A Photoactivatable Push−Pull Fluorophore for Single-Molecule Imaging in Live Cells

Abstract

We have reengineered a red-emitting dicyanomethylenedihydrofuran push-pull 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 proof-of-principle demonstration provides a new class of bright photoactivatable fluorophores, as are needed for super-resolution imaging schemes that require active control of single molecule emission.

Full text

A Photoactivatable Push-Pull Fluorophore for Single-Molecule Imaging in
Live Cells
Samuel J. Lord,
†
Nicholas R. Conley,
†
Hsiao-lu D. Lee,
†
Reichel Samuel,
‡
Na Liu,
‡
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 44240
Received April 18, 2008; E-mail: wmoerner@stanford.edu
Recent advances in optical imaging with single molecules beyond
the diffraction limit (e.g., PALM, FPALM, STORM)
1–3
have
introduced a new requirement for fluorescent labels: fluorophores
must be actively controlled (usually via photoswitching or photo-
activation) to ensure that only a single emitter is switched on at a
time in a diffraction-limited region. The location of each of these
sparse molecules is precisely determined, and a super-resolution
image is obtained from the summation of many successive rounds
of photoactivation. The ultimate spatial resolution is determined
by a number of factors, most importantly the total number of
photons detected from each individual molecule.
4
Super-resolution
imaging by these methods, and cell imaging in general, often use
photoswitchable fluorescent proteins, which have the advantage of
being genetically targeted;
5,6
however, they provide 10-fold fewer
photons before photobleaching than good small-molecule emitters.
7
Bright organic fluorophores that can be turned on and/or off are
therefore attractive; also smaller labels might be less perturbative
than fluorescent proteins, their chemistries and photophysics can
be more readily tailored, and they can be targeted using schemes
actively under development.
8–12
Here, we report a new, bright photoactivatable organic fluoro-
phore that can be imaged at the single-molecule level in living cells.
We use the DCDHF class of single-molecule cellular labels,
13–15
in which an amine donor is connected to a dicyanomethylenedi-
hydrofuran acceptor via a conjugated π-bonded network. We have
reengineered a red-emitting DCDHF to produce the fluorogen 1,a
molecule that is dark until photoactivated with a short burst of low-
intensity violet light. Photoactivation leads to conversion of the
azide moiety to an amine, which shifts the absorption to long
wavelengths and creates a bright, red emitter that is photostable
enough to be imaged on the single-molecule level in living cells.
Our proof-of-principle demonstration shows that photoactivation
of an azide-based DCDHF fluorogen provides a new class of labels
that would be useful for super-resolution imaging schemes that
require active control of single molecules.
A fluorescent label useful for live-cell imaging must be pumped
at wavelengths >500 nm to avoid cellular autofluorescence. Figure
1A shows that activating the fluorogen 1at 407 nm leads to loss
of the azido-DCDHF absorption at 424 nm and the formation of
long-wavelength absorption at 570 nm from 2. The fluorogen does
not absorb at 594 nm, but this wavelength does strongly pump the
emissive photoproduct 2. Thus, imaging with 594 nm produces no
emission until 407 nm is used to turn-on of fluorescence.
Drawing from the extensive work on the photochemistry of aryl
azides,
16
we expected that photoelimination of N2would produce
a highly reactive nitrene, which could subsequently react either by
inserting into bonds of surrounding molecules (2and Supporting
Information (SI)) or intramolecularly via a ring-expansion to an
azepine. Product 2is an amine, as is required to produce a
fluorescent red-shifted DCDHF; minor nonfluorescent products are
discussed in SI; no azepine products were found. Azide-based
fluorogens have been reported previously, but they require short
wavelengths and are not photostable enough to be applied to single-
molecule imaging.
17
Figure 1 and Scheme 1 may be rationalized as follows. First, in
1, replacing the usual amine donor group in DCDHF fluorophores
with the mildly electron-withdrawing azide (Hammett constants,
Table S1) disrupts the donor-π-acceptor push-pull character. This
explains the large (∼150-nm) blue-shift of the absorption wave-
length relative to the amine donor DCDHF. Second, electron-
withdrawing substituents (i.e., the DCDHF acceptor) on an aryl
azide stabilize the nitrene intermediate in an alcohol solvent, and
yield aniline products rather than azepines.
19
In fact, HPLC-MS
and NMR analysis of the photoproducts of 1confirm structure 2
as the major product in ethanol (see SI).
The efficiency of azide photolysis is measured by the quantum
yield of photoconversion (ΦP), which is calculated from the rate at
which 1disappears (Figure 1B and SI); photoconversion of 1is
†
Stanford University.
‡
Kent State University.
Figure 1. (A) Absorption curves in ethanol (bubbled with N2) showing
photoactivation of 1(λabs )424 nm) over time to fluorescent product 2
(λabs )570 nm). Different colored curves represent 0, 10, 90, 150, 240,
300, 480, and 1320 s of illumination by 3.1 mW/cm
2
of diffuse 407-nm
light. The sliding isosbestic point may indicate a build-up of reaction
intermediates.
18
Dashed line is the absorbance of pure, synthesized 2. (Inset)
Dotted line is weak pre-activation fluorescence of 1excited at 594 nm;
solid line is strong post-activation fluorescence resulting from exciting 2at
594 nm, showing >100-fold turn-on ratio. (B) Photoactivation kinetics from
data in A. The total yield of the reaction ([2]f/[1]i) is 69%. Photoconversion
data for 1were fit using two exponentials (τ)7.4 and 291 s); data for 2
were fit using one exponential (τ)353 s).
Scheme 1. Photochemical Activation of the Azido-DCDHF
Fluorogen
Published on Web 06/24/2008
10.1021/ja802883k CCC: $40.75 2008 American Chemical Society9204 9J. AM. CHEM. SOC. 2008,130, 9204–9205

