A Dual Fluorescence–Spin Label Probe for Visualization and Quantification of Target Molecules in Tissue by Multiplexed FLIM–EPR Spectroscopy

Article abstract of DOI:10.1002/anie.202012852

Simultaneous visualization and concentration quantification of molecules in biological tissue is an important though challenging goal. The advantages of fluorescence lifetime imaging microscopy (FLIM) for visualization, and electron paramagnetic resonance

Full text of DOI:10.1002/anie.202012852

A Journal of the Gesellschaft Deutscher Chemiker
Angewandte
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Accepted Article
Title: A Dual Fluorescence-Spin Label Probe for Visualization and
Quantification of Target Molecules in Tissue by Multiplexed FLIM
– EPR Spectroscopy
Authors: Pin Dong, Johannes Stellmacher, Lydia M. Bouchet, Marius
Nieke, Amit Kumar, Ernesto R. Osorio-Blanco, Gregor Nagel,
Silke B. Lohan, Christian Teutloff, Alexa Patzelt, Monika
Schäfer-Korting, Marcelo Calderón, Martina C. Meinke, and
Ulrike Alexiev
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To be cited as: Angew. Chem. Int. Ed. 10.1002/anie.202012852
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10.1002/anie.202012852
Angewandte Chemie International Edition
RESEARCH ARTICLE
A Dual Fluorescence-Spin Label Probe for Visualization and Quantification of Target
Molecules in Tissue by Multiplexed FLIM – EPR Spectroscopy
Pin Dong§[a,b], Johannes Stellmacher§[c], Lydia M. Bouchet§[d], Marius Nieke[c,e], Amit Kumar[d], Ernesto
R. Osorio-Blanco[d], Gregor Nagel[d], Silke B. Lohan[a], Christian Teutloff[c], Alexa Patzelt[a], Monika
Schäfer-Korting[b], Marcelo Calderón*[d,f,g], Martina C. Meinke*[a], and Ulrike Alexiev*[c]
Abstract:
Simultaneous
visualization
and
concentration
quantification of molecules in biological tissue is an important though
challenging goal. The advantages of fluorescence lifetime imaging
microscopy (FLIM) for visualization, and electron paramagnetic
resonance (EPR) spectroscopy for quantification are complementary.
Their combination in a multiplexed approach promises a successful
but ambitious strategy because of spin label-mediated fluorescence
quenching. Here, we solved this problem and present the molecular
design of a dual label (DL) compound comprising a highly fluorescent
dye together with an EPR spin probe, which also renders the
fluorescence lifetime to be concentration sensitive. The DL can easily
be coupled to the biomolecule of choice, enabling in vivo and in vitro
applications. This novel approach paves the way for elegant studies
ranging from fundamental biological investigations to preclinical drug
research, as shown in proof-of-principle penetration experiments in
human skin ex vivo.
Figure 1. Fluorescence label (Rhodamine B, RhoB) properties, intensity and
fluorescence lifetime, can be tuned to be both true concentration sensitive
when in conjugation with a spin label (3-Carboxy-Proxyl, PCA). Colors mark
the read-out parameters of each label. The resulting dual label (DL) probe
provides non-destructive target detection and quantification as well as
characterization of local environment in tissue via FLIM and EPR. Besides
tissue applications, a DL-labeled biomolecule enables in vitro spectroscopic
studies of the biomolecular conformation, structure, and dynamics.
