Journal of the American Chemical Society
Article
This function enables the separate emission collection at different and
limited wavelength ranges (for example, 10 nm) at each pixel. Each
wavelength range is given an image with a certain intensity and proper
pseudocolor as a function of fluorescence emission signal strength and
wavelength. As a result, by plotting pixel intensity versus wavelength,
the in situ fluorescence spectral profile of the dye can be readily
acquired. When all images consisting of different collected wavelength
bands were merged, a new image of nearly its real emission color (called
the real-color image) could be obtained. The real-color images and in
situ fluorescence spectra were acquired every 10 nm from 420 to 730
nm.
Cytotoxicity Measurements. The effects of PPC and EPC on cell
viability were carried out using the standard MTT assay. SiHa cells
growing in the log phase were seeded into 96-well plates (ca. 1 × 104
cells/well) and allowed to adhere for 24 h. Probes (100 μL/well) at
different concentrations (1, 4, and 10 μM) for 24 h or at 1 μM for
additional incubation times (2, 12, and 24 h) were placed in the wells of
the treatment groups, and 100 μL/well DMSO diluted in DMEM at
corresponding concentrations was added to the negative control group,
respectively. Then 10 μL of MTT was placed in each well. After
incubation for 4 h, the culture medium in each well was removed, and
DMSO (100 μL) was added to dissolve the purple crystals. After 20
min, the absorbance was measured at 570 nm with a microplate reader.
Finally, the cell survival rate could be calculated using the following
equation: survival rate = (Asample − Ablank)/(ADMSO − Ablank).
Tissue Staining. The various tissue blocks were directly removed
from just killed adult Wistar rats and placed at room temperature in H-
DMEM supplemented with 10% FBS and 1% penicillin and
streptomycin. Then, the tissue blocks were stained with SYTO 61
(10 μM, 30 min) and PPC or EPC (5 μM, 10 min) and imaged with a
Nikon A1R CLSM instrument. All of the above experiments were
performed according to the international, national, and institutional
rules concerning animal experiments, clinical studies, and biodiversity
rights.
Dynamic Monitoring of LD Formation. First, HeLa cells were
treated with 20 μM oleic acid and stained with 2 μM PPC or EPC
without washing. Then the cells were observed using confocal
microscopy in the Lambda mode. A real-color fluorescent image was
obtained every 10 s during a time course of 200 s.
structure, they could simultaneously label incellular LDs and the
ER. Among these probes, PPC snd EPC could achieve the two-
color fluorescence imaging of LDs and ER in the Lambda mode,
because of their supersensitive responses to slight water content
changes, resulting in distinct color changes revealed by the CIE
1931 color coordinates. Such remarkable sensitivity benefits
from their strong electron-donating substituents, which has been
1
demonstrated by the H NMR spectra and DFT calculations
using MNC and PC as control probes. As a result, LDs and the
ER in live cells and tissues have been clearly visualized by
utilizing PPC or EPC, revealing their close apposition as well as
specific morphology, volume, quantity, and distribution in
different tissues. To the best of our knowledge, this is the first
time that two-color fluorescence imaging of LDs and the ER in
live samples using SF-probes has been realized. Further, the
relationship between LD birth and the ER was investigated in
live cells via the in situ and real-time imaging experiments, which
reveal that the nascent LDs are generated near the ER regions
and share an identical fluorescence color with that of the ER,
probably providing a new and valuable proof for the hypothesis
that LDs originate in the ER. We expect that our probes PPC
and EPC can serve as novel and powerful imaging tools to
uncover further interplays between LDs and the ER and
decipher LD-ER related biological processes. Our work can also
shed new light on the design strategy of future fluorescent
probes for two-color imaging of other organelles and tracking of
their interactions using advanced Lambda mode based
fluorescnece imaging.
EXPERIMENTAL SECTION
■
General Information. Unless otherwise stated, all of the solvents
and reagents were commercially available and were used without
further purification. Silica column chromatography was conducted over
silica gel using the SepaBean machine U200 from Santai Technologies
Inc. MTT was purchased from Sigma. BODIPY 493/503, ER-Tracker
1
Immunofluorescence Method. HeLa cells were cultured on glass
coverslips for 24 h and then pretreated with 40 μM oleic acid for 2 h.
