Squaraine rotaxanes: Superior substitutes for Cy-5 in molecular probes for near-infrared fluorescence cell imaging

Article abstract of DOI:10.1002/anie.200701491

(Figure Presented) It's hip to be square: Squaraine rotaxanes have very similar photophysical properties to the commonly used Cy-5 fluorophore, but are substantially more photostable and resist self-quenching upon aggregation. Molecular probes containing

Full text of DOI:10.1002/anie.200701491

Communications
DOI: 10.1002/anie.200701491
Imaging Probes
Squaraine Rotaxanes: Superior Substitutes for Cy-5 in Molecular
Probes for Near-Infrared Fluorescence Cell Imaging**
James R. Johnson, Na Fu, Easwaran Arunkumar, W. Matthew Leevy, Seth T. Gammon,
David Piwnica-Worms, and Bradley D. Smith*
Recent breakthroughs in fluorescence microscopy are pro-
ducing notable improvements in imaging resolution.[1] These
technical advances should have a major positive impact on the
emerging field of cell imaging with single-molecule methods
and help merge the subdisciplines of cell and molecular
biology.[2] However, the full potential of many of these new
high-resolution imaging methods will only be achieved if they
employ extremely bright and highly stable luminescent
probes that do not undergo photobleaching or photoblinking.
Luminescent probes can be grouped into two major
classes: synthetic systems, such as organic dyes, inorganic
nanoparticles, and lanthanide coordination complexes,[3] and
biological systems, such as fluorescent and bioluminescent
proteins.[4] It is unlikely that any one luminescent system will
emerge as the universal solution for all optical applications,
and each class warrants further development. Herein we focus
on fluorescent organic dyes that emit in the near-infrared
(NIR) region (650–900 nm). NIR dyes are particularly
attractive for fluorescence imaging of biomedical samples
because there is very little undesired absorption and auto-
fluorescence by common biomolecules, and diminished
Rayleigh–Tyndall scattering of light.[5] Indeed, wavelengths
above 650 nm can penetrate through skin and tissue,[6] and
fluorescent NIR imaging probes are increasingly used for
in vivo optical imaging of live animals.[7] At present, the most
commonly used fluorescent NIR dyes are sulfonated carbo-
cyanine dyes (Cy-5, Cy-5.5, Cy-7) and their derivatives.[8]
Although they are quite popular, the performance of Cy
dyes is limited by molecular properties such as moderate to
poor photostability, undesired reactivity with nucleophiles,
and a propensity to self-quench upon dye aggregation.[9]
Furthermore, cyanine dyes are known to undergo photo-
blinking[10] and light-driven fluorescence switching,[11] which
makes them inherently unsuitable as FRET acceptors in
single-molecule studies (FRET= fluorescence resonance
energy transfer).[12] New classes of high-performance NIR
fluorophores are under development by various commercial
and academic research groups around the world.[13,14] In
particular, significant progress has been made with Bodipy,[15]
and Rylene[16] dyes, which exhibit excellent photostability;
however, most of these fluorophores have not been incorpo-
rated into molecular probes for cell imaging.
Our interest is in squaraine dyes, which have very
attractive photophysical properties for biological imaging.[17]
However, they suffer from two important limitations: chem-
ical instability as a result of attack by strong biological
nucleophiles and a tendency to form nonfluorescent self-
aggregates. We have discovered that both problems can be
greatly attenuated by encapsulating the dye as the thread
component inside an interlocked rotaxane structure.[18] The
synthetic strategy to produce squaraine rotaxanes has already
been described;[19] the key reaction is a templated Leigh-type
macrocyclization that connects five building blocks in a single
step. X-ray crystal structures show that the surrounding
macrocycle sits perfectly over both faces of the electrophilic
cyclobutene core of the squaraine thread and blocks nucle-
ophilic attack.[20] The steric protection provided by the
surrounding macrocycle also explains why there is no
aggregation-induced broadening of absorption or self-
quenching of fluorescence. Even when aggregated, the inner
squaraine chromophores are unable to get close enough to
interact. Furthermore, non-halogenated squaraine rotaxanes
are poor photosensitizers of singlet oxygen and do not react
readily with it;[21] thus, squaraine rotaxanes are likely to be
non-phototoxic and highly resistant to photobleaching.
