Fluorescence Lifetime-resolved Ion-selective Nanospheres for

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Fluorescence Lifetime-resolved Ion-selective Nanospheres for Simultaneous Imaging of Calcium ion in Mitochondria and Lysosomes Shuai Zhou, Xiao Peng, Haiyan Xu, Yu Qin, Dechen Jiang, Junle Qu, and Hong-Yuan Chen Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.8b00735 • Publication Date (Web): 04 Jun 2018 Downloaded from http://pubs.acs.org on June 4, 2018

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Analytical Chemistry

Fluorescence Lifetime-resolved Ion-selective Nanospheres for Simultaneous Imaging of Calcium ion in Mitochondria and Lysosomes

Shuai Zhou1+, Xiao Peng2+, Haiyan Xu1, Yu Qin1, Dechen Jiang1*, Junle Qu2*, Hong-Yuan Chen1

1

State Key Laboratory of Analytical Chemistry for Life Science, School of Chemistry and

Chemical Engineering, Nanjing University, Nanjing 210093, China. 2

Key Laboratory of Optoelectronic Devices and Systems of Ministry of Education and

Guangdong Province, College of Optoelectronic Engineering, Shenzhen University, Shenzhen, 518060, China.

+ These authors equally contributed to this work.

Phone: 86-25-89684846(D.J); 86-755-26538584 (J.Q) Fax: 86-25-89684846(D.J); 86-755-26538580 (J.Q) Email: [email protected] (D.J), [email protected] (J.Q)

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Abstract: In this paper, novel calcium-selective nanospheres incorporating Pluronic® F127 and (4-carboxybutyl) triphenylphosphonium bromide (TPP) as shell layers were designed to monitor the level of free calcium ion in mitochondria and lysosomes at living cells simultaneously. TPP as a target for mitochondria drove the nanospheres to bind intracellular mitochondria, while, the lipophilic F127 layer resulted in the partial accumulation of nanospheres in lysosomes. This dual feature of the shell layer led to the co-location of nanospheres in both mitochondria and lysosomes. Chromoionophore III (ETH 5350) was chosen as the chromoionophore in the nanospheres that had different fluorescence lifetimes in either mitochondria or lysosomes, and therefore, the locations of the nanospheres at these two cellular compartments were identified.

After the

stimulation of cells using ionomycin, a burst of calcium concentration in mitochondria was observed that was associated with almost constant calcium concentration in lysosomes. The simultaneous recording of calcium ions in both of the compartments using fluorescence lifetime-solved nanospheres offered a special strategy to spatially monitor sub-cellular fluctuation of ions in living cells.


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1. Introduction. Cellular compartments are specialized subunits within cells that have their own special functions for cellular activity.

Classically, they exist in different cellular micro-environments

and establish an interconnected network of signaling pathways to sense and interpret the environmental changes.1,2 Free ions, such as calcium and zinc ions, are one of important species in these physiological processes.3,4

For example, extracellular calcium induces calcium release

from endoplasmic reticulum (ER) where emptying of the ER stores causes the opening of calcium channels in plasma membrane to promote refilling of the ER stores.5 Therefore, monitoring the spatiotemporal dynamics of these ions involved in intracellular signaling pathways/compartments is significant for the deep understanding of their biological roles. Currently, fluorescence imaging is popular to determine the localization, level and movement of ions at subcellular levels due to its high selectivity, sensitivity and spatial resolution.6-8 To enable the investigation calcium ion in different cellular compartments, multiple color variants of fluorescent proteins or organic dyes are developed.9,10

For higher spatial and temporal resolution,

the caged chemical compounds and optogenetic tools are attempted to achieve local light-induced activation and measurement.11-14

Although these significant progress has been made in the last

few years, the special design and synthesis of these organic dyes and genetically encoded proteins to simultaneously recognize ions in different types of cellular compartments are challenging and time-consuming.2

Therefore, developing a simple and general strategy to achieve spatial

monitoring of sub-cellular fluctuation of ions is urgent for the biological study. Ion selective nanospheres are new type of optical sensors for the detection of ions, which are consist of special ionophore, ion exchanger and chromoionophore in the core and Pluronic® F127 (polyoxyethylene-polyoxypropylene block copolymer) as the shell layer.15-20

