Glucagon-secreting alpha cell selective two-photon fluorescent probe TP-α: For live pancreatic islet imaging

Article abstract of DOI:10.1021/ja5115776

Two-photon (TP) microscopy has an advantage for live tissue imaging which allows a deeper tissue penetration up to 1 mm comparing to one-photon (OP) microscopy. While there are several OP fluorescence probes in use for pancreatic islet imaging, TP imaging of selective cells in live islet still remains a challenge. Herein, we report the discovery of first TP live pancreatic islet imaging probe; TP-α (Two Photon-alpha) which can selectively stain glucagon secreting alpha cells. Through fluorescent image based screening using three pancreatic cell lines, we discovered TP-α from a TP fluorescent dye library TPG (TP-Green). In vitro fluorescence test showed that TP-α have direct interaction and appear glucagon with a significant fluorescence increase, but not with insulin or other hormones/analytes. Finally, TP-α was successfully applied for 3D imaging of live islets by staining alpha cell directly. The newly developed TP-α can be a practical tool to evaluate and identify live alpha cells in terms of localization, distribution and availability in the intact islets.

Full text of DOI:10.1021/ja5115776

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Article
Glucagon-Secreting Alpha Cell Selective Two-Photon
Fluorescent Probe TP-#: for Live Pancreatic Islet Imaging
Bikram Keshari Agrawalla, Yogeswari Chandran, Wut Hmone Phue, Sung-Chan
Lee, Yun-Mi Jeong, Si Yan Diana Wan, Nam-Young Kang, and Young-Tae Chang
J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/ja5115776 • Publication Date (Web): 13 Apr 2015
Downloaded from http://pubs.acs.org on April 20, 2015
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Glucagon-Secreting Alpha Cell Selective Two-Photon Fluorescent
Probe TP-α: for Live Pancreatic Islet Imaging.
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Bikram Keshari Agrawalla,♦ Yogeswari Chandran,♣ Wut-Hmone Phue,♦ Sung-Chan Lee,♣ Yun-
Mi Jeong,♣ Si Yan Diana Wan,♣ Nam-Young Kang ∗,♣ and Young-Tae Chang∗,♦,♣
♦Department of Chemistry & Medicinal Chemistry Program of Life Sciences Institute, National University of Singa-
pore, Singapore 117543
♣
Laboratory of Bio-imaging Probe Development, Singapore Bio-imaging Consortium, Agency of Science, Technolo-
gy and Research, Singapore 138667
Supporting Information Placeholder
cy. The conventional way of alpha cell imaging is the use of
ABSTRACT: Two-photon (TP) microscopy has an advantage
glucagon antibody for immunostaining or transgenic mouse
models which express a fluorescent reporter gene linked to
glucagon promoter.6 Furthermore, the metabolic status of
cells has been utilized for pancreatic islets studies by NADPH
auto-fluorescence imaging7 or cytosolic calcium imaging.8
Previously, our group reported an OP small molecule probe;
Glucagon Yellow (GY: Figure S2b) which stains alpha cells
selectively.9 Despite the fact that GY has alpha cell selectivity,
the negligible TP optical property (Figure S2C) has limited its
application as a TP imaging probe. As such TP microscopy
offers benefit over OP microscopy for deeper tissue imaging
due to the utilization of high power pulsed near-infra-red
excitation laser light.10 GY showed high photo bleaching
(Figure S2D), which further limits its suitability as a TP imag-
ing probe. Even though a few small molecule one photon
fluorescent probes has been published, till date no two-
photon alpha cell imaging probe has been reported for live
islet imaging.
for live tissue imaging which allows a deeper tissue penetra-
tion up to one millimeter comparing to one-photon (OP)
microscopy. While there are several OP fluorescence probes
in use for pancreatic islet imaging, TP imaging of selective
cells in live islet still remains a challenge. Herein, we report
the discovery of first TP live pancreatic islet imaging probe;
TP-α (Two Photon-alpha) which can selectively stain gluca-
gon secreting alpha cells. Through fluorescent image based
screening using three pancreatic cell lines, we discovered TP-
α from a TP fluorescent dye library TPG (TP-Green). In vitro
fluorescence test showed that, TP-α have direct interaction
with glucagon with a significant fluorescence increase, but
not with insulin or other hormones/ analytes. Finally, TP-α
was successfully applied for 3D imaging of live islets by stain-
ing alpha cell directly. The newly developed TP-α can be a
practical tool to evaluate and identify live alpha cells in terms
of localization, distribution and availability in the intact is-
lets.
