Near-infrared fluorescence lifetime pH-sensitive probes

Article abstract of DOI:10.1016/j.bpj.2011.02.050

We report what we believe to be the first near-infrared pH-sensitive fluorescence lifetime molecular probe suitable for biological applications in physiological range. Specifically, we modified a known fluorophore skeleton, hexamethylindotricarbocyanine, with a tertiary amine functionality that was electronically coupled to the fluorophore, to generate a pH-sensitive probe. The pK_a of the probe depended critically on the location of the amine. Peripheral substitution at the 5-position of the indole ring resulted in a compound with pK_a ～ 4.9 as determined by emission spectroscopy. In contrast, substitution at the meso-position shifted the pK_a to 5.5. The resulting compound, LS482, demonstrated steady-state and fluorescence-lifetime pH-sensitivity. This sensitivity stemmed from distinct lifetimes for protonated (～1.16 ns in acidic DMSO) and deprotonated (～1.4 ns in basic DMSO) components. The suitability of the fluorescent dyes for biological applications was demonstrated with a fluorescencelifetime tomography system. The ability to interrogate cellular processes and subsequently translate the findings in living organisms further augments the potential of these lifetime-based pH probes.

Full text of DOI:10.1016/j.bpj.2011.02.050

Biophysical Journal Volume 100 April 2011 2063–2072
2063
Near-Infrared Fluorescence Lifetime pH-Sensitive Probes
Mikhail Y. Berezin,† Kevin Guo,† Walter Akers,† Ralph E. Northdurft,† Joseph P. Culver,† Bao Teng,§
Olga Vasalatiy,§ Kyle Barbacow,§ Amir Gandjbakhche,{ Gary L. Griffiths,§ and Samuel Achilefu†‡*
†Department of Radiology and ‡Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, St. Louis,
Missouri; and §Imaging Probe Development Center, National Heart, Lung, and Blood Institute and {Physical Biology Program, Eunice Shriver
National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Washington, District of Columbia
ABSTRACT We report what we believe to be the first near-infrared pH-sensitive fluorescence lifetime molecular probe suitable
for biological applications in physiological range. Specifically, we modified a known fluorophore skeleton, hexamethylindotricar-
bocyanine, with a tertiary amine functionality that was electronically coupled to the fluorophore, to generate a pH-sensitive
probe. The pKa of the probe depended critically on the location of the amine. Peripheral substitution at the 5-position of the indole
ring resulted in a compound with pKa ~ 4.9 as determined by emission spectroscopy. In contrast, substitution at the meso-posi-
tion shifted the pKa to 5.5. The resulting compound, LS482, demonstrated steady-state and fluorescence-lifetime pH-sensitivity.
This sensitivity stemmed from distinct lifetimes for protonated (~1.16 ns in acidic DMSO) and deprotonated (~1.4 ns in basic
DMSO) components. The suitability of the fluorescent dyes for biological applications was demonstrated with a fluorescence-
lifetime tomography system. The ability to interrogate cellular processes and subsequently translate the findings in living
organisms further augments the potential of these lifetime-based pH probes.
INTRODUCTION
pH-sensitive fluorescent probes are widely used to report the
acidic status of a variety of cellular compartments (1). This
approach has been applied to in vivo imaging of diseases
associated with elevated acidity level (2,3). High acidity
levels (acidemia and acidosis) have been implicated in
a number of systemic pathologies (4), such as renal acidosis,
metabolic disorders, intoxications, diabetes, and emphy-
sema, as well as localized hyperacidity such as infections,
inflammation, and cancer. In particular, solid tumors with
pH ranging from 5.8 to 7.7 (5,6) on average are 0.5 units
lower than the pH of normal tissue due to a high level of
anaerobic metabolism. The tight control of intracellular
pH requires removal of the excess acidic products from
tumor cells to the extracellular-extravascular space due to
the poor lymphatic drainage in cancer tissue (7). This signif-
icant difference in pH between normal and surrounding
provides a unique opportunity to image the metabolic status
of cancerous tissue using optical imaging with a suitable pH
indicator. To be used in vivo, the indicator should preferably
be optically active within a relatively narrow near-infrared
(NIR) region (700–900 nm) and possess a pKa within phys-
iologically relevant range, which we loosely defined
between 4.8 and 7.8 (8). Interest in molecular probes with
these features has recently spurred the synthesis of novel
NIR pH indicators that have had success in vitro, with
potential for in vivo imaging applications (9–16).
The prevalent approach utilizes substantial enhancement
or quenching of fluorescence intensity or a shift of excita-
tion and/or emission wavelength peak that responds maxi-
mally at the pH close to a probes’ pKa value. However,
fluorescence intensity measurements are difficult to quantify
in heterogeneous tissue and recorded intensity changes may
be due to a concentration gradient instead of differences in
tissue pH values. Spectral shifts are more reliable and quan-
titative in microscopy but developing NIR fluorescent
probes with pH-induced distinct spectral shift between
700 and 900 nm represents a synthetic challenge. Moreover,
fluorescence intensity measurements for such molecular
systems are also subject to wavelength-dependent heteroge-
neities in biological tissues.