thousands of times more likely than bleaching pathways of the
photoproduct fluorophore 2(ΦP.ΦB, Table 1). Photoconversion
of 1requires only very mild illumination by violet light (407 nm),
which is several orders of magnitude lower irradiance than required
for activating PA-GFP or EYFP, and only slightly higher than
required for Dronpa or the Cy3/Cy5 photoswitch (Table S2).
5,6,12,20
This is important, because high doses of short-wavelength light
can kill cells, alter morphology, or create unwanted phenotypes.
Imaging single molecules in living cells has stringent prerequi-
sites, and foremost among them is photostability. An emitter that
delays permanent photobleaching will be bright longer and thus
be easier to image and precisely locate.
4
As metrics of photosta-
bility, we report both the probability of photobleaching per photon
absorbed (ΦB) and the total number of photons emitted per molecule
(Ntot,e), which is an average from hundreds of individual copies of
2(see SI). Most DCDHFs are photostable,
13–15
and the photoprod-
uct 2is no exception: each molecule emits millions of photons on
average before bleaching.
For quantitative analysis, single molecules were easily activated
and imaged in polymer films and aqueous gelatin. But a crucial
test is whether this fluorogen can also be photoactivated in living
cells. Figure 2 shows three Chinese hamster ovary (CHO) cells
growing on a glass slide and incubated with 1, which easily inserts
into and penetrates the plasma membrane; fluorescence in the
cytosol turns on only after a short flash of diffuse violet light. A
fraction of the fluorophores remained stationary at the activation
site (movie S1), presumably bioconjugated to relatively static
biomolecules (via nitrene insertion into C-C bonds). The remaining
untethered fraction was free to move throughout the cell: single
molecules were visible diffusing in the cell. (Figure S1 shows the
two-dimensional tracking of a single molecule.)
The photoactivatable DCDHF single-molecule fluorogen pre-
sented here is but one example of a larger class based on replacing
a donor group in a push-pull chromophore with a photoactivatable
azide group. Unlike the Cy3/Cy5 photoswitching system, photo-
activating the azido-DCDHF does not require other additives (i.e.,
oxygen-scavengers and exogenous thiol)
12,21,22
and thus may find
greater ease of use in living systems. The next step we are pursuing
with these photoactivatable DCDHFs is to apply specific targeting
schemes to direct the label to desired locations. These molecules
may also be used for fluorogenic photoaffinity labeling;
23
assuming
a binding pocket is engineered for the fluorogen, a flash of blue
light can simultaneously turn on fluorescence and form a covalent
bond between the DCDHF and the biomolecule. The azido-DCDHF
fluorogen described here is an example of a rich new class of
photoactivatable molecules, which should be a powerful tool for
single-molecule studies in the chemically and optically complex
medium of the cell.
Acknowledgment. This work was supported in part by the
National Institutes of Health through the NIH Roadmap for Medical
Research, Grant No. P20-HG003638-02.
Supporting Information Available: Experimental procedures,
chemical analysis, additional figures and movies, plus comparisons to
other photoswitchable fluorophores. This material is available free of
charge via the Internet at http://pubs.acs.org.