INTRODUCTION
Pancreatic alpha cells secrete glucagon in response to low
blood glucose level. Glucagon counter-regulates the hypogly-
cemic effect of insulin by glycogenolysis and gluconeogene-
sis.1 Studies over the last decade showed that the human
islets not only have architectural differences with the well-
studied rodent islets, but also varied endocrine cell popula-
tion.2 These studies have revealed that the proportion of beta
cells in human islet is much less than the murine islets (ap-
proximately 55% versus 77%). On the contrary, the fraction
of alpha cells is greater in human islet than murine islet (ap-
proximately 35% versus 15%).2 Together, the higher alpha cell
proportion and the unique association of alpha and beta cells
in human islets have drawn more research interests to study
pancreatic alpha cells for understanding their function and
influence on diabetes. Although several beta cell imaging
probes are in use,3-5 imaging of alpha cells is still in its infan-
Scheme 1. Solid-phase synthesis of TPG library. (I) 80 different
aromatic and aliphatic amines (Table S1, Supporting Information)
loaded on to the resin: DIEA, NMP, 70° C, 12 h. (II) Acid-amine cou-
pling: HATU, DIEA, DMF/DCM, RT, 24 h. (III) Cleavage of final
compound: 5% TFA in DCM in RT for 1 h gives the final TPG library
compounds.
OP microscopy requires shorter excitation wavelength
which limits its application in live deep tissue imaging due to
shallow penetration depth of excitation light. On the contra-
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ry, TP microscopy utilizes two near-infrared photons as
RESULTS
source of excitation and offers superior advantages for live
tissue imaging by means of a higher depth of penetration,
localized area of excitation and long observation of items.10
TP imaging technique has been assessed for pancreatic islets
either with auto-fluorescence NADPH imaging7 or with vari-
ous hydrophilic and hydrophobic agents.11 Development of
more novel two-photon probes suitable for live islet imaging
will therefore be of great value to future diabetic research.
Development of Two-photon fluorescent dye library. Here-
in, we report the synthesis of two-photon fluorescent dye
library by solid phase combinatorial synthesis using 2-acetyl-
6-(methyl-amino) naphthalene (ACEMAN) core (Scheme S1
and S2). We chose the ACEMAN scaffold due to its well-
known TP property (TP fluorescent action cross section is
about 100 GM), reasonable OP fluorescent quantum yield
(50%) and lower molecular mass (200 Da.).12 The synthesis of
TPG acid was done in solution phase based on previous re-
port.12 Diversity was introduced to the TPG scaffold (Scheme
1) by using 80 chemically different amine building blocks
(Table S1). These diverse TP fluorescent dyes were generated
without predesigned specificity towards any particular target.
Hence, subsequent unbiased fluorescent image based screen-
ing with various panel of cell lines may lead to the discovery
of totally novel TP fluorescent probes. We named this set of
80 compounds as Two-Photon-Green (TPG; Figure 1A and 2A)
library because of their emission wavelength range (λabs/λem
= 370/510 nm, in PBS buffer). Detailed synthetic procedure
and building block structures with characterization are avail-
able in Supporting Information.
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In this study, we report the first TP fluorescent probe for
islet imaging which has a selectivity of alpha cell and it is
denoted by TP-α (Two Photon Alpha). Through a fluorescent
cell based screening of TP fluorescent dye library TPG, we
found TP-α which can selectively stains alpha TC1 cells
against beta TC6 and acinar cell.
Figure 2. One-photon and two-photon optical property of TP-α. (A)
One-photon absorption and emission spectra for TP-α 1 μM in PBS
buffer (pH = 7.4). (B) Two-photon action cross section of TP-α
measured in ethanol at 10 μM concentration.