In contrast to intensity, the fluorescence lifetime of
a probe is largely independent from concentration and has
been introduced as in vivo optical imaging modality to
overcome the problem of concentration dependence and
also enhance contrast and alleviate some other typical prob-
lems associated with intensities including scattering (17),
sample turbidity, and autofluorescence (18–20). Recently,
several articles describing lifetime pH-sensitive probes
such as quantum dots (21), fluorescent proteins (22), and
the mechanism of their sensing have been published.
Despite the advantages of fluorescence lifetime and the
long lifetime appreciated by microscopists in cell imaging,
several limitations hinder the broad acceptance of the
method for in vivo imaging. In the past, the lack of correla-
tion between fluorescence lifetime of the probes and the
tissue environment, engineering challenges inherent to
deep tissue imaging, and long acquisition and data process-
ing time have hampered the recognition of fluorescent life-
time imaging as an in vivo method. Recently, significant
There are several different mechanisms by which a fluo-
rescent probe can indicate a change in environmental pH.
Submitted December 28, 2010, and accepted for publication February 23,
2011.
*Correspondence: achilefus@mir.wustl.edu
Editor: David E. Wolf.
Ó 2011 by the Biophysical Society
0006-3495/11/04/2063/10 $2.00
doi: 10.1016/j.bpj.2011.02.050

2064
Berezin et al.
advances in the design of fluorescent-environment-sensitive
lifetime probes (23–25), high-speed electro-optic instru-
mentation (26), and fast postacquisition time (27) have
turned fluorescence lifetime in vivo imaging into a practical
tool.
MATERIALS AND METHODS
Steady-state optical measurements
Absorption spectra were recorded on a DU 640 UV-visible spectropho-
tometer (Beckman-Coulter, Brea, CA) and fluorescence spectra were re-
corded on
a Fluorolog-3 spectrofluorometer (Horiba Jobin Yvon,
Herein, we report the design and synthesis of NIR fluores-
cence lifetime pH-sensitive fluorophores and lifetime pH
imaging using diffuse optical fluorescence lifetime tomog-
raphy. We demonstrate that two NIR fluorophores, LS479
and LS482 (Fig. 1), have marked pH-sensitivity through
steady-state and fluorescence lifetime measurements. The
pH sensitivity of LS479, initially synthesized as a fluoro-
genic metal chelator (28), was found by serendipity while
studying the pH stability of metal complexes. Although
LS479 was pH-sensitive, its low pKa value led to the
synthesis of LS482, which has a higher pKa value in the
physiologically important range. Moreover, LS482 ex-
hibited a large variance in lifetime between acidic and basic
forms, providing a foundation for pH lifetime imaging.
Using fluorescence lifetime diffuse optical tomography
(26), we demonstrate that phantoms composed of LS482
at three different pH values, implanted in a mouse, have life-
times similar to those obtained from in vitro measurements.
Thus, the combination of a pH-sensitive fluorescence life-
time molecular probe and a fluorescence lifetime diffuse
optical tomography (FL-DOT) system allowed us to estab-
lish the foundation for the practical application of fluores-
cence lifetime pH imaging in the NIR region.
Edison, NJ). For LS479 and LS482, excitation was at 675 nm, with emis-
sion scan from 690 to 950 nm. All fluorescence measurements were con-
ducted at room temperature. For titrations, the compounds were
predissolved in dimethylsulfoxide (DMSO) and added to water (30 mL)
so the total concentration of the dye did not exceed 0.2 absorbance units.
Titration was conducted with stirring under Ar atmosphere to exclude
CO2 interference. The solution was initially basified with dilute aqueous
NaOH and the desired pH was attained by titrating the solution with
diluted aqueous HCl. The acidification of the solution was conducted at
relatively constant ionic strengths (I ¼ 0.1 M provided by 0.1 M NaCl
in water). The pH of the solution was continuously measured using an
Accumet pH meter AB15 (Fisher Scientific, Pittsburgh, PA) equipped
with a liquid-filled, Ag/AgCl reference pH/ATC double-junction combina-
tion Accumet electrode.
At each pH point, absorption and fluorescent measurements were deter-
mined. The pKa values were calculated from the sigmoidal dose-response
curve fit implemented in software Prism 5.0 (GraphPad Software, La
Jolla, CA). Contour plots of fluorescence intensity versus excitation wave-
length versus emission wavelength of the dye solutions at different pH
were generated with excitation scanning from 560 to 780 nm with
2 nm intervals. The simultaneous changes in emission from 680 to
880 nm with 1 nm intervals at pH ¼ 3.0 and 8.0 were recorded and
the data were plotted using SigmaPlot 11.0 software (Systat Software,
Chicago, IL). Quantum yields at different representative pH were
measured relative to indocyanine green in DMSO (V ¼ 0.12) at
700 nm excitation (29).
Titrations using fluorescence lifetime
measurement
Fluorescence lifetimes (FLT) were measured using time-correlated
single-photon-counting (Horiba) with
a 700-nm excitation source
NanoLed (impulse repetition rate 1 MHz) at 90ꢀ to the detector (Hama-
matsu Photonics, Hamamatsu City, Japan). For the FLT measurements,
the dyes were dissolved in water and the absorbance of the measured
solutions was maintained below 0.15 at a 700-nm excitation wavelength.