Herein, we demonstrate that squaraine rotaxanes are versa-
tile, high-performance molecular probes for in vitro and
in vivo fluorescence imaging of cells. Three different types
of probe structures are shown to target different cellular
locations, namely, the organelle membrane, the internal
aqueous phase, and the exterior cell surface.
[*] J. R. Johnson, N. Fu, Dr. E. Arunkumar, Dr. W. M. Leevy,
Prof. Dr. B. D. Smith
Department of Chemistry and Biochemistry
University of Notre Dame
Notre Dame, IN 46556 (USA)
Fax: (+1)574-631-6652
E-mail: smith.115@nd.edu
Squaraine rotaxanes 1–4 have absorption and emission
profiles that closely match the Cy-5 chromophore in control
compound 5 (Table 1). Thus, all probes can be conveniently
imaged by using an epifluorescence microscope and a
standard Cy-5 filter set (exciter: HQ620/60X, dichroic:
660LP, emitter: HQ700/75m). We find that the relatively
nonpolar squaraine rotaxane 1 interacts with cells in a very
similar way to the well-known lipophilic stain Nile red.[22] As
shown in Figure 1, probe 1 rapidly accumulates at lipophilic
sites inside a living cell, such as the endoplasmic reticulum
Homepage: http://www.chem.nd.edu/faculty/detail/bsmith3
Dr. S. T. Gammon, Prof. Dr. D. Piwnica-Worms
Mallinckrodt Institute of Radiology
Washington University School of Medicine
510 South Kings Highway Boulevard, Campus Box 8225, St. Louis,
MO 63110 (USA)
[**] We warmly thankthe NIH (GM059078 and P50 CA94056) and Notre
Dame CEST for funding support.
Supporting information for this article is available on the WWW
under http://www.angewandte.org or from the author.
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Figure 1. Fluorescence-microscopy images of live mammalian cells
treated with 1: A) Endoplasmic-reticulum staining of a single CHO
cell. B) Lipid-droplet staining in A549 cells. C) Multicolor image of a
single COS-7 cell treated with H33342 (blue), FM1-43 (green), and 1
(red). D) MCF7 cells 144 h after a single treatment with 1. Single-color
images were left in grayscale to preserve image detail and contrast.
Scale bars corresponds to 20 mm.
obtained by co-staining the lipid droplets with Nile red, and
the ER with blue-emitting ER-Tracker Blue-White DPX
(Molecular Probes)[23] (see the Supporting Information). The
red emission band for probe 1 is quite narrow and permits the
acquisition of multicolor images. For example, shown in
Figure 1C is a monkey kidney (COS-7) cell that has been
treated with the DNA stain H33342 (blue), a lipophilic stain
administered to label the plasma membrane (FM1-43, green),
and probe 1 (red). Figure 1D illustrates the high chemical
stability and low toxicity of 1. Even after eight days, the
staining pattern in live human breast cancer (MCF7) cells
remains the same as obtained on day one, and the fluores-
cence intensity remains strong in all cells; thus, it appears that
the dye is transferred efficiently to subsequent daughter cells.
Another notable feature with probe 1 is that its staining is
unaffected by cell fixation with paraformaldehyde (see the
Supporting Information).
Table 1: Photophysical properties in water.
Compound
labs [nm]
lem [nm]
Ff[d]
The tetracarboxylic acid squaraine rotaxane 2 is soluble in
solutions at physiological pHand acts as an excellent
fluorescent marker of encapsulated aqueous phases inside
living cells. For example, treatment of COS-7 cells with 2 leads
to accumulation of the dye in small punctate compartments
within the cell that are trafficked quite rapidly. The high
photostability of 2 allows this fascinating trafficking process
to be monitored as a real-time movie, with constant sample
irradiation, over many minutes (see the Supporting Informa-
tion). This type of movie cannot be acquired with currently
available NIR fluorescent probes, such as the amphiphilic
styryl dye FM4-64 and water-soluble dextran Alexa-647
conjugate, because they are rapidly photobleached.[16]
1[a]
2[b]
3[c]
4
639
653
653
650
659
675
675
669
671
0.70
0.25
0.20
0.08
0.25
5
648, 603
[a] Measurements were carried out in THF. [b] Excited-state lifetime in
water was found to be 1.5 ns. [c] Molar absorptivity (e) in water was
found to be 121.052mÀ1 L (loge=5.1). [d] Solutions were excited at
optically matching wavelengths and emission monitored in the region
600–900 nm. Fluorescence quantum yields were determined using 4,4-
[bis(N,N-dimethylamino)phenyl]squaraine dye as the standard (Ff =
0.70 in CHCl3); error limit Æ5%.