The ionophores

with excellent selectivity are responsible to recognize the target ion. Upon the exposure of the nanospheres to the target ion, the ion exchange and the following binding of ion with the ionophore results in the release of hydrogen ions. Since the chromoionophore is a pH-dependent fluorescence indicator, the loss of hydrogen ion in the nanosphere induces the change of fluorescence intensity that is closely related to the ionic concentration.16

As compared with the

complicated synthesis of fluorescent probe, the nanospheres are formed spontaneously after the addition of water into tetrahydrofuran (THF) solution. In addition, the ionophores for most of



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ions and the chromoionophores are commercially available. Accordingly, these ions could be detected by simply choosing the proper ionophore in ion selective nanospheres.16 Based on this principle, our group has prepared and loaded calcium-selective nanospheres into the cells through free endocytosis21. The fluorescence intensity was correlated with the fluctuation of intracellular calcium in 24 h exhibiting the feasibility of these nanospheres for the continuous imaging of intracellular ions. The further development of spatial-resolved ion selective nanospheres will solve the challenging above mentioned. Here, fluorescence lifetime-resolved calcium-selective nanospheres were designed , as shown in Figure 1, to monitor the fluctuation of intracellular calcium in two cellular compartments, lysosome and mitochondria, simultaneously.

In this new design, calcium ionophore, ion

exchanger and chromoionophore were still incorporated as the core for the recognition of calcium. For special binding of nanosphere with mitochondria, triphenylphosphonium cation (TPP), a target for mitochondria, was linked with carboxylic functioned F127 at the outer layer of the nanosphere through the linker, polyetherimide (PEI).22 After the loading of these nanospheres into the cells, TPP at the surface of nanospheres induced the binding of some nanospheres with mitochondria. Meanwhile, insufficient TPP at the outer surface resulted in the exposure of lipophilic F127 layer to the cytosol. As a result, the other nanospheres were accumulated in the lysosomes.

To distinguish the location of the nanospheres in mitochondria or lysosome,

chromoionophore III (ETH 5350) was chosen as the chromoionophore that showed different fluorescence lifetimes in these two compartments.23

Eventually, intracellular calcium in

mitochondria and lysosomes could be monitored simultaneously using the fluorescence lifetime microscopy. The achievement of these spatial -resolved ion selective nanospheres should offer a general strategy for the measurement of intracellular ions in multiple cellular compartments.

2. Materials and Methods 2.1. Chemicals and Cell Culture Mito-Tracker Green, Hoechst 33342 and ionomycin were purchased from Beyotime Institute of Biotechnology (Nantong China). Lyso-Tracker Green DND 26 was purchased from Yeasen (Shanghai China). Spectra/Por(R) dialysis membrane (RC, MWCO = 50 KD, 28 mm width) and Spectra/Por(R) dialysis membrane (RC, MWCO = 10 KD, 24 mm width) were purchased from


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Sangon Biotech (Shanghai China). ETH5350 and the other chemicals were purchased from Sigma-Aldrich (Switzerland). Carboxylic functioned Pluronic F127 was kindly provided by Prof. Kai Xi (Nanjing University, China). Hela cells (Institute of Biochemistry and Cell Biology, Shanghai Institute for Biological Sciences) were cultured in high glucose Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum and 1% antibiotics (penicillin/ streptomycin) at 37℃ in a humidified incubator with 5% CO2. 2.2. Preparation of calcium-selective nanospheres. 8.0 mg of bis(2-ethylhexyl)sebacate (DOS), 5.0 mg of carboxylic F127, 1.2 mg of ETH 5350, 1.86 mg of cation-exchanger sodium tetrakis [3.5-bis(trifluoromethylphenyl) borate (NaTFPB), and 3.9 mg of calcium ionophore II were dissolved in 3.0 mL of THF to form a homogeneous solution. Then, 0.5 mL of solution was added into 4.5 mL of deionized water slowly on a vortex with a spinning speed of 800 r/min to form the nanospheres. Finally, the clear mixture was blown with N2 for 30 minutes to remove THF. 2 mL of resulting solution including nanospheres was mixed up with 20 µL of N-ethyl-N′(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC, 57.4 mM) and 20 µL of N-hydroxy succinimide (NHS, 60.8 mM) at room temperature for 1 h.24

In the mixture, the carboxyl group:

EDC: NHS was about 1: 20: 20. Then, 0.25 mL of PEI solution (PEI: F127 = 1:50) was added and the mixture was placed at room temperature overnight. Finally, the sample was dialyzed against water with a Spectra/Por dialysis membrane (MWCO = 50 KD) to remove non-cross-linked PEI and other small molecules. To form F127/PEI/TPP nanospheres, 100 µM TPP was activated by EDC/NHS following the reaction with F127/PEI nanospheres at room temperature overnight. The final product was dialyzed against water with a Spectra/Por dialysis membrane (MWCO = 10 KD) to remove the residual TPP. The density of nanospheres was estimated to be 30 µM according to the previous report.21 2.3 Characterization of nanospheres in the buffer. The morphology of synthesized nanospheres was recorded using JEOL JEM2800 transmission electron microscopy (Japan) operated at an acceleration voltage of 200 kV. The surface charge of nanospheres was measured using a Brookhaven ZetaPlus dynamic light



scattering instrument (DLS, Holtsville, NY).

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The formation of PEI-TPP structure was

determined using an UV spectrometer (Shimadzu, Japan).

The fluorescence intensity of

nanospheres solution was measured in extracellular buffer (ECB: 135 mM NaCl, 5 mM KCl, 10 mM HEPES, 2 mM MgCl2, and 2 mM CaCl2, pH 7.4) using a Hitachi fluorescence spectrometer (Japan). The viscosity of solution was adjusted by the addition of glycerol and measured using Brookfield DV-II viscometer (Brookfield engineering lab, USA). 2.4 Imaging of free calcium in mitochondria and lysosome using nanospheres. Hela cells were cultured in confocal dishes for 24 h initially.

After removing culture

medium, the cells were incubated in 10 mM phosphate buffer saline (PBS, pH 7.4) with 30 µM F127/PEI/TPP nanospheres for 1 h at 37℃. Then, the cells were recovered in high glucose DMEM containing 1% antibiotics at 37℃ overnight to achieve the distribution of nanospheres in both compartments.

To confirm the intracellular location of nanospheres, the cells were stained

with 100 nM Mito-tracker Green for 1h at 37℃ or 100 nM Lyso-tracker Green DND 26 for 1.5 h at 37℃. Before adjusting the level of free calcium inside the cells, the cells were placed in DMEM containing 1g/L D-glucose, L-glutamine, 110mg/L sodium pyruvate, 1% antibiotics and 2 mM CaCl2. Then, 10 µM ionomycin was added into the medium to elevate intracellular calcium concentration.25 2.5 Cytotoxicity of nanospheres Cells were incubated in 10 mM PBS containing 30 µM F127/PEI/TPP nanospheres (diluted in 10mM PBS, v/v = 1:1) for 1 h at 37 ℃ and then placed in serum-free DMEM at 37℃ overnight. After washed three times with 10 mM PBS, cells were treated with the medium containing Hoechest 33342 (5 µg/mL) for 20 min at 37℃. The control cells were loaded with Hoechest 33342 only. 2.6 Fluorescence image system. The fluorescence lifetime image was obtained from a self-assembled fluorescence lifetime microscopy and analyzed using TCSPC and SPCImage softwares.26

Confocal laser scanning

microscope (Leica SP3) equipped with an oil-immersion 63× objective was used to obtain fluorescence images. The excitation wavelengths were 488 nm for Mito-tracker Green and Lyso-tracker Green DND 26, and 633 nm for F127/PEI/TPP nanospheres. All cell images were


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analyzed by the ImageJ software.

3. Results and Discussion. 3.1 Characterization of nanospheres in the solution. The typical transmission electron microscopic (TEM) image of the prepared TPP/PEI/F127 nanospheres was shown in Figure 2A. The size of the nanospheres was 44.2 ± 7.7 nm. Since the attachment of PEI with F127 could induce the change of surface state from negative to positive charged, the surface charge at the nanospheres was evaluated.27,28

As expected, the surface

charge was changed from -27.2 ± 2.0 to 21.6 ± 3.6 mV, which suggested the assembling of PEI with carboxylic functioned F127 at the nanospheres.

After the introduction of TPP in the

preparation, a characteristic absorption peak from TPP at 270 nm appeared in the UV spectrum (Figure 2B) that supported the linkage of TPP at the nanospheres.29

All these results exhibited

the successful modification of PEI and TPP at the shell layer of nanospheres. The response of the synthesized nanospheres to aqueous calcium was characterized in ECB buffer. As shown in Figure 2C and Figure S1 (supporting information), a gradual decrease of fluorescence intensity was observed from the nanospheres in presence of aqueous calcium from 10 µM to 1 mM. This detection range was close to the feature from the unmodified nanospheres,21 which confirmed the monitor of free calcium using F127/PEI/TPP nanospheres.