The detector was set to 780 nm with a 26-nm bandpass and data
collected until the peak signal reached 10,000 counts. The FLT was re-
corded on a 50-ns scale. The instrument response function was obtained
using a Rayleigh scatter of Ludox-40 (0.05% in Milli-Q water; Sigma-
Aldrich, St. Louis, MO) in an acrylic transparent cuvette at 700-nm emis-
sion. Decay analysis software (DAS6 v6.1; Horiba) was used for lifetime
calculations. The goodness of fit was judged by c2 values, Durbin-Wat-
son parameters, as well as visual observations of fitted line, residuals,
and autocorrelation functions. For titrations, the dyes were dissolved in
DMSO and acidified with dilute trifluoroacetic acid (TFA) or basified
with dilute triethylamine (TEA). The resulting DMSO solutions were
diluted with 0.1 M NaCl aqueous solution (1:1 vol), the pH of the solu-
tion was measured (pHm) and corrected for dilution using the following
equation:
pH ¼ pHm ꢁ log102:
FIGURE 1 Structures of NIR dyes used in this study. LS479 is an HITC
analog with DTPA conjugated to the indole group while LS482 has DTPA
conjugated to the meso position.
Synthesis of pH-sensitive probes
Synthesis of LS479 was described in a previous publication (28).
Biophysical Journal 100(8) 2063–2072

Fluorescence Lifetime pH Probes
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SCHEME 1
For the preparation of LS482, benzylamine was N-alkylated with the
bromo derivative 1 (see Scheme 1) using n,n-diisopropylethylamine as
a base in acetonitrile to obtain Compound 2. Intermediate 3 was prepared
by removal of the benzyl protective group by hydrogenolysis with
hydrogen and 10% palladium on carbon in methanol. N-alkylation of
Specifically, we used an analytical solution to the diffusion equation that
incorporated absorption and scattering in the light model (33).
Lifetime tomography imaging
N,N00-tetrakis(t-butyloxycarbonylmethyl)-diethylenetriamine
3 with IR
Male nude mice 6–12 weeks in age were used for in vivo imaging.
Before scanning, each mouse was anesthetized with a mixture of ketamine
(87 mg/kg) and xylazine (13 mg/kg) via intraperitoneal and subcutaneous
injection for initial and sustained sedation, respectively. The mouse was
then suspended vertically within the fluid-filled imaging cassette for the
duration of the scan, as previously described. LS482 in acidic (DMSO-
TFA), neutral (neat DMSO), and basic (DMSO-TEA) solutions were loaded
into thin-walled sealed straws 3-mm in diameter and 10-mm long. Dye
concentration was selected so that all three tubes had comparable fluores-
cence emission at 830 nm. The tubes were implanted ~1 mm below the
skin surface in the subcutaneous space via a 5-mm incision on the dorsal
aspect of each flank. Skin incisions were closed with tissue adhesive (Nex-
abond; Abbott Animal Health, Chicago, IL). Twenty-four time gates over
a 20 ꢂ 3 source grid with 2-mm separation were imaged, covering a total
area of 4.0 cm ꢂ 0.6 cm. The acquisition time for the entire set of data
was ~5 min for excitation and 74 min for emission.
786 perchlorate using n,n-diisopropylethylamine as a base in anhydrous
dimethylformamide provided Compound 4. The yield of the reaction is
somewhat moderate (10%), which might be attributed to steric hindrance
around the secondary amine, as compared to the higher reaction yields
seen with a primary amine (76%) (30). Isolation of Compound 4 from
the reaction mixture was performed by preparative high-performance
liquid chromatography. Removal of t-butyl protective groups under stan-
dard conditions using TFA gave target LS482. Details of the synthesis
are given in the Supporting Material.
Fluorescence-lifetime tomography system
Animal studies were conducted on a recently developed temporally
resolved fluorescence tomography system (26). Briefly, a pulsed Ti:S laser
source with spectral filtering (80 MHz, 785 nm, <100-fs pulse width,
60 mW) was used for illumination. The beam, after passing through the
focusing lens, was steered by a pair of galvanometer scanning mirrors to
the source side of an imaging cassette. Light emitted from the detector
plane of the imaging cassette was collected by a lens and temporally gated
by an ultrafast gated image intensifier and detected by an electron-multi-
plying charge-coupled device camera for read-out. Optional filters in front
of the lens allow acquisition of either the transmitted excitation or fluores-
cence emission (832 5 10 nm). Timing between the source and the gated
detection was controlled by a programmable delay unit allowing a series of
images to capture the light emission as a function of time. A scan resulted in
a temporal profile of transmitted excitation and stimulated emission at each
detector location. From these data, we reconstructed a map of fluorescence
yield and lifetime simultaneously via a normalized Born approach (31,32).