resolved concentration quantification are highly desirable. While
EPR allows to quantify the absolute number of spin-labeled
molecules in tissue without homogenization and extraction, as
shown for nanocarriers and drugs in skin[2], it has a limited spatial
resolution of approximately 50-100 µm.[3] The use of conventional
Introduction
The quantification of molecules of interest in tissue usually
requires invasive procedures, including time-consuming steps of
tissue homogenization, and performing analysis such as chemical
extraction of the sample. Current methods in preclinical research
often lack a proof of translatability to human [1], which contributes
to the poor success rates in clinical studies. Method validation is
thereby often neglected while found to be essential as the real
concentration at the target site remains unknown.[1] Therefore,
spectroscopic techniques for direct, non-invasive, and spatially
fluorescence microscopy as
a direct quantitative tool for
concentration determination in tissue has been explored but
remains challenging, except in certain cases[4], mostly due to
scattering and absorption of the excitation and the emitted
fluorescence light, as well as the autofluorescence in the tissue.[5]
However, fluorescence lifetime imaging microscopy (FLIM) has
received a lot of attention in the recent years as a sensitive tool
for visualizing the spatial distribution of endogenous and
exogenous fluorescent molecules at the subcellular and
suborganelle level.[6] This sensitivity and signal specificity of FLIM
relies on the fluorescence lifetime as an intrinsic property of a
given fluorophore that is usually independent of fluorophore
concentration and optical loss in the sample[7], and inter alia
provides discrimination between background fluorescence from
molecules that naturally reside in tissue and the target
fluorescence.[5d, 8] Moreover, fluorescence lifetime is sensitive to
the environment[6b, 6c, 9], as is EPR.[10] EPR provides information
about the surrounding microenvironment of the spin-labeled
molecule, such as polarity, pH, and viscosity, which can be
inferred from the spectral shape, hyperfine coupling constant, g-
tensor, and rotational correlation time of the EPR signal.[2, 10-11]
The changes in fluorescence lifetime offer insight into the
microenvironment of the fluorophore, including pH, polarity,
viscosity, biomolecular interactions, or reactive oxygen species.[6,
9] However, this environmental sensitivity may also interfere with
concentration determination in FLIM.
[a]
Prof. Dr. M. C. Meinke, Dr. A. Patzelt, Dr. S. B. Lohan, Dr. P. Dong
Department of Dermatology, Venereology and Allergology, Charité
Universitätsmedizin Berlin, corporate member of Freie Universität Berlin,
Humboldt-Universität zu Berlin, and Berlin Institute of Health, Berlin,
Germany
E-mail: martina.meinke@charite.de
[b]
[c]
Prof. Dr. M. Schäfer-Korting, Dr. P. Dong
Freie Universität Berlin, Institute of Pharmacy, Berlin, Germany
Prof. Dr. U. Alexiev, J. Stellmacher, M. Nieke, Dr. C. Teutloff
Freie Universität Berlin, Institute of Experimental Physics, Department of
Physics, Berlin, Germany
E-mail: ulrike.alexiev@fu-berlin.de
[d]
Prof. Dr. M. Calderón, L.M. Bouchet, A. Kumar, E. R. Osorio-Blanco, G.
Nagel, Freie Universität Berlin, Institute of Chemistry and Biochemistry,
Berlin, Germany
[e]
[f]
M. Nieke
Humboldt-Universität zu Berlin, Institute of Biology, Berlin, Germany
Prof. Dr. M. Calderón
POLYMAT, Faculty of Chemistry, University of the Basque Country,
UPV/EHU, 20018 Donostia-San Sebastián, Spain.
E-mail: marcelo.calderon@polymat.eu
[g]
Prof. Dr. M. Calderón
Up to now, the biomolecule of interest in general is singly labeled:
IKERBASQUE, Basque Foundation for Science, 48013 Bilbao,
Spain.
P.D., J.S., and L.M.B. contributed equally.
Supporting information for this article is given via a link at the end of the
document.
a fluorophore label for studying biomolecular dynamics and
§
9d,
interactions[9a,
12], or visualization and tissue distribution by
FLIM[6c, 9c, 13]; and a spin label for quantification of target molecules
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10.1002/anie.202012852
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Figure 2. Scheme of DL synthesis. PCA (3) and Rhodamine B-piperazine (5) were covalently linked to Fmoc-Lys(Boc)-OH (1) yielding the DL conjugate (6). (2)
deprotected Lys(Boc)-OH, (4) Boc-Lys-PCA.
in tissue or their dynamics by EPR.[2, 10-11, 14] However, individual
labeling of the molecule of interest with a fluorophore or spin label
could change its physicochemical properties, such as the logP-
value, and thereby tissue penetration.[15] Combining the strength
of FLIM and EPR, we envisioned a dual label (DL) for multiplexed
FLIM and EPR measurements (Figure 1) overcoming those risks.
Herein, we developed a general platform for synthesizing a dual
label compound (DL), which realizes the simultaneous
investigation by FLIM and EPR. We present the design, synthesis
and photophysical properties of DL. The major challenge for a
dual fluorescence-spin label is fluorescence deactivation by the
unpaired electron of the spin label, a process which is usually
employed in fluorescence quenching studies.[9e, 16] We show that
evaluate the microenvironments of the DL in the different tissue
layers.