Discovery of TP-α: an alpha TC1 selective probe. The
cell-based screening with fluorescent dye library has led to
the discovery of various cell-selective imaging probes.13-18 To
expedite the discovery of useful probes from the synthesized
TPG library, we setup a cell-based screening system with
three commercially available pancreatic cell lines; glucagon
secreting alpha TC1 cells, insulin secreting beta TC6 cells and
exocrine acinar 266-6 cells as a model system (Figure 1A). All
three cell lines were seeded into 384 well plates and allowed
to settle overnight before use. These cells were incubated
Figure 1. (A) Overview of image based screening for TPG library
compounds. Three pancreatic cell lines alpha TC1, beta TC6 and
acinar 266-6 cells were used for discovery of pancreatic two photon
imaging probe with 80 TPG fluorescent dyes, lead to the discovery of
alpha TC1 cell selective probe: TP-α. (B) Selective staining of alpha
TC1 cell by TP-α, in comparison with beta TC6 and acinar cells. Live
cells were stained with 1 μM TP-α and incubated at 37° C for 1 h be-
fore imaging. Images were captured with Image Xpress Micro system
after PBS wash. Fluorescence data collected at 450-500 nm upon
excitation at 370 nm. Scale bar 50 μm.
with 1 ꢀM of TPG library-80 compounds for 1 h before image
acquisition. Images were analyzed based on fluorescence
intensity to seek out the alpha cell selective probe. Primary
image analysis based on relative fluorescent intensity re-
vealed that one TPG compound shows higher fluorescent
signal with alpha TC1 cells (glucagon secreting) in compari-
son to beta TC6 (insulin secreting) and exocrine acinar cells.
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3500
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A
1 mg/mL Glucagon
H2N
O
O
N
N
0 mg/mL
TP-
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Wavelenght (nm)
B
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0.12 mg/mL
0.25 mg/mL
0.5 mg/mL
1 mg/mL
Fluorescence spectra of TP-α
With 1 mg/mL of analytes
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Glucagon
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Insulin and
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other analytes
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0
2
420 450 480 510 540 570 600
Wavelength (nm)
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0
-1
*
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Figure 3. In vitro fluorescence response of TP-α. (A) Fluorescence spectra of TP-α (10 μM) upon incubation with different concentration of glu-
cagon (from 0 mg/mL to 1 mg/mL) in 10 mM phosphate buffer (1% DMSO, pH 7.4) under excitation of 370 nm light. Insert is the structure of TP-
α. (B) Selectivity of TP-α (10 μM) for glucagon in comparison with insulin, other proteins and small molecule analytes at 1 mg/mL concentration.
Macromolecules: IgG (human), Catalase, Cytochrome C, Transferrin (holo), Myoglobin, RNA, Insulin (bovine), Insulin (human), Glucagon and
small molecule analytes; Acetylcholine, Dopamine, Glucose, Fructose, Adenine, Guanine, Uridine, Cytosine, Ascorbic acid, Folic acid, Sod. Py-
ruvate, Glutathione, Glutathione reduced, β-Nicotinamide adenine dinucleotide (NAD), Flavin adenine dinucleotide (FAD), and H2O2
(*Hydrogen peroxide 30% solution in water). Insert shows the selective increase in fluorescent emission of 10 μM TP-α with 1 mg/mL glucagon in
comparison to other analytes at 1 mg/mL concentration in PBS buffer. Values are represented as mean and error bars are standard deviation.
(n=3).
After secondary and tertiary imaging tests (see Figure S1) the
compound, TPG-456 (λabs/λem=370/510 nm, TP fluorescence
action cross section = 107 GM, OP quantum yield Φ = 0.69)
was selected to be the final hit for glucagon secreting alpha
TC1 cells (designated by TP-α: Figure 1A, 1B, and 2 in the
manuscript, Figure S1, S2, 1H, 13C NMR and HRMS spectra in
the Supporting Information). The MTT assay using alpha TC1
and beta TC6 cells revealed that the TP-α staining either at
working (1 μM) or higher concentrations (2 μM) had minimal
interference with the cell viability even up to 24 h (Figure S3).
With these experiments, we confirmed the selectivity and
non-toxicity of TP-α in alpha TC1 and beta TC6 cells in the
working concentration range of the probe.
glucagon (Figure 3A). TP-α showed concentration dependent
increase in fluorescence emission with glucagon (up to 9 fold
at 1 mg/mL, (Figure 3A, 3B) with a KD of 65 μΜ (Figure S10).
The selectivity test revealed that TP-α has higher fluores-
cence response with glucagon in comparison to insulin (Fig-
ure 3B). Similar in-vitro fluorescence test with various pro-
teins, neurotransmitters, and analytes also shows that, TP-α
has notable selectivity towards glucagon (Figure 3B). The
glucagon selectivity of TP-α further supports the selectivity
of the probe towards glucagon secreting alpha TC1 cells.