Next, the cells were fixed with 4% formaldehyde for 30 min,
permeabilized with saponin, and blocked with 10% goat serum. After
this, primary (antihuman TIP47 rabbit polyclonal antiserum,
abbreviated as anti-TIP47 antiserum) and secondary (Alexa Fluor
594-conjugated goat antirabbit IgG) antibodies were placed sequen-
tially in the cells. Finally, the cells were stained with 1 μM PPC or EPC
for 2 min and imaged with a Nikon A1R CLSM instrument.
Red, and SYTO 61 were purchased from Molecular Probes. The H
NMR and 13C NMR spectra were recorded on a Bruker AVANCE III
400 MHz digital NMR spectrometer. The geometrically optimized
structures of the molecules were calculated withthe Gaussian 09
package at B3LYP/6-31G level. The UV−visible−near-IR absorption
spectra of dilute solutions were recorded on a Hitachi U-2910
spectrophotometer using a quartz cuvette of 1 cm path length. The
fluorescence spectra were obtained on a Hitachi F-2700 spectro-
fluorimeter equipped with a 450 W Xe lamp. The spectral testing
concentrations of PPC, EPC, and MNC were 2 μM. The CIE
chromaticity coordinates were based on the spectral data. By calculation
of the wavelength and corresponding intensity, the CIE chromaticity
coordinates could be directly given by an FLS1000 photoluminescence
spectrometer.
Cell Culture and Fluorescence Imaging. HeLa and SiHa cells
were cultured in H-DMEM supplemented with 10% fetal bovine serum
(FBS) and 1% penicillin and streptomycin. Mesenchymal stem cells
(MSCs) were grown in alpha-MEM supplemented with 10% FBS and
1% penicillin and streptomycin. All of the above cells were cultured in a
5% CO2 incubator at 37 °C. Before cell staining, all cells were plated on
glass coverslips and allowed to adhere for 24 h.
For live cell staining experiments, the adherent cells were stained
with PPC, EPC, or MNC (1 μΜ, 2 min) and then washed once with
PBS and imaged. For costaining experiments, cells were stained with
BODIPY 493/503 (5 μM, 30 min) or ER-Tracker Red (5 μM, 30 min)
followed by rinsing twice with PBS; then the cells were further stained
with PPC or EPC (1 μΜ, 2 min), washed once with PBS, and imaged.
Confocal fluorescence pseudocolor images and real-color images
were obtained by a Nikon A1R confocal laser scanning microscope
(CLSM) in the multichannel or Lambda mode. The Lambda scan
mode is based on the spectral imaging function of confocal microscopy.
Synthetic Details. A mixture of compound 2 (0.5 mL, 4.1 mmol)
and the equivalent compound 1 (1a−d) was added to a solution of
EtOH (15 mL) in a flask. Next, 8 equiv of NaOH dissolved in 2 mL of
water was placed in the flask. After the mixture was stirred for 24 h at
room temperature, compound 3 (3a−d) was formed. Then 2 equiv of
NaOH dissolved in 2 mL of water and 3 mL of 30% H2O2 were further
added to this mixture. This mixture was stirred for 15 min at room
temperature and then heated to 50 °C for 5 h. After it was cooled to
room temperature, the reaction system was neutralized to neutral pH
and then extracted with CH2Cl2 and washed with water three times. A
yellow powder product was obtained after purification by silica gel
chromatography and recrystallization with absolute EtOH.
1
Data for PPC (1.33 g, 80%) are as follows. H NMR (400 MHz,
DMSO-d6): δ (ppm) 9.47 (s, 1H), 8.23−8.00 (m, 3H), 7.84−7.75 (m,
1H), 7.71 (d, J = 8.4 Hz, 1H), 7.46 (t, J = 7.5 Hz, 1H), 7.38 (t, J = 7.7
Hz, 4H), 7.19−7.09 (m, 6H), 7.05 (d, J = 8.7 Hz, 2H). 13C NMR (100
MHz, DMSO-d6): δ (ppm) 172.97, 154.90, 149.12, 146.86, 146.05,
138.75, 133.94, 130.26, 129.47, 125.95, 125.58, 125.20, 124.93, 124.68,
124.43, 121.87, 121.18, 118.71. HR-MS: calculated for C27H20NO3+ m/
z 406.1438 ([M + H]+), found 406.1451.
1
Data for EPC (0.99 g, 78%) are as follows. H NMR (400 MHz,
DMSO-d6): δ (ppm) 9.15 (s, 1H), 8.10 (t, J = 8.3 Hz, 3H), 7.80−7.68
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J. Am. Chem. Soc. 2021, 143, 3169−3179