Time-resolved long-lived luminescence imaging method employing luminescent lanthanide probes with a new microscopy system

Article abstract of DOI:10.1021/ja073392j

Superior fluorescence imaging methods are needed for detailed studies on biological phenomena, and one approach that permits precise analyses is time-resolved fluorescence measurement, which offers a high signal-to-noise ratio. Herein, we describe a new fluorescence imaging system to visualize biomolecules within living biological samples by means of time-resolved, long-lived luminescence microscopy (TRLLM). In TRLLM, short-lived background fluorescence and scattered light are gated out, allowing the long-lived luminescence to be selectively imaged. Usual time-resolved fluorescence microscopy provides fluorescence images with nanosecond resolution and has been used to image interactions between proteins, protein phosphorylation, the local pH, the refractive index, ion or oxygen concentrations, etc. Luminescent lanthanide complexes (especially europium and terbium trivalent ions (Eu ³⁺ and Tb³⁺)), in contrast, have long luminescence lifetimes on the order of milliseconds. We have designed and synthesized new luminescent Eu³⁺ complexes for TRLLM and also developed a new TRLLM system using a conventional fluorescence microscope with an image intensifier unit for gated signal acquisition and a xenon flash lamp as the excitation source. When the newly developed luminescent Eu³⁺ complexes were applied to living cells, clear fluorescence images were acquired with the TRLLM system, and short-lived fluorescence was completely excluded. By using Eu ³⁺ and Tb³⁺ luminescent complexes in combination, time-resolved dual-color imaging was also possible. Furthermore, we monitored changes of intracellular ionic zinc (Zn²⁺) concentration by using a Zn²⁺-selective luminescent Eu³⁺ chemosensor, [Eu-7]. This new imaging technique should facilitate investigations of biological functions with fluorescence microscopy, complementing other fluorescence imaging methodologies.

Full text of DOI:10.1021/ja073392j

Published on Web 10/10/2007
Time-Resolved Long-Lived Luminescence Imaging Method
Employing Luminescent Lanthanide Probes with a New
Microscopy System
Kenjiro Hanaoka,† Kazuya Kikuchi,‡ Shigeru Kobayashi,§ and Tetsuo Nagano*,†
Contribution from the Graduate School of Pharmaceutical Sciences, The UniVersity of Tokyo,
7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Department of Materials and Life Sciences, Graduate
School of Engineering, Osaka UniVersity, 2-1 Yamada-oka, Suita City, Osaka 565-0871, and
Products DeVelopment Department 2, Micro-Imaging Systems DiVision, OLYMPUS
CORPORATION, 2951 Ishikawa-cho, Hachioji-shi, Tokyo 192-8507, Japan
Received May 13, 2007; E-mail: tlong@mol.f.u-tokyo.ac.jp
Abstract: Superior fluorescence imaging methods are needed for detailed studies on biological phenomena,
and one approach that permits precise analyses is time-resolved fluorescence measurement, which offers
a high signal-to-noise ratio. Herein, we describe a new fluorescence imaging system to visualize
biomolecules within living biological samples by means of time-resolved, long-lived luminescence microscopy
(TRLLM). In TRLLM, short-lived background fluorescence and scattered light are gated out, allowing the
long-lived luminescence to be selectively imaged. Usual time-resolved fluorescence microscopy provides
fluorescence images with nanosecond resolution and has been used to image interactions between proteins,
protein phosphorylation, the local pH, the refractive index, ion or oxygen concentrations, etc. Luminescent
lanthanide complexes (especially europium and terbium trivalent ions (Eu3+ and Tb3+)), in contrast, have
long luminescence lifetimes on the order of milliseconds. We have designed and synthesized new
luminescent Eu3+ complexes for TRLLM and also developed a new TRLLM system using a conventional
fluorescence microscope with an image intensifier unit for gated signal acquisition and a xenon flash lamp
as the excitation source. When the newly developed luminescent Eu3+ complexes were applied to living
cells, clear fluorescence images were acquired with the TRLLM system, and short-lived fluorescence was
completely excluded. By using Eu3+ and Tb3+ luminescent complexes in combination, time-resolved dual-
color imaging was also possible. Furthermore, we monitored changes of intracellular ionic zinc (Zn2+
)
concentration by using a Zn2+-selective luminescent Eu3+ chemosensor, [Eu-7]. This new imaging technique
should facilitate investigations of biological functions with fluorescence microscopy, complementing other
fluorescence imaging methodologies.
Introduction
the local pH, the refractive index, ion or oxygen concentrations,
etc.3 In this paper, we present a new system for time-resolved
Fluorescence imaging is a powerful tool for the visualization
of biomolecules in various biological environments and is
important for elucidating biological functions.1,2 Further, the
development of new fluorescent dyes with improved photo-
physical properties combined with technical progress in optical
devices for fluorescence microscopy will open the way to
superior fluorescence imaging technologies. Usual time-resolved
fluorescence microscopy exploits differences in fluorescence
lifetimes on the order of nanoseconds to monitor target
fluorescence, and this technique has been used to image
interactions between proteins or protein phosphorylation by
detecting fluorescence resonance energy transfer (FRET) and
to report on the local environment of fluorophores, for example,
long-lived luminescence microscopy (TRLLM), which utilizes
the extremely long luminescence lifetimes of luminescent
lanthanide complexes, on the order of milliseconds. This
approach is expected to allow imaging with complete elimination
of the short-lived background fluorescence,4-13 providing high
signal-to-noise ratios, by the introduction of an appropriate delay
(3) Suhling, K.; French, P. M. W.; Phillips, D. Photochem. Photobiol. Sci.
2005, 4, 13-22.
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Am. Chem. Soc. 2004, 126, 4888-4896.
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Parker, D.; Williams, J. A. G. J. Photochem. Photobiol., B 2000, 57, 83-
90.
† The University of Tokyo.
‡ Osaka University.
§ OLYMPUS CORPORATION.
(8) Phimphivong, S.; Saavedra, S. S. Bioconjugate Chem. 1998, 9, 350-357.
(9) de Haas, R. R.; van Gijlswijk, R. P. M.; van der Tol, E. B.; Veuskens, J.;
van Gijssel, H. E.; Tijdens, R. B.; Bonnet, J.; Verwoerd, N. P.; Tanke, H.