RESULTS
In vitro studies
pH-dependent optical probe LS479
Our initial efforts to develop an NIR lifetime probe focused
on pH-sensitive properties of the previously described
LS479 (28), a fluorescent dye with a covalently linked metal
chelating group (Fig. 1). The probe showed low toxicity in
mice, even at a relatively large dose of 15 mg/kg body
Biophysical Journal 100(8) 2063–2072

2066
Berezin et al.
weight. Previous studies showed that the molecular probes
do not have observable side effects in rodents during the
two-month study period (34). LS479 is structurally similar
to a known dye 1,10,3,3,30,30-hexamethylindotricarbocya-
nine (HITC), which is widely used in analytical chemistry
and biological optical imaging studies (35,36). The
chelating group of LS479 was derived from diethylenetria-
minepentaacetic acid (DTPA). A characteristic feature of
LS479 compared to HITC is the presence of an amine func-
tionality electronically coupled with the fluorophore. The
electron pair on the amine is known to affect fluorescence
and a number of pH-sensitive compounds, including NIR
dyes, have utilized amino groups as fluorescence intensity
pH-sensitive sensors (11,37–39).
Indeed, the steady-state titration absorbance and fluores-
cence spectra (Fig. 2, A and B) showed that LS479 was
pH-sensitive. A similar analysis of HITC is described below
and in the Supporting Material. An isosbestic point at
575 nm in the absorption spectra (Fig. 2 A) suggests the
presence of protonated and deprotonated forms (40). Under
basic (pH > 7) conditions, the absorption band exhibited
a moderate increase in peak intensity of ~25% from pH 3
to pH 8. In contrast, the fluorescence intensity increased
substantially (Fig. 2 B), indicating that the deprotonated
form of LS479 was more fluorescent than the protonated
form (see Table 1 for quantum yields). A plot of intensity
as a function of excitation and emission wavelength (con-
structed by scanning excitation wavelengths) demonstrated
the presence of only one fluorescent form (Fig. 3). Acid
dissociation constants (pKa), computed from titration
diagrams, were generated from acid-base absorption and
steady-state emission experiments (Fig. 2 C). The pKa in
the ground state was found to be 4.45 5 0.097 while the
pKa in the excited state was found to be 4.93 5 0.099, sug-
gesting moderate photobasicity (41,42). Although the pKa
of the ground state is lower than the pH of most cells, the
excited state pKa is at the fringe of lysosomal pH of 4.8.
However, these pH values narrows down the potential,
in vivo, application of LS479 to extremely low pH organ-
elles of cells.
The pH dependence of the probes was investigated in
dilute solutions (<100 mM). At higher concentrations,
a number of nonlinear optical effects, such as quenching
due to aggregation, inner filter effect, and reabsorption can
occur, causing a significant change in the emission lifetimes
(43). In a biological system, however, the aggregation is
minimal due to a variety of opsonization factors and the rela-
tively low concentration of molecules injected into animals.
To expand the application to other cellular compartments
and less acidic tissue environments, we explored the effect
of pH on the fluorescence lifetime of LS479. We measured
the lifetime in DMSO rather than in aqueous solutions. The
use of DMSO as a solvent simulates in vivo environment
more realistically than water because DMSO polarity is
closer to that of blood and tissues (44). Hence, the lifetime
of LS479 was measured in neat, acidified, and basified
A
B
0.12
0.10
0.08
0.06
0.04
0.02
0.00
pH increase 3.14-7.7
70000
60000
50000
40000
30000
20000
10000
0
pH increase 3.14-7.7
FIGURE 2 Absorption (A) and emission (B)
titration spectra of LS479 in 0.1 M NaCl aqueous
500
700
900
680
780
880
solutions, excitation/emission: 675:690–950 nm.
(C) Titration diagrams of LS479 were generated
from fluorescence and absorbance spectra. Acid
dissociation constants were computed with the
software Prism (GraphPad Software): pKa in the
ground state from the absorbance data is 4.45 5
0.097 and pKa* in the excited state from the emis-
sion data is 4.93 5 0.099. R values were >0.99.
Desired physiological range is shown in gray.
Wavelength, nm
Wavelength, nm
C
0.12
0.10
0.08
0.06
0.04
0.02
0.00
7.00E+04
6.00E+04
5.00E+04
4.00E+04
3.00E+04
2.00E+04
1.00E+04
0.00E+00
absorbance
fluorescence
3
4
5
6
7
8
pH
Biophysical Journal 100(8) 2063–2072

Fluorescence Lifetime pH Probes
2067
TABLE 1 Photophysical characteristics of studied dyes in
aqueous solutions (excitation/emission: 700:715–950 nm)
to the same set of steady-state and lifetime titration experi-
ments as LS479 and the results are given in Figs. 3 and 4 as
well as Tables 1 and 2. We found that LS482 displayed
pH-sensitivity in all three categories: absorption, fluores-
cence intensity, and—most importantly—fluorescence life-
time. The changes upon protonation-deprotonation were
reversible (Fig. S1 in the Supporting Material), indicating
chemical stability of both forms.
Similar to LS479, the absorbance and fluorescence of
LS482 decreased under acidic conditions and amplified
(ꢂ10) at higher pH. Compared to LS479, the meso-
substituted LS482 exhibited more intense fluorescence.