The 95%

response time was measured to be 0.92 ± 0.21 s. This value was slightly larger than that from the unmodified nanospheres (0.74 ± 0.22 s) suggesting the minor hindrance of the ions into the bulk in presence of TPP and PEI layers.21

The other three nanospheres with two molar ratios of PEI and

F127 (1:10 and 1:100) and without PEI were prepared, respectively. Their responses to aqueous calcium were shown in Figure S2 (supporting information). As compared with the response from unmodified nanospheres, the addition of PEI/TPP at the nanospheres generated less fluorescence change. While, for F127/PEI/TPP nanospheres, the ratio of PEI and F127 at 1:50 during the preparation offered the highest fluorescence change. Therefore, this ratio was used as the optimized condition for the preparation of the nanospheres. Meanwhile, the selectivity of the nanospheres to different ions was tested and the results were shown in Figure 2D. In comparison with 10 mM aqueous Na+, K+ or Mg2+, the normalized fluorescence intensity change of the nanospheres to 50 µM Ca2+ was significantly higher. These



good sensitivity and unique selectivity guaranteed the application of this new ion selective nanosphere for the following intracellular calcium measurement. 3.2 Fluorescence lifetime of ETH5350. Since mitochondria and lysosomes have different pH, viscosity and the retention ability for the nanospheres, the fluorescence lifetimes of ETH 5350 in the solutions with various viscosities and pHs were investigated. Considering the viscosities of mitochondria and lysosome were 12 and 65 cp30,31, the viscosity of solution was adjusted to be between 3 and 92 cp by the addition of glycerol. As shown in Figure 3A, longer fluorescence lifetime from ETH 5350 was recorded in the solutions with low viscosity. Moreover, increasing the concentration of the dye provided a near-exponential-decay of the lifetime in Figure 3B. However, adjusting aqueous pH in ECB buffer did not have obvious influence on fluorescence lifetime of the dye, as shown in Figure S3A (supporting information). Therefore, variable lifetime of ETH5350 could be obtained in the solutions with different viscosity or the dye’s concentration. The more in-depth study was performed to investigate the fluorescence lifetime of ETH 5350 incorporated nanospheres under these conditions. Similar to the trends obtained at the dye, lowering the viscosity or elevating the concentration of the nanospheres led to longer fluorescence lifetime, as shown in Figure 3C and D. Also, no effect of aqueous pH in ECB buffer on the lifetime of nanospheres was exhibited in Figure S3B (supporting information). Therefore, for better distinction of nanospheres in these two compartments, they should be designed with less TPP at the surface so that abundant lipophilic F127 regions were exposed resulting in more accumulation of nanospheres in lysosomes. The concentrated nanospheres in lysosomes with low viscosity should exhibit a shorter fluorescence lifetime than they did in mitochondria. Eventually, these nanospheres in these two cellular compartments could be successfully distinguished. 3.3 Location of F127/PEI/TPP at mitochondria and lysosome. The loading of F127/PEI/TPP nanospheres into the cells was performed by the incubation of nanospheres with the cells at 37℃ for 1 h. After the recovery overnight, the bright-field and fluorescence images of the cells loaded with nanospheres were shown in Figure 4A and B. The fluorescence observed at the cells in Figure 4B confirmed the loading of the nanospheres into the cells.

No obvious increase in fluorescence intensity from Hoechest 33342 was observed from the


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cells loaded with the nanospheres suggesting low cytotoxicity of nanospheres32. To investigate the intracellular location of the nanospheres, the cells loaded with the nanospheres were stained with mito-tracker Green, and then, imaged in Figure 4C. The overlapping of Figure 4 B and C, as exhibited in Figure 4D, displayed that all mitochondria compartments were stained with the nanospheres.

Some additional strong fluorescence regions were observed, which might be

ascribed to the binding of lysosomes with nanospheres. To support this hypothesis, the other group of cells was co-stained with Lyso-tracker Green DND 26 and the nanospheres.

For better visualization of lysosomes with strong fluorescence,

the voltage of photomultiplier tube (PMT) at the confocal fluorescence microscope was lowered so that the fluorescence at mitochondria was weak.