J. J. Histochem. Cytochem. 1999, 47, 183-196.
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74, 2210-2222.
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530.
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13502
J. AM. CHEM. SOC. 2007, 129, 13502-13509
10.1021/ja073392j CCC: $37.00 © 2007 American Chemical Society

Luminescence Imaging Employing Lanthanide Probes
A R T I C L E S
New fluorescent dyes with a long fluorescence lifetime are
needed for the TRLLM-based microscopic method. Typical
organic phosphorescent dyes, which possess high triplet quantum
yields, such as eosin or erythrosine or metal complexes showing
MLCT luminescence are considered to be good candidates for
the TRLLM-based microscopic method,9,14 because phospho-
rescence lifetimes are relatively long. However, these com-
pounds often transfer the energy of the triplet excited state to
surrounding biomolecules in cells and tissues through singlet
oxygen, acting as photosensitizers.14,15 Such light-activated
photosensitizing severely limits the use of these phosphorescent
dyes in TRLLM, resulting in cell death via apoptosis and/or
necrosis and photobleaching of the dyes. Luminescent lanthanide
complexes, in particular complexes of europium and terbium
trivalent ions (Eu3+ and Tb3+), possess extremely long lumi-
nescence lifetimes on the order of milliseconds, whereas typical
organic fluorescent compounds possess short fluorescence
lifetimes in the nanosecond region.16 Further, luminescent
lanthanide complexes are thought to be relatively insensitive
to photobleaching and the generation of singlet oxygen com-
pared with phosphorescent dyes.5 For these reasons, luminescent
lanthanide complexes have been recently exploited in various
bioassays with time-gating in the fields of medicine, biotech-
nology, and biological science.17,18 The lanthanide f-f transi-
tions have low absorbance, so a sensitizing chromophore
covalently bound to the ligand is desirable for high lumines-
cence.16 Absorption by the chromophore results in effective
population of its triplet level, and efficient intramolecular energy
transfer conveys the absorbance energy of the excited chro-
mophore to a chelated lanthanide metal ion, which becomes
excited to the emission state (Figure 1b). Therefore, a proper
chromophore design should afford luminescent lanthanide
complexes which possess appropriate photochemical properties
for TRLLM.16
Despite the tremendous amount of work on luminescent
lanthanide complexes, only a few complexes have yet been
employed for TRLLM,4,5,7,8,10-13,19-21 and most complexes are
used as labeling reagents for proteins. There are numerous
requirements to be fulfilled by luminescent lanthanide complexes
for usage in molecular imaging with TRLLM, and these include
kinetic stability in water and a sensitizing chromophore unit
that generates an efficient antenna effect. Further, it would be
desirable to move the excitation wavelength toward the visible
region to reduce the phototoxicity of the excitation light for
biological samples and to ensure compatibility with the optics
of standard fluorescence microscopes. Here, we report the design
and synthesis of new luminescent Eu3+ complexes for use as
long-lived luminescent dyes for TRLLM and the development
of a new TRLLM system, based on conventional fluorescence
Figure 1. Design of luminescent lanthanide complexes. (a) Principle of
time-resolved long-lived luminescence measurements. The short-lived
background fluorescence decays to negligible levels during an appropriate
delay time between a pulse of excitation light and the measurement of the
long-lived luminescence. The period for which the fluorescence is measured
is the gate time. The time-resolved long-lived luminescence measurement
obviates the short-lived background fluorescence. (b) Schematic view of a
sensitizing chromophore incorporated into a lanthanide emitter. Efficient
intramolecular energy transfer should occur from the excited chromophore
to the proximate lanthanide ion after excitation of the sensitizing chro-
mophore, and the metal ion becomes excited to the emission state. (c)
Structure of the synthesized Eu3+-polyaminocarboxylate complexes in-
corporating a covalently bound sensitizing chromophore. Eu3+ complexes
possess various substituents, H, F, CH3, CH3O, NHAc, and NH2, at the
6-position of quinoline ()R) as a light-harvesting moiety for efficient Eu3+
sensitization. Also shown is the structure of the Zn2+-selective luminescent
Eu3+ chemosensor [Eu-7]. Reprinted from ref 29. Copyright 2004 American
Chemical Society.
(12) Seve´us, L.; Va¨isa¨la¨, M.; Hemmila¨, I.; Kojola, H.; Roomans, G. M.; Soini,
E. Microsc. Res. Tech. 1994, 28, 149-154.
(13) Connally, R.; Veal, D.; Piper, J. Microsc. Res. Tech. 2004, 64, 312-322.
(14) Redmond, R. W.; Gamlin, J. N. Photochem. Photobiol. 1999, 70, 391-
475.
(15) Gorman, A.; Killoran, J.; O’Shea, C.; Kenna, T.; Gallagher, W. M.; O’Shea,
D. F. J. Am. Chem. Soc. 2004, 126, 10619-10631.
time between the pulsed excitation light and measurement of
the long-lived luminescence of the dyes (Figure 1a). Thus, this
TRLLM imaging has characteristics very different from those
of conventional time-resolved fluorescence imaging.
(16) Parker, D.; Williams, J. A. G. J. Chem. Soc., Dalton Trans. 1996, 3613-
3628.
(17) Hemmila¨, I.; Webb, S. Drug DiscoVery Today 1997, 2, 373-381.
(18) Petoud, S.; Cohen, S. M.; Bu¨nzli, J. C. G.; Raymond, K. N. J. Am. Chem.
Soc. 2003, 125, 13324-13325.
(19) Poole, R. A.; Bobba, G.; Cann, M. J.; Frias, J. C.; Parker, D.; Peacock, R.
D. Org. Biomol. Chem. 2005, 3, 1013-1024.
(11) Seveus, L.; Va¨isa¨la¨, M.; Syrja¨nen, S.; Sandberg, M.; Kuusisto, A.; Harju,
(20) Song, B.; Wang, G.; Tan, M.; Yuan, J. J. Am. Chem. Soc. 2006, 128,
13442-13450.
R.; Salo, J.; Hemmila¨, I.; Kojola, H.; Soini, E. Cytometry 1992, 13, 329-
338.