Thus, our novel platform for dual-labeled compounds will provide
unprecedented insight into the tissue distribution of biomolecules,
their concentration and microenvironment due to multiplexed
FLIM and EPR spectroscopy, both in fundamental and applied
research.
Results and Discussion
To develop a dual fluorescence-spin label compound for
accurate concentration determination and simultaneous
visualization of molecules in tissue (Figure 1), we first had to
establish a platform for synthesizing the DL. We describe the
synthesis and characterization of the tri-functional linker that
contains the spin label PCA, a fluorescent molecule, and a third
open labeling position for the conjugation to a biomolecule, drug
or nanocarrier of choice. As shown in Figure 2, the synthesis can
be split into two general processes: a) activation and conjugation
of PCA and b) modification and conjugation of a fluorescent dye
(the fully detailed synthesis conditions of each step can be found
in the SI Experimental procedures). Both, the conjugation of
PCA and the dye to lysine were conducted via a stable amide
linkage. In the first step, after Fmoc deprotection of 1 according to
in contrast to other tested spin label
- fluorescent dye
combinations, such as fluorescein and 3-Carboxy-Proxyl (PCA),
the combination of Rhodamine B (RhoB) and PCA (Figure 2)
yields high fluorescence and spin probe stability. Very importantly,
upon PCA conjugation RhoB is turned into a concentration
dependent fluorescence lifetime probe.
The characterization of this RhoB containing DL is further
accompanied by proof-of-principle experiments in skin tissue – a
tissue that is highly relevant in topical drug application and the
development of dermal and transdermal drug delivery systems.[2,
13b, 17] In the aged population, tissues including skin and mucous
membranes of the oral cavity are prone to tumors[9f, 18], but 96%
of anticancer drug candidates from preclinical studies fail in
human.[19] DL labeling for insight into spatially-resolved tissue
concentrations quantified by EPR and FLIM offers an approach to
method validation.[1] In particular, preclinical drug development
will take significant profit from a larger spectrum of analytical
methods with high spatial resolution and responding to
concentration and environmental changes.
literature (95% yield)[20]
, the NHS-activated PCA (3) was
introduced via amide bond formation to give 4 as a yellow oil (81%
yield). In the next step a fluorescent dye is covalently attached to
4. In a first trial, 6-amino-fluorescein was attached through an
amide bond to the carboxylic acid of compound 4, expecting that
the covalent attachment of 6-aminofluorescein yields
a
fluorescent conjugate.[21] Unfortunately, neither fluorescence
increase upon attachment nor an active spin-probe were
observed (Figure S1). This may be due to quenching effects on
the reactions of the PCA radical by the aminofluorescein
derivative. For a successful fluorescence labeling, we used a
piperazine linker between the fluorophore and the spin probe.
RhoB was functionalized with piperazine (5) to allow stable amide
bond formation with the COOH group of lysine and to avoid pH-
The experiments in tissue-mimicking agarose gel samples as well
as in skin tissue revealed a highly consistent determination of DL
concentration by both techniques, and the spatial distribution of
DL in different tissue layers. Furthermore, the combination of EPR
and FLIM in multiplexed FLIM – EPR experiments enabled us to
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10.1002/anie.202012852
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RESEARCH ARTICLE
driven cyclization that forms a non-fluorescent adduct. The
functionalization of 5 was performed using EDC-HCl, DIPEA, and
HOBt, during 16 h reaction at room temperature. Furthermore, the
fluorescent dye was covalently attached to 4. The DL (6) was
obtained as a purple solid (76% yield) after chromatographic
purification. Importantly, the tert-butanol ester bond in the DL
provides the third labeling position, after deprotection. All
1
characteristic signals of DL were identified in the H NMR and
mass spectra (Figure S2-11). The logP-value (log octanol-water
partition coefficient) of 0.07 ± 0.05 for DL reveals good water
solubility. Furthermore, we demonstrated the stability of DL
against endogenous tissue enzymes (Figure S12, Table S1).