TP-α stains primary alpha cell selectively. The gluca-
gon and alpha TC1 selectivity of TP-α further inspire us to
evaluate the probe in mouse primary islet cells. Alpha cells
are an integral component of the pancreatic islet of Langer-
hans. Islet tissues are scarce, and represent only about 1-2%
of the whole pancreas.19,20 The scarceness of these cells in
In-vitro fluorescence response of TP-α. To elucidate the
selectivity of TP-α towards glucagon secreting alpha TC1 cells,
we performed an in-vitro fluorescence response test with
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mice pancreatic islet makes selective alpha cell imaging a
In order to assure that this mantle staining is due to the
more challenging task. To avoid practical troubles of isolat-
ing alpha cells from mouse pancreatic islets, we decided to
use whole islets for further studies. As the attachment of
islets to the culture dish is known to be difficult, partially
dissociated islet culture technique was adopted for im-
munostaining purpose.21 The partially dissociated islets took
over a week time in culture to spread out as monolayer.
Staining with TP-α showed brightly stained cells at the edge
of these cultured dissociated islets (Figure 4A, 5A-B). The
glucagon antibody staining confirmed that TP-α bright cells
are glucagon positive alpha cells (Figure 4B).
selective staining of alpha cells and not due to impermeabil-
ity of the fluorescent probe, we stained both intact islets and
dissociated islet cells with TP-α and confirmed with flow
cytometry. For both intact and dissociated islet staining con-
ditions, we found similar TP-α stains similar sub-population
of 18 ± 5%. This revealed that, TP-α staining of these cells is
because of its intrinsic selectivity towards glucagon secreting
alpha cells and not due to surface localization of these cells.
By far, this is the first demonstration where an alpha cell
selective small molecule fluorescent probe was used for two-
photon imaging of alpha cells in live pancreatic islet (Figure
5G, 5H).
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The flow-cytometry analysis of TP-α stained islet cells
showed 10~25 % brighter sub-populations (Figure S7, S8)
which is pertinent to the reported alpha cell population.2,19
For quantitative analysis, we performed flow cytometry ex-
periments of isolated mouse islet cells after double staining
of TP-α with either glucagon or insulin antibody. The majori-
ty of TP-α staining cells are overlapped with glucagon anti-
body positive cells with an accuracy of 96% (Figure 4B) con-
firming that TP-α stains glucagon secreting alpha cells.
Whereas, 99% of TP-α negative cells are glucagon antibody
negative.
Another mouse pancreatic alpha cell marker GABAA recep-
tor antibody22 showed a similar result with glucagon anti-
body (Figure 9A). When tested with insulin antibody, less
than 1% of the TP-α positive cells were positive to insulin
antibody, demonstrating the high selectivity of TP-α for glu-
cagon producing alpha cells over other islet cells. The double
negative sub-populations may represent non-alpha, non-beta
cell populations such as delta cells (Figure S9B).
TP-α stains alpha cells in live fresh islet. Finally, we
applied TP-α staining for alpha cell imaging on freshly iso-
lated pancreatic islet. TP-α stained pancreatic islets (Figure
5C-F) showed three-dimensional distribution of brightly
stained alpha cells on the surface of mice pancreatic islets in
live condition (Figure 5G, H). We found that, 1 μM TP-α
staining for 1 h was the optimal staining for both OP and TP
imaging (Figure 5, Figure S4). By evaluating the optical prop-
erty of TP-α using TP spectral images, we found 750 nm and
430-530 nm to be the most suitable excitation light and emis-
sion channels respectively (Figure S5 and S6) for TP-α imag-
ing. Likewise, TP optical sections of TP-α stained live islet
even at the middle plane (~50 μm depth from the bottom of
islet on the coverslip) showed good contrast between bright
and dim cells (Figure 5C-5H). The discontinuous distribution
of TP-α stained alpha cells at the mantle position on the
islets displayed the native distribution of alpha cells (Figure
5C-5H) and matches with previous report.23
Figure 4. Antibody confirmation of TP-α selectivity by imaging and
flow-cytometry. (A) Cultured dissociated islets stained with 1 μM TP-
α (in green) followed by immunostaining with glucagon antibody (in
red). Merged image demonstrates the TP-α stained cells to be gluca-
gon positive alpha cells (white arrow). Scale bar is 50 μm. (B) Dual
parametric dot plot for TP-α and glucagon antibody. About 17,000
islet cell counted.