(21) Pandya, S.; Yu, J.; Parker, D. Dalton Trans. 2006, 2757-2766.
9
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A R T I C L E S
Hanaoka et al.
microscopy, for molecular imaging in living biological samples.
We also confirmed the usefulness of this TRLLM method in
cultured living cells.
Results and Discussion
Synthesis of Long-Lived Luminescent Dyes for TRLLM.
Various luminescent Eu3+ complexes (R ) H, F, CH3, CH3O,
NHAc, and NH2 in the 6-position of the quinoline) (Figure 1c),
which were developed as candidate long-lived luminescent dyes
for TRLLM, were synthesized via similar routes to examine
their suitability for microscopic measurement, which requires
strong luminescence and an excitation wavelength longer than
350 nm (because the optics of standard fluorescence microscopes
have only marginal transmittance below 350 nm). The synthetic
schemes and detailed descriptions of the synthetic procedures
for these Eu3+ complexes are shown in the Supporting Informa-
tion.
Luminescence and Chemical Properties of Synthesized
Long-Lived Luminescent Dyes. Quinolines with F, CH3, and
CH3O in the 6-position ()R) have been reported as efficient
sensitizing chromophores for Eu3+ 22-24
but the photophysical
,
properties of these Eu3+ complexes have not been analyzed well.
The integrity of the Eu3+ complexes must be maintained in vivo
to ensure nontoxicity, because the free metal ion and unchelated
chelators are generally more toxic than the complex itself.25
Chelators such as DTPA (diethylenetriamine-N,N,N′,N′′,N′′-
pentaacetic acid) and DOTA (1,4,7,10-tetraazacyclododecane-
1,4,7,10-tetraacetic acid) have very high stability constant values
for lanthanide ion,25 so we expected that our Eu3+ complexes
would not have serious toxicity. First, the time-delayed lumi-
nescence spectra and UV-vis absorption spectra of these Eu3+
complexes were measured in 100 mM HEPES buffer (pH 7.4).
When time-delayed luminescence spectra were measured with
a delay time of 50 µs and a gate time of 1.00 ms, these Eu3+
complexes (except R ) NH2) were highly luminescent (Figure
2a). The luminescence spectra displayed six bands, arising from
transitions from the emissive 5D0 level to the 7F0, 7F1, 7F2, 7F3,
7F4, and 7F5 levels of the ground states, respectively.16 Indeed,
aqueous solutions of Eu3+ complexes except R ) NH2 showed
bright pink luminescence upon excitation with a TLC plate
reader lamp (312 nm) (see the Supporting Information). The
UV-vis absorption spectra of these Eu3+ complexes showed
intense absorption bands due to the quinolyl substituent, which
is the sensitizing chromophore (Figure 2b,c). Figure 2c shows
the normalized absorption spectra of Eu3+ complexes (R ) H,
F, CH3, CH3O, NHAc, NH2) between 280 and 450 nm on the
basis of Figure 2b, indicating the comparison between the
dominant absorption bands of Eu3+ complexes at a concentration
of 50 µM in HEPES buffer (pH 7.4). The absorption spectra of
Eu3+ complexes with R ) H, F, and CH3 showed a peak around
320 nm, and introduction of a CH3O or NHAc substituent ()R)
resulted in an absorption peak at close to 330 nm, tailing out to
370 nm. The absorption spectra of the Eu3+ complex with R )
NH2 showed an absorption peak at 352 nm, tailing out to 420
nm. Although the Eu3+ complex with R ) NH2 showed the
Figure 2. Time-resolved luminescence spectra and absorbance spectra of
newly synthesized Eu3+ complexes. (a) Time-resolved luminescence spectra
of Eu3+ complexes (H, red; F, blue; CH3, green; CH3O, black; NHAc, pink;
NH2, orange) in HEPES buffer at pH 7.4. The bands arise from 5D0 f 7FJ
transitions; the J values of the bands are labeled. (b, c) Absorbance spectra
of the Eu3+ complexes in HEPES buffer at pH 7.4. (b) Absorbance spectra
and (c) normalized absorbance spectra over 280 nm. R ) H, red; F, blue;
CH3, green; CH3O, black; NHAc, pink; NH2, orange.
longest absorption wavelength among the synthesized Eu3+
complexes, this Eu3+ complex did not luminesce at all. The
luminescence and chemical properties of Eu3+ complexes are
listed in Table 1. These Eu3+ complexes were dissolved even
at 10 mM, so they were highly water-soluble. The luminescence
quantum yields (φ) of the Eu3+ complexes except R ) NH2 are
sufficiently large for luminescence detection in fluorescence
microscopy.4,7 The luminescence lifetimes of the above Eu3+
complexes except R ) NH2 were approximately 0.60 ms in
H2O and 2.00 ms in D2O. These values indicate that the number
of coordinated water molecules (q value)27 at the Eu3+ ion was
(22) Griffin, J. M. M.; Skwierawska, A. M.; Manning, H. C.; Marx, J. N.;
Bornhop, D. J. Tetrahedron Lett. 2001, 42, 3823-3825.
(23) Manning, H. C.; Goebel, T.; Marx, J. N.; Bornhop, D. J. Org. Lett. 2002,
4, 1075-1078.
(24) Kiefer, G. E.; Bornhop, D. J. PCT Int. Appl. WO03/035655 A1, 2003.
(25) Caravan, P.; Ellison, J. J.; McMurry, T. J.; Lauffer, R. B. Chem. ReV. 1999,
99, 2293-2352.
(26) Nakamaru, K. Bull. Chem. Soc. Jpn. 1982, 55, 2697-2705.
(27) Beeby, A.; Clarkson, I. M.; Dickins, R. S.; Faulkner, S.; Parker, D.; Royle,
L.; de Sousa, A. S.; Williams, J. A. G.; Woods, M. J. Chem. Soc., Perkin
Trans. 2 1999, 493-503.