In Figure 3, we present the spectroscopic characterization
of the DL and its precursors in aqueous solution. The addition of
the piperazine linker to RhoB at its carboxy-phenyl group (RhoB-
pip, 5) leads to a bathochromic shift compared to the RhoB
spectrum, yielding a ꢀꢁꢂꢃ ꢄ ꢅ567 ꢆ 1ꢇ ꢈꢉ and ꢀꢊꢋ ꢄ ꢅ592 ꢆ
1ꢇ ꢈꢉ (Figure 3A, B). A decrease of the fluorescence decay time
from ꢌ ꢄ 1.7 ꢈꢍ for RhoB[22] to ꢌ ꢄ 1.49 ꢈꢍ for RhoB-pip (5) was
observed (Figure 3C, Figure S13, Table S2) and consistent with
known effects on RhoB emission, quantum yield, and
fluorescence lifetime depending on the condition of the carboxy-
phenyl group.[23] The addition of the Boc-Lys linker and PCA
changed the absorption, emission, and fluorescence only slightly
(Figure 3A-C, Table S2). DL exhibits a ꢀꢁꢂꢃ ꢄ ꢅ566 ꢆ 1ꢇ ꢈꢉ and
ꢀꢊꢋ ꢄ ꢅ590 ꢆ 1ꢇ ꢈꢉ (Figure 3A, B), a fluorescence lifetime of ꢌ ꢄ
1.41 ꢈꢍ (Figure 3C, Table S2), and a quantum yield  = 0.18 ±
0.03 in water, which was determined using RhoB in ethanol[23a] as
reference. The main reduction of RhoB fluorescence in aqueous
solution stems from the conjugation to the piperazine linker
(Figure 3B). Compared to the RhoB fluorescence, the DL
fluorescence is reduced only by a factor of about two (Figure 3B),
taking the respective extinction coefficients of RhoB and DL into
account (SI Experimental procedures). Thus, DL exhibits good
fluorescence in phosphate buffered saline (PBS).
Figure 3D shows the EPR spectra of DL and PCA alone.
The spectra exhibit the typical three peaks of the nitroxide spin,
meaning that the chemical synthesis did not destroy the unpaired
electron of PCA. A quantitative EPR analysis reveals about 60%
of the spin labels being active after DL synthesis (SI
Experimental Procedures, Figure S14, S15). Since stability
tests of the spin label (Figure S16) show no further losses, the
DL concentrations from EPR in Figure 4 F-H were calculated by
taking the 60% active spin label into account (see SI
Experimental procedures). The high magnetic field peak of DL
had a lower peak-to-peak height in comparison to the unlabeled
PCA (Figure 3D). By simulating the EPR spectra of PCA and DL
(see SI Experimental procedures), the rotational correlation
time of DL was about ten times slower than for PCA free in
solution, indicating that the mobility of PCA in the DL was reduced
because of the neighboring covalently bound Fmoc or RhoB
moiety. Taken together, the results shown in Figure 3
demonstrate the establishment of a novel platform to synthesize
a functional dual fluorescence-spin label probe.
Figure 3. Characterization of DL in phosphate-buffered saline (PBS) solution
and in cryosections from tissue mimetics (agarose gels). (A) Normalized
absorbance spectra of Rhodamine B (Rho B), DL (6), Rhodamine B-piperazine
(5), and Boc-Lys-RhoB-piperazine for comparison. (B) Emission spectra of the
compounds shown in (A) at 10 or 12.6 µM (DL). (C) Fluorescence decay curves
of the compounds shown in (A). (D) EPR spectra of 504 µM DL (6) and PCA
dissolved in PBS. The correlation time is given. (E) Concentration dependence
of the fluorescence intensity of RhoB and DL from tissue mimetics using FLIM.