DISCUSSION
New technological advancement in the TP microscopic
techniques has created a high demand for the development
of novel TP bio-imaging probes.10,24 Although the fluorescent
proteins have TP fluorescent excitation property,25 their ex-
pression in target cells is expensive and tedious. Hence, small
molecule fluorescent probes offer a better alternative. Cell
based screening of fluorescent compound libraries has re-
sulted in the discovery of various useful bio-imaging
probes.13-18 In the present study, we report the development
of ACEMAN based TP fluorescent dye library and the discov-
ery of a novel alpha cell selective probe, TP-α. The two-
photon alpha cell probe (TP-α) labels alpha cells quickly and
effectively in live conditions and offers significant advantages
over conventional glucagon antibody labeling which requires
fixations and permeabilization.
The surface localization of alpha cells makes them suscep-
tible to fall off during the enzymatic (collagenase p) tissue
digestion during islet isolation from the mouse pancreatic
tissue.24 When the isolated islets were stained with TP-α, we
found that the over digested islets do not have TP-α bright
cells on its surface. The simple TP-α staining can be a useful
tool to examine the presence or absence of alpha cells on the
isolated islets.
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Figure 5. TP-α selectively satins mouse pancreatic alpha cells in live islet. (A) Selective staining of alpha cell by TP-α in the cultured dissociated
islet cells. (B) Merged image with bright field showing both the TP-α bright alpha cell (white arrow) and TP-α dim other islet cells (red arrow).
Scale bar 15 μm. (C) Overlay of bright field and TP fluorescence image of TP-α stained islets. Shows distribution of TP-α brightly stained alpha
cells on the edge of the islet. Arrow head indicates absence of alpha cells in the mantle position. (~50 μm depths representative images). (D) For
control DMSO was used. Scale bar 50 μm. (E) and (F) TP fluorescent optical section images of individual TP-α stained live islets. (G) 3D Surface
projection of the two-photon z-stack images for TP-α stained live islet (step size, 0.17 μm) using image processing software IMARIS (Supporting
Video S1). (H) Representative sectional overlaid TPM images of TP-α stained live islet at the depth range of 5 μm to 105 μm. It reveals the natural
distribution of alpha cells in the mouse pancreatic islet in live condition. Two-photon imaging was performed on 16 weeks old mouse pancreatic
islet. All images were taken with 40X water immersion objective using Leica TCS SP5X MP, Ex: 750 nm femtosecond (fs) laser light, Em: 430-530
nm (images are represented from n = 30 islets).
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The two-photon alpha cell probe (TP-α) labels alpha cells
CONCLUSION
quickly and effectively in live conditions and offers signifi-
cant advantages over conventional glucagon antibody label-
ing which requires fixations and permeabilization. TP-α ena-
bles real-time staining and observation of alpha cells in their
inherent condition in live intact islet with TP imaging. Since
TP-α have emission ranging from 430 nm to 530 nm, it can
be combined with other fluorescent proteins with yellow or
red emission in islet for dual color imaging.
In conclusion, we have synthesized a TP fluorescent dye li-
brary TPG-80 compounds using solid support. The image-
based screening of this library led to the discovery of a novel
TP imaging probe TP-α for live alpha cell imaging in intact
mice islets. TP-α is suitable for both TP islet imaging as well
as the flow cytometry study of scarce primary alpha cells
from mice pancreatic islet tissue. Furthermore, in vitro fluo-
rescence response test of TP-α with 23 relevant biological
analytes displayed an excellent selectivity towards glucagon.
The glucagon and alpha cell selective TP-α may serve as a
useful tool in pancreatic islet research for monitoring the
survival or distribution of alpha cells during islet isolation
and transplantation procedure. To facilitate dual color two-
photon imaging of pancreatic islets more strategic designs
are underway in our group to develop two-photon beta cell
imaging probe.
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TP-α was discovered from image based screening of fluo-
rescent dye library: TPG (containing 80 compounds) with
glucagon secreting alpha TC1, insulin secreting beta TC6 and
exocrine acinar 266-6 cell lines, based on relative fluorescent
intensity. TP-α stained glucagon secreting alpha TC1 cells,
showed significantly stronger fluorescent signal compared to
other cells. From the in-vitro fluorescence test we found that,
TP-α has more than 8 fold fluorescence intensity with Glu-
cagon in comparison with Insulin and other pancreatic sig-
naling molecules like acetylcholine,26 dopamine (Figure 3B).