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Luminescence Imaging Employing Lanthanide Probes
A R T I C L E S
Table 1. Luminescence and Chemical Properties
to be insensitive to photobleaching and inefficient generators
of singlet oxygen.5 Reactive oxygen species such as singlet
oxygen cause significant cellular damage. First, to evaluate the
photostability of Eu3+ complexes (R ) NHAc or CH3O),
photobleaching experiments with Eu3+ complexes and fluores-
cein were performed in 100 mM HEPES buffer at pH 7.4
(Figure S2, Supporting Information). Fluorescein is often used
as a fluorescent dye for fluorescence microscopy. The Eu3+
complexes (40 µM) and fluorescein (1 µM) were irradiated with
an external light at an appropriate wavelength, 325 nm (1.57
mW) for Eu3+ complexes and 492 nm (2.07 mW) for fluores-
cein. The luminescence intensity of Eu3+ complexes (R ) NHAc
and CH3O) without time-gating reached 114% and 111% of
the initial intensity after 420 min, respectively. The fluorescence
intensity of fluorescein was decreased to 48% of the initial
intensity after 420 min. The Eu3+ complexes appear to be more
insensitive to photobleaching than fluorescein. Furthermore, the
ability of Rose Bengal, fluorescein, and Eu3+ complexes (R )
NHAc or CH3O) to produce singlet oxygen was tested by
trapping with 1,3-diphenylisobenzofuran (DPBF), an efficient
quencher of singlet oxygen.15 Rose Bengal is commonly
exploited as a photosensitizer and possesses a high triplet
quantum yield as a phosphorescent dye.14 Fluorescein was
employed here as a reference sensitizer. The generation of
singlet oxygen was evaluated by following the disappearance
of the 410 nm absorbance of DPBF at the initial concentration
of 20 µM. We used a xenon lamp as a light source and an
irradiation wavelength of 555 nm for Rose Bengal, 492 nm for
fluorescein, and 325 nm for Eu3+ complexes. The values of the
relative rate of singlet oxygen generation were 0.89, 0.51, 1,
and 18 for Eu3+ complexes (R ) NHAc and R ) CH3O),
fluorescein, and Rose Bengal, respectively (details in Figure
S3, Supporting Information). Thus, the Eu3+ complexes had
relatively low singlet oxygen production, whereas Rose Bengal
showed efficient production of singlet oxygen compared with
a reference sensitizer, fluorescein.
Dual-Color Imaging with TRLLM. We investigated the
feasibility of dual-color imaging with TRLLM using long-lived
luminescent Eu3+ and Tb3+ complexes. Luminescent Tb3+
complexes also have advantages for TRLLM, such as long
luminescence lifetimes on the order of milliseconds, narrow
emission peaks, large Stokes shifts, high quantum yields, and
excellent water solubility, like luminescent Eu3+ complexes. The
main luminescence wavelength of Tb3+ complexes is between
480 and 630 nm,16 and that of Eu3+ complexes is between 570
and 720 nm (Figure 2a). The newly synthesized Eu3+ complex
(R ) NHAc) is used as a red emitter, and the Tb3+ complex
DTPA-cs124 with a chelated Tb3+ ion (Tb3+-DTPA-cs124) is
employed as a green emitter.28 This DTPA-cs124 chelator was
prepared using the reported procedure,28 and the complexation
of DTPA-cs124 with Tb3+ is described in the Supporting
Information. The Eu3+ complex (R ) NHAc) and Tb3+-DTPA-
cs124 were injected separately into cultured living HeLa cells.
The fluorescence microscope had optical windows centered at
617 ( 37 nm for the Eu3+ emission (commercially available
Cy3 filter) and at 528 ( 19 nm for the Tb3+ emission
(commercially available FITC filter) with the same excitation
wavelength at 360 ( 40 nm, which also utilized a commercially
available filter. In the time-resolved long-lived luminescence
b
c
φa
τH
O
2
τD
O
2
R
(%)
(ms)
(ms)
qd
H
F
Me
MeO
NHAc
NH2
5.9
4.2
4.2
2.4
2.6
NLe
0.59
0.60
0.60
0.59
0.60
NL
2.01
1.99
2.00
1.88
1.96
NL
1.14
1.10
1.10
1.10
1.09
NL
a Quantum yields were calculated using [Ru(bipy)3]Cl2 (bipy ) 2,2′-
bipyridine; φ ) 0.028 in water) as a standard and measured in 100 mM
HEPES buffer at pH 7.4, 25 °C.26 b The luminescence lifetimes were
measured in H2O. c The luminescence lifetimes were measured in
D2O. d q values were estimated using the equation qEu ) 1.2 (τH O
-
-1
2
-1
τD O - 0.25), which allows for the contribution of unbound water
2
molecules.27 e NL ) no luminescence.
approximately 1.10. Thus, the lanthanide hydration state was
hardly affected by the introduction of various substituents at
the 6-position of the quinolyl sensitizing chromophore of the
Eu3+ complexes. These results demonstrate that it is feasible to
modulate the photophysical properties of Eu3+ complexes, such
as the intensity of luminescence and the excitation wavelength,
and also indicate that the Eu3+ complexes with R ) NHAc or
CH3O are good candidates for long-lived luminescence dyes
for TRLLM because they have a sufficiently long excitation
wavelength of over 350 nm.
Time-Resolved Long-Lived Luminescence Imaging in
Living Cells. A schematic of the TRLLM system is shown in
Figure 3a. This new TRLLM system utilizes a conventional
fluorescence microscopy system equipped with an image
intensifier (I.I.) unit, a xenon flash lamp, and a timing controller.
Applications of mechanical choppers in TRLLM with the use
of luminescent lanthanide complexes have been reported by
some groups,4-6,8-12 and some groups have employed an I.I.
unit for the time resolution.7,13 In this study, we used an I.I.
unit for the time resolution for the following reasons. First, the
gate time (10 µs to 100 ms) and delay time (10 µs to 100 ms)
can be easily controlled with the I.I. unit. Second, the I.I. unit
improves the uniformity of the emission light, which can be
unsatisfactory with mechanical choppers. Third, the gain of the
luminescence signal can be adjusted easily. As regards the
excitation source, a powerful excitation light is required for time-
resolved long-lived luminescence measurements, so a xenon
flash lamp was selected. A laser might be preferable, but its
wavelength cannot be changed easily.