References
(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.
Science 2006,313, 1642–645.
(2) Hess, S. T.; Girirajan, T. P. K.; Mason, M. D. Biophys. J. 2006, 194–
4272.
(3) Rust, M. J.; Bates, M.; Zhuang, X. Nat. Methods 2006,3, 793–795.
(4) Thompson, R. E.; Larson, D. R.; Webb, W. W. Biophys. J. 2002,82, 2775–
2783.
(5) Ando, R.; Mizuno, H.; Miyawaki, A. Science 2004,306, 1370–1373.
(6) Patterson, G. H.; Lippincott-Schwartz, J. Science 2002,297, 1873–1877.
(7) Harms, G. S.; Cognet, L.; Lommerse, P. H. M.; Blab, G. A.; Schmidt, T.
Biophys. J. 2001,80, 2396–2408.
(8) Chen, I.; Ting, A. Curr. Opin. Biotechnol. 2005,16, 35–40.
(9) Prescher, J. A.; Bertozzi, C. R. Nat. Chem. Biol. 2005,1, 13–21.
(10) Adams, S. R.; Campbell, R. E.; Gross, L. A.; Martin, B. R.; Walkup, G. K.;
Yao, Y.; Llopis, J.; Tsien, R. Y. J. Am. Chem. Soc. 2002,124, 6063–6076.
(11) Fo¨lling, J.; Belov, V.; Kunetsky, R.; Medda, R.; Scho¨ nle, A.; Egner, A.;
Eggeling, C.; Bossi, M.; Hell, S. W. Angew. Chem., Int. Ed. 2007,46,
6266–6270.
(12) Bates, M.; Blosser, T. R.; Zhuang, X. Phys. ReV. Lett. 2005,94, 108101-
1–108101-4.
(13) Willets, K. A.; Nishimura, S. Y.; Schuck, P. J.; Twieg, R. J.; Moerner,
W. E. Acc. Chem. Res. 2005,38, 549–556.
(14) Nishimura, S. Y.; Lord, S. J.; Klein, L. O.; Willets, K. A.; He, M.; Lu,
Z. K.; Twieg, R. J.; Moerner, W. E. J. Phys. Chem. B 2006,110, 8151–
8157.
(15) Lord, S. J.; Lu, Z.; Wang, H.; Willets, K. A.; Schuck, P. J.; Lee, H.-L. D.;
Nishimura, S. Y.; Twieg, R. J.; Moerner, W. E. J. Phys. Chem. A 2007,
111, 8934–8941.
(16) Schriven, E. F. V., Ed. Azides and Nitrenes: ReactiVity and Utility;
Academic Press: Orlando, FL, 1984.
(17) Dockter, M. E. J. Biol. Chem. 1979,254, 2161–2164.
(18) Muller, P. Pure Appl. Chem. 1994,66, 1077–1184.
(19) Soundararajan, N.; Platz, M. S. J. Org. Chem. 1990,55, 2034–2044.
(20) Dickson, R. M.; Cubitt, A. B.; Tsien, R. Y.; Moerner, W. E. Nature 1997,
388, 355–358.
(21) Heilemann, M.; Margeat, E.; Kasper, R.; Sauer, M.; Tinnefeld, P. J. Am.
Chem. Soc. 2005,127, 3801–3806.
(22) Rasnik, I.; McKinney, S. A.; Ha, T. Nat. Methods 2006,3, 891–893.
(23) Kotzyba-Hibert, F.; Kapfer, I.; Goeldner, M. Angew. Chem., Int. Ed. Engl.
1995,34, 1296–1312.
JA802883K
Table 1. Photophysical Properties of 1and 2in Ethanol (unless
Otherwise Stated)
a
λabs (nm),
(M
-1
cm
-1
)λfl(nm) ΦFΦP
b
ΦB
c
SM Ntot,e
d
1424, 29100 552 n/a 0.0059 n/a n/a
2570, 54100 613 0.025, 0.39
e
n/a 4.1 ×10
-6
2.3 ×10
6
a
See SI for details on measurements and calculations.
b
Quantum
yield of photoconversion from azide with 407-nm illumination (see SI).
c
Bulk quantum yield of permanent photobleaching, measured in
aqueous gelatin.
d
Average number of photons emitted per molecule in
gelatin.
e
Fluorescence quantum yield in ethanol and PMMA,
respectively; rigidification increases the brightness.
13
Figure 2. (A) Three CHO cells incubated with fluorogen 1are dark before
activation. (B) The fluorophore 2lights up in the cells after activation with
a 10-s flash of diffuse, low-irradiance (0.4 W/cm
2
) 407-nm light. (False
color: red is the white-light transmission image and green shows the
fluorescence images, excited at 594 nm.) Scalebar: 20 µm. (C) Single
molecules of activated 2in a cell under higher magnification. Background
was subtracted and the image was smoothed with a 1-pixel Gaussian.
Scalebar: 800 nm.
J. AM. CHEM. SOC. 9VOL. 130, NO. 29, 2008 9205
COMMUNICATIONS