This in vitro fluorescence study with total 23 diverse biologi-
cal analytes revealed excellent glucagon selectivity of TP-
α (Figure 3B). To demonstrate the suitable application for
the glucagon selective TP-α, we envisioned to achieve selec-
tive staining of glucagon producing alpha cells. The pancre-
atic alpha cells are an integral component of islets and con-
stitute a scarce sub-population of it (10-25%). Thus, we de-
cided to isolate the pancreatic islet and stain them with TP-α
directly. We found that 1 h incubation with 1 μM TP-α gives
the best alpha cell selective staining in live fresh islets (con-
firmed by immunostaining, Figure 4). As anticipated, the TP
imaging of TP-α stained islets has revealed the three dimen-
sional distribution of alpha cells in the live islets.
MATERIALS AND METHODS
1
Characterization of TP-α. H NMR TP-α (TPG-456) (300
MHz, CD3OD), δ = 8.33 (dd, J=18, 1.8 Hz, 1H), 7.84 (dd, J=
10.5, 1.8 Hz, 1H), 7.80 (s, 1H), 7.56 (dd, J=8.4, 1.2 Hz, 1H), 7.05
(dd, J=9, 2.1 Hz, 1H), 6.95 (m, 1H), 6.85 (d, J=1.5 Hz, 1H), 6.74
(d, J=1.5 Hz, 1H), 4.44 (s, 2H), 3.16 (s, 3H), 3.08 (s, 5H), 2.64
(s, 3H), 2.11 (m, 4H), 1.26 (s, 6H), 1.17 (m, 14H), 1.11 (s, 3H),
0.966 (s, 3H). 13C NMR TPG-M456 (75 MHz, CD3OD), δ =
198.22, 170.60, 161.26, 160.81, 149.03, 146.33, 145.63, 144.95,
133.15, 129.82, 125.57, 125.19, 124.43, 122.86, 117.86, 114.97,
114.00, 104.52, 59.18, 39.95, 39.22, 38.97, 37.24, 36.95, 36.76,
36.47, 36.19, 35.24, 34.54, 32.64, 29.09, 28.60, 28.41, 24.48,
23.47, 22.45, 18.49, 16.09. ESI-MS m/z (M+H) calculated:
582.39, found 582.20. ESI-HRMS of TP-αm/z (M+H) calculat-
ed: 582.4060, and found 582.4062 (ESI HRMS Supporting
Information).
Till date, the reported methodology for TP islet imaging is
based on metabolic imaging (with glucose or other stimula-
tion conditions).7,8 Piston DW et al. has reported the TP im-
aging of islet for increased NADPH monitoring in beta cells
with glucose stimulation.7,27-29 However the NADPH TP im-
aging of islets can help visualize beta cells, but not alpha cells.
Whereas, cytosolic calcium imaging of islets with Indo-1
staining followed by glucose or amino acid stimulus condi-
tions can differentiate the alpha and beta cells.8 But the cal-
cium indicator Indo-1 has poor photo-stability30 and the pho-
to degradation of Indo-1 leads to the generation of a fluores-
cent but calcium insensitive probe.30 Additionally Indo-1
stains all the islet cells and needs a different stimulus condi-
tion to differentiate alpha and beta cells.8 However, TP-α can
directly and selectively stain only the alpha cells in live islets.
Also the selective staining of alpha cells with TP-α does not
require any glucose or other stimulation. Glucagon yellow is
another alpha cell selective one-photon fluorescent probe
published earlier from our group. However, while evaluating
the optical property we found that, GY have negligible TP
action cross-section in comparison to TP-α (Figure S2A). The
excellent photo stability of TP-α in contrast to GY (Figure
S2D) makes TP-α a more valuable TP alpha cell imaging
probe.
Quantum yield measurements. Quantum yield were calcu-
lated by measuring the integrated area of emission spectra
for TPG library compounds (including TP-α) in comparison
to the same measurement for Coumarin-1 (φ=0.58) in DMSO
as reference compound.31 Both TPG library compounds and
Coumarin 1 were excited at 370 nm and emission spectra
were collected from 420-600 nm. Quantum yields were cal-
culated using equation -1, where Φ is the quantum yield, F is
the area of fluorescent emission, η is reflective index of the
solvent, and Abs is absorbance at excitation wavelength se-
lected for standards and samples. Emission was integrated
between 420 to 600 nm.