As imaged in Figure 4 E-H, a good

overlapping between the fluorescence from nanospheres and Lyso-tracker Green inside the cells proved the location of nanospheres at the lysosomes, as well.

The strong fluorescence in

lysosomes supported more accumulation of nanospheres at lysosome than that in mitochondria, which facilitated the distinction of the nanospheres locations in the following analysis. The modification of sufficient TPP at the nanospheres was critical to drive their binding with mitochondria.

The control experiment was conducted at the nanospheres without the

modification of TPP.

The fluorescence images in Figure S4 A-D (supporting information)

exhibited that most of the nanospheres were retained into the lysosomes. When the ratio of PEI and F127 was 1:100, few nanospheres were observed to be retained at the lysosomes, as shown in Figure S4 E-H (supporting information). The continuous increase the ratio of PEI and F127 to 1:50 led to the distribution of the nanospheres in both of mitochondria and lysosome.

However,

the addition of more TPP at the nanospheres could not restrict all nanospheres in the mitochondria. With the ratio of PEI and F127 over 1:10, the significant cytotoxicity was observed. Therefore, the nanospheres with the maximal ratio of 1:10 were loaded into the cells and the distribution of the nanospheres in both of lysosome and mitochondria was still observed in Figure S4 I-L (supporting information). For the location of nanospheres in mitochondria or lysosomes, the fluorescence lifetime images were recorded at the cells labeled with mito-tracker Green/nanospheres or Lyso-tracker Green/nanospheres, as shown in Figure 5. Based on the location information provided from mito-tracker Green, the fluorescence lifetime of ETH 5350 in the nanospheres at mitochondria



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was between 2 and 2.3 ns; while the lifetime of ETH 5350 in lysosomes was mainly in the range of 1.2-1.5 ns. The shorter fluorescence lifetime observed in lysosomes was consistent with the data from the aqueous characterization, which was attributed to high viscosity of lysosomes and high concentrations of nanospheres accumulated in lysosomes. Accordingly, the nanospheres in mitochondria or lysosomes were distinguishable so that the simultaneous recording of calcium in both of the compartments was achievable. 3.4 Calcium fluctuation at mitochondria and lysosomes recorded by the nanospheres. Ionomycin is known to raise the intracellular level of calcium via allowing direct calcium influx across the cellular plasma membrane, which has been imaged using our previous nanospheres.21 The application of our new nanospheres should provide the information about the calcium fluctuation at these two compartments after the simulation.

Figure 6 A -C showed

real-time fluctuation of free calcium in mitochondria and lysosomes under the stimulation of cell using ionomycin. Figure 6 D displayed the fluorescence lifetime image of mitochondria and lysosomes, which were stained in purple/blue and yellow/green, respectively. Based on these locations, the fluorescence intensities at mitochondria and lysosomes after the stimulation were measured and listed in Figure 6E.

The drop in the fluorescence intensity at mitochondria

exhibited a burst of calcium after the stimulation, while, little change on lysosomal free calcium concentration was observed. The different trend of calcium level exhibited diverse responses of the two compartments after the stimulation, which was consistent with the literature.33, 34

As

compared with the other fluorescence probes to characterize the ion concentration at one special compartment, our nanosphere permitted the simultaneous recording of ion level in two compartments that should facilitate the study of calcium trafficking among the cellular compartments.

4. Conclusion. In this paper, fluorescence lifetime resolved calcium-selective nanospheres that could bind the mitochondria and lysosomes at living cells were successfully prepared to monitor the level of free calcium ion in these two compartments simultaneously. The different fluorescence lifetimes of ETH5350 incorporated nanospheres permitted the distinction of nanospheres within these two compartments, which provided a new strategy for the preparation of spatial-resolved ion selective


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probes for cellular detection.

Different trends of calcium level after the stimulation were

observed at mitochondria and lysosomes revealed the significance of subcellular observation. The future work will focus on the usage of other ionophores in the nanospheres for the detection of more types of ions within multiple cellular compartments.

Moreover, the optimization of

these nanospheres structure needs to be performed so that better visualization of calcium in these two compartments is realized.