We examined the application of two Eu3+ complexes with R
) NHAc or CH3O to conventional microscopy or TRLLM of
cultured living cells (HeLa cells). The fluorescence microscope
had an optical window centered at 617 ( 37 nm for the emission
due to Eu3+-based luminescence upon excitation at 360 ( 40
nm. The Eu3+ complex with R ) NHAc or CH3O was injected
into the cultured HeLa cells (Figure 3b). In the prompt
fluorescence images, the Eu3+ complex-injected cells were seen,
together with the Eu3+ complex-noninjected cells, which ap-
peared owing to their weak autofluorescence. In the TRLLM
images, the autofluorescence from the cells, which is short-
lived, was gated out, allowing the Eu3+ complex-injected cells
to be clearly distinguished.
We next investigated the usefulness of the luminescent Eu3+
complexes as long-lived luminescent dyes, since they were
expected to have advantages for biological applications. Unlike
triplet-state phosphorescent dyes, Eu3+ complexes are thought
(28) Li, M.; Selvin, P. R. J. Am. Chem. Soc. 1995, 117, 8132-8138.
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A R T I C L E S
Hanaoka et al.
Figure 3. Time-resolved, long-lived luminescence images provided by our new TRLLM system. (a) Schematic diagram of the optical apparatus used for
the TRLLM system. The excitation source is a xenon flash lamp. The excitation light passes through the excitation filter and is focused onto cells with
dichroic mirrors. The emission light passes through the emission filter. The I.I. unit passes the long-lived luminescence to the CCD camera, controlling the
delay time, the gate time, and the gain. The excitation and emission light can be easily selected by using appropriate excitation and emission filters. The
image is recorded on the CCD camera and then transferred to the computer for further processing with MetaFluor 6.1 software. (b) One or two cells were
injected with the Eu3+ complex (R ) NHAc or CH3O) in HBSS buffer. The fluorescence was measured at 617 ( 37 nm, which is the wavelength range of
the Cy3 emission filter, with excitation at 360 ( 40 nm. Bright-field transmission images (DIC), prompt fluorescence images (Promp), and time-resolved
long-lived luminescence images (Time-resolved) of living cells, including the Eu3+ complex-injected cells. (c) Time-resolved dual-color luminescence images
of living HeLa cells. One cell was injected with the Eu3+ complex (R ) NHAc) or Tb3+-DTPA-cs124, respectively. The fluorescence emission at 617 (
37 nm (the Cy3 emission filter) for Eu3+ or 528 ( 19 nm (the FITC emission filter) for Tb3+ was measured with excitation at 360 ( 40 nm. DIC: Bright-
field transmission image. Red and green colors in DIC show Eu3+ complex-injected and Tb3+-DTPA-cs124-injected cells, respectively. Cy3 filter: Time-
resolved long-lived luminescence image of DIC with the Cy3 emission filter (617 ( 37 nm). FITC filter: Time-resolved long-lived luminescence image of
DIC with the FITC emission filter (528 ( 19 nm).
image produced with the Cy3 filter, the luminescence of Eu3+
was clearly seen, whereas that of Tb3+ was only weakly detected
due to the difference in the range of the emission wavelengths
(Figure 3c). With the FITC filter, the Tb3+ luminescence was
strongly detected, while the Eu3+ luminescence was not
observed at all (Figure 3c). Thus, our TRLLM technique can
provide dual-color detection with appropriate emission filters,
in addition to discriminating against short-lived background
fluorescence. To our knowledge, this is the first report of dual-
color imaging in living cells with TRLLM.
various biological systems.30 This compound, [Eu-7], has a
sufficiently long excitation wavelength for fluorescence mi-
croscopy and is suitable for fluorescence microscopic measure-
ments of the Zn2+ concentration in living cells. In this study,
we examined the application of [Eu-7] to cultured living HeLa
cells by using our TRLLM system (Figure 4). Optical parameters
used for the fluorescence microscope were as follows: the
fluorescence microscope had an optical window centered at 617
( 37 nm for the emission due to Eu3+-based luminescence
excited at 360 ( 40 nm. The delay time, prior to initiation of
counting, and the gate time, during which counting takes place,
were set at 70 and 808 µs, respectively. Compound [Eu-7] was
Time-Resolved Long-Lived Luminescence Imaging of
Zn2+ in Living Cells. We previously reported a Zn2+-selective
luminescent Eu3+ chemosensor, [Eu-7] (Figure 1c).29 Zn2+ is
the second most abundant heavy metal ion after iron in the
human body, and chelatable Zn2+ plays an important role in
(29) Hanaoka, K.; Kikuchi, K.; Kojima, H.; Urano, Y.; Nagano, T. J. Am. Chem.
Soc. 2004, 126, 12470-12476.
(30) Vallee, B. L.; Falchuk, K. H. Physiol. ReV. 1993, 73, 79-118.
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Luminescence Imaging Employing Lanthanide Probes
A R T I C L E S
Figure 4. Time-resolved long-lived luminescence imaging of intracellular Zn2+ in living HeLa cells. The luminescence at 617 ( 37 nm, excited at 360 (
40 nm, was measured at 30 s intervals. The time-resolved long-lived luminescence images were measured with our TRLLM system using a delay time of
70 µs and a gate time of 808 µs. The HeLa cells were injected in HBSS buffer with the [Eu-7] solution. (a) Bright-field transmission image (0 min). (b)
Time-resolved long-lived luminescence image of (a) (0 min). (c) Time-resolved long-lived luminescence image (7 min) following addition of 5 µM pyrithione
(zinc ionophore) and 50 µM ZnSO4 to the medium at 5 min. (d) Time-resolved long-lived luminescence image (17 min) following addition of 100 µM TPEN
to the medium at 15 min. Time-resolved long-lived luminescence images (b-d) correspond to the luminescence intensity data in (e), which shows the
average intensity of the corresponding area or cell area in (a) (1, extracellular region; 2, intracellular region of the injected cell; 3, 4, intracellular regions
of noninjected cells).
injected into a single cultured HeLa cell in the central part of
the field of view in Figure 4a,b. A prompt increase of
intracellular luminescence was induced when Zn2+ (50 µM) and
pyrithione (2-mercaptopyridine N-oxide, 5 µM), which is a zinc-
selective ionophore, were added to the medium at 5 min (Figure
4c). Further, the luminescence intensity decreased immediately
upon the extracellular addition of the cell-membrane-permeable
chelator TPEN (N,N,N′,N′-tetrakis(2-picolyl)ethylenediamine)
(100 µM) at 15 min (Figure 4d). Clear images of the intracellular
Zn2+ concentration changes were obtained with the TRLLM
system (Figure 4e). To explore further the utility of TRLLM,
we tested whether long-lived luminescence could be well
distinguished from the short-lived fluorescence of rhodamine
6G as an artificial source of short-lived background fluorescence.