F(sample)
η
(sample) Abs (ref)
=
Φ(sample)
(1)
Φ(ref)
F(ref) η (ref) Abs (sample)
In vitro fluorescence screening. TP-α (10 μM) was used for
fluorescence property measurement and in vitro screening in
10 mM phosphate buffer (pH 7.3, 1% DMSO). Fluorescence
intensity was measured using Spectra MaxM2 plate reader in
96 well plate. We used 370 nm wavelength light for excita-
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tion and emission was observed from 420 nm to 600 nm. All
The cells were incubated with 0.5 and 1 μM of TPG library-80
analytes were tested in four concentrations with serial dilu-
tion ranging from 1 mg/mL to 0.12 mg/mL. We used both
macromolecules: IgG (human), Catalase, Cytochrome C,
Transferrin (holo), Myoglobin, RNA, Insulin (bovine), Insulin
(human), Glucagon and small molecule analytes: Acetylcho-
line, Dopamine, Glucose, Fructose, Adenine, Guanine, Uri-
dine, Cytosine, Ascorbic acid, Folic acid, Sod. Pyruvate, Glu-
compounds for 1 h. Wells were washed one time with fresh
media to reduce the background before image acquisition.
Automated imaging system ImageXpress® Micro with 10X
objectives was used to capture the images. Images were ana-
lyzed based on fluorescence intensity to seek out the alpha
cell selective probe. An alpha cell selective fluorescent probe
will show higher fluorescent signal in the alpha TC1 cells in
comparison to beta TC6 and acinar cells. TPG-456 (TP-α)
showed consistently higher fluorescent signal in alphaTC1
cells then the beta and acinar cells in the secondary and ter-
tiary tests (see Figure S1).
9
tathione, Glutathione reduced, β-Nicotinamide adenine di-
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nucleotide (NAD), Flavin adenine dinucleotide (FAD), and
30% Hydrogen peroxide in the selectivity test. After the addi-
tion of dye the 96 well plates were incubated on ice for 30
min before fluorescence measurement.
OP and TP imaging. All confocal and TP imaging was done
using Leica TCS SP5X MP (Leica Microsystems CMS GmbH,
Mannheim, Germany) with 405 nm laser light for OP excita-
tion and 750 nm femtosecond (fs) pulsed laser for TP excita-
tion for TP-α. Images were taken using 10X dry, 40X water
and 100X oil objectives. The TP excitation of TP-α was
achieved by titanium-sapphire laser light set at wavelength
750 nm and an output power of 2710 mW. To obtain the
probe signal, internal photomultiplier tube was set at 430 to
530 nm. Image analysis and intensity measurements were
carried out by Leica Application Suite Advanced Fluores-
cence (LAS AF). 3D projection of Z-stack TP images were
processed with IMARIS software (Bitplane AG, Switzerland)
and the intensity is depicted as false color.
Two-photon absorption cross sections measurement. The
TP emission spectra of TPG library compounds were deter-
mined over a broad spectral region by the two-photon in-
duced fluorescence (TPF) method with Rhodamine B in
methanol as a reference. A PTI, Quanta MasterTM spectro
fluorimeter and femtosecond (fs) Ti: Sapphire laser (Mira
900F, 220 fs pulse width, 76 MHz repetition rate, tuning
range 740 - 840 nm, Coherent (USA) were used. Two-photon
fluorescence measurements were performed in 10 mm fluo-
rometric quartz cuvettes with 10 μM TP-α in Methanol, and
10 μM Rhodamine B as reference in same solvent. The exper-
imental fluorescence excitation and detection conditions
were conducted with negligible reabsorption processes
which can affect TPA measurements. The two-photon ab-
sorption cross section of the probes was calculated at each
wavelength according to equation 2.