The brightness of LS482, measured as a product of molar
absorptivity and quantum yield (3 ꢂ j), was 2.5–4.6 times
higher than LS479 at corresponding pH values based on
data from Table 1. Higher brightness is beneficial in optical
imaging in vivo because it allows for lower dosage of a probe
and the use of lower laser output. Analysis of the titration
spectra (for the ground and excited states diagram; see
Fig. 4 C) revealed that the acid dissociation constant of
LS482 was shifted toward a more physiologically relevant
range with ground (pKa) and the excited (pKa*) states pKa
of ~5.5.
labs max,
nm
3 ꢂ 10ꢁ3
,
lem max,
nm
Entry
pH*
Mꢁ1 cmꢁ1y
jz
LS479
3
8
3
8
3
8
750
747
760
710
736
736
83
105
105
84
762
762
760
793
771
771
0.008
0.042
0.037
0.101
0.113
0.106
LS482
HITC
250
250
*Buffers, pH
3 and pH 8 (Hydrion; Micro Essential Laboratory,
Brooklyn, NY).
yMolar absorptivities at the absorption maximum.
zQuantum yield measured relative to indocyanine green in DMSO
(j ¼ 0.12).
DMSO. Fluorescence decays in neat and basified DMSO
solutions were largely monoexponential (>96% of one
component from two-exponential fit) and exhibited similar
calculated fluorescence lifetimes of 0.77–0.78 ns (Table
2). Unfortunately, we were not able to determine the fluores-
cence lifetime at lower pH accurately because of a very
weak fluorescence signal below the pKa level.
In contrast to LS479, LS482 exhibited absorption (50 nm)
and fluorescence (33 nm) bathochromic shifts upon acidifi-
cation, suggesting the presence of two distinct fluorescence
species, as illustrated by emission versus excitation contour
plots (Fig. 3). The shift in spectra indicated a prominent
alteration in chromophore structure upon protonation. Fluo-
rescence lifetime measurements at different pH levels in
LS482 as a pH-dependent optical probe
with biologically suitable pKa
To improve the low pKa of LS479 and overcome potential
problems associated with low fluorescence signal of proton-
ated species, we synthesized LS482, where DTPA was
placed in the meso position. This compound was subjected
LS479, pH 3
LS479, pH 8
750
700
650
600
750
700
650
600
FIGURE 3 Contour plots representa-
tions emission versus excitation of
0.0
3.0e+5
6.0e+5
9.0e+5
1.2e+6
1.5e+6
1.8e+6
2.1e+6
2.4e+6
0
LS479 and LS482 in two different
aqueous media, acidic (pH ¼ 3) and
1e+5
2e+5
3e+5
4e+5
5e+5
6e+5
basic (pH ¼ 8). The intensity of the
fluorescence signals are shown in
pseudo colors in the online version.
700
750
800
850
700
750 800
Emission, nm
850
Note that the scale for LS479 at pH 3
is four-times higher for better visualiza-
tion. Excitation was scanned within
560–780 nm range with 2 nm steps;
emission was collected from 680 to
880 nm with 1-nm intervals. Rayleigh
signals were digitally removed (a stripe
20-nm-wide corresponding to Rayleigh
scattering is seen in upper-left corner of
each plot).
Emission, nm
LS482, pH 3
LS482, pH 8
750
700
750
700
.
2.0e+4
0.0
2.0e+4
4.0e+4
6.0e+4
8.0e+4
1.0e+5
1.2e+5
1.4e+5
1.6e+5
1.8e+5
4.0e+4
6.0e+4
8.0e+4
1.0e+5
1.2e+5
1.4e+5
1.6e+5
1.8e+5
600
600
700
750 800
Emission, nm
850
700
750 800
Emission, nm
850
Biophysical Journal 100(8) 2063–2072

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Berezin et al.
TABLE 2 Fluorescence lifetime of LS482 and HITC in DMSO
solvent of differing acidity (excitation/emission: 700:780 nm,
two-exponential fit)
time titration curve (Fig. 4 D) and estimate the pKa*t solely
based on the fluorescence lifetime. The determined pKa*t ¼
5.39 was similar to the pKa values determined by steady-
state methods.
~
Entry
pH
Species tAH, ns fAH, % tA, ns fA, % t, ns c2
3.70*
AH
n/dy
LS479 4.95
8.33
3.94
AHþA
0.78
0.77
1.18
1.25z
—
96.8
97.0
98. 1
98.1
—
—
—
—
—
—
0.78 1.35
0.77 1.29
1.18 1.25
1.25 1.33
A
AH
AHþA
A
—
—
Fluorescence lifetime tomography
Having established that LS482 possesses lifetime pH-sensi-
tive properties, we tested the feasibility of measuring
pH-dependent lifetime using previously reported time-
resolved diffuse optical tomography, which can simulta-
neously measure fluorescence intensity (yield) and lifetime
(26). Due to the differential phase method, the system was
able to measure short fluorescence lifetimes typical
for NIR cyanine probes 0.35–2 ns with high precision
(55 ps).
To demonstrate the feasibility of measuring fluorescence
lifetimes at different pH levels in animals, we adopted a
known approach of implanting phantoms into mice
(14,26). Following this approach, we encapsulated solutions
of LS482 in aqueous DMSO at pH 2.3, 5.7, and 8.1. The
capsules were placed in the hindquarters of mice. The
results of the imaging conducted in intensity and lifetime
modes are shown in Fig. 5, A and B, correspondingly. The
intensity map revealed that three phantoms had no func-
tional information regarding the local milieu. In contrast,
fluorescence lifetime imaging clearly indicated the presence
LS482 5.52
8.60
2.50
—
1.39
98.3 1.39 1.31
A
—
—
—
—
1.43 101.5 1.43 1.06
1.42 100.0 1.42 1.08
1.42 101.2 1.42 1.08
HITC
5.30
11.5
A
A
—
—
*The pH of DMSO solution was determined after diluting it with 0.1 M
NaCl(aq) (1:1 vol).
yFluorescence intensity in acidic DMSO was too low for lifetime measure-
ment.
zThe values correspond to a mixture of protonated AH and deprotonated
A forms.