(F) Concentration dependence of the fluorescence lifetime of RhoB and DL in
cryosections from tissue mimetics using FLIM. The errors bars are ± SD (n= 3).
investigated the DL stability by comparing the absorbance and
emission of the DL solution under conditions mimicking the skin
penetration experiment (Figure S17). We found a decrease in
fluorescence intensity over time upon the exposure of the DL
solution to normal air, probably due to oxidation. Fluorescence
intensity of the DL decreased by a factor of Fr = 1.8 after 2 h of
incubation at 32 °C, while the fluorescence lifetime remained
unchanged (Figure S17). To correlate the fluorescence read-out
with concentration, we prepared varying concentrations of DL and
RhoB from 10 to 253 µM in agarose gels, which simulate tissue
samples (Figure S18). The fluorescence intensity increased
linearly with concentration for RhoB and DL (Figure 3E). The
fluorescence lifetime of RhoB was almost invariant to its
concentration in the range up to 250 µM, in agreement with
literature data for RhoB.[24] However, DL exhibited a shorter
fluorescence lifetime with increasing DL concentrations in the
same concentration range (Figure 3F and Figure S18). Thus, in
addition to FLIM-based visualization, we can utilize the DL
fluorescence intensity and lifetime as a concentration measure in
tissue. DL concentration was calculated from the fluorescence
intensity of the FLIM images according to equation 1:
Next, we performed proof-of-principle experiments in
human skin. The determination of DL concentration in skin via
fluorescence requires the following two factors to be known: a)
the stability of DL upon incubation with skin, and b) the correlation
of fluorescence intensity and lifetime with DL concentration. We
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ꢑꢒꢓꢔ/ #ꢕꢖꢗꢘꢙ ꢖꢈ ꢚꢛꢑ ꢜ ꢖꢉꢝꢞꢘ ꢕꢖꢗꢘꢙ
(Eq. 1),
ꢅ ꢇ
ꢑ
ꢎꢏꢐ
ꢄ
∗ MW ∗ ꢠ
ꢡ
ꢟ
where ꢑꢒꢓꢔ is the fluorescence intensity in the region of interest
(ROI), such as whole skin, viable epidermis or dermis, ꢢꢣ is the
molecular weight, and ꢟ and ꢤ are slope and intercept of a linear
fit of the concentration calibration data (Figure S18) with ꢤ ꢄ 0
for Eq. 1. The FLIM lifetime data were analyzed by a cluster
algorithm[5d, 8], which allows us to separate image pixel according
to discrete lifetime clusters. Using equation 2, we calculated the
concentration from the lifetime data:
ꢌ
ꢧꢨꢩꢃꢪꢊꢡ ꢫ ꢤ
ꢟ
ꢭꢮꢭꢝꢙ #ꢕꢖꢗꢘꢙ ꢖꢈ ꢚꢛꢑ
ꢦ
ꢬ ∗ #ꢕꢖꢗꢘꢙꢧꢨꢩꢃꢪꢊꢡ
(Eq. 2),
ꢎꢏꢐꢅꢌꢇ ꢄ
ꢥ
∗ ꢢꢣ ∗ ꢠ
ꢡ
ꢧꢨꢩꢃꢪꢊꢡ
where ꢌꢧꢨꢩꢃꢪꢊꢡ is the mean fluorescence lifetime of a given
fluorescence cluster (see SI Experimental procedures) and
#ꢕꢖꢗꢘꢙꢧꢨꢩꢃꢪꢊꢡ are the corresponding number of pixels of this cluster,
ꢟ and ꢤ are slope and intercept of a linear fit to the lifetime-based
concentration calibration data (Figure S18).
For efficient DL penetration in skin, barrier disrupted human
skin was used whose stratum corneum was removed by
cyanoacrylate glue (Figure S19-20). Figure 4A depicts the
sample preparation for FLIM and EPR measurements. In EPR,
whole tissue blocks were measured, while cryosections of the skin
sample were used for FLIM measurements. The EPR spectrum
of DL in skin represents a superposition of narrow and broad
spectral lines, indicating that the DL resides in different
microenvironments in the skin. Figure 4B-D visualizes DL skin
penetration using FLIM, providing a spatially resolved image of
DL distribution in the tissue (Figure 4D). Cluster-FLIM enhances
the image contrast as well as statistical significance[5d, 8], thereby
resolving detailed skin structures (Figure 4D). The highest DL
concentration is depicted by the red fluorescence decay cluster in
the false-color coded image with the fastest fluorescence decay
time, while the lowest concentration is shown in blue with the
slowest fluorescence decay time (Figure 4D, E). The
concentration gradient from red to blue as determined from the
calibration (Figure S18) follows the penetration profile, i.e., the
highest concentration is found in the upper layers of the viable
epidermis (VE), while low but significant concentrations are found
in the dermis. As faster fluorescence lifetimes are also found in
filament-like structures in the dermis (DE), presumably collagen
and elastin fibers, we assume DL accumulation at these sites.