Rhodamine 6G fluorescence directly interferes with Eu3+
luminescence because rhodamine 6G has excitation and emission
wavelength ranges similar to those of [Eu-7]. As a result, the
time-resolved long-lived luminescence images were not affected
at all by rhodamine 6G fluorescence (Figure S4, Supporting
Information); i.e., [Eu-7] could be used to monitor changes of
the intracellular Zn2+ concentration even in cells stained with
rhodamine 6G.
reagents for biological molecules. We have shown here that the
long-lived luminescence of luminescent lanthanide complexes
can be clearly distinguished from the short-lived background
fluorescence such as autofluorescence, present in most biological
systems as well as scattered excitation light, and even the strong
short-lived fluorescence of rhodamine 6G by using our TRLLM
system.
By using both luminescent Eu3+ and luminescent Tb3+
complexes, dual-color imaging was possible with our micro-
scope system. For example, it was possible to discriminate red
and green images of luminescent Eu3+ and Tb3+ complexes with
our microscope system using commercially available Cy3 and
FITC filters, while excluding short-lived fluorescence. More-
over, we could monitor intracellular Zn2+ concentration changes
in living cells by using the TRLLM system with our Zn2+
-
sensitive luminescent lanthanide sensor probe [Eu-7]. This paper
is the first to describe imaging of the concentration changes of
intracellular metal ion, including Zn2+, in living cells as time-
resolved long-lived luminescence images obtained with a
luminescent lanthanide sensor probe. The next step should be
the development of a range of superior long-lived luminescent
lanthanide sensor probes, which possess extremely high quantum
yields, long excitation wavelengths over 400 nm, and cell
permeability.31,32 Efforts are under way to develop new long-
lived luminescent lanthanide sensor probes which switch
between weak and strong luminescence in the absence and the
Conclusions
We have developed a new methodology, i.e., the TRLLM
technique with our new luminescent lanthanide complexes, for
fluorescence imaging in living biological samples such as live
cells. Time-resolved fluorescence imaging has been achieved
using other TRLLM systems and luminescent lanthanide
complexes, but these complexes were mostly used as labeling
(31) Yang, C.; Fu, L. M.; Wang, Y.; Zhang, J. P.; Wong, W. T.; Ai, X. C.;
Qiao, Y. F.; Zou, B. S.; Gui, L. L. Angew. Chem., Int. Ed. 2004, 43, 5010-
5013.
(32) Allen, M. J.; MacRenaris, K. W.; Venkatasubramanian, P. N.; Meade, T.
J. Chem. Biol. 2004, 11, 301-307.
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A R T I C L E S
Hanaoka et al.
presence of specific biological molecules, in parallel with efforts
to improve our TRLLM system. We consider that our TRLLM
imaging technique using luminescent lanthanide sensor probes
should be useful as a complement to, rather than a replacement
for, existing fluorescence imaging methodologies.
I ) I0 exp(-t/τ)
(2)
time t ) 0 and time t, respectively, and τ is the luminescence emission
lifetime. Lifetimes were obtained by monitoring the emission intensity
at 614 nm (λex ) 318 nm (R ) H), 320 nm (R ) F), 323 nm (R )
CH3), 330 nm (R ) CH3O), 325 nm (R ) NHAc)).
Determination of the Number of Water Molecules Bound to the
Inner Coordination Sphere of Eu3+. The number of coordinated water
molecules (q value) at the Eu3+ ion is determined by the following
equation:27
Experimental Section
Materials. DTPA bisanhydride was purchased from Aldrich Chemi-
cal Co. Inc. (St. Louis, MO). All other reagents were purchased from
either Tokyo Kasei Kogyo Co., Ltd. (Japan) or Wako Pure Chemical
Industries, Ltd. (Japan). All solvents were used after distillation. Silica
gel column chromatography was performed using BW-300 and Chro-
matorex-ODS (all from Fuji Silysia Chemical Ltd.).
q
Eu ) 1.2(τH O-1 - τD O-1 - 0.25)
(3)
2
2
1
Instruments. H and 13C NMR spectra were recorded on a JEOL
where τH O or τD O is the luminescence lifetime of the complex in H2O
2
2
or D2O, respectively.
JNM-LA300. Mass spectra were measured with a JEOL-T100LC
AccuTOF (ESI+ or ESI-). HPLC purification was performed on a
reversed-phase column (GL Sciences (Tokyo, Japan), Inertsil Prep-
ODS 30 mm × 250 mm) fitted on a Jasco PU-1587 system. Time-
resolved luminescence spectra were recorded on a Perkin-Elmer LS55
luminescence spectrometer (Beaconsfield, Buckinghamshire, England).
The slit width was 5 nm for both excitation and emission. A delay
time of 50 µs and a gate time of 1.00 ms were used. UV-vis spectra
were obtained on a Shimadzu UV-1650PC (Tokyo, Japan) or an Agilent
8453 (Agilent Technologies, Waldbronn, Germany) UV-vis spectros-
copy system. The fluorescence intensities without a delay time were
also recorded on a Perkin-Elmer LS55 luminescence spectrometer
(Beaconsfield). The slit width was 10 nm for both excitation and
emission.