DMSO solution confirmed the two components with signif-
icantly different lifetimes of ~1.16 ns for protonated (AH,
pH < 4) and ~1.40 ns (A, pH > 8) for nonprotonated
species. As expected, the structurally similar HITC, which
lacks an amine-coupled chromophore system, did not
show any lifetime pH sensitivity (Table 2).
The fluorescence of both protonated and deprotonated
forms of LS482 allowed us to construct a fluorescence life-
A
pH increase 3.14-7.7
0.07
0.06
0.05
0.04
0.03
0.02
0.01
0.00
pH increase 3.3-8.8
300000
250000
200000
150000
100000
50000
0
B
FIGURE 4 Absorption (A) and emission (B) spectra of
LS482 in 0.1 M NaCl aqueous solutions at varying pH,
excitation/emission: 675:690–900 nm. Steady-state titra-
tion diagram (C) was generated from absorption and emis-
500
700
900
680
780
Wavelength, nm
880
Wavelength, nm
sion spectra. Fluorescence lifetime titration diagram (D)
was generated from measuring fluorescence lifetime of
LS482 in acidic, neat, and basic DMSO. (Calculated
pKa: from absorption pKa ¼ 5.47 5 0.053, R2 ¼ 0.99,
from emission pKa* ¼ 5.46 5 0.059, R2 ¼ 0.99, from
lifetime pKa,*t ¼ 5.39 5 0.09, R2 ¼ 0.98.) Prism 5.0
(GraphPad Software) was used to determine the pKa. Phys-
iological range is shown in gray.
0.07
0.06
0.05
0.04
0.03
0.02
0.01
0.00
300000
250000
200000
150000
100000
50000
0
C
D
1.40
1.35
1.30
1.25
1.20
1.15
absorbance
fluorescence
3
4
5
6
7
8
2
3
4
5
6
7
8
9
pH
pH
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Fluorescence Lifetime pH Probes
2069
the fluorophore lifetime. In a previous study, we demon-
strated that in vivo lifetime measurements of NIR fluoro-
phores were within 10% of their values in solution (26).
DISCUSSION
Mechanism of pH sensitivity
At extreme pH environments, cyanine dyes typically display
pH-sensitivity through large spectral shifts but these
extremes are not valuable for biological imaging. Recent
studies have shown that some cyanine dyes can exhibit
significant changes in fluorescence intensity as a function
of pH but we are not aware of studies reporting the develop-
ment and use of NIR fluorescent lifetime pH-sensitive dyes.
To delineate the contribution of cyanine structural frame-
work to the observed pH-response of both LS479 and
LS482, we analyzed the pH-dependent properties of their
parent dye, HITC (Fig. 1). The three dyes share the same
hexamethylindotricarbocyanine skeleton responsible for
their NIR fluorescence.
However, the HITC structure does not have similar acid-
base sensitive functionalities or solvation centers as LS479
and LS482. Consequently, it is not expected to be pH-sensi-
tive within a physiologically relevant range. Indeed, the
pH-sensitivity of HITC was negligible in the NIR region
(Fig. S2, A and B). This resulted in a marginal change in
quantum yield (Table 1). Consequently, the fluorescence
decays of HITC at three different media (acidic, neutral,
and basic DMSO) remained primarily monoexponential
(~100% of one component from a two-exponential fit)
with the same lifetime 1.42–1.43 ns (Table 2). The lack of
pH-sensitivity by HITC supports the hypothesis that the
presence of amine functionality on LS479 and LS482
conferred steady-state fluorescence and, in the case of
LS482, lifetime pH-sensitivity.
FIGURE 5 Fluorescence lifetime tomography imaging of three phan-
toms (Ac, acidic; N, neutral; and B, basic) implanted into a mouse, excita-
tion/emission: 785:830 nm. A vertical slice from the DOT reconstructed
yield (Panel A) and lifetime (Panel B) is overlaid here on a white-light
image of the mouse. A threshold of 50% of local maximum has been
applied to isolate each tube location. Average lifetimes for the targets are
0.95 ns (Ac, acidified DMSO), 1.25 ns (N, neat DMSO), and 1.46 ns (B,
basified DMSO).The fluorescence lifetime of the signals are shown in
pseudo colors in the online version.
of three different lifetimes, suggesting three distinct envi-
ronments (Fig. 5 B). Quantitative analysis of lifetime bands
for each phantom showed good agreement with in vitro
results (Table 2). The lifetime of the acidic phantom, deter-
mined tomographically, was shortest at ~0.96 ns, followed
by an intermediate lifetime of ~1.25 ns in neat DMSO
(pH ¼ 5.7), and ~1.46 ns in the basic tube.