The absolute concentrations of DL in whole skin as well as
in the viable epidermis and dermis (cVE and cDE, respectively) were
quantified by EPR and FLIM (Figure 4F-H). Both fluorescence-
based methods are in good agreement with each other and for
each donor in whole skin. We found the concentrations of DL in
the skin based on EPR and fluorescence approaches to be
consistent with each other within 2ꢯ, except for donor #3. At
lower DL concentrations within the dermis (Figure 4H), the
fluorescence lifetime-based concentration determination is in very
good agreement with EPR quantification. We also compared the
relative distribution of the DL concentration in the different skin
layers, i.e., viable epidermis and dermis, in a normalized plot of
concentration (Figure 4I, J). Here, the data reveal a very good
Figure 4. Localization and quantification of DL in human skin by EPR and
FLIM. (A) Sample preparation of skin after DL penetration, EPR measured
whole tissue blocks, while for FLIM measurements cryosections were used.
(B) Brightfield, (C) fluorescence intensity, and (D) FLIM image with
penetration profile. (E) Fluorescence lifetime curves from the FLIM cluster
image in (D). The concentration calibration is shown in Figure 3E,F and
Figure S18. Comparison of DL concentration in (F) whole skin, (G) viable
epidermis, and (H) dermis, determined by EPR and fluorescence lifetime and
intensity from FLIM. Error bars are ± SD (n = 4). (I) and (J) DL concentrations
from (G) and (H) normalized to whole skin.
agreement between EPR and FLIM in the viable epidermis for all
three donors (Figure 4I). We estimated that minimum
concentrations in the lower µM to 100 nM range can be
determined from EPR and FLIM using the DL.[9e, 25]
The apparent heterogeneous inter-donor penetration
behavior of donor #3 may be due to the fact that sample
penetration heterogeneities would be more pronounced in
cryosections, as used for FLIM, than in the integral concentration
determination in EPR (Figure 4A). Another reason could be
follicular penetration, which was so far unaccounted for in the
cryosections. As shown in Figure 5A-B, the follicular and
transfollicular penetration was significant (18-fold higher
concentration compared to surrounding tissue) for the barrier
disrupted skin used in this work. Compared to the FLIM
measurements on cryosections, the EPR measurements on
tissue blocks could involve DL accumulation within the hair
follicles and its surroundings. However, even if considerably
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RESEARCH ARTICLE
(see SI Experimental procedures)[27] of 9 ns, representing DL
with low mobility in a slightly apolar environment, compatible to a
restricted microenvironment with lipids, hydrophobic proteins, and
less water content. In the other microenvironment, DL had an azz
of 105 MHz and a smaller tcorr of 0.3-2 ns, indicating DL with high
mobility in a hydrophilic microenvironment (DLHydro). The tcorr of
DLHydro in the DE was shorter (0.3 ns) than in the VE (tcorr = 2 ns),
indicating a higher water content and thus enabling a higher
mobility. DLHydro was found with (4 ± 3)% in the viable epidermis,
and this fraction increased in the dermis (Figure 5F, Table S3).
The less hydrophilic environment in the viable epidermis is well in
line with previous data.[28] Thus, the environmental sensitivity of
the spin label was compatible with the main distribution of DL in
the viable epidermis and within dermal collagen, as shown by
FLIM (Figure 5C, D).
Conclusion
This work realizes the functional chemical synthesis of both a
fluorophore and a spin marker into one compound, that we call
dual label. This strategy provides the possibility of multiplexing
FLIM and EPR in tissue investigations, e.g. in skin penetration
experiments for drug delivery. The amount of DL penetrating into
different skin layers quantified by FLIM was in very good
agreement with EPR. The spatial distribution of DL based on FLIM
and its visualized accumulation sites yields a holistic image of the
DL diffusion and interaction pattern when combined with EPR
spectra simulations of DLs microenvironment. This is, for instance,
of utmost importance when investigating anticancer drugs in
preclinical studies. 3D tissue models, e.g. tumor models to
replace animal models are getting more important. Here, the dual
label could be a valuable tool to understand drug and carrier
distribution which could give information with respect to treatment
efficiency and development of treatment resistance; in particular,
for macromolecular drugs for which the penetration into tissue is
Figure 5. Sites of DL accumulation. (A) Brightfield and (B) FLIM images of a
hair follicle of the same ROI. (C) The viable epidermis and (D) the dermis
shown by FLIM. (E) EPR spectrum of DL in whole skin is a linear combination
of pure spectra of DL in a hydrophilic, fast mobility (DLHydro) or less polar, low
mobility environment (LME). (F) Experimental (solid lines) and simulated
(dotted lines) EPR spectra of DL in the viable epidermis (VE) and dermis
(DE). All spectra were normalized to the maximum peak intensity. The
fraction of DLHydro and the corresponding rotational correlation time (tcorr) in
different skin layers are indicated.