Islet isolation and imaging. Primary pancreatic islets were
isolated from the 16 week old C57BL/6 (WT) male mice. The
animal handling and tissue harvesting is in accordance with
the Institutional Animal Care and Use Committee of Singa-
pore Bio-imaging Consortium (Agency of Science, Technolo-
gy and Research, Singapore). Pancreatic islets were isolated
by Collagenase P digestion and islet picking methods as de-
scribed earlier.21 Briefly, fresh pancreas was cut into small
pieces and digested with 1 mg/mL collagenase P (Roche, In-
dianapolis, IN, USA) solution in Hank’s Balanced Salt Solu-
tion (HBSS; Invitrogen) for 10 minutes at 37° C on a shaker,
followed by 4 times buffer washing to remove the digested
exocrine tissue. Manual picking of pancreatic islets was car-
ried out using Zeiss Stemi DV4 stereomicroscope. Islets were
maintained in DMEM with 4500 mg/L glucose + 10% FBS and
1% penicillin-streptomycin (GIBCO, Life Technologies,
Carlsbad, CA, United States) for 24 h before TP imaging.
Islets were stained with 1 μM TP-α for 1 h at 37° C and were
transferred to fresh media for TP image acquisition.
2
2
Φ(ref) I(sample) C(ref) η(sample) P(ref)
=
δsample δreference
(2)
2
2
Φ(sample) I(ref) C(sample) η(ref) P(sample)
Where I is the integrated fluorescence intensity, C is the
concentration, η is the refractive index, Φ is the quantum
yield, and P is the incident power on the sample, subscript
‘ref’ stands for reference samples, ‘sample’ stands for samples.
The uncertainty in the measured cross sections was about ±
10%.
Cell culture condition for screening. Insulin producing be-
ta-TC6 (ATCC® CRL-11506™), glucagon producing alpha TC1
clone 6 (ATCC® CRL-2934™) and exocrine acinar cells 266-6
(ATCC® CRL-2151™) were obtained from the American Type
Culture Collection (ATCC) and maintained according to
ATCC protocols. Beta and acinar cells were cultured using
Dulbecco’s Modified Eagle’s Medium (DMEM) with 4500
mg/L D-glucose, supplemented with 10% fetal bovine serum
(FBS) and 1% penicillin-streptomycin. Alpha TC1 cells were
cultured in DMEM with 1000 mg/L D-glucose, supplemented
with 15% heat inactivated FBS, 2.0 g/L D-glucose, 15 mM
HEPES, and 0.1 mM non-essential amino acids (GIBCO, Life
Technologies, Carlsbad, CA, United States).
Dissociation and culture of islet. Primary pancreatic islet
cells were dissociated by trypsinization and trituration
method.21 Approximately 50 - 100 islets were dissociated by
incubating with 0.25% Trypsin-EDTA (1X), phenol red
(GIBCO) at 37° C for 2 minutes followed by trituration with
repeated pipetting. Finally, the dissociated islet cells were
transferred to the 4500 mg/L D-glucose DMEM with 10% FBS
and 1% penicillin-streptomycin (GIBCO) for culture. Dissoci-
ated cells were cultured for 1 week with media change every
2-3 days. The cells were stained with 1 μM TP-α for 1 h at 37°
C and were transferred to fresh media before TP image ac-
quisition.
Image-based screening and hit selection. A primary screen-
ing platform was established with the alpha TC1, beta-TC6
and acinar 266-6 cells. All three cell lines were seeded into
384 well plates and allowed to settle overnight before use.
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Immunostaining. Primary cells and islets were fixed in 4%
with editing the manuscript. The author also thanks to Ms
Paraformaldehyde and permeabilized with 0.1% Triton-X 100.
Fixed Cells were identified by primary antibodies for over-
night at 4° C at the respective dilutions. For alpha cell - Glu-
cagon antibody (Monoclonal Anti-Glucagon antibody pro-
duced in mouse, G2654, Sigma-Aldrich 3050 Spruce St. St.
Louis, USA) 1:2000. Another mice pancreatic alpha cell
Tong Yan and Keshmarathy D/O Sacadevan of NUS Centre
of Bio-Imaging Science; for Two-Photon imaging facility.
This study was supported by intramural funding from
A*STAR (Agency for Science, Technology and Research, Sin-
gapore) Biomedical Research Council, and Oasis Grant from
KRICT, South Korea.
8
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marker GABAA receptor antibody (Anti-GABA A Receptor β
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9957;
e-mail: kang_nam_young@sbic.a-star.edu.sg
Professor Young-Tae Chang, S9-03-01, 3-Science Drive 2, De-
partment of Chemistry, National University of Singapore,
Singapore 117543; Tel: (+65) 6478 8736; Fax: (+65) 6478 9957
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