Previously, we reported that complexation of Zn2þ to
LS479 significantly enhanced the fluorescence quantum
yield of the fluorophore. We explained the enhancement
through an excited-state charge transfer (ESCT) mechanism
(46). According to the proposed mechanism, Zn2þ acts as
a Lewis acid and attracts a lone pair, suppressing ESCT.
Protonation of the amine group was also expected to suppress
ESCT and enhance fluorescence (14,47,48). However, the
opposite trend was actually observed: protonation of
LS479 and LS482 caused significant reduction in quantum
yields and, in the case of LS482, in the fluorescence lifetime.
Similar effect was also noticed by Peng et al. (38), although it
was not clear whether that was due to fluorescence quenching
or change in absorbance. Preliminary computational analysis
(not shown here) revealed that upon nitrogen protonation in
LS479 and LS482, the negative charge on the nitrogen
atom in the ground state becomes larger, suggesting that
the electron pair migrates closer to nitrogen. Enlarged elec-
tron density could participate in ESCT and effectively
To determine the lifetimes, we modeled light propagation
using the diffusion equation, explicitly taking into account
the background scattering and absorption. The reconstruc-
tion process essentially assessed the amount of light (yield
dominant) and phase delay (lifetime dominant) in each
measurement and from a group of such measurements,
determined the most likely three-dimensional map of fluoro-
phore properties. At the regions where the tubes were placed
in the mouse, we assumed homogeneous tissue optical prop-
erties (45). Although this approach produced a reasonable
lifetime map, incorporation of the local map of heteroge-
neous tissue optical properties would more accurately
delineate the in vivo lifetimes. As long as the estimates of
optical properties are correct, the reconstruction method
used in this study deconvolves the scattering process from
Biophysical Journal 100(8) 2063–2072

2070
Berezin et al.
suppress fluorescence. The computational work is currently
under way and will be reported separately.
Thus, for a protolytic reaction, the concentrations of the
protonated and deprotonated forms in the excited state are
Due to the presence of metal chelating group, the
compounds LS479 and LS482 might not be suitable for
in vivo imaging applications of tissues with substantial
level of metals. Chelation is likely to affect the pKa of the
dyes; for example, LS479-metal complexes are insensitive
to the changes in pH in physiological range (data are not
shown). Hence, the design of the probes has to be further
tuned to eliminate potential complexation of the dyes with
metals.
½AHꢃꢄð3AH ꢂ jAH
Þ
fAH
¼
;
(3)
(4)
½AHꢃꢄð3AH ꢂ jAHÞ þ ½Aꢃꢄð3A ꢂ jAÞ
½AHꢃꢄð3A ꢂ jAÞ
;
fA
¼
½AHꢃꢄð3AH ꢂ jAHÞ þ ½Aꢃꢄð3A ꢂ jAÞ
where 3AH and 3A are molar absorptivity of protonated and
deprotonated forms at the wavelength of excitation, and
jAH and jA are fluorescence quantum yields of protonated
and deprotonated forms.
Dividing Eq. 4 by Eq. 3, we have
Strategies for developing lifetime
pH-sensitive probe
fAH
fa
½AHꢃꢄð3AH ꢂ jAH
½Aꢃꢄð3A ꢂ jAÞ
Þ
Þ
¼
:
:
(5)
(6)
(7)
For a compound to be a lifetime pH-sensitive probe, certain
criteria have to be met:
Rearranging of Eq. 5 gives:
1. The fluorescence has to be pH-sensitive, requiring the
formation of two emission species.
2. The fluorescence lifetime of protonated and deproto-
nated species have to be distinct.
3. The lifetime should not be affected by other species, like
metals.
4. Both protolytic forms must emit in the same range,
ideally with almost complete spectral overlap.
½AHꢃꢄ
½Aꢃꢄ
fAHð3AH ꢂ jAH
¼
fað3A ꢂ jAÞ
Combining Eqs. 1 and 6:
ꢀ
ꢁ
3A ꢂ jA
fAH
fA
pH ¼ pKꢃa ꢁ log10
ꢂ
:
3AH ꢂ jAH
The measured fluorescence lifetime ~t from a two-exponen-
tial fit could be expressed by Eq. 8, as
Although the former three requirements are obvious, the
last one necessitates an additional explanation.
Consider the Henderson-Hasselbach equation (49) for
a pH-sensitive fluorophore formed as a result of a protolytic
reaction in the excited state,
~
t ¼ fAtA þ fAHtAH; fA þ fAH ¼ 1;
(8)
where fA and fAH are the fractional contributions of the
deprotonated and protonated forms, tA and tAH are the
fluorescence lifetimes for each emitting state.
It can readily be shown from Eq. 8 that the ratio of the two
fractional contributions can be expressed as given in Eq. 9:
ꢁH4
AHꢃ
Aꢃ;
þH4
ꢀ
ꢁ
½AHꢄꢃ
pH ¼ pKꢃa ꢁ log
;
(1)
~
fAH
fA
t ꢁ tA
½Aꢄꢃ
¼
:
(9)
~
tAH ꢁ t
where [AH]* is the concentration of a protonated form in the
excited state, [A]* is the concentration of a deprotonated
form in the excited state, and pKa* is the acid dissociation
constant in the excited state.