critical. In-depth insight into drug diffusivities and physical
[28]
parameters determining drug distribution in tissue
could be
obtained in the future from DL concentration data. Besides the
quantification and target detection in tissue and drug delivery, the
applications of combined FLIM and EPR measurements are
versatile and future applications are numerous. The DL platform
can be applied to any system, where spatial resolution combined
with knowledge about concentration and local environment is
required. For instance, the deactivation of the EPR label could act
as a sensor for the production of reactive oxygen species[29], while
the FLIM label allows the simultaneous localization of the DL
within the cellular environment.[30] Also, photodynamic therapy in
conjunction with the use of photosensitizers are feasible.[31]
Similarly, we envision the use of the DL compound in in vitro
experiments to provide new and unprecedented insight into
biomolecular dynamics, structure, and interactions. Thus, we
hope that this multi read-out labeling platform paves the way for
future studies in which the complementary information from FLIM
and EPR enables comprehensive characterization of any target
system.
higher DL (Figure 5A, B) can be found in hair follicles, they only
contribute to 1-14% of the skin surface depending on body site[26]
such that DL penetration would be increased by about 5%. Thus,
everything considered, our quantitative results of DL skin
penetration were consistent between EPR and FLIM.
,
Besides spin label counting, EPR provides information
about the microenvironment of the spin label. Combined with the
high spatial resolution and contrast enhancement of FLIM, we
were also able to obtain details about the environment of the DL
at the subcellular level. Figure 5C shows the localization of the
DL in the viable epidermis, both in the intercellular space and the
cytoplasm of keratinocytes, which was verified in monolayer
experiments (Figure S21). In the dermis, collagens and elastins
exhibit a notably strong DL fluorescence signal, while DL was not
identified in local skin cells, i.e., fibroblasts or macrophages
(Figure 5D). By simulating the EPR spectrum (SI Experimental
procedures, Table S3), DL was found to be surrounded by two
different microenvironments (Figure 5E).
In the first microenvironment, DL had a hyperfine coupling
Acknowledgements
constant (azz) of 100 MHz and a rotational correlation time (tcorr
)
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This work was financially supported by the German Research
Foundation (Collaborative Research Center 1112, Project B01 to
P.D., S.B.L, and M.C.M, B03 to U.A., C02 to M.S.K, and A04 to
M.C, and partially by Collaborative Research Center 1078,
Project A2 to U.A.). We would like to acknowledge the assistance
of the Core Facility BioSupraMol supported by the DFG and thank
Dr. A. Springer. M. C. acknowledges IKERBASQUE-Basque
Foundation for Science and MINECO project RTI2018-099227-B-
I00 for financial support. E.R.O.-B. gratefully thanks the Dahlem
Research School for a scholarship. P.D. thanks the China
Scholarship Councel for financial support.
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Keywords: multiplexed FLIM-EPR spectroscopy, concentration
determination in tissue, dual label, Rhodamine B, 3-Carboxy-
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This article is protected by copyright. All rights reserved.

10.1002/anie.202012852
Angewandte Chemie International Edition
RESEARCH ARTICLE
Entry for the Table of Contents
A novel multi-label synthetic platform for a functional dual fluorescence-spin label probe enables the non-destructive simultaneous
quantification and visualization of molecules in biological tissue. Multiplexed FLIM and EPR spectroscopy avoids analytical
inconsistencies between both techniques. Beside tissue applications, molecular spectroscopic studies of biomolecular conformation,
structure, dynamics, and microenvironment are feasible.
This article is protected by copyright. All rights reserved.