Acknowledgements This work was supported by National key research and development program (2016YFA0201203), the National Natural Science Foundation of China (nos. 21575060, 31771584), Science Foundation of SZU (No.000193) and Key Laboratory of Analytical Chemistry for Life Science (nos. 5431ZZXM1803）

Supporting Information Available: This material is available free of charge via the Internet at http://pubs.acs.org. Relation of fluorescence intensity with calcium concentration, fluorescence lifetime of ETH 5350 and ETH 5350 incorporated nanospheres in buffer with different pHs, and the intracellular location of the nanospheres.

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Figure Caption. Figure 1. The preparation process of F127/PEI/TPP incorporated calcium-selective nanospheres. Figure 2. Characterization of F127/PEI/TPP incorporated calcium-selective nanospheres in the solution.

(A) TEM image of the nanospheres;

(B) UV spectrums of F127/PEI/TPP

nanospheres, F127/PEI nanospheres and TPP; (C) fluorescence spectrum of F127/PEI/TPP nanospheres exposure to ECB (pH 7.4) containing calcium with the concentrations of 10, 50, 100, 500 and 1000 µM; (D) the relative fluorescence unit (R.F. U) of the nanospheres exposed to multiple ions (50 µM Ca2+. 10 mM Na+, K+ or Mg2+).

The fluorescence response of the

nanospheres to 50 µM Ca2+ was normalized to be 100%. The error bar presented the standard deviation from three independent measurements. Figure 3. The fluorescence lifetime of (A) ETH 5350 and (C) ETH 5350 incorporated nanospheres in glycerol-water solution with different viscosity, and (B) ETH 5350 and (D) ETH 5350 incorporated nanospheres with different concentrations. The error bar presented the standard deviation from three independent measurements. Figure 4. Location of F127/PEI/TPP nanospheres in the cells. (A-D): Bright-field image of cells (A); fluorescence images of nanospheres (B) and mito-tracker Green (C) at the cells; (D) the overlapping image from B and C; (E-H): bright-field image of cells (E); fluorescence image of nanospheres (F) and lyso-tracker Green (G) at the cells collected using lower PMT voltage; (H) the overlapping image from F and G.

The voltage of PMT at the confocal fluorescence

microscope was lowered so that the fluorescence at mitochondria was weak in image E-H. Figure 5. (A, C) Fluorescence lifetime images of F127/PEI/TPP nanospheres in the cells. (B) fluorescence image of mito-tracker Green at the cells in image A; (D) fluorescence image of lyso-tracker Green at the cells in image C. Figure 6. (A-C) The selected fluorescence images of F127/PEI/TPP nanospheres in the cells before and after the simulation using 10 µM ionomycin; (D) the fluorescence lifetime image of F127/PEI/TPP nanospheres at mitochondria and lysosome; (E) the statistic change of fluorescence intensity at the mitochondria and lysosome before and after the stimulation. The error bar presented the standard deviation from four cells. .


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Figure 1.



Figure 2.

AA

3

F127/PEI/TPP nanospheres TPP F127/PEI nanospheres

B

Abs

2

1

0 230

50 nm C

330

430 530 Wavelength /nm

630

D

100 10 µM

200

R.F.U (%)

300 Fluorescence intensity /a.u.

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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1 mM 100

50

0

0 660

680 700 720 Wavelength /nm

740

Ca

K


Na

Mg

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Figure 3.

2.0

A

Fluorescence lifetime /ns

Fluorescence lifetime /ns

2.0

1.5

B 1.5

1.0

1.0 20

40

60

80

1

100

ƞ/cp 1.6

1.2

0.8

10 100 Conc/ µM

1000

2.0

C

Fluorescence lifetime/ns

Fluorescencence lifetime/ns

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


0

20

40

ƞ/cp

60

80

100

D 1.5

1.0 0.01

0.1

1 Conc /µM


10


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Figure 4.

A

B

10 µm

E

C

10 µm

F

10 µm

D

10 µm

10 µm

G

10 µm


H

10 µm

10 µm

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Figure 5.

B

A

1000-2500 ps

10 µm

C

10 µm

D

10 µm 1000-2500 ps


10 µm


Figure 6. A

B

0s

60 s

(A)

20 µm

20 µm

C

D 120 s

1000-2500 ps

20 µm

E 100 R. F. I (%)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 20 of 21

80 mitochondria lysosome

60 40 0

20

40

60

80

100 120 140

Time/s


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For TOC only


Fluorescence Lifetime-resolved Ion-selective Nanospheres for

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