 103, 26–
32.
(42) Hassner, A.; Birnbaum, D.; Loew, L. M. Charge-Shift Probes of
Membrane Potential. Synthesis. J. Org. Chem. 1984, 49, 2546–2551.
(43) Thompson, R. E.; Larson, D. R.; Webb, W. W. Precise Nanometer
Localization Analysis for Individual Fluorescent Probes. Biophys. J. 2002,
82, 2775–2783.
(44) Ober, R. J.; Ram, S.; Ward, E. S. Localization Accuracy in Single-
Molecule Microscopy. Biophys. J. 2004, 86, 1185–1200.
(45) Nyquist, H. Certain Topics in Telegraph Transmission Theory.
Trans. AIEE 1928, 47, 617–644.
(58) Angelides, K. J. Application of Photoactivatable Fluorescent Active-
Site Directed Probes to Serine-Containing Enzymes. Biochim. Biophys. Acta
Protein Struct. 1981, 669, 149–156.
(46) Shannon, C. E. Communication in the Presence of Noise. Proc.
IRE 1949, 37, 10–21.
(47) Shroff, H.; Galbraith, C. G.; Galbraith, J. A.; Betzig, E. Live-Cell
Photoactivated Localization Microscopy of Nanoscale Adhesion Dynamics.
Nat. Methods 2008, 5, 417–423.
JP907080R