Fluorescent probes 3–5 have appended zinc(II)–dipicolyl-
amine (Zn–DPA) coordination centers as cell-targeting
ligands. We have recently demonstrated that fluorescent
conjugates with attached Zn–DPA groups will selectively
associate with the anionic surfaces of bacterial cells in
preference to the zwitterionic membrane surfaces of healthy
(ER) and intracellular lipid droplets. For example, probe
localization with Chinese hamster ovary (CHO) cells is
primarily in the ER (Figure 1A), whereas, with human lung
carcinoma (A549) cells, uptake is mainly in lipid droplets
(Figure 1B). Confirmation of these localization sites was
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Communications
animal cells.[24] To compare the targeting ability and photo-
illustrated in Figure 3, both sites of bacterial inoculation are
very apparent, because the fluorescence emission intensities
are about 100 times greater than the background signal from
other anatomical parts of the mouse.
stability of a squaraine rotaxane directly with a related Cy-5
probe, we prepared three fluorophore Zn–DPA conjugates;
squaraine rotaxane 3 with two Zn–DPA targeting ligands,
squaraine rotaxane 4 with one Zn–DPA targeting ligand, and
Cy-5 probe 5 with one Zn–DPA targeting ligand. As
expected, all three probes strongly associate with the periph-
ery of bacterial cells, which allowed photobleaching experi-
ments to be conducted. Three separate samples of bacterial
cells (E. coli), each stained with equal amounts of probes 3, 4,
or 5, were irradiated continuously with light (l = (620 Æ
30) nm) from an X-cite 120 fluorescence illumination
system through a Nikon TE2000-U epifluorescence micro-
scope. The observed photobleaching half-lives were: 1080 s
for 3, 197 s for 4, and 11 s for Cy-5 conjugate 5. Thus, direct
comparison between squaraine rotaxane 4 and Cy-5 probe 5
suggests that the squaraine rotaxane is almost 20 times more
photostable. The additional fivefold increase in half-life with
doubly valent squaraine rotaxane 3 is attributed to its stronger
cell-surface affinity, leading to a slower off rate for the probe.
In other words, there is diminished signal loss with 3 as a result
of slower probe diffusion away from the cell surface.
Figure 3. Optical image of a live mouse with subcutaneous injections
of S. aureus and E. coli that were prelabeled with 3. The entire animal
was irradiated with filtered light at l=(625Æ40) nm and an image of
emission intensity at l=(670Æ20) nm was collected by a CCD
camera during a 5-s acquisition period.
The remarkable stability of probe 3 permits fluorescence-
imaging experiments that are impossible with probes based
on NIR cyanine dyes. For example, we have acquired real-
time fluorescence-microscopy movies of bacteria undergoing
cell division. Shown in Figure 2 is a montage of images at
In summary, squaraine-rotaxane dyes can be readily
converted into extremely bright and highly stable NIR
fluorescent probes for in vitro and in vivo optical imaging of
live and fixed cells. The probes can be structurally modified
for targeting of quite different cellular locations. Squaraine
rotaxanes are likely to be superior substitutes for Cy-5 in
many biotechnology and imaging applications that require
NIR fluorescent dyes.
Received: April 5, 2007
Published online: June 22, 2007
Keywords: cell recognition · dyes/pigments · fluorescence ·
.
imaging agents · rotaxanes
Figure 2. Binary fission of E. coli cells stained with 3. The cells were
imaged every 5 s for 30 min by using fluorescence microscopy. The
times stated in each panel correspond to the movie time point and the
scale bar represents 2 mm.
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