The fraction contribution of both forms can be expressed
via their excited concentrations, molar absorptivities, and
quantum yields from Eq. 2,
Combining Eqs. 7 and 9 gives Eq. 10 for measuring pH from
steady-stateparametersand fluorescencelifetimesdetermined
for each individual protonated and deprotonated species:
ꢀ
ꢁ
ꢀ
ꢁ
~
3A ꢂ jA
t ꢁ tA
pH ¼ pKꢃa ꢁ log10
ꢁ log10
:
~
3AH ꢂ jAH
tAH ꢁ t
(10)
½ fiꢄð3i ꢂ jiÞ
fi ¼
;
(2)
From here, we can postulate the expression of Henderson-
Hasselbach equation for lifetime measurements given in
Eq. 11. Good correlation between the predicted curve
from Eq. 11 and experimental data observed (Fig. S4)
support our postulate, with
n
P
½ fiꢄð3i ꢂ jiÞ
i
where [ fi] values are the concentrations of each component
in the excited state, fi is the fractional component obtained
from fluorescence n-exponential fit decay analysis, n is the
number of fractional components, 3i is the molar absorp-
tivity of each component at the wavelength of excitation,
and ji is the fluorescence quantum yield.
ꢀ
ꢁ
~
t ꢁ tA
pH ¼ pKꢃa;t ꢁ log10
;
(11)
~
tAH ꢁ t
where
Biophysical Journal 100(8) 2063–2072

Fluorescence Lifetime pH Probes
2071
ꢀ
ꢁ
substitution at the 5-position of the indole ring resulted in
a compound (LS479) with pKa ~ 4.9, which is less optimal
for biological imaging studies. However, shifting the substi-
tution to the meso-position produced a fluorophore (LS482)
with a pKa of 5.5. Additionally, LS482 demonstrated life-
time pH-sensitivity originating from distinct lifetimes for
the protonated (~1.16 ns in acidic DMSO) and deprotonated
(~1.40 ns in basic DMSO) components. Although the design
of the probes has to be further tuned to eliminate potential
complexation of the dyes with endogenous metals, the
feasibility of the lifetime sensitivity in vivo imaging was
demonstrated. The simulated mouse model was developed
and the lifetime values measured with FL-DOT system
were in a good agreement with the values obtained in
cuvettes. Such agreement provides a unique opportunity to
develop optimal NIR molecular probes and FL-DOT system
for detecting and diagnosing acidosis-related diseases.
3A ꢂ jA
pKꢃa;t ¼ pKaꢃ ꢁ log10
3AH ꢂ jAH
and where pK*a,t is the acid dissociation constant deter-
mined from lifetime measurements.
Similar to the acid dissociation constant for the excited
state pK*a, the pK*a,t depends on the fluorescence bright-
ness of the protolytic forms (3i ꢂ ji), and therefore on the
wavelength of excitation. For molecules with similar bright-
ness values, both pKa parameters become equal (pK*a,t
pK*a) and the pH of the unknown system can be determined
directly from the measured lifetimes.
z
Equation 11 has an important implication in predicting
whether the probe is suitable as a lifetime pH-sensitive
probe. If one of the protolytic forms is significantly shifted
(>200 nm as with norcyanines (50,51)), then the molar
absorptivity at the wavelength of excitation would be insig-
nificant (3i / 0), leading to a very large discrepancy
between pK*a,t and pK*a and loss of lifetime sensitivity.
Similarly, if one of the forms is nonfluorescent (quantum
yield ji / 0), the lifetime sensitivity is also negligible.
LS482 has a relatively small bathochromic spectral shift
upon protonation with both forms absorbing and emitting
within narrow NIR range, and hence exhibits fluorescence
lifetime sensitivity.
Molecular probes that meet the above requirements have
additional advantages for in vivo molecular imaging.
Because the spectral shifts are small, leading to significant
fluorescence spectral overlap between protonated and non-
protonated forms, in vivo ratiometric measurements will
be difficult. In this case, the unique characteristic of lifetime
parameter conveniently allows the two species to be delin-
eated with ease. Moreover, single excitation and detection
wavelengths can be used for imaging the pH status of tissue.
This strategy minimizes the complexity of instrumentation
needed for FL-DOT studies. It must be noted that the
absorption and emission properties of dye LS482 are not
optimal under the in vivo experimental conditions utilized
in this work. To optimize the fixed excitation wavelength
of our present FL-DOT system, future studies will use
pH-sensitive FLT dyes prepared for excitation at 785 nm.
An alternative strategy is to use lasers with different excita-
tion outputs, although such systems tend to be more
expensive.
SUPPORTING MATERIAL
One diagram and four figures are available at http://www.biophysj.org/
biophysj/supplemental/S0006-3495(11)00302-X.
This work was supported primarily by the National Institute of Biomedical
Imaging and Bioengineering under grant No. RO1EB007276, and in
part by other funds by the National Institutes of Health under grants
No. R01 EB008111, No. R01 EB001430, No. R01 EB008458, No. R01
CA109754, No. R33 CA123537, and No. R21 CA149814 as well as the
the NIH Roadmap for Medical Research as funding source for the
IPDC, and the Intramural Research Program of Eunice Shriver NICHD.
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