Photobleaching of Eu3+ Complexes. Photobleaching of luminescent
Eu3+ complexes (R ) NHAc or CH3O) (40 µM) and fluorescein (1
µM) was performed in 100 mM HEPES buffer (pH 7.4) at 24 °C by
using a xenon lamp (UXL-500D-0, USHIO, Tokyo, Japan) as an
excitation source in an SM-5 system (Bunkoh-Keiki Co., Ltd., Tokyo,
Japan). The wavelength and the energy of the irradiation light were
325 nm and 1.57 mW for Eu3+ complexes and 492 nm and 2.07 mW
for fluorescein. The absorbance values of Eu3+ complexes (R ) NHAc
or CH3O) (40 µM) and fluorescein (1 µM) solution were 0.16 at 325
nm, 0.15 at 325 nm, and 0.08 at 492 nm, respectively. The fluorescence
intensities at 618 nm (for Eu3+ complexes) and 516 nm (for fluorescein)
were recorded on a Perkin-Elmer LS55 luminescence spectrometer
(Beaconsfeld) and plotted.
Comparative Singlet Oxygen Generation Measurements. Reaction
of 1,3-diphenylisobenzofuran (DPBF) with singlet oxygen was moni-
tored in terms of the reduction in the intensity of the absorbance at
410 nm. An aerated solution of a photosensitizer and DPBF (20 µM)
in 100 mM HEPES buffer (pH 7.4)/MeOH ) 1/1 was irradiated with
a xenon lamp (UXL-500D-0, USHIO) in an SM-5 system (Bunkoh-
Keiki Co., Ltd.) at 24 °C. Rose Bengal (1 µM), fluorescein (1 µM), or
a Eu3+ complex (R ) NHAc or CH3O) (100 µM) was used as a
photosensitizer. The wavelength and the energy of the irradiation light
were 555 nm and 3.0 mW for Rose Bengal, 492 nm and 3.09 mW for
fluorescein, and 325 nm and 2.55 mW for Eu3+ complexes (R ) NHAc
or CH3O). The UV-vis spectra were recorded on an Agilent 8453 UV-
vis spectroscopy system (Agilent Technologies). The absorbance of a
Rose Bengal solution (1 µM), a fluorescein solution (1 µM), and Eu3+
complex (R ) NHAc and CH3O) solutions without DPBF were 0.08
(at 555 nm), 0.07 (at 492 nm), 0.40 (at 325 nm), and 0.41 (at 325 nm),
respectively. Irradiation at 555 or 492 nm of a 20 µM DPBF solution
in 100 mM HEPES buffer (pH 7.4)/MeOH ) 1/1 produced no change
in the intensity of the 410 nm absorption band, which was derived
from DPBF. No changes in the absorbance spectra of the photosensi-
tizers were observed during the irradiation, indicating that the photo-
sensitizers were not photobleached in these experiments. However,
irradiation at 325 nm of a 20 µM DPBF solution without photosensitizer
produced a reduction in the intensity of the 410 nm absorption band of
DPBF. Therefore, the rates of oxygenation of a 20 µM DPBF solution
without photosensitizer in 100 mM HEPES buffer (pH 7.4)/MeOH )
1/1 on irradiation at 325, 555, or 492 nm were also measured as
background rates, and we subtracted the rates of oxygenation of a
solution of DPBF (20 µM) only from the rates of oxygenation of a
solution of the Eu3+ complex (R ) NHAc or CH3O), Rose Bengal, or
fluorescein containing DPBF (20 µM) when irradiated at 325 nm (for
Eu3+ complexes), 555 nm (for Rose Bengal), or 492 nm (for
fluorescein). Details are shown in the Supporting Information.
Preparation of Cells. HeLa cells were cultured in Dulbecco’s
modified Eagle’s medium (DMEM) (Invitrogen Corp., Carlsbad, CA),
supplemented with 10% fetal bovine serum (Invitrogen Corp.), 1%
penicillin, and 1% streptomycin (Invitrogen Corp.) at 37 °C in a 5%
CO2/95% air incubator. The cells were grown on an uncoated 35 mm
Time-Delayed Luminescence Spectral Measurements. The time-
delayed luminescence spectra of Eu3+ complexes (50 µM) were
measured in 100 mM HEPES buffer at pH 7.4, 24 °C (excitation at
318 nm (R ) H), 320 nm (R ) F), 323 nm (R ) CH3), 330 nm (R )
CH3O), 325 nm (R ) NHAc), and 352 nm (R ) NH2)). The slit width
was 5 nm for both excitation and emission. A delay time of 50 µs and
a gate time of 1.00 ms were used.
UV-Vis Absorption Spectrum Measurements. The absorption
spectra of Eu3+ complexes (50 µM) were measured at 24 °C in an
aqueous solution buffered to pH 7.4 (100 mM HEPES buffer). UV-
vis spectra in Figure 2b,c were recorded on a Shimadzu UV-1650PC.
Quantum Yield Measurements. The luminescence spectra were
measured with a Hitachi F4500 spectrofluorometer (Tokyo, Japan). The
slit width was 2.5 nm for both excitation and emission. The photo-
multiplier voltage was 950 V. The luminescence spectra of Eu3+
complexes (5 µM) were measured in 100 mM HEPES buffer at pH
7.4, 25 °C, with irradiation at 300 nm. The quantum yields of Eu3+
complexes were evaluated using a relative method with reference to a
luminescence standard, [Ru(bpy)3]Cl2 (φ ) 0.028 in air-equilibrated
water).26 The quantum yields of Eu3+ complexes can be expressed by
eq 1,33 where Φ is the quantum yield (subscript “st” stands for the
2
Φx/Φst ) [Ast/Ax][nx /nst2][Dx/Dst]
(1)
reference and “x” for the sample), A is the absorbance at the excitation
wavelength, n is the refractive index, and D is the area (on an energy
scale) of the luminescence spectra. The samples and the reference were
excited at the same wavelength (300 nm). The sample absorbance at
the excitation wavelength was kept as low as possible to avoid
fluorescence errors (Aexc < 0.03).
Luminescence Lifetime Measurements. The luminescence lifetimes
of the Eu3+ complexes were recorded on a Perkin-Elmer LS-55
luminescence spectrometer (Beaconsfield). The data were collected with
10 µs resolution in H2O and D2O and fitted to a single-exponential
curve obeying eq 2, where I0 and I are the luminescence intensities at
(33) Chen, Q. Y.; Feng, C. J.; Luo, Q. H.; Duan, C. Y.; Yu, X. S.; Liu, D. J.
Eur. J. Inorg. Chem. 2001, 1063-1069.
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A R T I C L E S
diameter glass-bottomed dish (MatTek, Ashland, MA) and washed twice
with Hank’s balanced salt solution (HBSS) buffer (Invitrogen Corp.),
and then the medium was replaced with HBSS buffer before imaging.
The Eu3+ complexes (2 or 10 mM) were dissolved in microinjection
buffer (HBSS buffer) and injected into cells with an Eppendorf injection
system (Transjector 5246).
Prompt Fluorescence Microscopy and Imaging Methods. The
imaging system consisted of an inverted microscope (IX71, Olympus)
with a cooled CCD camera (Cool Snap HQ; Roper Scientific, Tucson,
AZ). The microscope was equipped with a xenon lamp (AH2-RX,
Olympus), a 40× objective lens (UApo 40× oil/340, NA 1.35;
Olympus), and a dichroic mirror (420DCLP, Omega). The whole system
was controlled using MetaFluor 6.1 software (Universal Imaging,
Media, PA). The fluorescence microscope had an optical window
centered at 617 ( 37 nm (S617/73m, Chroma) for the emission due to
Eu3+-based luminescence upon excitation at 360 ( 40 nm (D360/40×,
Chroma).
TRLLM and Imaging Methods. For time-resolved long-lived
luminescence imaging we used the above conventional fluorescence
microscopy system with the following modifications, as shown in Figure
3a. The microscope was equipped with an auxiliary 50 Hz pulsed
excitation source (60 W xenon flash lamp (L6784), Hamamatsu
Photonics K. K., Shizuoka, Japan) triggered by a timing controller
(model 555-2C, BNC (Berkeley Nucleonics Corp.)). The delay time
and gate time were controlled by an I.I. and I.I. controller (C9016-02,
Hamamatsu Photonics K. K.) located in the emission light pathway
and synchronized with the xenon flash lamp using a timing controller.
The excitation light was delivered into a dual port (U-DPLHA;
Olympus, Tokyo, Japan) via an optical fiber (A7632, Hamamatsu
Photonics K. K.). The excitation source was easily switched from a
xenon flash lamp to a xenon CW lamp. The I.I. device can strongly
amplify the fluorescence signal. The amplified emission signal was
delivered via a relay lens (A4539, Hamamatsu Photonics K. K.) to a
cooled CCD camera (Cool SNAP HQ, Roper Scientific). Time-resolved
long-lived luminescence images were acquired with a 52 or 70 µs delay
time between the application of a pulse of excitation light to the sample
and the acquisition of the luminescence signal, while no delay was
used for the acquisition of prompt fluorescence images; to obtain a
good signal-to-noise ratio, the overall acquisition time for each image
was 1 s. The gate time, which is the acquisition time of the luminescence
signal, was 808 µs. Quantitative evaluation of the luminescence images
was done using the MetaFluor imaging analysis software package
(Universal Imaging Corp.).
Time-Resolved Long-Lived Luminescence Imaging of Intracel-
lular Zn2+ in Living Cultured HeLa Cells. A Zn2+-selective
luminescent Eu3+ chemosensor, [Eu-7] (2 mM), was dissolved in
microinjection buffer (HBSS buffer) and injected into living HeLa cells.
The time-resolved long-lived luminescence images were acquired every
30 s. A delay time of 70 µs and a gate time of 808 µs were used.
Rhodamine 6G Loading Conditions and Imaging Method. HeLa
cells were grown on an uncoated 35 mm diameter glass-bottomed dish
(MatTek, Ashland, MA) and washed twice with HBSS buffer (Invit-
rogen Corp.); then the cells were incubated with rhodamine 6G (4 µM)
in HBSS buffer for dye loading for 30 min at room temperature, which
was a sufficient time for intracellular accumulation of rhodamine 6G
judging from the fluorescence seen in the intracellular regions (Figure
S4b). The stained cells were washed twice with HBSS buffer, and the
medium was replaced with HBSS buffer before imaging. Then [Eu-7]
was injected into a cultured living HeLa cell in the same manner as
the above experimental method of time-resolved long-lived lumines-
cence imaging of intracellular Zn2+ in living cultured HeLa cells. The
fluorescence microscope had an optical window centered at 617 ( 37
nm (S617/73m, Chroma) for the emission due to rhodamine 6G-based
fluorescence upon excitation at 360 ( 40 nm (D360/40×, Chroma).
In time-resolved long-lived luminescence imaging, images with a delay
time of 70 µs were free of contributions from prompt fluorescence of
rhodamine 6G, while a delay time of 52 µs was not sufficiently long
to eliminate this prompt fluorescence from the time-resolved long-lived
luminescence images.
Acknowledgment. This work was supported by the Ministry
of Education, Culture, Sports, Science and Technology of Japan
(Grants for The Advanced and Innovational Research Program
in Life Sciences, 16370071 and 16659003 to T.N., 15681012,
17035019, 17036012, 017048006, and 17651119 to K.K.). T.N.
was also supported by the Hoh-ansha Foundation. K.K. was
also supported by the Sankyo Foundation, by the Kanagawa
Academy of Science, and by the Suzuken Memorial Foundation.
K.H. was the recipient of Research Fellowships of the Japan
Society for the Promotion of Science for Young Scientists.
Supporting Information Available: Detailed descriptions of
synthetic procedures for luminescent Eu3+ complexes and the
luminescent Tb3+ complex, photograph of solutions of lumi-
nescent Eu3+ complexes, photobleaching profiles of solutions
of Eu3+ complexes and fluorescein, comparative singlet oxygen
generation plots of Rose Bengal, fluorescein, and Eu3+ com-
plexes, and time-resolved long-lived luminescence images of
intracellular Zn2+ in HeLa cells which were stained with
rhodamine 6G. This material is available free of charge via the
Internet at http://pubs.acs.org.
Time-Resolved Dual-Color Imaging Methods. The synthesized
luminescent Eu3+ complex (R ) NHAc) (10 mM) or Tb3+-DTPA-
cs124 (2 mM) was dissolved in microinjection buffer (HBSS buffer)
and injected into cultured living HeLa cells in HBSS buffer. A delay
time of 70 µs and a gate time of 808 µs were used. An optical window
centered at 528 ( 19 nm (S528/38m, Chroma) was employed for Tb3+
-
based luminescence.
JA073392J
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